<%BANNER%>

Description of Osteoprogenitor Gene Expression in Periodontal Soft Tissues

Permanent Link: http://ufdc.ufl.edu/UFE0022238/00001

Material Information

Title: Description of Osteoprogenitor Gene Expression in Periodontal Soft Tissues
Physical Description: 1 online resource (48 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: bone, cells, osteoprogenitor, periodontal, periodontics, stem
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Recent work has shown that cells outside of periodontal ligament, including those found in granulation tissue may also have the regenerative potential to induce new bone formation by the expression of specific proteins and transcription factors.1 It was therefore the purpose of this investigation to determine if periodontal granulation tissues possess the specific proteins and gene expression pattern necessary for osteoblast differentiation in an effort to determine if this granulation tissue should therefore be retained in the surgical treatment of periodontal defects. In order to accomplish this, granulation tissue and healthy gingival tissue samples were harvested during periodontal surgeries, after which, polymerase chain reaction (PCR) was used to determine the expression of BMP-2, Cbfa1 and Osterix; three genes involved in bone development. In order to demonstrate a potential model of how these genes could be regulated/modulated under inflammatory conditions, an in-vitro stimulation assay using a human monocytic cell line (THP-1) along with a source of BMP-2, specifically an osteosarcoma cells line (SoaS) was also performed. Our results demonstrate the genes for BMP-2, Cbfa1 and Osterix are expressed at similar levels in both granulation as well as healthy gingival tissues. Furthermore, the in vitro stimulation assay was unable to demonstrate that under inflammatory conditions these genes were modulated.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Aukhil, Ikramuddin.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022238:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022238/00001

Material Information

Title: Description of Osteoprogenitor Gene Expression in Periodontal Soft Tissues
Physical Description: 1 online resource (48 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: bone, cells, osteoprogenitor, periodontal, periodontics, stem
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Recent work has shown that cells outside of periodontal ligament, including those found in granulation tissue may also have the regenerative potential to induce new bone formation by the expression of specific proteins and transcription factors.1 It was therefore the purpose of this investigation to determine if periodontal granulation tissues possess the specific proteins and gene expression pattern necessary for osteoblast differentiation in an effort to determine if this granulation tissue should therefore be retained in the surgical treatment of periodontal defects. In order to accomplish this, granulation tissue and healthy gingival tissue samples were harvested during periodontal surgeries, after which, polymerase chain reaction (PCR) was used to determine the expression of BMP-2, Cbfa1 and Osterix; three genes involved in bone development. In order to demonstrate a potential model of how these genes could be regulated/modulated under inflammatory conditions, an in-vitro stimulation assay using a human monocytic cell line (THP-1) along with a source of BMP-2, specifically an osteosarcoma cells line (SoaS) was also performed. Our results demonstrate the genes for BMP-2, Cbfa1 and Osterix are expressed at similar levels in both granulation as well as healthy gingival tissues. Furthermore, the in vitro stimulation assay was unable to demonstrate that under inflammatory conditions these genes were modulated.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Aukhil, Ikramuddin.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022238:00001


This item has the following downloads:


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101109_AAAACV INGEST_TIME 2010-11-10T00:30:03Z PACKAGE UFE0022238_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 7305 DFID F20101109_AABTUR ORIGIN DEPOSITOR PATH mendro_r_Page_03.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
0559f0645e3ffdb6e5dab51b293849b3
SHA-1
ae0fc81213c3ce85fd13d0a4e1339b3e5365c839
11252 F20101109_AABTZP mendro_r_Page_10.pro
058af104e9a402b0d1be2b6693affec9
15880ee574b5fb9122fc1636ec7f3e03f45c1d0d
805 F20101109_AABUBP mendro_r_Page_27.txt
7151149a678d03b1d44f6ae5bfbb281f
8acfbd9b9eb2cc798d5702dbaf3d3665b86d7543
24878 F20101109_AABTUS mendro_r_Page_04.jpg
889bae012340da689eeca08abe4591a8
e705319f93126ed9b847faca331cad86743100eb
51931 F20101109_AABTZQ mendro_r_Page_11.pro
a49d6bcc11e3704baa8bac49cf55eaaa
05e02adce5c7229f023723981a761c7d1c3191e3
894 F20101109_AABUBQ mendro_r_Page_28.txt
01ef0f9eb4eac98b1f989ad62e99fa53
acefa358dd3e375e6ab8fc23b6145cb8725d6c15
111657 F20101109_AABTUT mendro_r_Page_05.jpg
d0921e1a2ed2ecc63b088e0c01df161d
41179e3fe2d81ed1cc60d4c1abb726aa745e4d61
55781 F20101109_AABTZR mendro_r_Page_14.pro
84b461eda3676e4c994bf9a2c26e21f6
7d54aa8beeb75db4d09a4e95155d96e9329cbefd
2105 F20101109_AABUBR mendro_r_Page_29.txt
2472a055b62d6e11583d19cccd9bcfbe
1bfc31034d7e63d679465abb9812cc52bf46537d
12595 F20101109_AABTUU mendro_r_Page_06.jpg
97db33d7661544959e38ef90f5ef2e5d
d7147c6a12aca8664715a72e446fe7443f457d9c
55967 F20101109_AABTZS mendro_r_Page_15.pro
fb8814476607a16cc2d9d72e35ada0d4
1632913a45008098b840352ecf9bb1c0a41a57d9
2229 F20101109_AABUBS mendro_r_Page_32.txt
171631654c7b05819a3513dd513b5dcd
f0f65f5a070aff762ad50fe8e228a235328e6409
74797 F20101109_AABTUV mendro_r_Page_07.jpg
f724dff4bb43914476116bf26707718a
35396ff6a221da6ad43d61f81c4d058cc9c00a78
57833 F20101109_AABTZT mendro_r_Page_16.pro
e6eade7dda56af57f3a2f969dc02d3b8
7ed14316c9e7404b7021269b86335278ca87bfb7
2094 F20101109_AABUBT mendro_r_Page_33.txt
e241ced478d4619aaa3bbabcfa2f3f4e
3d28e156e46385203d92445f1b9b05bba2969610
812 F20101109_AABUBU mendro_r_Page_34.txt
077a2e5103148cfb867e5011c4fbf45d
f03a4f3fe52ed2aeaa6e9d46c7ea68c5e5b772b5
109125 F20101109_AABTUW mendro_r_Page_09.jpg
3b0e48740d06045b3813434b2832ea70
e43087a6ab3da12f558bf6d24cf373d75cc484d1
55275 F20101109_AABTZU mendro_r_Page_17.pro
cd73b260159fb3043db2557f486d34b3
5ad6f2b2ae47556d0fb8e17026e9f874a5b2557a
929 F20101109_AABUBV mendro_r_Page_35.txt
46b98904aca0de2a2fe25812490471b5
66d0e8bd62a643e2a7baa5554bbabbdedb6d96ae
25548 F20101109_AABTUX mendro_r_Page_10.jpg
a9d0e578d2dc8d23702fe3c147df4d59
9c82b26777d8b78b3aed8a77562c86cb17e0400c
13266 F20101109_AABTZV mendro_r_Page_18.pro
f48059fab2d8dafd9b111a4154ada13f
0c41dd28589fe62f35cb67e9a13f1b1298cb6cdc
1020 F20101109_AABUBW mendro_r_Page_36.txt
417febf8b4c78600846a086c1087872a
34709cb0bacde8d94d6c288082a4650de64d873c
105848 F20101109_AABTUY mendro_r_Page_11.jpg
3c8058ff4d7b7840ee207ccdbce6e840
9b1001fd1d59b3bfdf727b65a48e2b03cdbb0382
13267 F20101109_AABTZW mendro_r_Page_19.pro
93381f9430c6f7175a44006fc6df2051
894421f535d10f91822cf9e5afae449aea0c5808
916 F20101109_AABUBX mendro_r_Page_37.txt
4812854d3ec575302dc607539322a142
923ea7bb10b00b695b9c8e1fb076f71072dd2e50
100236 F20101109_AABTUZ mendro_r_Page_12.jpg
c5f382790f41853ebb0fe8b7d47e7d51
a0a10263abfccdf9ac740dd93667985108feaa2e
2714 F20101109_AABTZX mendro_r_Page_20.pro
16d02910df914b64ea7064353a43ef08
7569e718bc6c8abd81d64b18599139d60e60c1d1
1356 F20101109_AABUBY mendro_r_Page_38.txt
7bb55e6f40e37875051fbef13e296d21
e85834dda6c06764764607ffd057e732f447913c
495845 F20101109_AABTXA mendro_r_Page_28.jp2
fb07258cb3b2b1cfca96e2b52fb96c76
070c49deffbd58539cc26e0ee63353abd0eef06c
3251 F20101109_AABTZY mendro_r_Page_21.pro
fc8bf65b91c02104c06ffc5f682ee611
dd689cb41b72d1d6c659f29d84703692b924e979
1100 F20101109_AABUBZ mendro_r_Page_39.txt
f91bcbbbbf3b8eee72afb86d8f687d9f
b42b0b5831c9597f6523efd25dbc05149b675658
110480 F20101109_AABTXB mendro_r_Page_29.jp2
6243413250880661fa66396639a87fa8
c7bb7b2831348273b360d425f820b9be5af160b2
15199 F20101109_AABTZZ mendro_r_Page_22.pro
8a3cea459ddcc08874c4f4da7c5c511b
4e20ef8d300190fc792903ad19e81882d8f9d0dd
120362 F20101109_AABTXC mendro_r_Page_30.jp2
d861afe6b9d3615a7dc53eef46a396d8
77feb7e7054350ee5276e69930e34c879f9a7329
116437 F20101109_AABTXD mendro_r_Page_31.jp2
c0a2457240e3bdba9bf4994b585a2a47
7e4271edced47f71ff7c24413bff7ad0e4f575f1
37019 F20101109_AABUEA mendro_r_Page_14.QC.jpg
4b2b7d854a3c6244c5a370202a64ebe0
62a6d88583206c1e124fe50eeb997c5535107ea3
121306 F20101109_AABTXE mendro_r_Page_32.jp2
fc2accfb94262be8d8023739754eb05a
878cb61b64f000625c9f06a237cf9e289b29cd49
35387 F20101109_AABUEB mendro_r_Page_43.QC.jpg
d5319feee24a266479f860a046a0c740
0d7b4dda72acef4142afab1a76dbbd8c8ff4f8dd
46573 F20101109_AABTXF mendro_r_Page_34.jp2
695e28bba1f6739dd14f9d95ed643307
4cb8bd37e1a395c9fb3155587970b35c7ca2e7cb
34597 F20101109_AABUEC mendro_r_Page_09.QC.jpg
9a8337db9ffee6c934ad91206e9fd659
0ca433f802c161fe7abe18c2c980ae058bab3dde
542408 F20101109_AABTXG mendro_r_Page_35.jp2
b5557150c00a73ccd7af09e0339d2a58
9b6b79ba4307a28c9138a0665ab6d349c90612ce
1274 F20101109_AABUED mendro_r_Page_44thm.jpg
eeca601c1ba32a50677cf9a840bf1a2a
49806a9dff2624d8c45f4bdb59f65a74bf36431c
543985 F20101109_AABTXH mendro_r_Page_36.jp2
257fc83d39d6e3faa4b1cd6db133e3d8
b31331226f011050a7fd6e3e8623b196f871a8c6
30875 F20101109_AABUEE mendro_r_Page_41.QC.jpg
f5c94e9d57bacf852f68d0072924e5a4
712a7fba705ae45b954e667e09251ce2da0cf447
445139 F20101109_AABTXI mendro_r_Page_38.jp2
b4c88b10ab7cfa65671b808a55a21506
bcb52c7c38c6b8ebf8ac8191d52b253ace2ddf50
8795 F20101109_AABUEF mendro_r_Page_30thm.jpg
db192213e6f9862a320b1ce453008b8d
799211cd5b20c2c39dd1dbe340b0f96490a68943
646818 F20101109_AABTXJ mendro_r_Page_39.jp2
d8f1ce0e6064bac1dde92bbe7fd5ad84
8f0b70bec4764f757eb5896bd4ce942b17240353
9526 F20101109_AABUEG mendro_r_Page_46thm.jpg
47581412d9a1031dab425838bc466e64
f211abf0c7ce7637a1bcc63f68ffa597bd60ecc3
129671 F20101109_AABTXK mendro_r_Page_40.jp2
0f8561287d96f17016369c6a6cf4ad55
abe406271d9dda6f04e6f1b5e335f1f39d9a2352
8958 F20101109_AABUEH mendro_r_Page_17thm.jpg
77704fc9f3682d305b54a5536b51e0b8
d41e0ce0f0e2fd0f4b3b75a473521f22afcd74b6
103895 F20101109_AABTXL mendro_r_Page_41.jp2
c256b3afe00016ee6baf3b1100b035ed
8c2689391ee8ea932a437fd7c6618185e515820d
1051924 F20101109_AABTSN mendro_r_Page_33.jp2
9cb19f4dd450f56db27a42922ac247dd
98d002ff28a2171dcc25b51c0691add06984ba7a
1927 F20101109_AABUEI mendro_r_Page_40thm.jpg
77ce7dfb2aa604884fdb70af9585637d
cef89d3d53ff050ddc78766a7485bd36fecc8dab
120011 F20101109_AABTXM mendro_r_Page_42.jp2
0e6321f0773be5421fd11b6065ba0a6f
bbed09b38d874b3a86b742e02185b35e092d9b3b
1051917 F20101109_AABTSO mendro_r_Page_07.jp2
3f4e61fd6c2a8b7708ec79f07e8f098c
0fcc3ccbf56319f95180716f08287a9752f9f217
4900 F20101109_AABUEJ mendro_r_Page_44.QC.jpg
18b334cb85c180b975c0a3fff9ff1cda
f72436e578d0b43e7f3648c8498e0bf665ddc650
114840 F20101109_AABTXN mendro_r_Page_43.jp2
619967b316ccda5ee9b5777cc48d4620
94c88515da29693fe69bfdadf9be7b79c508cf84
37191 F20101109_AABTSP mendro_r_Page_25.QC.jpg
5ced8d46c573789293352614e790b1f1
8c00a8d8e562ecbbe67e2caced6020d5dabc7976
25356 F20101109_AABUEK mendro_r_Page_47.QC.jpg
d3378a93f2ead03fd4390d24e93ab4e2
c5b105c5d48a5712ffc4209e969c3a7af9913784
13711 F20101109_AABTXO mendro_r_Page_44.jp2
33e3c55813f7b4fd4e90ef09d28694fc
fbc614eba88668a4595c292c749e3a124582bf81
532456 F20101109_AABTSQ mendro_r_Page_37.jp2
f6cbdff363b522bbb2059aefef43ee51
ef398a6c89e902eacbac174a950184a317b55c35
3903 F20101109_AABUEL mendro_r_Page_28thm.jpg
c734966f8a2d76740088a54f665951c2
eb0701e2f3135a21b3e72866b617bd7aa8827d72
126934 F20101109_AABTXP mendro_r_Page_45.jp2
d4899ae3267c8526a45aa89617ddb342
98ece330af00b2eab82edea9556208fc7abbb303
2209 F20101109_AABTSR mendro_r_Page_14.txt
e4962e34719f37a2e1ca10e04898f6b0
852827e26d05cc6137763f75aad0ba46df5ef173
36664 F20101109_AABUEM mendro_r_Page_13.QC.jpg
0a92751dc19fa6d8e5148dac5d32b4ec
54752be0d3ce4e249a7ddb6237fa790a8fc7a867
143614 F20101109_AABTXQ mendro_r_Page_46.jp2
a95a0f5d761408c61fcac02a243107b7
b68d2bb87539f7a37e76ed679e8a85e47ea1157a
10086 F20101109_AABTSS mendro_r_Page_18.QC.jpg
57ec336dc43404051385bab6b8a1214e
bb0a3754c27407e3eca80624dc92976cda0d173a
37950 F20101109_AABUEN mendro_r_Page_32.QC.jpg
829861eef6500c972499adc579dd95d0
f8bc9cd46a0a5e34129ff68d213c0f37247dc112
94355 F20101109_AABTXR mendro_r_Page_47.jp2
0c6fd0a07412136e88d8d248e99c1491
585b5f51f78abbb3faca99f051ee641ea5daac9c
47709 F20101109_AABTST mendro_r_Page_26.pro
9849f64fa79abf096491ae5fca30e557
7975d828bf5a091d300e1300b95ec93bb6e0fe87
9018 F20101109_AABUEO mendro_r_Page_16thm.jpg
26e4709daeee76500165f0b3aaf7ba06
30743cc55619100de34fa5580df16de7484dcbd6
1053954 F20101109_AABTSU mendro_r_Page_04.tif
850f24bcf9a2ba187daa4726bc5bfaf9
8938d6f858492a6dcda82df1c95b287eb9d43c95
14791 F20101109_AABUEP mendro_r_Page_38.QC.jpg
d3e7ec28e14416e5ae07ba202fbef071
a395747381ea4dbc070eb34ccc6125dc016f0cee
8829 F20101109_AABTSV mendro_r_Page_14thm.jpg
a29516149f23cb545fb9eaf176f63c7e
30b4e2d0c256f9b1980688d2dea72f9033a146b0
38609 F20101109_AABUEQ mendro_r_Page_46.QC.jpg
9b4ad456a521db47f983d84108ce4ed1
dc55c364ae52f4d8db4a618d28a75a56f96a94aa
34003 F20101109_AABTXS mendro_r_Page_48.jp2
59b0fe98b5d867c6ac2e3940ac60e1b2
c39083cdd0ae6fb8b2e99cc15c1038e77005e3f9
99531 F20101109_AABTSW mendro_r_Page_08.jpg
709f4d5a04ed3d281657e924cf0a1755
34fd67f5ea9bc04f5e025aa1643796eb9ce1d6b9
3694 F20101109_AABUER mendro_r_Page_27thm.jpg
2a0c98e6d7a80d460726bc44de435f82
b3fd25fca01ec099cc250f132969e149be2431e1
25271604 F20101109_AABTXT mendro_r_Page_06.tif
4806761637a587b37c44b145bee21a75
a93bd9a187ee24273e1ad1198858a8844d909fb0
49465 F20101109_AABTSX mendro_r_Page_12.pro
7f65cc32099ae24056a91e731a866336
2b876aef0ed24ca61032d70cecaa7e2e80054945
74822 F20101109_AABUES UFE0022238_00001.xml FULL
fad8047399267a936fbb47240541a56c
77e68ebc395e27f43290b5d8ee399644d910d2b6
F20101109_AABTXU mendro_r_Page_07.tif
7f762e73663477f68b65a3aa867fbf09
6901bdb83519149440507166b92dc36c7d12a659
108818 F20101109_AABTSY mendro_r_Page_12.jp2
b7362b9a3c40a93caa398e5c791b4080
74f34a9a252aa4d8330f291bc45eb75915aa5bba
1946 F20101109_AABUET mendro_r_Page_03.QC.jpg
84a78cc2a991891bb4196c664628467c
ebeadb36aedaed92a384fda6c6291645cea2907d
F20101109_AABTXV mendro_r_Page_08.tif
b84737768a16d7d3ebe409bf793e5a31
4d90b7f8b789422ce846b9251d21f4b7b990db47
24172 F20101109_AABTSZ mendro_r_Page_01.jpg
59b580d5e25858bcf03ac5c73b0e7948
7aa29e8e32d97297d9beecc72ce2053b31b1dec9
7940 F20101109_AABUEU mendro_r_Page_04.QC.jpg
66cec62124d50cf4309a07739f4d728b
83ed39ae95ecd54ad11d47ee1b899e684786513f
F20101109_AABTXW mendro_r_Page_09.tif
493847b9b3e9cbe57b607d3d5bfadfc2
1c385d21c0df9c40600f674db20334e65fd259fd
1951 F20101109_AABUEV mendro_r_Page_04thm.jpg
bb5bb59ab24e910ba467155eb8a305bd
779c5d93b34a68371b4282f8d196c9c26b24f8e0
F20101109_AABTXX mendro_r_Page_10.tif
d9e6c22a8d0f0e71d6de4ea948b71838
d4d386a770cd12b1d927a1dd11886b5432f7a76b
6078 F20101109_AABUEW mendro_r_Page_05thm.jpg
8b89210002b2ebb60b4f295e16618b66
46e5de9e740b4ce3dd79186fa0ec815926a8a2e4
112102 F20101109_AABTVA mendro_r_Page_14.jpg
b38a04886a7fedadb61c2e79f84e00a2
ff423409498f29ef1526c0a887bf774f4f06b8c6
F20101109_AABTXY mendro_r_Page_11.tif
5549ce097f78afb1144c8e468ff7de26
ac42122985ea1b5b819a2288b19d97592f786319
34203 F20101109_AABUEX mendro_r_Page_11.QC.jpg
03aac23b91be80bf0db42b1a0827b67d
41f6abf6750c962ac879e67e7d1d156d2ba16efc
112052 F20101109_AABTVB mendro_r_Page_15.jpg
6f59bcf8c8c13b642fd4d2be2b01367a
695bdb5ba8643b3463ffb3d6ec7ffe0760c25423
F20101109_AABTXZ mendro_r_Page_12.tif
7286a2b5239f36f7ea24cd4dc6afda26
2b7ee2ae607008c29f38d5771db16026b997f824
8827 F20101109_AABUEY mendro_r_Page_15thm.jpg
d24b42f6e39227d22a589f6ceccf9351
53085a484712a7919abf2f39e58e69181e4d8ecc
117482 F20101109_AABTVC mendro_r_Page_16.jpg
004a86382716162236860a5a032e4826
f5ac5eafabae8cc35828669303bd4b62062b0506
254 F20101109_AABUCA mendro_r_Page_40.txt
145508d4fba0e8a17842580f71cd39b4
3f677dabdda9748be7a6a6857abf801ea61d61f9
2510 F20101109_AABUEZ mendro_r_Page_18thm.jpg
5d64075bc1dacf43dd6ba72a07aa8673
787cc07f96db4efd42ce4b72e120163fb7a4b525
113267 F20101109_AABTVD mendro_r_Page_17.jpg
6fe4cc82b70764bc6d1882621a7df6c5
ca86ef0d85b19b9fd706e99fb372611bf6a52b8a
1997 F20101109_AABUCB mendro_r_Page_41.txt
868d612e1f9d94b9f17579ed13edefd9
19fd1e482aed0c6771f512d529dc42fa3d9e6555
29820 F20101109_AABTVE mendro_r_Page_18.jpg
4ed996f43e4f97c3583fa8c2a1bbb411
f5cba2a3271f4c7a0c6d84b6c25d32f1a5023e05
2225 F20101109_AABUCC mendro_r_Page_42.txt
c3c9e6e21e048e700e686754e640da9e
74acb50ed7f9b6b9145352f8cdd1df8d36749294
58173 F20101109_AABTVF mendro_r_Page_19.jpg
ef0bfcf6ab62e052361c49e1b7a064e0
d589aa06bf52a28b691ac4db7632776c81fa6bcf
2110 F20101109_AABUCD mendro_r_Page_43.txt
d04afd0a82d14517222f370d9176bf0e
31aee63fe2ddf89f70552a4b3c1d3f8130b3c3a2
40827 F20101109_AABTVG mendro_r_Page_21.jpg
c3fe172381b60dc7dbf807d2c3119395
beb28f1a6746484d904fadcfc1391d5daac89b37
200 F20101109_AABUCE mendro_r_Page_44.txt
0b0ca3789d66803b0216e605916f81b1
feb5c8cea7437fa2e1387ce898a81248218e105a
102808 F20101109_AABTVH mendro_r_Page_24.jpg
95a0ab9cd6d1fd5cdbd51b904bdc5278
e0c6e278d4d46324128fa2733da2a9a9051747a2
2423 F20101109_AABUCF mendro_r_Page_45.txt
cf2ff37477607a5430b7f7264aee21fb
170e4de0d50be6d894b336791e2456552e2c0696
110054 F20101109_AABTVI mendro_r_Page_25.jpg
4575c101423ca23b2fe2d3a70b5c2af8
455b481f91183383367314faa70d4eb62cc44a28
2724 F20101109_AABUCG mendro_r_Page_46.txt
f810c504d5e9061cd646121442618bb6
7e4c36f72fb438660e61a18e24330c5ca4c795d4
107153 F20101109_AABTVJ mendro_r_Page_26.jpg
a4251c3fc067ab6d9ec01e937337e2f3
d8e728baddac83247e5d93055f4f0b682f19cd15
1775 F20101109_AABUCH mendro_r_Page_47.txt
b8f8707f7b9e223c24f5390478823ad9
b6ade0650a37c5201bd275d8683421a7e5a9e786
105070 F20101109_AABTVK mendro_r_Page_29.jpg
beccb65efa3fb3cc36d2d7288f1d4794
dfea0aa6ffc7853490d4ed92a96006f9fc448fea
586 F20101109_AABUCI mendro_r_Page_48.txt
b6ec3bc0d67e383914fb5d9561e84c41
3356777a4bb40d61664b33abbfa40e4ac62ee37d
113814 F20101109_AABTVL mendro_r_Page_30.jpg
5e10f3bead6ccfe8d8628e40991f17cd
fdd02e4c8a4a883cfd0d2fd13db81bc53db37f45
2022 F20101109_AABUCJ mendro_r_Page_01thm.jpg
c1b860cfb1e887c373afd0194da487eb
388ee030c7eb9c347fa04b84991772431736c517
108539 F20101109_AABTVM mendro_r_Page_31.jpg
bce712910cfa29922895ea7e56c31468
d2e7d375ed084965e0a32d45e416a7d143954cbd
3454320 F20101109_AABUCK mendro_r.pdf
496c8f18d4cbfaa1be4f6306b46834a4
fc353acecd57eb42b32e66989e10d6aa1ee4545c
113868 F20101109_AABTVN mendro_r_Page_32.jpg
b2558ec88236f8dcf660307501d9a164
e235fd54539b1f7c9b12334d428ca689ae3afd99
10802 F20101109_AABUCL mendro_r_Page_48.QC.jpg
8ed72c264e34dad3a0539680ac8b4c20
ba2be66ab9420706b4b09142f3737e70759bf372
105986 F20101109_AABTVO mendro_r_Page_33.jpg
71f69ee669b651114bb282cf99907e89
1531bfea35925b671626e51ae793352ea0dbb0ed
8439 F20101109_AABUCM mendro_r_Page_24thm.jpg
1ccb6c46a955f6c351f6715244943581
daf033a04fc6fbf384b0a51701b61a21036a2449
42988 F20101109_AABTVP mendro_r_Page_34.jpg
2fbbdee3d262ed6592250b44f2068269
cb133ea33ce9ef2971412da689c20a0d8ad1e589
8733 F20101109_AABUCN mendro_r_Page_31thm.jpg
f6f294b4a79faf865192100bc94a5902
119bf31d70168f4794f98f77732754cff47a6ac5
4950 F20101109_AABUCO mendro_r_Page_19thm.jpg
dfa9dd51a809c0c54090a871033da6c7
c529b4d084731010677c1677aaca9ac3ebfad5bb
55837 F20101109_AABTVQ mendro_r_Page_35.jpg
4d39adc9d476cff66a9be806f10b572b
d37eb9b8af39fa600485ff663db525081588a4a0
1202 F20101109_AABUCP mendro_r_Page_06thm.jpg
f5f83fb014be5b67eb844f1378ea24f7
ab34cc6f3ab8e90149d058aa4c05ce518a78516f
56985 F20101109_AABTVR mendro_r_Page_36.jpg
60b567812d6c194a04b4b79b2589872f
7ef1452693af6cb417e2d48d80471a97594c8b1f
4999 F20101109_AABUCQ mendro_r_Page_37thm.jpg
b570bc55cee9248f61dfeadc164970fb
ebf02c6d398e8c57bbb703d5856a96d3280d1f48
56058 F20101109_AABTVS mendro_r_Page_37.jpg
0d44cbafc5807827a2acff541bab27f6
76f9cb5e915a3aa00bae61a28cca1cf25500060f
7222 F20101109_AABUCR mendro_r_Page_20.QC.jpg
7b5ae0074a777badf7cdcc966c434eb6
8dfae3f74a7c9cd3ae78499844daabee27f39720
47773 F20101109_AABTVT mendro_r_Page_38.jpg
d36a493e22614f1c3d436b1203fbff76
f10e2ea64c7b0385ddab82e781b4f3304357e140
543 F20101109_AABUCS mendro_r_Page_02thm.jpg
5dbbaacb09fb0a9e1040cf4bc5879987
e50e358e605a2a7c40edef3b1751705de975051f
64816 F20101109_AABTVU mendro_r_Page_39.jpg
5599e158fb7dbddb2b31813d2253b44d
4af016063b1bca400d111404d87aace626e036a7
34915 F20101109_AABUCT mendro_r_Page_26.QC.jpg
5d288a50c2266e019c388eb1bf0c19ba
140c4c922bc06738740232708595c9ecd60ccd33
16289 F20101109_AABTVV mendro_r_Page_40.jpg
b7cae864d2516e61c76f58ac751b38b3
ada6644d937ac810c99251e35510b45866ceac30
38834 F20101109_AABUCU mendro_r_Page_16.QC.jpg
384ea16738862ae0ffc1e0a94d038743
7c9f103ddfc8d5cf367c3e979b29c1d5d51ac82d
97752 F20101109_AABTVW mendro_r_Page_41.jpg
a6087f3964e82a0a6a82e93659513dcf
adc4ceccdd09d69e035aab79111a94a784ec3ef3
1181 F20101109_AABUCV mendro_r_Page_02.QC.jpg
72f024173e773dbef685f8599827c22f
73db1d6e7ad069988917691e790f1a011c5ad6a9
108375 F20101109_AABTVX mendro_r_Page_43.jpg
f4f630d5b4dc82b823f1cf416e88fef4
b7929550263a6843f9c27f0ecf3a654e08a79d20
F20101109_AABTTA mendro_r_Page_27.tif
db2c7cc996f3fb03176a4fd5fbc032c6
bd79e138f3649b5c37834a693edba9dab3597d9f
13143 F20101109_AABTVY mendro_r_Page_44.jpg
5ed7eb8199b29e38978d728f655b91b2
9efc1622174687839865ddad60c01f16665716c8
5820 F20101109_AABUCW mendro_r_Page_40.QC.jpg
0db8b3101767b848de25c4bddb525c88
83b751c23f8159eac64c975fed3a78467545cc45
8517 F20101109_AABTTB mendro_r_Page_26thm.jpg
b21c58d887eacff0f747a5c3af752c7b
e2a8bd9d7f5d42db25a8b92801d434dce319cc50
115724 F20101109_AABTVZ mendro_r_Page_45.jpg
60ae142395f09c5b2a952d8376f8bf77
74da2a7cd9a9e845239f600715edf28bf2d776df
3651 F20101109_AABUCX mendro_r_Page_06.QC.jpg
38ce63fe0ea8f09f65a671df889d6067
4bf92d09762e1cd70ac21a5e4a1ef71725ed364c
8940 F20101109_AABUCY mendro_r_Page_42thm.jpg
90aa8306d233040039288d3c5f3b9e41
f2cae4bce8323a18b441d03ae35e98c7693362f4
14004 F20101109_AABTTC mendro_r_Page_22.QC.jpg
5e3868cb4c27cc49e21d2331ef1015c6
dff38f2d6d24cc79c29584eefbd7e7aab3c84cba
F20101109_AABTYA mendro_r_Page_13.tif
75815b044f78ed40106668885a9bc7fe
d1b95fa52ed1562ec36bc3e36afa5bffd2753437
48253 F20101109_AABUAA mendro_r_Page_23.pro
0ea7327b1bd5d481a6eaf2241819f272
56280e36a74519eb72b152e4af37fb8bbf3d0a13
2354 F20101109_AABUCZ mendro_r_Page_10thm.jpg
9be06142c3aaa1c7f421cd88e8e0c37e
38aeb21f5705edfa5c728651b9c594f07c94c9c0
2074 F20101109_AABTTD mendro_r_Page_24.txt
470c8b551780101b60e1a586c16c2209
4359b9928061bf6ffaf1702e3d5f42b0066119f2
F20101109_AABTYB mendro_r_Page_14.tif
9b90cd1fdfe44bf2126477f7000b0346
6213530209e4b3c9e897cd6bf401cd78e117d7f7
51027 F20101109_AABUAB mendro_r_Page_24.pro
40ce26ecf4d812714250e9ef3c700074
a6944192d0a496d926b55f847eba0d098575f162
113119 F20101109_AABTTE mendro_r_Page_42.jpg
083383b0f4896280f252bf2e2a591c97
9857de1ceee46faaccb74e716627e60fc130343d
F20101109_AABTYC mendro_r_Page_15.tif
41c173aab0d00a9fbf82f130edbf8fb9
f583db348e63746798bcdcc1fd21d5c491bb1916
52341 F20101109_AABUAC mendro_r_Page_25.pro
2f0b3ec94e00c7582ccdc8ac2508933e
e65139945e97dd60be7c7095b49d66ea864abbcc
4538 F20101109_AABTTF mendro_r_Page_38thm.jpg
a463c25f2acc6228462361219827c206
054f8b672672a7712a1929f36c82e78e83ba78d0
4296 F20101109_AABUFA mendro_r_Page_21thm.jpg
41872b73f43c053f0e722476e6dbdc2f
df6d44bb0364b737b6c5d08551e958b21f20ad91
F20101109_AABTYD mendro_r_Page_16.tif
0a02798f486ca80da0757ff5f90e26b7
899d0c4090fa0cb98e82d56dd66bba67dc9f62ec
19275 F20101109_AABUAD mendro_r_Page_27.pro
05e947fc2420e3c7ec3b4dbee18eb498
83b3f3371990703668026d9b3719615a4572b7aa
F20101109_AABTTG mendro_r_Page_03.tif
edcddaa325d6ce76ad862370707b3008
1879c426ed2d10f64cba997e6d077ac66bf5342e
4161 F20101109_AABUFB mendro_r_Page_22thm.jpg
ecfcb4548751d2a17f1385c9a570678c
516c5d7fd064d3b9f7153a029e08a8a0086241af
F20101109_AABTYE mendro_r_Page_17.tif
38098114595db817996b8e62cf190367
a41a3bcd5fa317dae10e7c5b4e269b5d2cde17e1
19711 F20101109_AABUAE mendro_r_Page_28.pro
284fa94cb9b171b63eab5368b4f6e1c1
c13f9ffcd8b26841d4ab550d598b79b473eb5390
8314 F20101109_AABTTH mendro_r_Page_12thm.jpg
a0627f0615255ec16544ba151473805c
1d1993b99a2cc1682bb9e162034a7f7379cd2145
33579 F20101109_AABUFC mendro_r_Page_23.QC.jpg
89f1e612968fa029a192097f8d029183
5fa672b665578d9597beaf29ed9188e63032bba6
F20101109_AABTYF mendro_r_Page_18.tif
5fb4aed9655cfed5d316bc72955de87e
209a68a47b116a568ad03e0edc6843fa4742ccee
56292 F20101109_AABUAF mendro_r_Page_30.pro
cbfe82e611ce44b9ddc0298abc969741
4fd47c5951ad57ce2447ad5b0bff64cdf086f2e5
56471 F20101109_AABTTI mendro_r_Page_42.pro
6f35f4ab95160e40555e492faefe9a7d
f59cf3da73ef4d79595780f2692ea3093659c2ed
13927 F20101109_AABUFD mendro_r_Page_27.QC.jpg
f4930ef4b67a88f174b695e6477b2f4f
8621525059b177b4246cac9fc29d61edaedee35c
F20101109_AABTYG mendro_r_Page_19.tif
6947c1de963d162b00c2af4fbecf7810
a81d821ba2c60838222b79e940ee84e51003193f
56260 F20101109_AABUAG mendro_r_Page_32.pro
49a0a14d6d476ca81f1f91f71e5e801e
baffafbcffb325ce3437f147eea972ebefdd3545
17182 F20101109_AABTTJ mendro_r_Page_19.QC.jpg
028650e54ac50d927e4136d97fe153af
6f67d52dc9f817529782e7ac00546a3732049cd9
14517 F20101109_AABUFE mendro_r_Page_28.QC.jpg
111784f4bf27940639f6968da852b462
16d81f264cd1369d6e477d602f4868fe0bfc7e6e
F20101109_AABTYH mendro_r_Page_20.tif
196694ec9b51d4503b443323eba357ac
01310c61b5cfff23bf182f3eac3c6dec9d5fad4b
51121 F20101109_AABUAH mendro_r_Page_33.pro
24fac939851a561da1a194fb985cb429
4fa25b0f6e873960e6b82e17ea97252eb6fd9535
113886 F20101109_AABTTK mendro_r_Page_13.jpg
e51da788de509bc3344db6ec6eea2862
8b8220d75ce85f6273140e7b0a3d34bf8bb25592
34534 F20101109_AABUFF mendro_r_Page_29.QC.jpg
ebfcffb8dd74bba0ad8b059f28199212
268ca78d79c6b43b8919d739cfbf99303cf4cf29
F20101109_AABTYI mendro_r_Page_21.tif
ffd0f43fc75d5164b29f2106d753d579
697056065a7f544f63384017ba1f7d6eb477365d
19396 F20101109_AABUAI mendro_r_Page_34.pro
00c24b47b0221d6c81fa0788aba5e0ce
a827a8ac11a98577a720efcdb8a93a577683072d
48867 F20101109_AABTTL mendro_r_Page_28.jpg
7cd65efe48cc22b21e3899c9544e2194
0c56acd8a99046da858de3d2a1b567e97aee7813
9218 F20101109_AABUFG mendro_r_Page_32thm.jpg
b6fa84bf07924926ece85881750bc5e6
d280ca72768b14d2a509b61c7e0dd5f617fe06f3
F20101109_AABTYJ mendro_r_Page_22.tif
a34a1f27c98914aef0e834edc28aeb99
831e3573a3cede1debe7b3ee0d596295e5e28a83
22143 F20101109_AABUAJ mendro_r_Page_35.pro
c5dda12fd9742de00132d432dc9c9661
b94dd758d4e4dcba9fc916905110c23fa85387a4
50983 F20101109_AABTTM mendro_r_Page_29.pro
c3a8f13e946281ee5b3aff2d2e05b335
aa5f9d8c64eec539ec7d0f9fec666832d70b58ac
14182 F20101109_AABUFH mendro_r_Page_34.QC.jpg
a9984f3658331f103e80ef0914b99b4c
7389b1e9ef67a45db954697fcd279e1cdc207c63
F20101109_AABTYK mendro_r_Page_24.tif
12726938def0706239922f4ce00d7e98
d098ee477f826168edc4524c1a3e453a5259fb6d
23548 F20101109_AABUAK mendro_r_Page_36.pro
a62473b278a9d0ed37e6cf038350af74
824bf6644b0ff43e645ca33c04412e3f63bf4c79
35278 F20101109_AABTTN mendro_r_Page_24.QC.jpg
5d4340775b3234a039de70413626feb5
bc32e2409f17db00c019a8a8ee034f8773172352
3761 F20101109_AABUFI mendro_r_Page_34thm.jpg
05a4deae6afb425fc32aaa151e1f4ffa
d16a9d1a973ac5d73cae09f6dc85f8877b328e54
F20101109_AABTYL mendro_r_Page_25.tif
d2ee41da71b6bd8a2f093a4973e31935
64492f8e2d644d95b4a26297c7f80553b83ac4fd
22606 F20101109_AABUAL mendro_r_Page_37.pro
9fc24ad0e090ccac85cba7ce1f2a3fe2
be1a97b2bd95e40ebc62052e7d89d7efe6d76c48
5434 F20101109_AABUFJ mendro_r_Page_35thm.jpg
db6dbe7f92c27fc9e669d398b93c9afc
939fa23f6a6e8c74ee21ce778b7e010ebda064df
F20101109_AABTYM mendro_r_Page_26.tif
aad75f8264a742f709882e1ce6296a67
aee8ff0ecc24c37425246d1a7ca5d8fff1cba329
17512 F20101109_AABUAM mendro_r_Page_38.pro
e032a6aa25ca786393e13c3136b3bd14
f3fd6a3016516123bbafd4ad401801a72bf21608
56313 F20101109_AABTTO mendro_r_Page_13.pro
dfff14db359783842575c5fb0a287196
1a21fb83013a84404bb55d369dc837e26d6dc838
17226 F20101109_AABUFK mendro_r_Page_36.QC.jpg
6ac74fb3588bad3c555b100ef1711636
c341721fc95527db39c6c682a86445c08e43e09c
F20101109_AABTYN mendro_r_Page_28.tif
4a966158f3d123ac3cec6848a733eafa
d38edf404c16c61bea2ea6e3b9d0986e5e3e74b0
25865 F20101109_AABUAN mendro_r_Page_39.pro
292d3a1c926754505eb2ec1ccb7f1689
66fa124bb265e5d1da80f5daf609b7f47c47c553
625 F20101109_AABTTP mendro_r_Page_22.txt
0d9401d2d0ee05cb27a3c380347990a8
3df5991723b011cc27abf91c58eb570428e61644
20132 F20101109_AABUFL mendro_r_Page_39.QC.jpg
fc2840528cd7047ab973540e0ac2903c
070928582cfabd3c8724bbcbebfe5f6d1790b639
F20101109_AABTYO mendro_r_Page_30.tif
1c2d426f6c62357d8a650991c516a89f
b55ee016cb283257365d29b88ffb1750f33149b8
5495 F20101109_AABUAO mendro_r_Page_40.pro
c0e66f307ba4819483624906d41dcffe
fb50210f867090ee6e0c0902aba94e3bdd458b35
124024 F20101109_AABTTQ mendro_r_Page_16.jp2
ff626de3bd52aa35ac96bfb4a86b6bf8
dc6756d70f42b729fa1829a13ac7284bed3a3063
7666 F20101109_AABUFM mendro_r_Page_41thm.jpg
a3e0bc4abc0ca5477a83dabee809aab4
cdeadebc3682ab806d334e0a049b5ecca1eb7a54
F20101109_AABTYP mendro_r_Page_31.tif
0718e3eae7880a6de187506ce56ecf36
179afd5e168d73ddf94e2b7a8298b0a98e534c0f
48205 F20101109_AABUAP mendro_r_Page_41.pro
ea5356691f3a57a470d53c6e841c3a07
9880d82f0a03e13a71e3cdb1baa1f6cd2a84e0c0
F20101109_AABTTR mendro_r_Page_02.tif
c09ede9e1b4908fec6ab9184ebe29d8f
11441c901c8f569f871f8d877d1d82561c599b23
36351 F20101109_AABUFN mendro_r_Page_42.QC.jpg
deafd42604376b2d56b2d6700290a095
d755c57eeec5196c8ce418f87358bfc09cdaffed
F20101109_AABTYQ mendro_r_Page_32.tif
030e2bdfa9397f62441b33361a6dbf55
e95e38b20964d6cdeee82cdcb388212ffca95cbc
53682 F20101109_AABUAQ mendro_r_Page_43.pro
fa14665a748f14240bf71cb639b2acb3
23e528767b6be1065283bd2c2a20b9036663fc6d
460 F20101109_AABTTS mendro_r_Page_10.txt
bf560c21f36e47ed58a1ce2298969edc
01966c6c31ca45aca73a7d8facfa8ce6a59ca33d
8916 F20101109_AABUFO mendro_r_Page_45thm.jpg
d74cbdd598f3074b97cacc5daf2e0c54
144417a130712bd4d1b8820c07dc66cff1fa7bb2
F20101109_AABTYR mendro_r_Page_33.tif
dec3914d5fb9d2875ee95da2758a5ce1
4ced54edc63c321818e08bbd097c1e857f6a78c6
4978 F20101109_AABUAR mendro_r_Page_44.pro
2f43e46bb989d0391b94d868d51371a4
fa8b262826d5eb792d628dbb393e680a6f978493
115382 F20101109_AABTTT mendro_r_Page_25.jp2
3b0927195619de81031d62003c3b9fb2
9cb1f3c8b52b092ad54a79f9426e74edf70f5892
6239 F20101109_AABUFP mendro_r_Page_47thm.jpg
57734d8f48f43a8d8c385e34e41fbfd6
54962831f0e4ecff85d7f69f0c0296b5e97f6ef2
F20101109_AABTYS mendro_r_Page_34.tif
4cdb0ca9e6c5a8b185e347253f3e9519
48fdc1d1b1faf28d773e0eb1ff3135767ae60783
58343 F20101109_AABUAS mendro_r_Page_45.pro
ce3a2eef7b43ede585bbd34ed8e222dd
25cf8ec44e786f32a6ad7c4c34819b3f50a5d7a3
F20101109_AABTTU mendro_r_Page_05.tif
58bba073aa81b46c46103ba8fe9a5ec6
53f82b096a8b731f1685a3015fdbf6d0a21727f1
66802 F20101109_AABUAT mendro_r_Page_46.pro
63542a9aea4f3c0a507700c034c8bf59
4def1c38852f9792f17ee69992d5b582ace0a9b6
43706 F20101109_AABTTV mendro_r_Page_27.jpg
4f174791ce006b8a77aa80967890d4c4
255ff15d098431690b3a57df1332851671f443e0
F20101109_AABTYT mendro_r_Page_35.tif
65d10c53d898007c03625cd6002d2f92
e361dcf022a3b7fcfa024e13417d129fd6b305d5
42732 F20101109_AABUAU mendro_r_Page_47.pro
ab876cafd110c0c7f8a57865065cb876
c64f44c07f20aac2ead0e239cff16b6167474e5d
F20101109_AABTTW mendro_r_Page_29.tif
508fcbef9c46bfca7f19892e6065b209
f5ce527c0c91130cff1e288952ae281995e3d01c
F20101109_AABTYU mendro_r_Page_36.tif
cea6458d3fbb87a2e3f389f13dc23ea0
c8240396713b26187a22d590b23452df1cec75a3
13737 F20101109_AABUAV mendro_r_Page_48.pro
5cb7ef5e77b53b506db5a81bcfef4b2f
b2938ac66b7c3674be369289b68ade43035455bb
100153 F20101109_AABTTX mendro_r_Page_23.jpg
38b291844f6398b38c007e922dbe4348
78e25f7bb983de8a6adadeba5b32b76491b33366
F20101109_AABTYV mendro_r_Page_37.tif
81d356dfa71466839c4398891e750cb1
844d5136708ed63b2d8a99b0e64c276f3840e5a2
484 F20101109_AABUAW mendro_r_Page_01.txt
2d4fcb2d644b0c737923ee00de3a0723
b8f522848b504347d6547cf189d71150e69c4fdf
2240 F20101109_AABTTY mendro_r_Page_13.txt
9e29c4af54455c8eded1b136e905851e
409067c67bc27a058383f04b85988ef1583f020e
F20101109_AABTYW mendro_r_Page_38.tif
e0ac7da6838860257018479d962045de
024ce7958b9af3c6503f5916f812af8a0b3a8b68
90 F20101109_AABUAX mendro_r_Page_02.txt
a23d4c891f56cf4cb6a28cd4ecfed3da
8a7d9d5ce9b1f98e48d5335856d3f57e7a7ff9d4
2159 F20101109_AABTTZ mendro_r_Page_31.txt
8cf03c5b835f1f1b724cafbb5e3a7b9a
9af2ba1c707bd0e5d10365e36c97ba99459f62b7
F20101109_AABTYX mendro_r_Page_39.tif
201beca88e799e54db6505932ebab2c6
47967910a06fef9ebd4a69b8f0b788d02bf49dcb
448 F20101109_AABUAY mendro_r_Page_04.txt
0768b6e5cb8903e07550a5d99d2de0e7
2fda0f66ccc5f906fbd7c1c78b253853e0343c84
F20101109_AABTYY mendro_r_Page_40.tif
2a5aa5e883fd4bd0a5fb4893bad6bf27
a8f44803e71398e0e11b45dbb0d4dcf463d4dde1
3001 F20101109_AABUAZ mendro_r_Page_05.txt
1a82bce00bb308e3f619d6e32ce8be26
088660b9f4393153686b60403e488b728eaf9519
131348 F20101109_AABTWA mendro_r_Page_46.jpg
853b80455737660395fe4763694ee3ac
93ce983d253fb381da6dc7b5ede1bf9de0dad00f
F20101109_AABTYZ mendro_r_Page_41.tif
4414c0dd6b4b2e16014923342d830ec5
7e47a263845ce21ad32f19fc85963cb58b4dea89
87783 F20101109_AABTWB mendro_r_Page_47.jpg
c8f366be61085242a362404a18fb5c35
15074ffbf03f2bfd59f433dcabbd48e51286bdfe
32880 F20101109_AABTWC mendro_r_Page_48.jpg
259243a5b8ce6af716619f8ff8c8583e
1bdeb581ce632d2004e53c173e42988004e972c4
8668 F20101109_AABUDA mendro_r_Page_13thm.jpg
18adb62a5fdc0c12eeaad00466229cf2
3dab0f7961c329857ca862a58663acc9740def72
24081 F20101109_AABTWD mendro_r_Page_01.jp2
c55856f18d52acbd729609a2f9794d7f
63ed3ec82d25b360c682a1a925818e407e9f6239
36729 F20101109_AABUDB mendro_r_Page_17.QC.jpg
1df6045e145a706689cd3c1c3ea71dd3
a1af082c3c5d6b0888950f1b74f9a3e1f76bc3c3
5054 F20101109_AABTWE mendro_r_Page_02.jp2
66b90ce90fcfeedbe14e14a6890e8cff
f0b726a8825f04ae2135c4dd435dfedcb2d452e7
8848 F20101109_AABUDC mendro_r_Page_10.QC.jpg
453725a755be55f915de0aa9ee2de119
89b1d68918b9742d6c0671141707e47ded922c0d
8718 F20101109_AABTWF mendro_r_Page_03.jp2
41a937e6ee4a7e49a05e4facef3b2137
18f5e56b9c92ff4b20b46738ac37da457f5a640d
5348 F20101109_AABUDD mendro_r_Page_07thm.jpg
c99e50f2a01832718dfc462c67649908
998dbf7bea683026d4958c04c568f8b999db6c3e
25475 F20101109_AABTWG mendro_r_Page_04.jp2
54c8ff396d78001d020bbfadec19f27c
3e1f23da67f201f537eb197bec3b03f967695fd2
2674 F20101109_AABUDE mendro_r_Page_48thm.jpg
28deccfdc1deb984755e461293cb2148
3faca012ab574952da5dd35ccde201c34bdd7f55
1051980 F20101109_AABTWH mendro_r_Page_05.jp2
291c7c54dad876dd88470e28f29c718c
7b3d8127d6fcdc43dfa48a3e7234e4c5a21aaa5f
31187 F20101109_AABUDF mendro_r_Page_08.QC.jpg
810137c061848e9c94c2f9631a1fd505
d10636ede9539d824ee303d96c551bfe4528877a
161205 F20101109_AABTWI mendro_r_Page_06.jp2
925f5058c976636265084dc7317d76b7
0f01198fa2b8cc357d35fb5589d5a38fab2b7c9e
9062 F20101109_AABUDG mendro_r_Page_43thm.jpg
4653c8c72cbd7d3a90699cd99f7ba167
65b54b1877244934ad313b4dd1d5f79084d04531
103572 F20101109_AABTWJ mendro_r_Page_08.jp2
b4e867ed3bd34e67a11ff0eea6406a22
fb96d7d854f79fdd446e5c2c12136bda51516be2
34205 F20101109_AABUDH mendro_r_Page_45.QC.jpg
33b5bf42b72fc3cf82c34d6792b89376
792b9cf5e0795374e57fa2466ef5dd2a60b7ff03
115153 F20101109_AABTWK mendro_r_Page_09.jp2
2239fc489f28515867c1ac6323cf0dc9
d3240f90b51a0bf06821531a9492cc68f1a40a1c
33147 F20101109_AABUDI mendro_r_Page_12.QC.jpg
7c30390c5b8524aff21b1f296896e192
11fbcf93c1edab8c9cd91967c540c9bee88ddc40
27342 F20101109_AABTWL mendro_r_Page_10.jp2
ba0193b6decd1ba92bc03dc2d0f7d5b5
01c2ee888595a307b18b9bba384a9a72c588529d
17179 F20101109_AABUDJ mendro_r_Page_37.QC.jpg
054423f42397db93923e8199fa527cdf
1c1e1d5b352b327606d9e583cd46d31c38b089ce
109917 F20101109_AABTWM mendro_r_Page_11.jp2
e78f61c9fe7a6490d4152516e6e9a43a
6b9e9bc1f02935d96951fe3d8898d776b7ca0336
8698 F20101109_AABUDK mendro_r_Page_09thm.jpg
e9978196afc10c523ad1772bcedf137e
da33862d439b9f4a689bbc413d8ef7414f319e2e
119991 F20101109_AABTWN mendro_r_Page_13.jp2
4251717764ac39f24f6e35a3a20fa6d2
12dd4b0041d397f6f0c849d330ca91b60b9f166b
37044 F20101109_AABUDL mendro_r_Page_30.QC.jpg
bf889fbf20535051497c0c71728ea597
7306ce297bae8978c0ef12c7b959342dc1a53ad3
118501 F20101109_AABTWO mendro_r_Page_14.jp2
fcd08df8faaccb2daa4e66cddb594a00
05a41f42eedaf653dacbb468422711c7848ae0d3
8425 F20101109_AABUDM mendro_r_Page_33thm.jpg
8e6c401f384309759fce16eb2c61c6f3
d5c3a0887fc3b60de5921533713e863fd7783ee6
119103 F20101109_AABTWP mendro_r_Page_15.jp2
50e93b35765cea28fc44710b5f3b00f4
3ace6e3910085d8314385b36bf848d94d441e63c
36367 F20101109_AABUDN mendro_r_Page_15.QC.jpg
593652aa6c2da5c45e7d298d406716a2
8fa187744c4de0392455a1db7dfc01417bb50dfb
119031 F20101109_AABTWQ mendro_r_Page_17.jp2
a1722c4a49d53caaf9caad369f897e37
26364152395831c842446cb871096c40945ef064
7958 F20101109_AABUDO mendro_r_Page_01.QC.jpg
19c5c371b86941b3da53e0ff9e74b384
8fb46a0931a0a79646b19aa76715d9287206b6c6
20753 F20101109_AABUDP mendro_r_Page_07.QC.jpg
ffc31ffaf23005b0bff5cc08f12df7d4
598d3e031179e17203fa349ef15588560577a55c
32336 F20101109_AABTWR mendro_r_Page_18.jp2
3ac044e0c35bba1f4baee25e47ede64a
ea14dc350fc757561923bd1f031a32d642b3adc8
5090 F20101109_AABUDQ mendro_r_Page_36thm.jpg
e35b40471b783deba23f237eaeabf561
48df08a70a93a68b0f75ddbacf9262bf247066f0
822122 F20101109_AABTWS mendro_r_Page_19.jp2
1ff5685d38502609ce12ae52fff813d7
b08c6e56890c67cb897676bdd702239fdf63592c
2623 F20101109_AABUDR mendro_r_Page_20thm.jpg
90383a99518205934148d8f5948d2e15
f06f01a85d44095412c4228a3ed8da4a64fe1be1
203967 F20101109_AABTWT mendro_r_Page_20.jp2
cb59f17d145d78a5cfb2ef5e4a13c112
4dba45b0ee1a95021ffb60c06d6d7b24d4f5bc45
8817 F20101109_AABUDS mendro_r_Page_25thm.jpg
d09a7cf563d698d32a9ec24e5d1111fc
9dede8c1e7ec589bda8a6e6e86ddd338301aeaa7
598303 F20101109_AABTWU mendro_r_Page_21.jp2
b234254060a1d0d997ebb05bd7a3108e
6b782180fffc8b131131069b6bf958775e128e0b
24607 F20101109_AABUDT mendro_r_Page_05.QC.jpg
33efb0946929aad42b71bd5e8435a46f
70a25bbc687ac070c9e99a820309913dc80f5984
562059 F20101109_AABTWV mendro_r_Page_22.jp2
4344fb7ca640af90f50d23d07842f52b
c21aca985d976cb1330bf3c6989fcaecb75094cd
7902 F20101109_AABUDU mendro_r_Page_08thm.jpg
d2ef7b6f4eb9b943c218637be42c1aab
323e0528e3e445ae186c3d3e8b937a06f72e5818
107147 F20101109_AABTWW mendro_r_Page_23.jp2
0378fe25cb94a70f32f296c2c199ca0e
b66f56551a5fa67656e269b365773b4d66a9c46d
842 F20101109_AABUDV mendro_r_Page_03thm.jpg
92f37604409e0dbb4995d2d62b916170
b098a031832709cce26f3c4019937ef757ad013e
110396 F20101109_AABTWX mendro_r_Page_24.jp2
740dac0fb770c1ec90c8472c78017835
4f77499ae69a2c38432bfa8f0eb9e7a76d758176
35161 F20101109_AABUDW mendro_r_Page_33.QC.jpg
9d06773df623b67c4d0526149dc48096
06813278e020241478afe2ed21042c8811468ea9
17076 F20101109_AABTUA mendro_r_Page_35.QC.jpg
64eb026b2785de2d1699a290b10afe30
16e8bec739818ff8166a0265208b648f24b10d1e
1051972 F20101109_AABTWY mendro_r_Page_26.jp2
2d886768e060b19978f9edd35c19d154
54138b24c24e9a4051c5b04f5a035a2c249fa870
8388 F20101109_AABUDX mendro_r_Page_23thm.jpg
680dd491d1497175f13deb2ee9bd9fcd
b199eaa7886cd0d817dfca256a8ed50054fb11b0
829 F20101109_AABTUB mendro_r_Page_02.pro
0243f7d88d110ba083c1529e4032ce92
59350cf11188ba89cc43af46ad1917b67a8cfe0f
45834 F20101109_AABTWZ mendro_r_Page_27.jp2
75b459a83a6b8e147740efcf45ef8da7
3ece53c63175f63313001b2afdf1bf74c5a77416
14598 F20101109_AABUDY mendro_r_Page_21.QC.jpg
c4b87d58ce88ee15fe636ecd542587b1
bd916bb742accdb7cc1b95264eed24a6bd9447e2
47467 F20101109_AABTUC mendro_r_Page_22.jpg
bb6a3f34eb9785d09081c834ceed7df9
f6d40ba3d90b64ebc2b3db32c251123acf076be2
332 F20101109_AABUBA mendro_r_Page_06.txt
db13b46d28dd9a55a4e2bee6ab1d9d42
93fc8026fe7a8522c417da1f15cb67b6d55a624b
8514 F20101109_AABUDZ mendro_r_Page_11thm.jpg
47c925aa8cca65954f8c9dd66b5a8a6f
24f4c98dcde98e14c94f48c49961b7b633011942
5939 F20101109_AABTUD mendro_r_Page_39thm.jpg
55a1c6c0228b33e2aef36e791bde2589
1c4dc57328d099dc53292bc7b428747e3e517c86
F20101109_AABTZA mendro_r_Page_42.tif
e1bf5eb1ac6c57369e4330d4f35a433d
9a627d1c52e006747c5f41234e7b2d9fcb3ed0df
2244 F20101109_AABTUE mendro_r_Page_30.txt
e5691cedea5dbf040ec19cf4b05b71f3
efcc1a116993d52a73b4479150b223e132218b91
F20101109_AABTZB mendro_r_Page_43.tif
f43d348600b036d85ae74e97722bf463
b6fbd80ae2b19666059e867229dcbf8d9ba99670
1576 F20101109_AABUBB mendro_r_Page_07.txt
9e3ffe0a7e7fb91f41942be2e04b548d
175c76ce407875d765e9947d77ad759dad4a32c2
21538 F20101109_AABTUF mendro_r_Page_20.jpg
ed5b72dd57aeddeafe397b1ad05f7572
8a444e7f6424ce29cb7da0cfcbccbd7d7fdf50c2
F20101109_AABTZC mendro_r_Page_44.tif
11d15338fdf0496353e18101d0106e9b
1804ba4dc04d7d84e0cf3fe8134467486b4ee73a
2082 F20101109_AABUBC mendro_r_Page_08.txt
c0131f306ef637dfe40b937369d14f08
91aad9a81987d32559ee1072ee7e1a35dd20db8a
54086 F20101109_AABTUG mendro_r_Page_31.pro
f58ebe1bbcbe2427c64184849cac6ab3
3770c35cfab4a3b9f5e1e14a759555fa0c121c16
F20101109_AABTZD mendro_r_Page_45.tif
c83f8beab0aa280b62da9e8617c7d405
fe8c8f2e6fcf258a57bda08eceb177c06b1275b8
2168 F20101109_AABUBD mendro_r_Page_09.txt
883d849257ccc60dfc8ea250d0c29a24
bb38d2062176519344de6a8e36d794bc38fdc600
2153 F20101109_AABTUH mendro_r_Page_11.txt
77172c098f84722d063fd98122550db4
eae9f69b3716c1dfbdf67915f726187f9b9441f0
F20101109_AABTZE mendro_r_Page_46.tif
aabe26a7dc0d5f3ddfcc0f0701b7d711
6e705a4292a8f0b6b671182c43cb872182eeb9a2
2000 F20101109_AABUBE mendro_r_Page_12.txt
b4276db2b86df4defb5ce94a36ad6b99
eec9d5219d3fab6eac7d825e19f33bdad39dc3e2
F20101109_AABTUI mendro_r_Page_01.tif
4032d35e059a845fb99b99ec4e88a490
fe3d4f88b644d92afeeb047603f251ebea07d4a0
F20101109_AABTZF mendro_r_Page_47.tif
0c5c13b24844e57cd1c27ea5cf4f80c3
0e6d1608c342bfd4b82f6119abfc07e24682e483
2199 F20101109_AABUBF mendro_r_Page_15.txt
a84be927751ad7684a13324d59553693
3aa3926eb5a23bb3c220f1acd1f496f8d3e5e55c
8581 F20101109_AABTUJ mendro_r_Page_29thm.jpg
a644e53f4f3c1c91d14cb7c93998a946
568912c8b1437ef0e07bc4560c8f1287d4122589
F20101109_AABTZG mendro_r_Page_48.tif
9b75ec4d2b00c3a6502492d8d1d19333
aff24c9151073d3a7dfbd83460669d6ef1a2fdab
2296 F20101109_AABUBG mendro_r_Page_16.txt
37393ca856f00a5b09ae3ce109b49ea2
52242c056edfb389f09ab0620a06d7c18febd781
151 F20101109_AABTUK mendro_r_Page_03.txt
2fd677b172248d041c1733ad4f0f34b0
c2fb6b6b257498779d6bd9600d32f0f265559913
7813 F20101109_AABTZH mendro_r_Page_01.pro
7ced2cfbce3f9d7feb820c21ddde5766
b441b1ece80cffc18c0c12bbaae53d8315ecf5eb
2206 F20101109_AABUBH mendro_r_Page_17.txt
6c213dc95e51c76f8d7378ccbb7f7a8a
bfb814112c1702f3233b068c7435ada098a8ba16
F20101109_AABTUL mendro_r_Page_23.tif
5584aa2fefd537ca7dd51eb208daecf8
bf59d56e55ec56f60d07d372777f5f5c0734580e
2363 F20101109_AABTZI mendro_r_Page_03.pro
8041ec1e77f49a61341b39b12966e1fd
7e44848493ee4931796415dc582fffbe4d4bf2ea
529 F20101109_AABUBI mendro_r_Page_18.txt
f9e96aeb8928c7a30d14c24b94276227
92a99b7a6f6d204f69928bddf0e8157c805bbc46
36745 F20101109_AABTUM mendro_r_Page_31.QC.jpg
47fa472e084ae9503f96caf9f87f2c94
49e6b03251bbd99b0ae7470c833c0eb32fe22597
10223 F20101109_AABTZJ mendro_r_Page_04.pro
10c0be092df9e39ff11d3e65b4782726
bfa5772b207397abd59456c942cbf457a00c73c8
569 F20101109_AABUBJ mendro_r_Page_19.txt
93392b94a43f181b3ce2c7f75d92c081
a60ad272b804e4e1b9f5e51f8fe3d6a5783fead7
58217 F20101109_AABTUN UFE0022238_00001.mets
137e3dfa75c6c2327465748a749c333e
0d0acd5ed07560822540eff08bed0efbf1d09d9d
68252 F20101109_AABTZK mendro_r_Page_05.pro
e7117af105faae414c39bb373452539d
886fee2363cbc69ff98efe1520e623429582f6c1
149 F20101109_AABUBK mendro_r_Page_20.txt
92d3d52d202d419899b69bc232b9a7fb
72b042d1f44cd060e2732ad28a70417176b4ecbc
4524 F20101109_AABTZL mendro_r_Page_06.pro
27aeb3e225fd1eae4a137b7d152caf5b
46a901ccc45c276b03faf46d913c78dfe0f24a81
198 F20101109_AABUBL mendro_r_Page_21.txt
f22e4132245e2bdaef1e5c6297877539
c899aa0dd099bd3b197b4d938d98aa38de495554
37125 F20101109_AABTZM mendro_r_Page_07.pro
e0649b129f79e191e178a52f537b6e90
3c98e73ebea6bb057316d0207738904a8857fc22
2066 F20101109_AABUBM mendro_r_Page_23.txt
1c7fa7cf738615497673b8e89feddb03
424b79a8ddf9e25b1dd4593d834e9281cfdd04cc
46986 F20101109_AABTZN mendro_r_Page_08.pro
c69369c3efc09572516edcf6c965ad20
0c3dd56e12cdbdfb97b97ad4a83e097c7ee036e6
2124 F20101109_AABUBN mendro_r_Page_25.txt
eda140fbc91f9a915ccb6aee8362ae72
10c86b0fc67ef3bd70871008a1cd3dd566c62dd1
3887 F20101109_AABTUQ mendro_r_Page_02.jpg
61a4949aaa14f62038bbc002a5f89bd0
cb1c293bd086c73401e07989989a176092ebe8ce
53198 F20101109_AABTZO mendro_r_Page_09.pro
713ef6e244ee6b28d0f789c8cb1faebe
aef2206cad7a8afc251fba49744e8bcb946efce2
1880 F20101109_AABUBO mendro_r_Page_26.txt
99bc9d70d61dec8471c45cb0e0bc0bc5
77d08c631ae900f29bba780d1531a2284df6cf15







DESCRIPTION OF OSTEOPROGENITOR GENE EXPRESSION INT PERIODONTAL SOFT
TISSUES

















By

RYAN L. MENDRO


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008



































O 2008 Ryan L. Mendro




































To my family, instructors, fellow residents, and friends who made this possible.









ACKNOWLEDGMENTS

I would like to thank the faculty members of the University of Florida, Department of

Periodontics who faithfully and tirelessly strive to improve the future of this great profession.

Specifically, I would like to thank Shannon Wallet, Tord Lundgren, Theofilos Koutouzis,

Ikramuddin Aukhil, and Dennis Davis for their contributions to my education and this thesis.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF FIGURE S .............. ...............7.....


AB S TRAC T ......_ ................. ............_........8


CHAPTER


1 INTRODUCTION ................. ...............9.......... ......


2 BACKGROUND ................. ...............11.......... .....


Healthy Periodontium ................. ...............11........_ .....
Periodontal Disease .............. ...............12....
Periodontal Tissue Destruction ........._._._..... ..... ...............13....
Treatment of Chronic Periodontitis .............. .....................14
Mesenchymal stem cells............... ...............16.
Bone Development .............. ...............17....

3 MATERIALS AND METHODS .............. ...............23....


Participant Population ................. ...............23........._.....
Surgical Procedure ................. ...............23........ ......
Tissue Preparation and Storage .............. ...............24....
Ribonucleic Acid (RNA) Isolation ................ ...............24................
Reverse Transcription (RT) .............. ...............25....
Polymerase Chain Reaction (PCR) ................. ...............25......___....
In Vitro Stimulation Assay .............. ...............27....

4 RE SULT S ................. ...............29....... ......


Expression of Bone Morphogenetic Protein in Granulation Tissue ................ .........._.._.. .29
Expression of Cbfal in Granulation Tissue .....___.....__.___ .......____ ...........3
Expression of Osterix in Granulation Tissue ........._...... .. ......___ ....___ ...........3
Comparison of Expression Patterns for BMP-2, Cbfal1 and Osterix............. .... ........._._._32
In Vitro Induction of Osteoblast Associated Genes .............. ...............33....
In Vitro Induction of Bone Morphogenetic Protein .............. ...............33....
In Vitro Induction of Cbfal .............. ...............33....
In Vitro Induction of Osterix .............. ...............34....


5 DI SCUS SSION ........._.._ ..... .___ ...............4 1....











6 LIST OF REFERENCES............... ...............4

7 BIOGRAPHICAL SKET CH ................. ...............48................


































































6










LIST OF FIGURES


Figure page

2-1 Periodontium ........... ..... .._ ...............19...

2-2 Radiographic detection of an intrabony defect. .............. ...............20....

2-3 Surgical treatment of an intrabony defect ................. ...............21........... ..

2-4 Factors regulating osteoblast differentiation from mesenchymal stem cells.. ...................22

3-1 Experimental design of in-vitro stimulation assay. ................ ..............................28

4-1 Bone morphogenic proein-2 gene expression in periodontally diseased granulation
tissue samples (D1-D7) and matched healthy, non-diseased, control, gingival tissue
sampl es (C 1, C 4, C 5, C6) .............. ...............3 5....

4-2 Core binding factor al gene expression in periodontally diseased granulation tissue
samples (D1-D7) and matched healthy, non-diseased, control, gingival tissue
samples (C1, C4, CS, C6) .............. ...............36....

4-3 Osterix gene expression in periodontally diseased granulation tissue samples (D1-
D7) and matched healthy, non-diseased, control, gingival tissue samples (C1, C4,
CS C 6) ................ ...............37................

4-5 Bone morphogenic protein-2, Cbfal and Osterix gene expression under bone
inducing conditions of SoaS .............. ...............39....









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

DESCRIPTION OF OSTEOPROGENITOR GENE EXPRESSION INT PERIODONTAL SOFT
TISSUES

By

Ryan L. Mendro
May 2008

Chair: Ikramuddin Aukhil
Major: Dental Sciences

Recent work has shown that cells outside of periodontal ligament, including those found

in granulation tissue may also have the regenerative potential to induce new bone formation by

the expression of specific proteins and transcription factors.l It was therefore the purpose of this

investigation to determine if periodontal granulation tissues possess the specific proteins and

gene expression pattern necessary for osteoblast differentiation in an effort to determine if this

granulation tissue should therefore be retained in the surgical treatment of periodontal defects.

To accomplish this, granulation tissue and healthy gingival tissue samples were harvested

during periodontal surgeries, after which, polymerase chain reaction (PCR) was used to

determine the expression of Bone morphogenic protein-2 (BMP-2), Core binding factor al

(Cbfal) and Osterix; three genes involved in bone development. To demonstrate a potential

model of how these genes could be regulated under inflammatory conditions, an in-vitro

stimulation assay using a human monocytic cell line (THP-1) along with a source of BMP-2,

specifically an osteosarcoma cells line (SoaS) was also performed.

Our results demonstrate that BMP-2, Cbfal and Osterix are expressed at similarly in both

granulation as well as healthy gingival tissues. Furthermore, the in vitro stimulation assay was

unable to demonstrate that under inflammatory conditions these genes were modulated.









CHAPTER 1
INTRODUCTION

Periodontitis is an inflammatory disease in which the cementum, periodontal ligament

(PDL) and alveolar bone surrounding teeth are destroyed. This destruction of the alveolar bone

and supporting periodontal tissues can cause the formation of intrabony periodontal defects

adj acent to teeth. These defects contain a granulomatous tissue that fills the void where the bone

was lost. This tissue is typically excised and discarded in traditional surgical treatment of

periodontal defects. However, many cell types are contained within this granulation tissue

including osteoprogenitor stem cells (OSC). These OSCs are capable of differentiation into the

osteoblast cell lineage, which contribute to bone regeneration and periodontal lesion healing.

This differentiation requires the stimulation of the OSCs by specific proteins and expression of

specific genes necessary for bone formation. This study's aim was to use molecular and

immunohistochemical techniques to examine periodontal granulation tissue for the proteins and

gene expression pattern necessary for osteoblast differentiation to determine if this granulation

tissue is capable of bone regeneration.

To accomplish this, granulation tissue and healthy gingival tissue samples were harvested

during periodontal surgeries, after which, polymerase chain reaction (PCR) was used to

determine the expression of BMP-2, Cbfal and Osterix; three genes involved in bone

development. Here comparisons in gene expression were made between tissues from a non-

inflammatory site and the inflammatory granulation tissue. In order to demonstrate a potential

model of how these genes could be regulated/modulated under inflammatory conditions, an in-

vitro stimulation assay using a human monocytic cell line (THP-1) along with a source of BMP-

2, Cbfal and Osterix; specifically an osteosarcoma cell line (SoaS) was also performed.

Our results here demonstrate the genes for BMP-2, Cbfal and Osterix are expressed at similar










levels in both granulation as well as healthy gingival tissues. Furthermore, the in vitro

stimulation assay was unable to demonstrate that under inflammation conditions these genes

were modulated.

Hypothesis: Granulation tissue is a beneficial component in periodontal lesion healing

due to the presence of both cells with osteoblastic potential as well as the molecules required for

their osteoblastic differentiation.









CHAPTER 2
BACKGROUND

Healthy Periodontium

Healthy periodontium consists of all the supporting structures of the tooth, including the

cementum, periodontal ligament (PDL), alveolar bone and gingiva. Cementum is found on the

surface of tooth roots and serves as anchorage for the principal fibers of the PDL. PDL is

specialized, non-mineralized connective tissue, which attaches the tooth to the alveolar bone.

Additionally, the PDL contains cells including osteoblasts and osteoclasts, monocytes and

macrophages, undifferentiated mesenchymal cells, cementoblasts, odontoclasts and fibroblasts.

These cells are important for tissue homeostasis and repair of the periodontium. For example, it

has been shown in animal models that the fibroblast population in the PDL remains at a constant

state with the number of new fibroblast cells produced by mitosis always equaling the number of

cells that die or migrate.2,3 Fibers of the PDL course through an extracellular ground substance.

This substance comprised of approximately 70% water is thought to be important for distributing

forces applied to the tooth.4

Opposite the tooth, the PDL fibers attach to an outer cortical layer of bundle bone which

forms the bony socket around the teeth. This bone as well as the central lamellar component of

the bone form the alveolar process. Gmngiva covers the bony surface and consists of an outer

junctional epithelium (JE) and an underlying connective tissue. JE consists of nondifferentiated,

stratified squamous epithelial cells that attach to the tooth via a hemidesmosomal attachment.

Monocytes are found within JE which secrete a- and P-defensins, cathelicidin LL-37, interleukin

(1L)-8, IL-la and -10, tumor necrosis factor-a, intercellular adhesion molecule-1, and

lymphocyte function antigen-3.4 These molecules in addition to the JEs structural integrity help

to serve as the first line of defense to invading microorganisms and periodontal disease









progression. In health, a small crevice, the gingival sulcus is formed adjacent to the tooth,

extending from the crest of the gingiva to the JE attachment (Figure 2-1A).

Periodontal Disease

Periodontal disease, specifically chronic periodontitis, is an inflammatory disease which

affects all of the tissues of the periodontium. It is initiated by oral bacteria that infect the

gingival sulcus around the teeth. Proliferating bacteria can cause inflammation of the gingiva,

gingivitis, which can subsequently lead to the destruction of the underlying connective tissue

attachment, PDL, cementum and bone known as periodontitis. Clinically, the sulcus depth

increases and the JE begins to migrate apically as the underlying connective tissue and bone are

destroyed, forming a periodontal pocket (Figure 2-1B). Chronic periodontitis is characterized as

a continuous process with episodes of local exacerbation and remission.' Progression of the

disease can lead to continued loss of supporting structures and eventual tooth loss. This

destructive process can vary greatly and is largely influenced by differing host responses.6

Bacteria associated with chronic periodontitis vary significantly, but are often gram-negative,

anaerobes. Some of the primary bacteria associated with periodontitis include Porphyromona~s

gingivalis, Prevotella intermediate, BacteroidesJ;,i sythis, Aggregatibacter (Actinobacillus)

actinomycetemcomitans and Treponema denticola.

Almost one quarter of the United States is affected with at least a mild form of

periodontitis and approximately 13% of adults over 30 years of age have a moderate or severe

form of the disease.8 Periodontitis has both a subject and site predilection and does not affect all

teeth similarly.9 For example, one study showed that 70% of sites with advanced destruction

occurred in just 12% of the population.10









Periodontal Tissue Destruction

In health, the JE forms a protective band around the neck of the teeth along the

cementoenamel junction. However, the JE can be compromised by periodontal microorganisms

and their byproducts. Once the JE is breached, microorganisms can spread quickly and begin to

damage the underlying connective tissue and PDL by destroying cellular and extracellular

sub stances through the production of toxins including lipopolysaccharide. 1 Subsequently, an

inflammatory cascade is initiated by the host tissues which begin to produce inflammatory

mediators including proteases, cytokines and prostaglandins to fight off the pathogens.12 The

resultant inflammatory response is responsible for damaging the connective tissue and can

quickly spread into adj acent tissues.4 As the connective tissue and PDL are destroyed, the

rapidly proliferating epithelial cells begin to migrate apically along the root of the tooth,

preventing complete healing of the pre-existing connective tissue and PDL.4

As the inflammatory process progresses, it can extend from vessels in the gingival tissues

into the alveolar bone.13 Subsequently, the bone is resorbed by an increased amount of pro-

inflammatory mediators including interleukin 1 (IL-1) and tumor necrosis factor a (TNF-a) and

an increase in osteoclastic activity.4 Depending on the anatomy of the dentition and site

specificity of the disease process the bone loss may occur horizontally, that is parallel to the

cementoenamel junction; vertically, along a vector of the long axis of the tooth or a

combination of both.14 It has been shown that minimal thickness of alveolar bone, vasculature

and distance between tooth roots is associated with vertical bone loss.l

Predominately vertical bone loss creates the formation of intrabony pockets adj acent to teeth,

also known as intrabony or intraosseous defects (Figure 2-2). Clinically they are classified by

the number of intact bony walls that are present.16 These voids created by the vertical loss of

bone are simultaneously filled with newly forming granulation tissue. Granulation tissue is









defined as tissue formed in ulcers and in early wound healing and repair, composed largely of

newly growing capillaries. While granulation tissue is typically indicative of a repair process,

the granulation tissue found chronic periodontitis if left untreated will not ever fully repair or

create a new attachment to the tooth. Subsequently, if the bony defects are treated surgically, the

granulation tissue is typically excised in toto (Figure 2-3).16

More recent evidence suggests however, that this highly vascularized tissue which contains

various inflammatory cells, fibroblasts and stromal cells may be of value in the healing of

periodontal defects.l

Treatment of Chronic Periodontitis

Aims for treating chronic periodontitis are to reduce inflammation and to create an

improved environment for oral hygiene access in order to prevent or reduce disease

reoccurrences. Clinically, treatment outcomes are often measured in pocket depth reduction and

gain in clinical attachment of the soft tissues to the root surface. There are two primary

modalities for the treatment of chronic periodontitis; non-surgical and surgical. Non-surgical

treatment includes scaling and root planing; a procedure in which hand, ultrasonic, rotating or

laser instrumentation is used to cleanse the surface of the teeth without intentional displacement

of the gingival tissues. Scaling is defined as supra or subgingival debridement aimed at

removing the bacterial plaque and their associated mineralized accretions, calculus, from the

tooth surfaces. Root planing is the intentional removal of "diseased" cementum which has been

exposed to cytotoxic byproducts from the periodontal bacteria. It has been shown however, that

intentional aggressive instrumentation to remove all cementum is not necessary to achieve

periodontal health." Scaling and root planing reduces the depth of the pockets and subsequently

the bacterial reservoir by two mechanisms. First, removal of the bacteria decreases the amount

of inflammation present, allowing the gingival tissues to reduce in size and constrict towards the









base of the pocket, which is clinically measured as recession of the gingival tissues.

Additionally, the removal of accretions from the root surfaces can promote a new connective

tissue or long junctional epithelial attachment to form on the root surface coronal to its existing

level, clinically measured as a gain in tissue attachment from the base of the pocket. The main

limitation of non-surgical treatment is that is conducted without tissue reflection, therefore,

visualization of the tooth surface is impaired and subsequently complete root surface cleaning

can not be predictably achieved in even moderately, 5mm deep pockets.l

Surgical intervention is a second modality for treatment of chronic periodontitis. The

objectives of surgical treatment are the similar to non-surgical treatment. However, with surgical

intervention the soft tissue is reflected away from the tooth and bone for better visualization and

access for tooth root instrumentation. Surgical intervention can be accomplished by several

different techniques. First, is resective treatment. Here, mucoperiosteal flap reflection allows

access to the underlying bone, and the contours of the bone can be adjusted to further reduce

periodontal pocketing.19 Additionally, any granulation tissue present is removed, in a process

known as degranulation. Reasons for degranulation are largely empirical, however, it can be

noted that thorough removal of this tissue does undoubtedly provided better visualization and

access to the tooth and bone surfaces and does decrease the amount of residual pocketing

immediately following surgery by decreasing the total thickness of soft tissue.

Another form of surgical treatment of periodontitis is known as guided tissue

regeneration (GTR). Guided tissue regeneration is similar to surgical resective treatment in that,

a surgical flap is first elevated to expose the underlying tooth and bone followed by

degranulation and root instrumentation. However, in addition GTR utilizes the placement of

barrier membranes over the bony defects. Barrier membranes helps to exclude the cells of the









gingival connective tissue and epithelium and gives preference for repopulation by PDL cells in

the area of the defects which may be able to regenerate all of the structures lost in the disease

process, including cementum, PDL, and bone.20 Despite evidence that suggests that regeneration

of these structures may be possible, complete regeneration is not predictable.21 How PDL cells

function in tissue regeneration is not well understood. Some studies support the concept that the

PDL has progenitor cells capable of differentiating into bone forming osteoblasts, while others

suggest that the preexisting osteoblasts are responsible for wound repopulation and new bone

formation.21 22 More recent evidence suggests however, that there may be another source of

osteoblast for wound repair and bone regeneration found within the granulation tissue.l Our

hypothesis is that cells found within the granulation tissue are capable of bone formation and

posses the gene expression pattern necessary for new bone development.

Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are bone marrow derived, self-renewing multipotent

progenitor cells that can be found throughout development.23 Mesenchymal stem cells can

differentiate into various mesenchymal cell lineages including adipocytes, chondrocytes,

hepatocytes, cardiomyocytes, neurons, as well as osteoblasts, the cells responsible for bone

development.24 Stem cells have several key features. First, they must be able to undergo cell

division. Additionally, they must be able to differentiate into multiple cell types. Lastly, when

transplanted into a foreign site, they must possess the ability to reform the cells specific to the

transplant tissue. These stem cells have been isolated in various dental tissues including the

dental pulp and PDL.25, 26 More recent evidence suggests that periodontal granulation tissue may

also contain MSCs.l In order for the MSCs to differentiate into osteoblasts, first an intermediary

cell lineage between the mesenchymal stem cell and the osteoblast known as a pre-osteoblast or

osteoprogenitor cell is expressed. Ultimately, given proper signaling and gene expression, the









osteoprogenitor cells can differentiate into the osteoblasts which are necessary for new bone

formation. If these stem cells are present in granulation tissue it is plausible that they may be

able to regenerate the bone lost from periodontitis.27 Therefore, we hypothesize that the gene

expression pattern necessary for osteoblast differentiation and subsequent new bone formation

can be found in periodontal granulation tissue.

Genes involved in bone development can also be induced experimentally in-vitro,

without the presence of the MSCs. For example, osteosarcoma cell lineages have been shown to

express BMPs and are therefore capable of inducing bone formation.28 Here we use these

osteosarcoma cells in an experimental model of osteoblastic gene induction.

Bone Development

Bone is comprised mainly of hydroxyapatite and extracellular matrix proteins which

include type I collagen, osteocalcin, osteonectin, osteopontin, bone sialoprotein and

proteoglycans.29 It is produced by osteoblasts, specialized cells derived from mesenchymal stem

cells. In order for osteoblast differentiation to occur, the mesenchymal cells must be influenced

by several key regulatory factors (Figure 2-4). One such factor is bone morphogenic protein

(BMP).30 Bone morphogenic protein was discovered in 1965, when it was found that the protein

could ectopically induce bone formation if implanted into muscle.31 Currently, at least 15

different genes of BMPs have been identified.32 Bone morphogenic protein is the only known

growth factor known capable of ectopic bone formation. Signaling of BMP is initiated upon its

binding to two distinct transmembrane receptors.32 Once BMP is bound, the expression of

several other transcription factors are required for further differentiation into an osteoblast

lineage. One such factor, core binding factor al, Cbfal, has been shown to be a primary

transcriptional activator that controls the expression of the maj or structural proteins of the bone

matrix.33 This became evident when it was demonstrated that Cbfal null mice did not produce









any osteoblasts or bone.34 COre binding factor al has also been recognized as a gene responsible

for cleidocranial dysplasia an autosomal-dominant disease with bone abnormalities.35 In

addition, Osterix, a zinc finger-containing transcription factor, has also been shown as a

necessary factor for bone development. Experiments have shown that while Cbfalwas expressed

in Osterix null mice, Osterix is not expressed in Cbfal null mice, thus confirming that Osterix is

located downstream of Cbfal.36

































Figure 2-1.


Periodontium A) Healthy Periodontium. A. Cementum, B. Periodontal Ligament,
C. Alveolar Bone, D. Gingiva, E. Junctional Epithelium, F. Connective Tissue,
G. Gingival Sulcus
B) Chronic Periodontitis. Apical migration of the junctional epithelium occurs as
bacterial inflammation destroys the underlying connective tissue attachment and
bone. Consequently, the sulcus depth increases and a periodontal pocket (G*) is
formed. Granulation tissue fills the void where the bone was lost.



























on of an intrabony defect. Intrabony defect present on mesial of


Radiographic
first molar.


























A B
Figure 2-3. Surgical treatment of an intrabony defect. A) Before and B) after granulation tissue
was removed.













cbfal

Osterix


BMP-2


M~esenchymal Stem Cell


Osteoprogenitor Cell


Figure 2-4.


Factors regulating osteoblast differentiation from mesenchymal stem cells.
Undifferentiated mesenchymal stem cells are influenced by unknown mechanisms to
differentiate towards an osteoblast lineage. An intermediary osteoprogenitor cell is
first formed, upon which BMP-2 binds. After successful binding of BMP-2 several
transcription factors activate the immature osteoprogenitor cell to differentiate into a
fully functioning osteoblast capable of bone formation.


suP-Z


,









CHAPTER 3
MATERIALS AND IVETHODS

We conducted a prospective, observational study to determine if granulation tissue

removed from intrabony periodontal defects contains cells which express key genes necessary

for differentiation of an osteoprogenitor cell lineage capable of producing bone. Specifically, the

quantification of the genes for BlVP-2, Cbfal and Osterix was performed via polymerase chain

reaction (PCR). In addition, an in-vitro experiment was conducted to determine if the expression

of these genes could be modulated by a laboratory model of inflammation.

Participant Population

Seven patients were recruited from the University Of Florida College Of Dentistry,

Department of Periodontology. All patients consented to the study following Institutional

Review Board approval. Inclusion criteria were as follows: a diagnosis of severe periodontal

disease, completion of prior scaling and root planning, an age range between 18-65 years old and

the presence of at least one, 2-3 wall intraosseous periodontal defect with a coronal apical bone

depth of at least 4mm that required surgical treatment. Patients were excluded if they had a

history of severe acute or chronic systemic disease, uncontrolled or poorly controlled diabetes,

were pregnant or lactating or were taking medications known to affect the gingiva.

Prior to the surgery all enrolled patients received a comprehensive oral and periodontal

examination, oral hygiene instructions and scaling and root planing. Surgical intervention was

performed as needed, after a clinical re-evaluation, at least 6 weeks following the completion of

scaling and root planing.

Surgical Procedure

Under local anesthesia by one examiner (RIV), a standard surgical protocol for

periodontal regenerative therapy was completed. Buccal and lingual full-thickness,










mucoperiosteal flaps were elevated beyond the depth of the intraosseous defect. Upon full

reflection, granulation tissue was excised from within the osseous defect. Granulation tissue was

sectioned and immediately placed in Trizol for later processing. In 4 subjects a small piece of

"healthy", control tissue was also excised from a clinically non-inflamed area. Control tissue

included for example, tissue from within the secondary flap or distal wedge tissue. No attempt

was made to harvest control tissue in sites that were not indicated to undergo surgery. This

control tissue was also sectioned and placed into formalin and Trizol. After complete

degranulation of the defect, all teeth in the surgical site were scaled and root planed as needed

with ultrasonic and hand instruments. Next, the defects were filled with freeze dried,

mineralized, bone allograft. At the surgeon's discretion, resorbable membranes were placed over

the bone graft as needed. Flaps were then repositioned and sutured with tension free, primary

closure. Post-operative instructions and antibiotics (1000 mg Amoxicillin at time of surgery,

followed by 500 mg Amoxicillin q8h for 7 days) were administered. Patients were seen for

regular follow-up approximately 2 weeks, 1 month and 3 months post-surgery. Plaque

debridement and oral hygiene instructions were completed as needed at the follow up

appointments .

Tissue Preparation and Storage

After the surgery equal portions of both healthy tissue and granulation tissue were placed

in Trizol. After which, the specimens were frozen at -80oF until RNA harvesting could be

performed.

Ribonucleic Acid (RNA) Isolation

Ribonucleic acid was isolated from each individual tissue sample using a standardized

Trizol protocol. 0.75mL of Trizol LS reagent was added for each 0.25 ml of thawed sample.

The homogenized samples were incubated for 5 minutes at room temperature to permit complete









dissociation of the nucleoprotein complex. 0.2ml of chloroform was added to each sample.

Sample tubes were shaken vigorously by hand for 15 seconds and then incubated at room

temperature for 15 minutes. Next, the samples were centrifuged at 12,000 X g for 10 minutes at

40C. Then, the aqueous phase was transferred to a clean tube. Five hundred microliters of

isopropyl alcohol was added to each tube and gently mixed. Samples were then incubated at

room temperature for one hour. Next, they were centrifuged at 12,000 X g for 10 minutes at 40

After which, the supernatant was decanted. Then the samples were vortexed following the

addition of 1ml of 75% ethanol. Tubes were then centrifuged at 7,500 X g for 5 minutes at 40C.

The remaining pellet was dried for 10 minutes. Then, 25ul of RNase/DNase free water was

added and the samples were incubated for 20 minutes at 600C. Resulting RNA samples, were

frozen at -200C until reverse transcription (RT) could be performed. Subsequently, the

concentration of RNA for each sample was determined using a conventional spectrophotometer.

Reverse Transcription (RT)

Next cDNA was transcribed through reverse transcription of the RNA in the following

manner. First, a master mix containing 5X buffer, ImM DTTs, 2.5mM dNTPs, RT, and

oligopeptides was prepared and aliquoted for each sample. Next, extracted and normalized

concentrations of RNA from each sample were added to the tubes and the RT reaction was

brought to a final volume of 25ul using RNase/DNase free water. All steps were performed on

ice. After which, the RNA was reverse transcribed in a conventional thermocycler under the

following conditions: 400C for 40 minutes, 700C for 15 minutes and held at 40C. All cDNA

was stored at 40C until PCR could be performed.

Polymerase Chain Reaction (PCR)

Polymerase chain reaction was used to amplify the genes of interest from the cDNA. To

run the PCR, first a master mix was made containing 10X PCR buffer, 25mM MgC1,









2.5mMdNTP mix, Taq and 20uM primers for the genes of interest. Primers used included:

BMP -2, "'C GTC AAGC CAAAC ACAAAC AG"' (forward) and

"GAGCCACAATCCAGTCATTCC" (reverse); Cbfal, "CAGTCACCTCAGGCATGTCC"

(forward) and "GAGATATGGAGTGCTGCTGGTG" (reverse); Osterix,

"GGTACAAGGCAGGCATCCATG" (forward) and "AGTGTCCCTTGCAGCCCATC"

(reverse). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was

used as an internal control. Glyceraldehyde 3-phosphate dehydrogenase is constitutively

expressed in all cells and therefore allows for normalization of the total DNA isolated from the

PCR reactions. The primers for GAPDH were, "ACCACAGTCCATGCCATCAC" (forward)

and "TCCACCACCCTGTTGCTGTA" (reverse). Next, the master mix was aliquoted for each

cDNA sample along with RNase/DNase free water. All steps were performed on ice. After

which, the genes of interest were amplified in a conventional thermocycler under the following

conditions: 950C for 4 minutes, 940C for 1 minute, 550C for 45 sec for 30 cycles, at 720C for 2

minutes, 560C for 1 minute, and 720C for 5 minutes. All PCR products were stored at 40C for

until further analysis could be performed. In addition to the study samples, the same PCR

protocol was ran on two control samples known not to express the genes of interest, Human

Immortalized Gingival Keratinocytes (HIGK) and Human Umbilical Vein Endothelial Cells

(HUVEC). Any positive result in the controls would be indicative of either DNA contamination

or non-specificity of the primers designed for the experiment.

Lastly, to visualize the amplified genes, electrophoresis was run on a 2% agarose gel.

This gel was then viewed on a BioRad ChemiDoc and densitometric analysis was performed

using Quantity One (BioRad) software to semi-quantify the genes of interest.









In Vitro Stimulation Assay

The lx105 human monocytic THP-1 cells were plated on a 24 well fibronectin spotted

plate and allowed to adhere and differentiate for 24 hours. Non-adherent cells were removed and

the wells washed 3 times with 2mls of phosphate buffered saline (PB S). After which, in some

wells, lx105 osteosarcoma cells (SaoS2) were plated. THP-1 and SaoS2cells were maintained

in RPMI1640, 10% FBS with .05mM P2-ME during the co-culture. After co-incubation of 24

hours, the cells were harvested and the RNA isolated. RT-PCR was performed and gene

expression was quantified as described above. In some wells, THP-1 cells and SaoS2 cells were

incubated alone to serve as baseline gene expression controls (Fig 3-1).




























Harvest RNA


SPCPfol genre erplession of BMP-2, CbfalandOsterix


1


2


3


Plates sported with fibroneetin Tor ~dl
adhesion




~5 IYla~fnF-lni*r;ri~Taraddrd




CJ 1WO'~ OReosarcoma cells added


1 ho~ur for platesto dry










24 hour
InCUDWOODn


Experimental design of in-vitro stimulation assay. The lx105 human monocytic
THP-1 cells were plated on a 24 well fibronectin spotted plate and allowed to adhere
and differentiate for 24 hours. After which, in some wells, lx105 osteosarcoma cells
(SaoS2) were plated. After co-incubation of 24 hours, the RNA was isolated, RT-
PCR was performed and gene expression was quantified. In some wells, THP-1
cells and SaoS2 cells were incubated alone to serve as baseline gene expression
controls.


Figure 3-1.









CHAPTER 4
RESULTS

7 subj ects participated in this study, from which 7 granulation tissue samples and 4

gingival tissue samples were collected. All of the tissue samples were analyzed via PCR for the

gene expression of BMP-2, Cbfal and Osterix. These genes were used because all three have

been previously shown to be key regulators necessary for osteoblast differentiation and therefore

bone formation.

Expression of Bone Morphogenetic Protein in Granulation Tissue

Bone morphogenetic proteins (BMPs) are secreted signaling molecules which belong to

the transforming growth factor-beta (TGF-P) superfamily of growth factors. Bone morphogenic

proteinss were originally identified by their ability to induce ectopic bone formation when

implanted under the skin of rodents. This indicated that these molecules could play important

roles during bone formation. To date over 15 BMPs have been identified and their expression is

widespread and dynamic as development in general proceeds. Therefore, BMPs can have a

broad range of physiologic functions, including the control of osteoblast differentiation. Several

BMPs can induce osteoblast specific gene expression in vitro.

As previously mentioned, periodontal disease results in bone loss. Subsequently,

granulation tissue is formed in the resulting intrabony defect (Fig 2-3). Traditional periodontal

treatment calls for the removal of this tissue. Previous work done in our laboratory suggests that

this tissue may harbor bone regenerating potentially In order to support this hypothesis here, we

evaluated the gene expression of BMP-2 in granulation tissue to determine if this osteoblast

inducing factor could be present. In addition, we compared this expression level to that of

healthy tissue in order to elucidate if the retention of granulation tissue and thus BMP-2 activity

would be beneficial in healing and bone remodeling of the periodontal lesion.










Specifically, RT-PCR was performed on seven granulation tissue samples as well as four

matching healthy tissues from the same patients. bmp-2 specific primers were used (Figure 4-1A)

to determine gene expression along with GAPDH specific primers (Figure 4-1B) as a

normalization control Finally, as a negative control RT-PCR was also performed on primary

endothelial (HUVEC) cells and keratinocytes (HIGK), cells known not to express the genes of

interest (Figure 4-1C). Once the data was normalized for total cDNA content, densitometric

analysis demonstrated there was no significant difference in the expression levels of bmp-2

among the granulation tissues from our individual participants (Figures 4-1D, 4-4).

Interestingly, there was also no significant difference in the expression levels of bmp-2 in the

granulation tissue and gingival tissue from the same participant. Therefore, while our results do

determine that granulation tissue does have the potential for BMP-2 activity and therefore the

induction of osteoblastic differentiation, it also insinuates that healthy gingival tissues contain

the same properties and potential with regards to BMP-2. (Figures 4-1, 4-4) Additional studies

need to be performed to corroborate this evidence.

Expression of Cbfal in Granulation Tissue

Core binding factor al is the first isolated osteoblastic-specific transcription factor. It is

the earliest and most specific marker for osteogenesis and is capable of inducing osteoblast-

specific gene expression in various cell lines including fibroblasts as well as myoblasts.33, 37

Using immunohistochemi stry, work in our laboratory has shown Cbfal positive cells were also

present in granulation tissue retrieved from periodontal defects.l

As a second step in the investigation of this hypothesis we evaluated the gene expression

of Cbfal in granulation tissue to confirm if this osteoblast-specific transcription factor was

present, which would suggest the presence of osteoblastic cell populations. In addition, we

compared the expression level of Cbfal to that of healthy tissue in order to elucidate if the









retention of granulation tissue and osteoblastic cell populations would be beneficial in healing

and bone remodeling of the periodontal lesion.

Again, RT-PCR was performed on seven granulation tissue samples as well as 4

matching healthy tissues from the same patients. Cbfa-1 specific primers were used (Figure 4-

2A) to determine gene expression along with GAPDH specific primers (Figure 4-2B) as a

normalization control. Finally, as a negative control RT-PCR was also performed on primary

endothelial (HUVEC) cells and keratinocytes (HIGK), known not to express the gene of interest

(Figure 4-2C). The data was again normalized for total cDNA content and densitometric

analysis demonstrated there was no significant difference in the expression levels of cbfal

among the granulation tissues from our individual participants (Figures 4-2D, 4-4).

Interestingly, there was also no significant difference in the expression levels of cbfal in the

granulation tissue and gingival tissue from the same participant. Therefore, while our results do

determine that granulation tissue does have the potential for Cbfal activity and therefore the

induction of osteoblastic differentiation, it also insinuates that healthy gingival tissues contain

the same properties and potential with regards to Cbfal. (Figures 4-2, 4-4). Additional studies

need to be performed to corroborate this evidence.

Expression of Osterix in Granulation Tissue

Experiments have shown that Osterix, a zinc finger-containing transcription factor is also

required for osteoblast differentiation and bone development (36). Little is known about the

mediators of Osterix with regard to osteoblast differentiation; however research has shown that

BMP-2 induces Osterix expression.38

As a final step in investigation of this hypothesis we evaluated the gene expression of

Osterix in granulation tissue to determine if this osteoblast-specific transcription regulator could

be present. This, similar to the presence of Cbfa-1 would suggest the presence of osteoblastic









cell populations. In addition, we compared the expression level of Osterix to that of healthy

tissue in order to elucidate if the retention of granulation tissue and osteoblastic cell populations

would be beneficial in healing and bone remodeling of the periodontal lesion.

Again, RT-PCR was performed on seven granulation tissue samples as well as 4

matching healthy tissues from the same patients. Osterix specific primers were used (Figure 4-

3A) to determine gene expression along with GAPDH specific primers (Figure 4-3B) as a

normalization control. Finally, as a negative control RT-PCR was also performed on primary

endothelial (HUVEC) cells and keratinocytes (HIGK), known not to express the gene of interest

(Figure 4-3C). The data was again normalized for total cDNA content and densitometric

analysis demonstrated there was no significant difference in the expression levels of Osterix

among the granulation tissues from our individual participants (Figures 4-3D, 4-4).

Interestingly, there was also no significant difference in the expression levels of Osterix in the

granulation tissue and gingival tissue from the same participant. Therefore, while our results do

determine that granulation tissue does have the potential for Osterix activity and therefore the

induction of osteoblastic differentiation, it also insinuates that healthy gingival tissues contain

the same properties and potential with regards to Osterix. (Figures 4-3, 4-4). Additional studies

need to be performed to corroborate this evidence.

Comparison of Expression Patterns for BMP-2, Cbfal and Osterix

Figure 4-4A illustrates a concurrent overlay of the expression of the BMP-2, Cbfal and

Osterix. No discernable pattern of gene expression was noted between the genes or between the

granulation tissue group and the gingival tissue group. However, it was noted that for all

samples, expression of Cbfal was greater than the expression of Osterix.

In figure 4-4B, the average gene expression of BMP-2, Cbfal and Osterix is compared

between granulation and gingival tissues. On average, Cbfa-1 and Osterix are expressed more in










granulation tissue than gingival tissue. Alternatively, a trend of increased expression of BMP-2

was noted in gingival tissue.

In Vitro Induction of Osteoblast Associated Genes

Our results from the previous gene expression analysis clearly demonstrate that

osteoprogenitor gene expression for BMP-2, Cbfal and Osterix are found in both clinically

inflamed granulation tissues as well as clinically healthy, non-inflamed gingival tissues.

Subsequently, we sought to determine a plausible model to represent how, if at all, these genes

could be modulated under the inflammatory conditions as would be seen in a chronic periodontal

defect. To accomplish this, an in-vitro stimulation assay was performed using a source of

inflammatory cells, a human monocytic cell line (THP-1), along with a source bone producing

cells, specifically an osteosarcoma cell line (SoaS). The osteosarcoma cells express BMP-2,

Cbfal and Osterix, all of which are needed for bone development.

In Vitro Induction of Bone Morphogenetic Protein

We used bmp-2 specific primers to determine gene expression along with GAPDH

specific primers as a normalization control (Figure 4-5A, D). For BMP-2, the presence of

inflammatory mediators, monocytes, alone, showed the greatest gene expression. Interestingly,

the addition of BMP-2 expressing osteosarcoma cells, to the monocytes did not further increase

the overall BMP-2 gene expression. Osteosarcoma cells alone, had a BMP-2 gene expression

pattern similar to that of monocytes and osteosarcoma cells combined.

In Vitro Induction of Cbfal

Core binding factor al specific primers were used to determine gene expression along

with GAPDH specific primers as a normalization control (Figure 4-5B, D). For Cbfal, the

presence of inflammatory mediators, monocytes alone, showed the greatest gene expression.

Interestingly, the addition of Cbfal expressing osteosarcoma cells, to the monocytes did not










further increase the overall Cbfal gene expression. Osteosarcoma cells alone, had a Cbfal gene

expression pattern similar to that of monocytes and osteosarcoma cells combined.

In Vitro Induction of Osterix

Osterix specific primers were used to determine gene expression along with GAPDH

specific primers as a normalization control (Figure 4-5C, D). For Osterix, the presence of

inflammatory mediators, monocytes alone, showed the greatest gene expression. Interestingly,

the addition of Osterix expressing osteosarcoma cells, to the monocytes did not further increase

the overall Osterix gene expression. Osteosarcoma cells alone, had an Osterix gene expression

pattern similar to that of monocytes and osteosarcoma cells combined.











~r anulati on Ti ssue


Gingi val
Tissue









C1 C4 CS C6


D1 D2 DB D4 DS DG 07


G;APDH


D1 D2 D3 D4 DS DG D7
B


C1 C4 C5 C6


HIG;K andl HUV\;EC


Figure 4-1.


Bone morphogenic proein-2 gene expression in periodontally diseased granulation
tissue samples (D1-D7) and matched healthy, non-diseased, control, gingival tissue
samples (C1, C4, CS, C6). A) Polymerase chain reaction product from amplification
of cDNA using bmp2 specific primers. B) Polymerase chain reaction product from
amplification of cDNA using GAPDH specific primers. C) Polymerase chain
reaction product from amplification of bmp2 specific primers on HIGK and HUVEC
controls. D) Densitometric analysis of(A) normalized to GAPDH amplification in
(B).


BM P-2


BM P-2


3000




200
D1 D2 D3D 5D 7 1C 5C
pain ape














































IIIII IIIIII


Grlanul ati on Tissue ?


Gjingrival
Tissue


D1 D2 03 04 DE DG D7


C1 CQ C5 C6


G;APD!H


Y~ Y1 V~ VY Y3 Y~ VI


Cbfal1


4000
3500
,3000
S2500
2 000

ii
"CI


D1 D2 D3 D4 D5 D6 D7
patient samples


C1 C4 C5 C6


Figure 4-2.


Core binding factor al gene expression in periodontally diseased granulation tissue
samples (D1-D7) and matched healthy, non-diseased, control, gingival tissue
samples (C1, C4, CS, C6). A) Polymerase chain reaction product from amplification
of cDNA using cbfal specific primers. B) Polymerase chain reaction product from
amplification of cDNA using GAPDH specific primers. C) Polymerase chain
reaction product from amplification of cbfal specific primers on HIGK and HUVEC
controls. D) Densitometric analysis of(A) normalized to GAPDH amplification in
(B).


Cbfa l


-
-
-
-
-


II III


III1













~r anul ati on Ti ssue


~i ngival
Tissue


Osterix


01 Da DS D4 05 D6 D7


LI C4 C5 C6


G;APDH


D1 D2 D3 D4 DS


D6 07
B


C1 C4r CS C6


Osto rix


2500

,2000
31500

ii1000
500
0r


D1 D2 D3 D4 D5 D6 D7
patient samples


C1 C4 C5 C6


Figure 4-3.


Osterix gene expression in periodontally diseased granulation tissue samples (D1-
D7) and matched healthy, non-diseased, control, gingival tissue samples (C1, C4,
CS, C6). A) Polymerase chain reaction product from amplification of cDNA using
osterix specific primers. B) Polymerase chain reaction product from amplification of
cDNA using GAPDH specific primers. C) Polymerase chain reaction product from
amplification of osterix specific primers on HIGK and HUVEC controls. D)
Densitometric analysis of(A) normalized to GAPDH amplification in (B).














BMP-2
4000 Cbfal
3500 -1 DOtenlx

cn 3000-
3 2500-

O 2000-









3500-


patHeranulation




3000 -Tissue
niGingival
Tissue
ar 2500-


.2 2000 -

S1500-

S1000-


500-



RMP7 Chfe-1 OSterlx

B


Figure 4-4. Bone morphongenic protein-2, Cbfal and Osterix gene expression. A)
Densitometric analysis for concomitant gene expression of BMP-2, Cbfal and
Osterix in periodontally diseased granulation tissue samples (D1-D7) and healthy,
non-diseased, gingival tissues samples (C1, C4, CS, C6). B) Average gene
expression of BMP-2, Cbfal and Osterix in periodontally diseased granulation tissue
samples and healthy, non-diseased, gingival tissue samples.











3500
3P000




2500


Cbfal





12 3


OsteearomaCells
(1) (2)


Cnly
(3)


n
hn~nrcvtrr kleresr~ronlu om~ru~~mlcllr
ORl~rr~~ma(ellr 4nlv
II) (1) I'li
stimulnbn 4~nrp


Figure 4-5.


Bone morphogenic protein-2, Cbfal and Osterix gene expression under bone
inducing conditions of SoaS. A) Polymerase chain reaction products from
amplification of cDNA using bmp-2 and GAPDH specific primers and
accompanying densitometric analysis. B) Polymerase chain reaction products from
amplification of cDNA using cbfal and GAPDH specific primers and accompanying
densitometric analysis. C) Polymerase chain reaction products from amplification of
cDNA using osterix and GAPDH specific primers and accompanying densitometric
analysis. (D) Comparison of BMP-2, Cbfa-1 and Osterix gene expression under co-
or mono- culture conditions.


OdteesrcomaCells C iI
(1) (2) rl
Stimurlation Assay


GAL.*H


2500
Qsterix 000

S1500
1000


123
~,,,,,













4500
4000
3500
3000
2500
2000
1500
1000
500
0


BM P-2
m Cbfa-1
0 Osterix


Monocytes+ MonocytesOnly Osteosarcoma
Osteosarcoma Ctlls Only
Cells


Stimulation Assay
D


Figure 4-5. Continued









CHAPTER 5
DISCUS SION

Primarily, the objective of periodontal regenerative therapy is to exclude epithelial cells

from the areas where bone loss has occurred in an effort to selectively re-populate the defect with

cells from the PDL, which are presumed to be necessary for new bone and new PDL

attachment.20 More recent work has shown that other cells, including those found in granulation

tissue may also have the regenerative potential to induce new bone formation by the expression

of specific proteins and transcription factors.' It was therefore the purpose of this investigation

to determine if granulation tissue is a beneficial component in periodontal lesion healing due to

the presence of both cells with osteoblastic potential as well as the molecules required for their

osteoblastic differentiation.

Genetic expression of the protein BMP-2 and the transcription factors Cbfal and Osterix

are required for osteoprogenitor cells to differentiate into fully functioning osteoblasts.33, 34, 37

Using PCR, gene expression for BMP-2, Cbfal and Osterix was determined in granulation and

gingival tissue samples. All three genes were expressed in all of the granulation tissue samples.

Additionally, all three genes were also expressed in all gingival tissue samples in levels

comparable to that of the granulation tissue.

Granulation tissue is associated with a healing response. However, in untreated chronic

periodontal disease, the inflammatory insult is not resolved, and thus the granulation tissues

present do not appear to have the ability to spontaneously heal. As such, periodontal granulation

tissue may more appropriately be labeled a "granulomatous" tissue, as it is a tissue that shares

many similarities with granulation tissue, but lacks the distinct ability to repair the damaged

peri odontium.









While traditional surgical treatment of periodontal intrabony defects includes the

meticulous removal of all granulomatous tissues, our research sought to determine if complete

degranulation is necessary.1, 14 We have shown that this highly vascularized tissue may actually

possess the genetic potential to regenerate the bone lost to periodontal disease. If future

treatment could be aimed at shifting the environment within the granulation tissue to an optimal

condition for osteoblast differentiation, it is conceivable that this tissue may in fact be capable of

this differentiation and subsequent new bone formation.

Interestingly, our results found the presence of the three genes of interest for osteoblast

induction BMP-2, Cbfal and Osterix present in not only inflamed granulomatous tissue where

bone was once present, but also in healthy, non-inflamed gingival tissue samples. These results

were supported in a recent study by Zhou et al., who demonstrated that gene expression profiles

for BMPs, Cbfal and Osterix were similar in osteoblasts as well as gingival fibroblasts.39

Despite the presence of these osteogenic markers, the gingival fibroblasts lacked the ability to

induce osteogenesis.39 This was also illustrated in earlier immunohistochemistry work, which

examined only Cbfal, but found that Cbfal positive staining cells were present in granulation

tissues, but not gingival tissue samples.l Therefore, it is evident that even if osteoprogenitor and

osteoblast expression profiles are present, their activation is dependent upon additional unknown

factors which could include for example, cell surface receptors or environmental cues.

One limitation of this study was that the sample size was small and subsequently any

statistical analysis would have been of limited value. Also, while samples were taken either

from granulation or gingival tissues there exists the inherent heterogeneity, not only between

individuals, but between sample sites which may further confound any specific conclusions.

Additionally, while great care was take to retrieve only granulation or gingival tissue samples, it









is impossible to rule out the possibility that the cells from the existing adj acent bone and or

overlying periosteum were included in the tissue samples. Due to the sensitivity of the PCR

amplification process any amount cDNA from bone or periosteum that was present in the

samples may have had a significant impact on the overall gene expression patterns found within

the granulation or gingival tissues.

Our in vitro assay failed to show that the genes necessary for bone development would be

up-regulated under inflammatory conditions. Our model, perhaps an overly simplistic one, may

not have accurately reflected the "inflammatory" state found in periodontal lesions. While,

monocytes are undoubtedly found in periodontal lesions, their presence alone on a plate does not

necessarily create an environment comparable to the largely anaerobic, bacteria laden

periodontal defect environment which is affected by literally dozens of other chemokines,

cytokines, regulatory molecules, etc., all of which were unaccounted for in this model.

Furthermore, by using monocytes and osteosarcoma cells in combination, causality of the

changes in gene expression can not be determined. That is, from our model it can not be

determined if changes in gene expression in BMP-2, Cbfal and Osterix were related to changes

in the expression pattern of the osteosarcoma cells, or if the monocytes themselves, were induced

into an osteoblastic lineage.

Clearly, the process of osteoblast induction is a complex one, which while dependent on

the presence of the three genes BMP-2, Cbfal and Osterix, is not exclusive to all cells expressing

them. Further studies should be aimed at the examination of other possible factors that may be

ultimately responsible for the induction of osteoblasts. While our results do not offer clinical

results suggesting that leaving granulation tissue in periodontal defects may be advantageous to

defect healing, our results do offer a plausible explanation of how this tissue may be of value in









the future. Currently, further laboratory and clinical research is needed to determine if

granulation tissue should be removed during the surgical treatment of periodontal defects.









LIST OF REFERENCES

1. Davis D. Multipotent stem cells isolated from the granulation tissue of intrabony
periodontal defects. Masters Thesis 2007. Gainesville: University of Florida.

2. McCulloch C., Melcher A. Cell density and cell generation in the periodontal ligament of
mice. American Journal ofAnatomy 1983; 167: 43-58.

3. Schellens J, Everts V, Beersten W. Quantitative analysis of connective tissue resoption
in the supra-alveolar region of the mouse incisor ligament. Journal ofPeriodontal
Research 1982; 17: 407-422.

4. Nanci A, Bosshardt D. Structure of periodontal tissues in health and disease.
Periodontology 2000 2006; 40: 11-28.

5. Socransky S, Haffajee A, Goodson J, Lindhe J. New concepts of destructive periodontal
disease. Journal of Clinical Periodontology 1984; 11: 21-32.

6. Kinane D. Causation and pathogenesis of periodontal disease. Periodontology 2000 2001;
25: 8-20.

7. Zambon JJ. Periodontal diseases: microbial factors. Annals ofperiodontology/ The
American Acad'emy ofPeriodontology 1996; 1: 879-925.

8. Albandar J, Brunelle J, Kingman A. Destructive periodontal disease in adults 30 years of
age and older in the United States, 1988-1994. Journal ofPeriodontology 1999; 70: 13-
29.

9. Papapanou P, Wennstrom J, Grondahl K. Periodontal status in relation to age and tooth
type. A cross-sectional radiographic study. Journal ofClinicalPeriodontology 1988; 15:
469-478.

10. Lindhe J, Okamoto H, Yoneyama T, Haffajee A, Socransky S. Periodontal loser sites in
untreated adult subjects. Journal of ClinicalPeriodontology 1989; 16: 671-678.

11. Darveau R, Tanner A, Page R. The microbial challenge in periodontitis. Periodontology
2000 1997; 14: 12-32.

12. Kornman K, Page R, Tonetti M. The host response to the microbial challenge.
Periodontology 2000 1997; 14: 33-53.

13. Weinmann J. Progression of gingival inflammation in the supporting structures of the
teeth. Journal ofPeriodontology 1941; 12: 71-82.

14. Prichard J. Advanced Periodontal Disease: Surgical and Prosthetic Management. 2ed.
1965; 265.

15. Tal H. Relationship between the interproximal distance of roots and the prevalence of
intrabony pockets. Journal ofPeriodontology 1984; 55(1): 604-7.










16. Goldman H, Cohen DW. The infrabony pocket: Classification and treatment Journal of
Periodontology 1958; 29: 272-291.

17. Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of "diseased" root cementum
in healing following treatment of periodontal disease. Journal ofPeriodontalResearch
1986; 21: 496-503.

18. Waerhaug J. Healing of the dento-epithelial junction following subgingival plaque
control. As observed on extracted teeth. Journal ofPeriodontology 1978; 49: 119-134.

19. Schluger S. Osseous resection- a basic principle in periodontal surgery. Oral surgery,
Oral medicine, Oral Pathology, Oral Radiology,~RRR~~RRR~~RR and Endodontics 1949; 2: 3-12.

20. Gottlow J, Nyman S, Lindhe J, Karring T, Wennstroim J. New attachment formation in
the human periodontium by guided tissue regeneration. Case reports. Journal of Clinical
Periodontology 1986; 13: 604-616.

21. Melcher A. On the repair potential of periodontal tissues. Journal ofPeriodontology
1976; 47(5): 255-260.

22. Laurell L, Gottlow J, Zybutz M, Persson R. Treatment of intrabony defects by different
surgical properties. A literature review. Journal ofPeriodontology 1998; 69: 303-313.

23. Mackay-Sim A, Silburn P. Stem cells and genetic disease. CellProbiferation 2008; 41: 85-
93.

24. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human
mesenchymal stem cells. Science 1999: 284: 143-147.

25. Seo B, Miura M, Gronthos S. Investigation of multipotenet postnatal stem cells from
human periodontal ligament. Lancet 2004: 364:149-155.

26. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human
bone marrow and dental pulp. Journal of Bone and Mineral Research : The Official
Journal of the American Society for Bone and Mineral Research 2003; 1 8: 696-704.

27. Marie P. Transcription factors controlling osteoblastogenesis. Archives ofBiochemistry
and Biophysics 2008; In press.

28. Hanamura H. Solubilization and purification of bone morphogenic protein (BMP) from
Dunn osteosarcoma. Clinical Oi therve~l'~ iL \ and Relaedt Research 1980; 153: 232-240.

29. Young M, Kerr J, Ibaraki K. Heegard A, Robey P. Struture expression and regulation of
the maj or noncollagenous matrix proteins of bone. Clinical Oi they wed'tiL \ 1992; 281: 275-
294.

30. Ryoo H, Lee M, Kim Y. Critical molecular switches involved in BMP-2- induced
osteogenic differentiation of mesenchymal cells. Gene 2006; 366: 51-57.










31. Urist M. Bone:formation by sutoinduction. Science 1965; 150: 893-899.

32. Katagari T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast
differentiation. OralDiseases 2002; 8: 147-159.

33. Ducy P, Zhang R, Geoffrey V, Ridall A, Karsenty G. Osf2/Cbfal: a transcriptional
activator of osteoblast differentiation. Cell 1997; 89: 647-654.

34. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson
RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T.
Targeted disruption of Cbfal results in a complete lack of bone formation owing to
maturational arrest of osteoblasts. Cell 1997; 89: 755-764.

35. Lee B, Thirunavukkarasu K, Zhou L, Pastore L, Baldini A, Hecht J, Geoffroy V, Ducy P,
Karsenty G. Missense mutations abolishing DNA binding of osteoblast-specific
transcription factor OSF2/CBFAl in cleidocranial dysplasia. Nature Genetics 1997; 16:
307-310.

36. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe
B. The novel zinc finger-containg transcription factor osterix is required for osteoblast
differentiation and bone formation. Cell 2002; 108(1): 17-29.

37. Ducy P. The osteoblast: A sophisticated fibroblast under central surveillance. Science
2000; 289(5484): 1501-1504.

38. Celil A, Hollinger J, Campbell P. Osx transcriptional regulation is mediated by additional
pathways to BMP2/Smad signaling. Journal of Cellular Biochemistry 2005; 95(3): 518-
528.

39. Zhou Y, Hutmacher D, Sae-Lim VZ. (2008). Osteogenic and Adipogenic Induction
Potential of Human Periodontal Cells. Journal ofPeriodontology 2008; 79(3): 525-534.









BIOGRAPHICAL SKETCH

Dr. Ryan L. Mendro received his Bachelor of Science in Food science and human

nutrition at the University of Florida, where he graduated in the spring of 2001. He then

attended dental school at Columbia University where he received his Doctor of Dental Surgery

degree in the summer 2005. Currently, Ryan Mendro is completing his postdoctoral residency in

periodontics at the University of Florida. Upon graduation in the summer 2008, Ryan will begin

practicing periodontics in Orlando, Florida.





PAGE 1

1 DESCRIPTION OF OSTEOPROGENITOR GENE EXPRESSION IN PERIODONTAL SOFT TISSUES By RYAN L. MENDRO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Ryan L. Mendro

PAGE 3

3 To my family, instructors, fellow resident s, and friends who made this possible.

PAGE 4

4 ACKNOWLEDGMENTS I would like to thank the faculty members of the University of Florida, Department of Periodontics who faithfully and tirele ssly strive to improve the futu re of this great profession. Specifically, I would like to thank Shannon Wa llet, Tord Lundgren, Theofilos Koutouzis, Ikramuddin Aukhil, and Dennis Davi s for their contributions to my education and this thesis.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF FIGURES ............................................................................................................... ..........7 ABSTRACT ...................................................................................................................... ...............8 CHAPTER 1 INTRODUCTION ................................................................................................................ ....9 2 BACKGROUND .................................................................................................................. ..11 Healthy Periodontium .......................................................................................................... ...11 Periodontal Disease ........................................................................................................... .....12 Periodontal Tissue Destruction ...............................................................................................1 3 Treatment of Chronic Periodontitis ........................................................................................14 Mesenchymal stem cells ........................................................................................................ .16 Bone Development .............................................................................................................. ...17 3 MATERIALS AND METHODS ...........................................................................................23 Participant Population ........................................................................................................ .....23 Surgical Procedure ............................................................................................................ ......23 Tissue Preparation and Storage ..............................................................................................24 Ribonucleic Acid (RNA) Isolation .........................................................................................24 Reverse Transcription (RT) .................................................................................................... 25 Polymerase Chain Reaction (PCR) .........................................................................................25 In Vitro Stimulation Assay .................................................................................................... .27 4 RESULTS ..................................................................................................................... ..........29 Expression of Bone Morphogenetic Protein in Granulation Tissue .......................................29 Expression of Cbfa1 in Granulation Tissue ............................................................................30 Expression of Osterix in Granulation Tissue ..........................................................................31 Comparison of Expression Patterns for BMP-2, Cbfa1 and Osterix ......................................32 In Vitro Induction of Osteoblast Associated Genes ...............................................................33 In Vitro Induction of Bone Morphogenetic Protein ...............................................................33 In Vitro Induction of Cbfa1 ................................................................................................... .33 In Vitro Induction of Osterix ................................................................................................. .34 5 DISCUSSION .................................................................................................................. .......41

PAGE 6

6 6 LIST OF REFERENCES ........................................................................................................45 7 BIOGRAPHICAL SKETCH ..................................................................................................48

PAGE 7

7 LIST OF FIGURES Figure page 2-1 Periodontium .............................................................................................................. ........19 2-2 Radiographic detection of an intrabony defect. .................................................................20 2-3 Surgical treatment of an intrabony defect ..........................................................................21 2-4 Factors regulating osteob last differentiation from me senchymal stem cells.. ...................22 3-1 Experimental design of in-vitro stimulation assay.............................................................28 4-1 Bone morphogenic proein-2 gene expre ssion in periodontally di seased granulation tissue samples (D1-D7) and matched healthy, non-diseased, contro l, gingival tissue samples (C1, C4, C5, C6) ..................................................................................................35 4-2 Core binding factor 1 gene expression in periodontal ly diseased granulation tissue samples (D1-D7) and matched healthy, nondiseased, control, gingival tissue samples (C1, C4, C5, C6) ..................................................................................................36 4-3 Osterix gene expression in periodontally diseased granulati on tissue samples (D1D7) and matched healthy, non-diseased, c ontrol, gingival tiss ue samples (C1, C4, C5, C6) ....................................................................................................................... ........37 4-5 Bone morphogenic protein-2 Cbfa1 and Osterix gene expression under bone inducing conditions of SoaS ..............................................................................................39

PAGE 8

8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DESCRIPTION OF OSTEOPROGENITOR GENE EXPRESSION IN PERIODONTAL SOFT TISSUES By Ryan L. Mendro May 2008 Chair: Ikramuddin Aukhil Major: Dental Sciences Recent work has shown that cells outside of periodontal ligament, including those found in granulation tissue may also have the regene rative potential to induce new bone formation by the expression of specific protei ns and transcription factors.1 It was therefore the purpose of this investigation to determine if periodontal granulation tissues possess the specific proteins and gene expression pattern necessary fo r osteoblast differentiation in an effort to determine if this granulation tissue should therefore be retained in the surgic al treatment of pe riodontal defects. To accomplish this, granulation tissue and heal thy gingival tissue samples were harvested during periodontal surgeries, after which, pol ymerase chain reaction (PCR) was used to determine the expression of Bone morphogenic protein-2 (B MP-2), Core binding factor 1 (Cbfa1) and Osterix; three genes involved in bone development. To demonstrate a potential model of how these genes could be regulated under inflammatory conditions, an in-vitro stimulation assay using a human monocytic cel l line (THP-1) along with a source of BMP-2, specifically an osteosarcoma cells line (SoaS) was al so performed. Our results demonstrate that BMP-2, Cbfa1 and Osterix are expressed at similarly in both granulation as well as healthy gi ngival tissues. Furthermore, the in vitro stimulation assay was unable to demonstrate that under inflammato ry conditions these genes were modulated.

PAGE 9

9 CHAPTER 1 INTRODUCTION Periodontitis is an inflammatory disease in which the cementum, periodontal ligament (PDL) and alveolar bone surrounding teeth are destroyed. This de struction of the alveolar bone and supporting periodontal tissues can cause th e formation of intra bony periodontal defects adjacent to teeth. These defects contain a granulomatous tissue th at fills the void where the bone was lost. This tissue is typically excised and discarded in traditional surgical treatment of periodontal defects. However, many cell types are contained within th is granulation tissue including osteoprogen itor stem cells (OSC). These OSCs ar e capable of differentiation into the osteoblast cell lineage, which cont ribute to bone regeneration a nd periodontal lesion healing. This differentiation requires the stimulation of the OSCs by speci fic proteins and expression of specific genes necessary for bone formation. This study’s aim was to use molecular and immunohistochemical techniques to examine periodont al granulation tissue for the proteins and gene expression pattern necessary for osteoblast differentiation to determine if this granulation tissue is capable of bone regeneration. To accomplish this, granulation tissue and heal thy gingival tissue samples were harvested during periodontal surgeries, after which, pol ymerase chain reaction (PCR) was used to determine the expression of BMP-2, Cbfa1 a nd Osterix; three genes involved in bone development. Here comparisons in gene e xpression were made between tissues from a noninflammatory site and the inflamma tory granulation tissue. In order to demonstrate a potential model of how these genes could be regulated/mo dulated under inflammatory conditions, an invitro stimulation assay using a human monocytic cell line (THP-1) along w ith a source of BMP2, Cbfa1 and Osterix; specifically an osteosar coma cell line (SoaS) was also performed. Our results here demonstrate the genes for BMP2, Cbfa1 and Osterix are expressed at similar

PAGE 10

10 levels in both granulation as well as healthy gingival tissues. Furt hermore, the in vitro stimulation assay was unable to demonstrate that under inflammation conditions these genes were modulated. Hypothesis: Granulation tissue is a beneficial component in peri odontal lesion healing due to the presence of both cells with osteoblastic potential as well as the molecules required for their osteoblastic differentiation.

PAGE 11

11 CHAPTER 2 BACKGROUND Healthy Periodontium Healthy periodontium consists of all the supporti ng structures of th e tooth, including the cementum, periodontal ligament (PDL), alveolar bone and gingiva. Cementum is found on the surface of tooth roots and serves as anchorage fo r the principal fibers of the PDL. PDL is specialized, non-mineralized connect ive tissue, which attaches the tooth to the alveolar bone. Additionally, the PDL contains cells including osteoblasts and osteoclasts, monocytes and macrophages, undifferentiated mesenchymal cells, cementoblasts, odontoclasts and fibroblasts. These cells are important for tissu e homeostasis and repair of th e periodontium. For example, it has been shown in animal models that the fibrob last population in the PDL remains at a constant state with the number of new fibr oblast cells produced by mitosis always equaling the number of cells that die or migrate.2,3 Fibers of the PDL course through an extracellular ground substance. This substance comprised of approximately 70% wa ter is thought to be im portant for distributing forces applied to the tooth.4 Opposite the tooth, the PDL fibers attach to an outer cortical la yer of bundle bone which forms the bony socket around the teeth. This bone as well as the central lamellar component of the bone form the alveolar process. Gingiva co vers the bony surface and consists of an outer junctional epithelium (JE) and an underlying connec tive tissue. JE consis ts of nondifferentiated, stratified squamous epithelial cells that attach to the tooth via a hemidesmosomal attachment. Monocytes are found within JE which secrete and -defensins, cathelicid in LL-37, interleukin (IL)-8, IL-1 and -1 tumor necrosis factor, intercellular adhesion molecule-1, and lymphocyte function antigen-3.4 These molecules in addition to the JEs structural integrity help to serve as the first line of defense to invading microorganisms and periodontal disease

PAGE 12

12 progression. In health, a small crevice, the gi ngival sulcus is formed adjacent to the tooth, extending from the crest of the gingiva to the JE attachment (Figure 2-1A). Periodontal Disease Periodontal disease, specifically chronic periodontitis, is an inflammatory disease which affects all of the tissues of the periodontium. It is initiated by oral bacteria that infect the gingival sulcus around the teeth. Proliferating b acteria can cause inflammation of the gingiva, gingivitis, which can subsequently lead to the destruction of the underlying connective tissue attachment, PDL, cementum and bone known as periodontitis. Clinically, the sulcus depth increases and the JE begins to migrate apica lly as the underlying connective tissue and bone are destroyed, forming a periodontal pocket (Figure 2-1B ). Chronic periodontitis is characterized as a continuous process with episodes of local exacerbation and remission.5 Progression of the disease can lead to continued loss of supporting structures and eventual tooth loss. This destructive process can vary greatly and is largely influenced by differing host responses.6 Bacteria associated with chr onic periodontitis vary significantly, but are often gram-negative, anaerobes. Some of the primary bacteria associated with periodontitis include Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Aggregatibac ter (Actinobacillus) actinomycetemcomitans and Treponema denticola.7 Almost one quarter of the United States is affected with at least a mild form of periodontitis and approximately 13% of adults over 30 years of age have a moderate or severe form of the disease.8 Periodontitis has both a subject and s ite predilection and does not affect all teeth similarly.9 For example, one study showed that 70 % of sites with advanced destruction occurred in just 12% of the population.10

PAGE 13

13 Periodontal Tissue Destruction In health, the JE forms a protective ba nd around the neck of the teeth along the cementoenamel junction. However, the JE can be compromised by periodontal microorganisms and their byproducts. Once the JE is breached, mi croorganisms can spread quickly and begin to damage the underlying connective tissue and PDL by destroying cellular and extracellular substances through the production of toxins including lipopolysaccharide.11 Subsequently, an inflammatory cascade is initiated by the host tissues which begin to produce inflammatory mediators including proteases, cytokines and pr ostaglandins to fight off the pathogens.12 The resultant inflammatory response is responsib le for damaging the connective tissue and can quickly spread into adjacent tissues.4 As the connective tissue and PDL are destroyed, the rapidly proliferating epithelial cells begin to migrate apically along th e root of the tooth, preventing complete healing of the pr e-existing connective tissue and PDL.4 As the inflammatory process progresses, it can extend from vessels in the gingival tissues into the alveolar bone.13 Subsequently, the bone is resorb ed by an increased amount of proinflammatory mediators including interleu kin 1 (IL-1) and tumor necrosis factor (TNF) and an increase in osteoclastic activity.4 Depending on the anatomy of the dentition and site specificity of the disease process the bone loss ma y occur horizontally, th at is parallel to the cementoenamel junction; vertically, along a vector of the long axis of the tooth or a combination of both.14 It has been shown that minimal th ickness of alveolar bone, vasculature and distance between tooth roots is associated with vertical bone loss.15 Predominately vertical bone loss creates the fo rmation of intrabony pockets adjacent to teeth, also known as intrabony or intraosseous defects (F igure 2-2). Clinically they are classified by the number of intact bony walls that are present.16 These voids created by the vertical loss of bone are simultaneously filled with newly formi ng granulation tissue. Granulation tissue is

PAGE 14

14 defined as tissue formed in ulcers and in early wound healing and repair, composed largely of newly growing capillaries. While granulation tissue is typically indicative of a repair process, the granulation tissue found chronic periodontitis if left untreated will not ever fully repair or create a new attachment to the tooth. Subsequent ly, if the bony defects ar e treated surgically, the granulation tissue is typically excised in toto (Figure 2-3).16 More recent evidence suggests however, that this highly vascularized tissue which contains various inflammatory cells, fibroblasts and stro mal cells may be of va lue in the healing of periodontal defects.1 Treatment of Chronic Periodontitis Aims for treating chronic periodontitis are to reduce inflammation and to create an improved environment for oral hygiene access in order to prevent or reduce disease reoccurrences. Clinically, treatment outcomes ar e often measured in pocket depth reduction and gain in clinical attachment of the soft tissu es to the root surface. There are two primary modalities for the treatm ent of chronic periodontitis; non-surgical and surgical. Non-surgical treatment includes scaling and root planing; a procedure in which hand, ultrasonic, rotating or laser instrumentation is used to cleanse the su rface of the teeth without intentional displacement of the gingival tissues. Scaling is defined as supra or subgingival debridement aimed at removing the bacterial plaque and their associat ed mineralized accretions, calculus, from the tooth surfaces. Root planing is the intentiona l removal of “diseased” cementum which has been exposed to cytotoxic byproducts from the periodont al bacteria. It has been shown however, that intentional aggressive instrumentation to rem ove all cementum is not necessary to achieve periodontal health.17 Scaling and root plani ng reduces the depth of the pockets and subsequently the bacterial reservoir by two mechanisms. Firs t, removal of the bacter ia decreases the amount of inflammation present, allowing the gingival tissues to reduce in size and constrict towards the

PAGE 15

15 base of the pocket, which is clinically m easured as recession of the gingival tissues. Additionally, the removal of accretions from th e root surfaces can promote a new connective tissue or long junctional epithelia l attachment to form on the root surface coronal to its existing level, clinically measured as a gain in tissue at tachment from the base of the pocket. The main limitation of non-surgical treatment is that is conducted without tissue reflection, therefore, visualization of the toot h surface is impaired and subsequent ly complete root surface cleaning can not be predictably achieved in even moderately, 5mm deep pockets.18 Surgical intervention is a second modality for treatment of chronic periodontitis. The objectives of surgical treatment are the similar to non-surgical treatment. However, with surgical intervention the soft tissue is re flected away from the tooth and bone for better visualization and access for tooth root instrumentation. Surgic al intervention can be accomplished by several different techniques. First, is resective treatment. Here, mucoperiosteal flap reflection allows access to the underlying bone, and the contours of the bone can be adjusted to further reduce periodontal pocketing.19 Additionally, any granulation tissu e present is removed, in a process known as degranulation. Reasons for degranulat ion are largely empirical, however, it can be noted that thorough removal of this tissue does undoubtedly provided bett er visualization and access to the tooth and bone surfaces and doe s decrease the amount of residual pocketing immediately following surgery by decreasi ng the total thickness of soft tissue. Another form of surgical treatment of periodontitis is known as guided tissue regeneration (GTR). Guided tissu e regeneration is similar to surgical resective treatment in that, a surgical flap is first el evated to expose the underly ing tooth and bone followed by degranulation and root instrumentation. Howe ver, in addition GTR u tilizes the placement of barrier membranes over the bony def ects. Barrier membranes helps to exclude the cells of the

PAGE 16

16 gingival connective tissue and epithelium and gi ves preference for repopulation by PDL cells in the area of the defects which may be able to regene rate all of the structur es lost in the disease process, including cementum, PDL, and bone.20 Despite evidence that suggests that regeneration of these structures may be possible, co mplete regeneration is not predictable.21 How PDL cells function in tissue regeneration is not well understood. Some studies support the concept that the PDL has progenitor cells capable of differentiating into bone form ing osteoblasts, while others suggest that the preexisting osteoblasts ar e responsible for wound repopulation and new bone formation.21, 22 More recent evidence suggests howeve r, that there may be another source of osteoblast for wound repair and bone regenera tion found within the granulation tissue.1 Our hypothesis is that cells found within the granul ation tissue are capable of bone formation and posses the gene expression pattern n ecessary for new bone development. Mesenchymal stem cells Mesenchymal stem cells (MSCs) are bone marrow derived, self-renewing multipotent progenitor cells that can be found throughout development.23 Mesenchymal stem cells can differentiate into va rious mesenchymal cell lineages including adipocytes, chondrocytes, hepatocytes, cardiomyocytes, neurons, as well as osteoblasts, the cells responsible for bone development.24 Stem cells have several key features. First, they must be able to undergo cell division. Additionally, they must be able to diffe rentiate into multiple cell types. Lastly, when transplanted into a foreign site, they must possess the ability to reform the cells specific to the transplant tissue. These stem cells have been isolated in various dent al tissues including the dental pulp and PDL.25, 26 More recent evidence suggests th at periodontal granulation tissue may also contain MSCs.1 In order for the MSCs to differentiate into osteoblasts, first an intermediary cell lineage between the mesenchymal stem cell and the osteoblast known as a pre-osteoblast or osteoprogenitor cell is expressed. Ultimately, given proper signaling and gene expression, the

PAGE 17

17 osteoprogenitor cells can differen tiate into the osteoblasts wh ich are necessary for new bone formation. If these stem cells are present in gran ulation tissue it is plausible that they may be able to regenerate the bone lost from periodontitis.27 Therefore, we hypothesize that the gene expression pattern necessary for osteoblast diffe rentiation and subsequent new bone formation can be found in periodontal granulation tissue. Genes involved in bone development can also be induced experimentally in-vitro, without the presence of the MSCs. For example, osteosarcoma cell lineages have been shown to express BMPs and are therefore cap able of inducing bone formation.28 Here we use these osteosarcoma cells in an experimental model of osteoblastic gene induction. Bone Development Bone is comprised mainly of hydroxyapatite and extracellular matrix proteins which include type I collagen, osteocalcin, oste onectin, osteopontin, bone sialoprotein and proteoglycans.29 It is produced by osteoblasts, speciali zed cells derived from mesenchymal stem cells. In order for osteoblast differentiation to occur, the mesenchymal cells must be influenced by several key regulatory factors (Figure 2-4). One such factor is bone morphogenic protein (BMP).30 Bone morphogenic protein was discovered in 1965, when it was found that the protein could ectopically induce bone forma tion if implanted into muscle.31 Currently, at least 15 different genes of BMPs have been identified.32 Bone morphogenic protein is the only known growth factor known capable of ectopic bone fo rmation. Signaling of BMP is initiated upon its binding to two distinct transmembrane receptors.32 Once BMP is bound, the expression of several other transcription fact ors are required for further differentiation into an osteoblast lineage. One such factor, core binding factor 1, Cbfa1, has been shown to be a primary transcriptional activator that cont rols the expression of the major structural proteins of the bone matrix.33 This became evident when it was demonstrated that Cbfa1 null mice did not produce

PAGE 18

18 any osteoblasts or bone.34 Core binding factor 1 has also been recognize d as a gene responsible for cleidocranial dysplasia an autosomaldominant disease with bone abnormalities.35 In addition, Osterix, a zinc finger-c ontaining transcription factor has also been shown as a necessary factor for bone development. Experime nts have shown that while Cbfa1was expressed in Osterix null mice, Osterix is not expressed in Cbfa1 null mice, thus confirming that Osterix is located downstream of Cbfa1.36

PAGE 19

19 A B Figure 2-1. Periodontium A) Healthy Periodontium A. Cementum, B. Periodontal Ligament, C. Alveolar Bone, D. Gingiva, E. Juncti onal Epithelium, F. Connective Tissue, G. Gingival Sulcus B) Chronic Periodontitis Apical migration of the j unctional epithelium occurs as bacterial inflammation destroys the unde rlying connective tiss ue attachment and bone. Consequently, the sulcus depth incr eases and a periodont al pocket (G*) is formed. Granulation tissue fills the void where the bone was lost.

PAGE 20

20 Figure 2-2. Radiographic detec tion of an intrabony defect. Intrabony defect pres ent on mesial of first molar.

PAGE 21

21 A B Figure 2-3. Surgical treatment of an intrabony defect. A) Before and B) after granulation tissue was removed.

PAGE 22

22 Figure 2-4. Factors regulating osteoblast differentiation fr om mesenchymal stem cells. Undifferentiated mesenchymal stem cells are influenced by unknown mechanisms to differentiate towards an osteoblast lineage. An intermediary osteoprogenitor cell is first formed, upon which BMP-2 binds. After successful binding of BMP-2 several transcription factors activate the immature osteoprogenitor cell to differentiate into a fully functioning osteoblast cap able of bone formation.

PAGE 23

23 CHAPTER 3 MATERIALS AND METHODS We conducted a prospective, observational study to determine if granulation tissue removed from intrabony periodonta l defects contains cells whic h express key genes necessary for differentiation of an osteopr ogenitor cell lineage cap able of producing bone Specifically, the quantification of the genes for BMP-2, Cbfa1 an d Osterix was performed via polymerase chain reaction (PCR). In addition, an in-vitro experiment was conducte d to determine if the expression of these genes could be modulated by a laboratory model of inflammation. Participant Population Seven patients were recruited from the University Of Florida College Of Dentistry, Department of Periodontology. All patients c onsented to the study following Institutional Review Board approval. Inclusion criteria were as follows: a diagnosis of severe periodontal disease, completion of prior scaling and root planning, an age range between 18-65 years old and the presence of at least one, 23 wall intraosseous periodontal def ect with a coronal apical bone depth of at least 4mm that required surgical tr eatment. Patients were excluded if they had a history of severe acute or chronic systemic di sease, uncontrolled or poorly controlled diabetes, were pregnant or lactating or were taking medications known to affect the gingiva. Prior to the surgery all enro lled patients received a comp rehensive oral and periodontal examination, oral hygiene instructions and scali ng and root planing. Su rgical intervention was performed as needed, after a clin ical re-evaluation, at least 6 weeks following the completion of scaling and root planing. Surgical Procedure Under local anesthesia by one examiner (RM), a standard surgical protocol for periodontal regenerative therapy was comp leted. Buccal and lingual full-thickness,

PAGE 24

24 mucoperiosteal flaps were elevated beyond the depth of the intraosse ous defect. Upon full reflection, granulation tissue was ex cised from within the osseous defect. Granulation tissue was sectioned and immediately placed in Trizol for later processing. In 4 subjects a small piece of “healthy”, control tissue was also excised from a clinically non-inflamed area. Control tissue included for example, tissue from within the seconda ry flap or distal wedge tissue. No attempt was made to harvest control tissue in sites that were not indicated to undergo surgery. This control tissue was also sectioned and placed in to formalin and Trizol. After complete degranulation of the defect, all te eth in the surgical site were s caled and root planed as needed with ultrasonic and hand instruments. Next the defects were filled with freeze dried, mineralized, bone allograft. At the surgeon’s discretion, resorbable membranes were placed over the bone graft as needed. Flaps were then repos itioned and sutured with tension free, primary closure. Post-operative instructions and antib iotics (1000 mg Amoxicillin at time of surgery, followed by 500 mg Amoxicillin q8h for 7 days) were administered. Patients were seen for regular follow-up approximately 2 weeks, 1 month and 3 months post-surgery. Plaque debridement and oral hygiene instructions were completed as needed at the follow up appointments. Tissue Preparation and Storage After the surgery equal portions of both heal thy tissue and granulati on tissue were placed in Trizol. After which, the specimens were frozen at -80oF until RNA harvesting could be performed. Ribonucleic Acid (RNA) Isolation Ribonucleic acid was isolated from each indivi dual tissue sample using a standardized Trizol protocol. 0.75mL of Trizol LS reagent was added for each 0.25 ml of thawed sample. The homogenized samples were incubated for 5 minut es at room temperature to permit complete

PAGE 25

25 dissociation of the nucleoprotein complex. 0.2ml of chloroform was added to each sample. Sample tubes were shaken vigorously by hand for 15 seconds and then incubated at room temperature for 15 minutes. Next, the samples were centrifuged at 12,000 X g for 10 minutes at 4C. Then, the aqueous phase was transferred to a clean tube. Five hundred microliters of isopropyl alcohol was added to each tube and gen tly mixed. Samples were then incubated at room temperature for one hour. Next, they were centrifuged at 12,000 X g for 10 minutes at 4. After which, the supernatant was decanted. Th en the samples were vortexed following the addition of 1ml of 75% ethanol. Tubes were then centrifuged at 7,500 X g for 5 minutes at 4C. The remaining pellet was dried for 10 minutes Then, 25ul of RNase/DNase free water was added and the samples were incubated for 20 mi nutes at 60C. Resulting RNA samples, were frozen at -20C until reverse transcription (RT) could be performed. Subsequently, the concentration of RNA for each sample was determ ined using a conventional spectrophotometer. Reverse Transcription (RT) Next cDNA was transcribed through reverse tr anscription of the RNA in the following manner. First, a master mix containing 5X buffer, 1mM DTTs, 2.5mM dNTPs, RT, and oligopeptides was prepared and aliquoted for each sample. Next, extracted and normalized concentrations of RNA from each sample were added to the tubes and the RT reaction was brought to a final volume of 25ul using RNase/DNase free water. All steps were performed on ice. After which, the RNA was reverse transc ribed in a conventional thermocycler under the following conditions: 40C for 40 minutes, 70C fo r 15 minutes and held at 4C. All cDNA was stored at 4C until PCR could be performed. Polymerase Chain Reaction (PCR) Polymerase chain reaction was used to amplify the genes of interest from the cDNA. To run the PCR, first a master mix was made containing 10X PCR buffer, 25mM MgCl,

PAGE 26

26 2.5mMdNTP mix, Taq and 20uM primers for the ge nes of interest. Primers used included: BMP-2, “CGTCAAGCCAAACAC AAACAG” (forward) and “GAGCCACAATCCAGTCATTCC” (reverse); Cbfa1, “CAGTCACCTCAGGCATGTCC” (forward) and “GAGATATGGAGTGCTG CTGGTG” (reverse); Osterix, “GGTACAAGGCAGGCATCCATG” (forwa rd) and “AGTGTCCCTTGCAGCCCATC” (reverse). Glyceraldehyde 3-phosphate dehydr ogenase (GAPDH), a housekeeping gene, was used as an internal control. Glyceral dehyde 3-phosphate dehydrogenase is constitutively expressed in all cells and therefore allows for normalization of the total DNA isolated from the PCR reactions. The primers for GAPDH we re, “ACCACAGTCCATGCCATCAC” (forward) and “TCCACCACCCTGTTGCTGTA” (reverse). Next the master mix was aliquoted for each cDNA sample along with RNase/DNase free water. All steps were performed on ice. After which, the genes of interest were amplified in a conventional thermocy cler under the following conditions: 95C for 4 minutes, 94C for 1 minute 55C for 45 sec for 30 cycles, at 72C for 2 minutes, 56C for 1 minute, and 72C for 5 minutes. All PCR products were stored at 4C for until further analysis could be performed. In addition to the study samples, the same PCR protocol was ran on two control samples known not to express the genes of interest, Human Immortalized Gingival Keratinoc ytes (HIGK) and Human Umb ilical Vein Endothelial Cells (HUVEC). Any positive result in the controls would be indicativ e of either DNA contamination or non-specificity of the primers desi gned for the experiment. Lastly, to visualize the amplified genes, elec trophoresis was run on a 2% agarose gel. This gel was then viewed on a BioRad ChemiD oc and densitometric analysis was performed using Quantity One (BioRad) software to semi-quantify the genes of interest.

PAGE 27

27 In Vitro Stimulation Assay The 1x105 human monocytic THP-1 cel ls were plated on a 24 well fibronectin spotted plate and allowed to adhere and differentiate for 24 hours. Non-a dherent cells were removed and the wells washed 3 times with 2mls of phosphate buffered saline (PBS). After which, in some wells, 1x105 osteosarcoma cells (SaoS2) were plated. THP-1 and SaoS2cells were maintained in RPMI1640, 10% FBS with .05mM 2-ME during the co-culture. After co-inc ubation of 24 hours, the cells were harvested and the RNA isolated. RT-PCR was performed and gene expression was quantified as descri bed above. In some wells, THP-1 cells and SaoS2 cells were incubated alone to serve as baseline gene expression controls (Fig 3-1).

PAGE 28

28 1 2 3 Figure 3-1. Experimental design of in-vitro stimulation assay. The 1x105 human monocytic THP-1 cells were plated on a 24 well fibronectin spotted pl ate and allowed to adhere and differentiate for 24 hours. Af ter which, in some wells, 1x105 osteosarcoma cells (SaoS2) were plated. After co-incubati on of 24 hours, the RNA was isolated, RTPCR was performed and gene expression was quantified. In some wells, THP-1 cells and SaoS2 cells were incubated alone to serve as baseline gene expression controls. 24 hou r

PAGE 29

29 CHAPTER 4 RESULTS 7 subjects participated in this study, from which 7 granulation tissue samples and 4 gingival tissue samples were coll ected. All of the tissue sample s were analyzed via PCR for the gene expression of BMP-2, Cbfa1 and Osterix. Th ese genes were used because all three have been previously shown to be key regulators n ecessary for osteoblast diffe rentiation and therefore bone formation. Expression of Bone Morphogeneti c Protein in Granulation Tissue Bone morphogenetic proteins (BMPs) are s ecreted signaling molecules which belong to the transforming growth factor-beta (TGF) superfamily of growth f actors. Bone morphogenic proteinss were originally identified by thei r ability to induce ectopic bone formation when implanted under the skin of rodents. This indi cated that these molecules could play important roles during bone formation. To date over 15 BMPs have been identified and their expression is widespread and dynamic as development in general proceeds. Therefore, BMPs can have a broad range of physiologic functions including the control of oste oblast differentiation. Several BMPs can induce osteoblast specifi c gene expression in vitro. As previously mentioned, periodontal dis ease results in bone lo ss. Subsequently, granulation tissue is formed in the resulting intrabony defect (Fig 2-3). Traditional periodontal treatment calls for the re moval of this tissue. Previous work done in our laborat ory suggests that this tissue may harbor bone regenerating potential.1 In order to support this hypothesis here, we evaluated the gene expression of BMP-2 in granul ation tissue to determine if this osteoblast inducing factor could be present. In addition, we compared this expression level to that of healthy tissue in order to elucidate if the reten tion of granulation tissue and thus BMP-2 activity would be beneficial in h ealing and bone remodeling of the periodontal lesion.

PAGE 30

30 Specifically, RT-PCR was performed on seven gr anulation tissue samples as well as four matching healthy tissues from the same patients. bmp-2 specific primers were used (Figure 4-1A) to determine gene expression along with GAPDH specific primers (Figure 4-1B) as a normalization control Finall y, as a negative control RT-PCR was also performed on primary endothelial (HUVEC) cells and keratinocytes (HIGK), cells k nown not to express the genes of interest (Figure 4-1C). Once the data was normalized for tota l cDNA content, densitometric analysis demonstrated there was no significan t difference in the expression levels of bmp-2 among the granulation tissues from our indivi dual participants (Figures 4-1D, 4-4). Interestingly, there was also no significan t difference in the expression levels of bmp-2 in the granulation tissue and gingival tissu e from the same participant. Therefore, while our results do determine that granulation tissu e does have the potential for BMP-2 activity and therefore the induction of osteoblastic differentiation, it also in sinuates that healthy gi ngival tissues contain the same properties and potential with regards to BMP-2. (Figures 4-1, 4-4) Additional studies need to be performed to corroborate this evidence. Expression of Cbfa1 in Granulation Tissue Core binding factor 1 is the first isolated osteoblastic-specific transcription factor. It is the earliest and most specific marker for oste ogenesis and is capable of inducing osteoblastspecific gene expression in various cell lines including fibroblasts as well as myoblasts.33, 37 Using immunohistochemistry, work in our laborat ory has shown Cbfa1 positive cells were also present in granulation tissue re trieved from periodontal defects.1 As a second step in the investigation of th is hypothesis we evaluated the gene expression of Cbfa1 in granulation tissue to confirm if this osteoblast-specific transcription factor was present, which would suggest the presence of osteoblastic cell populations. In addition, we compared the expression level of Cbfa1 to that of healthy tissue in orde r to elucidate if the

PAGE 31

31 retention of granulation tissue a nd osteoblastic cell populations w ould be beneficial in healing and bone remodeling of th e periodontal lesion. Again, RT-PCR was performed on seven granulation tissue samples as well as 4 matching healthy tissues from the same patients. Cbfa-1 specific primers were used (Figure 42A) to determine gene expression along with GAPDH specific primers (Figure 4-2B) as a normalization control. Finall y, as a negative control RT-PCR was also performed on primary endothelial (HUVEC) cells and keratinocytes (HIGK), known not to express the gene of interest (Figure 4-2C). The data was again normalized for total cDNA content and densitometric analysis demonstrated there was no significan t difference in the expression levels of cbfa1 among the granulation tissues from our indivi dual participants (Figures 4-2D, 4-4). Interestingly, there was also no significan t difference in the expression levels of cbfa1 in the granulation tissue and gingival tissu e from the same participant. Therefore, while our results do determine that granulation tissu e does have the potential for Cbfa1 activity and therefore the induction of osteoblastic differentiation, it also in sinuates that healthy gi ngival tissues contain the same properties and potential with regards to Cbfa1. (Figures 4-2, 4-4). Additional studies need to be performed to corroborate this evidence. Expression of Osterix in Granulation Tissue Experiments have shown that Osterix, a zinc fi nger-containing transcri ption factor is also required for osteoblast differentiation and bone development (36). Little is known about the mediators of Osterix with regard to osteoblast differentiation; however re search has shown that BMP-2 induces Osterix expression.38 As a final step in investigation of this hypothesis we evaluated the gene expression of Osterix in granulation tissue to determine if th is osteoblast-specific tran scription regulator could be present. This, similar to the presence of Cb fa-1 would suggest the presence of osteoblastic

PAGE 32

32 cell populations. In addition, we compared the expression level of Osterix to that of healthy tissue in order to elucidate if th e retention of granulat ion tissue and osteobla stic cell populations would be beneficial in h ealing and bone remodeling of the periodontal lesion. Again, RT-PCR was performed on seven granulation tissue samples as well as 4 matching healthy tissues from the same patients. Osterix specific primers were used (Figure 43A) to determine gene expression along with GAPDH specific primers (Figure 4-3B) as a normalization control. Finall y, as a negative control RT-PCR was also performed on primary endothelial (HUVEC) cells and keratinocytes (HIGK), known not to express the gene of interest (Figure 4-3C). The data was again normali zed for total cDNA content and densitometric analysis demonstrated there was no significan t difference in the expression levels of Osterix among the granulation tissues from our indivi dual participants (Figures 4-3D, 4-4). Interestingly, there was also no significan t difference in the expression levels of Osterix in the granulation tissue and gingival tissu e from the same participant. Therefore, while our results do determine that granulation tissue does have the potential for Os terix activity and therefore the induction of osteoblastic differentiation, it also in sinuates that healthy gi ngival tissues contain the same properties and potential with regards to Osterix. (Figures 4-3, 4-4). Additional studies need to be performed to corroborate this evidence. Comparison of Expression Patterns for BMP-2, Cbfa1 and Osterix Figure 4-4A illustrates a concurrent overlay of the expression of the BMP-2, Cbfa1 and Osterix. No discernable pattern of gene expres sion was noted between the genes or between the granulation tissue group and the gingival tissue group. However, it was noted that for all samples, expression of Cbfa1 was greater than the expression of Osterix. In figure 4-4B, the average gene expression of BMP-2, Cbfa1 and Osterix is compared between granulation and gingival tissues. On aver age, Cbfa-1 and Osterix are expressed more in

PAGE 33

33 granulation tissue than gingival ti ssue. Alternatively, a trend of increased expression of BMP-2 was noted in gingival tissue. In Vitro Induction of Oste oblast Associated Genes Our results from the previous gene expr ession analysis clearl y demonstrate that osteoprogenitor gene expression for BMP-2, Cbfa 1 and Osterix are found in both clinically inflamed granulation tissues as well as clinically healthy, non -inflamed gingival tissues. Subsequently, we sought to determine a plausible model to represent how, if at all, these genes could be modulated under the inflammatory conditi ons as would be seen in a chronic periodontal defect. To accomplish this, an in-vitro stimul ation assay was performed using a source of inflammatory cells, a human monocytic cell lin e (THP-1), along with a source bone producing cells, specifically an osteosarcoma cell line (SoaS). The osteosarcoma cells express BMP-2, Cbfa1 and Osterix, all of which are needed for bone development. In Vitro Induction of Bo ne Morphogenetic Protein We used bmp-2 specific primers to determine ge ne expression along with GAPDH specific primers as a normalization control (F igure 4-5A, D). For BMP-2, the presence of inflammatory mediators, monocytes, alone, showed the greatest gene expression. Interestingly, the addition of BMP-2 expressing osteosarcoma cel ls, to the monocytes di d not further increase the overall BMP-2 gene expression. Osteosarcoma cells alone, had a BMP-2 gene expression pattern similar to that of monocytes and osteosarcoma cells combined. In Vitro Induction of Cbfa1 Core binding factor 1 specific primers were used to determine gene expression along with GAPDH specific primers as a normalizatio n control (Figure 4-5B, D). For Cbfa1, the presence of inflammatory mediators, monocytes alone, showed the greatest gene expression. Interestingly, the addition of Cbfa1 expressing osteosarcoma cells, to the monocytes did not

PAGE 34

34 further increase the overall Cbfa1 gene expressi on. Osteosarcoma cells alone, had a Cbfa1 gene expression pattern similar to that of m onocytes and osteosarcoma cells combined. In Vitro Induction of Osterix Osterix specific primers were us ed to determine gene expression along with GAPDH specific primers as a normalization control (Fig ure 4-5C, D). For Os terix, the presence of inflammatory mediators, monocytes alone, showed the greatest gene expression. Interestingly, the addition of Osterix expressi ng osteosarcoma cells, to the m onocytes did not further increase the overall Osterix gene expressi on. Osteosarcoma cells alone, had an Osterix gene expression pattern similar to that of monocytes and osteosarcoma cells combined.

PAGE 35

35 A B C D Figure 4-1. Bone morphogenic proein-2 gene ex pression in periodontally diseased granulation tissue samples (D1-D7) and matched healthy, non-diseased, contro l, gingival tissue samples (C1, C4, C5, C6). A) Polymerase chain reaction product from amplification of cDNA using bmp2 specific primers. B) Polymera se chain reaction product from amplification of cDNA using GAPDH specific primers. C) Polymerase chain reaction product from amplification of bmp2 specific primers on HIGK and HUVEC controls. D) Densitometric analysis of (A) normalized to GAPDH amplification in (B).

PAGE 36

36 A B C D Figure 4-2. Core binding factor 1 gene expression in periodontal ly diseased granulation tissue samples (D1-D7) and matched healthy, nondiseased, control, gingival tissue samples (C1, C4, C5, C6). A) Polymerase chain reaction product from amplification of cDNA using cbfa1 specific primers. B) Polymera se chain reaction product from amplification of cDNA using GAPDH specific primers. C) Polymerase chain reaction product from amplification of cbfa1 specific primers on HIGK and HUVEC controls. D) Densitometric analysis of (A) normalized to GAPDH amplification in (B).

PAGE 37

37 A B C D Figure 4-3. Osterix gene expres sion in periodontally diseased granulation tissue samples (D1D7) and matched healthy, non-diseased, c ontrol, gingival tiss ue samples (C1, C4, C5, C6). A) Polymerase chain reaction product from amplification of cDNA using osterix specific primers. B) Polymerase chai n reaction product from amplification of cDNA using GAPDH specific primers. C) Polymera se chain reaction product from amplification of osterix specific prim ers on HIGK and HUVEC controls. D) Densitometric analysis of (A) normalized to GAPDH amplification in (B).

PAGE 38

38 A B Figure 4-4. Bone morphongeni c protein-2, Cbfa1 and Oste rix gene expression. A) Densitometric analysis for concomitant gene expression of BMP-2, Cbfa1 and Osterix in periodontally dis eased granulation tissue samples (D1-D7) and healthy, non-diseased, gingival tissues samples (C1, C4, C5, C6). B) Average gene expression of BMP-2, Cbfa1 and Osterix in periodontally diseased granulation tissue samples and healthy, non-diseas ed, gingival tissue samples.

PAGE 39

39 A B C Figure 4-5. Bone mo rphogenic protein-2 Cbfa1 and Osterix gene expression under bone inducing conditions of SoaS. A) Poly merase chain reaction products from amplification of cDNA using bmp-2 and GAPDH specific primers and accompanying densitometric analysis. B) Polymerase chain reaction products from amplification of cDNA using cbfa1 and GAPDH specific primers and accompanying densitometric analysis. C) Polymerase chai n reaction products from amplification of cDNA using osterix and GAPDH specific primers and accompanying densitometric analysis. (D) Comparison of BMP-2, Cbfa -1 and Osterix gene expression under coor monoculture conditions.

PAGE 40

40 D Figure 4-5. Continued

PAGE 41

41 CHAPTER 5 DISCUSSION Primarily, the objective of peri odontal regenerative therapy is to exclude ep ithelial cells from the areas where bone loss has occurred in an effort to selectively re -populate the defect with cells from the PDL, which are presumed to be necessary for new bone and new PDL attachment.20 More recent work has shown that other cells, incl uding those found in granulation tissue may also have the regenerative potential to induce new bone form ation by the expression of specific proteins a nd transcription factors.1 It was therefore the pur pose of this investigation to determine if granulation tissue is a benefici al component in periodonta l lesion healing due to the presence of both cells with os teoblastic potential as well as the molecules required for their osteoblastic differentiation. Genetic expression of the protein BMP-2 and the transcription factors Cbfa1 and Osterix are required for osteoprogenitor cells to diffe rentiate into fully functioning osteoblasts.33, 34, 37 Using PCR, gene expression for BMP-2, Cbfa1 a nd Osterix was determined in granulation and gingival tissue samples. All thr ee genes were expressed in all of the granulation tissue samples. Additionally, all three genes were also expressed in all gingi val tissue samples in levels comparable to that of the granulation tissue. Granulation tissue is associated with a healing response. Ho wever, in untreated chronic periodontal disease, the inflammatory insult is not resolved, and thus the granulation tissues present do not appear to have the ability to spon taneously heal. As such, periodontal granulation tissue may more appropriately be labeled a “granulomatous” tissue, as it is a tissue that shares many similarities with granulation tissue, but l acks the distinct ability to repair the damaged periodontium.

PAGE 42

42 While traditional surgical treatment of periodontal intrabony defects includes the meticulous removal of all granulomatous tissues, our research sought to determine if complete degranulation is necessary.1, 14 We have shown that this highly vascularized tissue may actually possess the genetic potential to re generate the bone lost to pe riodontal disease. If future treatment could be aimed at shifting the environm ent within the granulati on tissue to an optimal condition for osteoblast differentiati on, it is conceivable that this ti ssue may in fact be capable of this differentiation and subsequent new bone formation. Interestingly, our results found the presence of the three genes of in terest for osteoblast induction BMP-2, Cbfa1 and Osterix present in not only inflamed granulomatous tissue where bone was once present, but also in healthy, non-inf lamed gingival tissue samples. These results were supported in a recent study by Zhou et al., who demonstrated that gene expression profiles for BMPs, Cbfa1 and Osterix were similar in osteoblasts as well as gingival fibroblasts.39 Despite the presence of these osteogenic markers, the gingival fibroblasts lacked the ability to induce osteogenesis.39 This was also illustrated in earli er immunohistochemistry work, which examined only Cbfa1, but found that Cbfa1 positive staining cells were present in granulation tissues, but not gingival tissue samples.1 Therefore, it is evident th at even if osteoprogenitor and osteoblast expression profiles are present, their activation is dependent upon additional unknown factors which could include for example, ce ll surface receptors or environmental cues. One limitation of this study was that the sa mple size was small and subsequently any statistical analysis would have been of limited value. Also, while samples were taken either from granulation or gingival ti ssues there exists the inherent heterogeneity, not only between individuals, but between sample sites which may further confound any specific conclusions. Additionally, while great care was take to retrieve only granulation or ging ival tissue samples, it

PAGE 43

43 is impossible to rule out the possibility that the cells from the existing adjacent bone and or overlying periosteum were included in the tissue samples. Due to the sensitivity of the PCR amplification process any amount cDNA from bone or periosteum that was present in the samples may have had a significant impact on the overall gene expression patterns found within the granulation or gingival tissues. Our in vitro assay failed to show that the genes necessary for bone development would be up-regulated under inflammatory conditions. Our model, perhaps an overly simplistic one, may not have accurately reflected the “inflammat ory” state found in periodontal lesions. While, monocytes are undoubtedly found in periodontal lesi ons, their presence alone on a plate does not necessarily create an envir onment comparable to the larg ely anaerobic, bacteria laden periodontal defect environment which is affected by literally dozens of other chemokines, cytokines, regulatory molecules, etc., all of which were unaccounted for in this model. Furthermore, by using monocytes and osteosarco ma cells in combination, causality of the changes in gene expression can not be determin ed. That is, from our model it can not be determined if changes in gene expression in BM P-2, Cbfa1 and Osterix were related to changes in the expression pattern of the osteosarcoma cells or if the monocytes themselves, were induced into an osteoblastic lineage. Clearly, the process of osteoblast induction is a complex one, which while dependent on the presence of the three genes BMP-2, Cbfa1 and Osterix, is not ex clusive to all cells expressing them. Further studies should be aimed at the ex amination of other possible factors that may be ultimately responsible for the induction of osteobl asts. While our results do not offer clinical results suggesting that leaving gr anulation tissue in pe riodontal defects may be advantageous to defect healing, our results do offer a plausible expl anation of how this tissue may be of value in

PAGE 44

44 the future. Currently, further laboratory and cl inical research is needed to determine if granulation tissue should be removed during th e surgical treatment of periodontal defects.

PAGE 45

45 LIST OF REFERENCES 1. Davis D. Multipotent stem cells isolated from the granulation tissue of intrabony periodontal defects Masters Thesis 2007. Gainesvill e: University of Florida. 2. McCulloch C., Melcher A. Cell density and ce ll generation in the pe riodontal ligament of mice. American Journal of Anatomy 1983; 167: 43-58. 3. Schellens J, Everts V, Beersten W. Quan titative analysis of connective tissue resoption in the supra-alveolar region of the mouse incisor ligment. Journal of Periodontal Research 1982; 17: 407-422. 4. Nanci A, Bosshardt D. Structure of pe riodontal tissues in health and disease. Periodontology 2000 2006; 40: 11-28. 5. Socransky S, Haffajee A, Goodson J, Lindhe J. New concepts of de structive periodontal disease. Journal of Clinical Periodontology 1984; 11: 21-32. 6. Kinane D. Causation and pathoge nesis of periodontal disease. Periodontology 2000 2001; 25: 8-20. 7. Zambon JJ. Periodontal dis eases: microbial factors. Annals of periodontology/ The American Academy of Periodontology 1996; 1: 879-925. 8. Albandar J, Brunelle J, King man A. Destructive periodontal di sease in adults 30 years of age and older in the United States, 1988-1994. Journal of Periodontology 1999; 70: 1329. 9. Papapanou P, Wennstrom J, Grondahl K. Period ontal status in rela tion to age and tooth type. A cross-secti onal radiographic study. Journal of Clinical Periodontology 1988; 15: 469-478. 10. Lindhe J, Okamoto H, Yoneyama T, Haffajee A, Socransky S. Periodontal loser sites in untreated adult subjects. Journal of Clinical Periodontology 1989; 16: 671-678. 11. Darveau R, Tanner A, Page R. The microbial challeng e in periodontitis. Periodontology 2000 1997; 14: 12-32. 12. Kornman K, Page R, Tonetti M. The hos t response to the microbial challenge. Periodontology 2000 1997; 14: 33-53. 13. Weinmann J. Progression of gingival infl ammation in the supporting structures of the teeth. Journal of Periodontology 1941; 12: 71-82. 14. Prichard J. Advanced Periodontal Disease: Surgical and Prosthetic Management. 2ed. 1965; 265. 15. Tal H. Relationship between the interproxi mal distance of roots and the prevalence of intrabony pockets. Journal of Periodontology 1984; 55(1): 604-7.

PAGE 46

46 16. Goldman H, Cohen DW. The infrabony pocket: Classification and treatment Journal of Periodontology 1958; 29: 272-291. 17. Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of "diseased" root cementum in healing following treatment of periodontal disease. Journal of Periodontal Research 1986; 21: 496-503. 18. Waerhaug J. Healing of the dento-epithe lial junction followi ng subgingival plaque control. As observed on extracted teeth. Journal of Periodontology 1978; 49:119-134. 19. Schluger S. Osseous resectiona ba sic principle in periodontal surgery. Oral surgery, Oral medicine, Oral Pathology Oral Radiology, and Endodontics 1949; 2: 3-12. 20. Gottlow J, Nyman S, Lindhe J, Karring T, Wennstrm J. New attachment formation in the human periodontium by guided ti ssue regeneration. Case reports. Journal of Clinical Periodontology 1986; 13: 604-616. 21. Melcher A. On the repair potential of periodontal tissues. Journal of Periodontology 1976; 47(5): 255-260. 22. Laurell L, Gottlow J, Zybutz M, Persson R. Treatment of intrabony defects by different surgical properties. A literature review. Journal of Periodontology 1998; 69: 303-313. 23. Mackay-Sim A, Silburn P. Stem cells and gentic disease. Cell Proliferation 2008; 41: 8593. 24. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 1999: 284: 143-147. 25. Seo B, Miura M, Gronthos S. Investigation of multipotenet postnatal stem cells from human periodontal ligament. Lancet 2004: 364:149-155. 26. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral Research 2003; 18: 696-704. 27. Marie P. Transcription factors controlling osteoblastogenesis. Archives of Biochemistry and Biophysics 2008; In press. 28. Hanamura H. Solubilization and purifica tion of bone morphogenic protein (BMP) from Dunn osteosarcoma. Clinical Orthopaedics and Relaedt Research 1980; 153: 232-240. 29. Young M, Kerr J, Ibaraki K. Heegard A, Robe y P. Struture expre ssion and regulation of the major noncollagenous matrix proteins of bone. Clinical Orthopedics 1992; 281: 275294. 30. Ryoo H, Lee M, Kim Y. Critical molecu lar switches involved in BMP-2induced osteogenic differentiation of mesenchymal cells. Gene 2006; 366: 51-57.

PAGE 47

47 31. Urist M. Bone:formation by sutoinduction. Science 1965; 150: 893-899. 32. Katagari T, Takahashi N. Regulatory m echanisms of osteoblast and osteoclast differentiation. Oral Diseases 2002; 8: 147-159. 33. Ducy P, Zhang R, Geoffrey V, Ridall A, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteobl ast differentiation. Cell 1997; 89: 647-654. 34. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a co mplete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997; 89: 755-764. 35. Lee B, Thirunavukkarasu K, Zhou L, Pastore L, Baldini A, Hecht J, Geoffroy V, Ducy P, Karsenty G. Missense mutations abolis hing DNA binding of osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nature Genetics 1997; 16: 307-310. 36. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-contai ng transcription factor osteri x is required for osteoblast differentiation and bone formation. Cell 2002; 108(1):17-29. 37. Ducy P. The osteoblast: A sophisticat ed fibroblast under central surveillance. Science 2000; 289(5484): 1501-1504. 38. Celil A, Hollinger J, Campbell P. Osx transc riptional regulation is mediated by additional pathways to BMP2/Smad signaling. Journal of Cellular Biochemistry 2005; 95(3): 518528. 39. Zhou Y, Hutmacher D, Sae-Lim VZ. ( 2008). Osteogenic and Adipogenic Induction Potential of Human Periodontal Cells. Journal of Periodontology 2008; 79(3): 525-534.

PAGE 48

48 BIOGRAPHICAL SKETCH Dr. Ryan L. Mendro received his Bachelor of Science in F ood science and human nutrition at the University of Florida, where he graduated in the spring of 2001. He then attended dental school at Columbia University where he rece ived his Doctor of Dental Surgery degree in the summer 2005. Currently, Ryan Mendr o is completing his post doctoral residency in periodontics at the University of Florida. U pon graduation in the summer 2008, Ryan will begin practicing periodontics in Orlando, Florida.