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Analysis of cells and matrix in cyclosporine-induced gingival overgrowth 2005

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Analysis of Cells and Matrix in Cyclosporine-induced Gingival Overgrowth By: Masoumeh Nouri DDS, Shahid Beheshti University of Medical Sciences 1985 Dip. Perio, Shahid Beheshti University of Medical Sciences 1989 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCEINCE in THE F A C U L T Y OF GRADUATE STUDIES DENTAL SCIENCE THE UNIVERSITY OF BRITISH COLUMBIA March 2005 © Masoumeh Nouri, 2005 Abstract - B a c k g r o u n d a n d objec t ive : G i n g i v a l overgrowth is one o f the side effects o f cyc lospor ine A , an immunosuppressant agent. Cyc lospor ine A is used c l i n i ca l l y to counteract rejection f o l l o w i n g organ transplantation. The a i m o f the present study was to characterize the tissue changes that occur i n g ing iva l tissue i n patients rece iv ing this medicat ion, compared to control patients ' tissues. - M a t e r i a l s a n d M e t h o d s : G i n g i v a l biopsies were taken from 18 k idney transplant patients referred to L a b a f i hospi tal i n Tehran/ Iran. Per iodontal flap approach was a part o f the procedure to treat their g ing iva l overgrowth. In order to compare these samples w i t h normal tissues, 12 g ing iva l tissue samples were obtained f rom healthy ind iv idua ls i n the process o f c r o w n lengthening that took part i n the school o f dentistry i n S H B Unive r s i ty , Tehran / Iran. The patients from the two groups were as sex and age-matched as m u c h as possible . In a l l cases, patients gave informed consent for the use o f their tissue samples for research. Tissue co l lec t ion , f ixat ion, h is to logical and his tochemical stainings were performed under the same condi t ions i n both groups. T h e antibodies u t i l i zed for immunohis tochemica l stainings compr i sed o f 1 A 4 , P C N A , L C 2 , 2 B 1 and C D 4 4 . These antibodies target ct-smooth muscle act in, prol i ferat ing cel ls , c terminal and core o f vers ican and hyaluronan receptors, respectively. - R e s u l t s : C o m p a r e d to controls, h i s to logica l features from g ing iva l overgrowth samples revealed changed retepegs, some degrees o f acanthosis, hyperkeratosis and parakeratosis, increased vascular iza t ion, and severe inf lammatory infi l t rat ion. The inf lammatory infiltrate inc luded h igh number o f macrophages. M a t r i x changes included prominent accumula t ion o f g lycosaminog lycans ( G A G s ) and proteoglycans (main ly versican). i i C o n c l u s i o n : B o t h ep i the l ium and connect ive tissue showed changes i n g ing iva l overgrowth samples. G i n g i v a l overgrowth tissues showed increased in f lammat ion i n w h i c h C D 4 4 pos i t ive ce l l s (macrophages) predominated. M o r e proliferat ive act ivi ty and changes i n retepegs were observed i n epi thel ium. The presence o f myofibroblasts was noted i n connect ive tissue. V e r s i c a n accumula t ion was also observed i n connective tissue, i n g ing iva l overgrowth samples. O u r studies are consistent w i t h a macrophage-driven accumula t ion o f vers ican-r ich connect ive tissue, associated w i t h epithelial prol iferat ion i n g ing iva l overgrowth , secondary to cyclospor ine therapy. i i i Table of Contents Abstract ii Table of contents iv Acknowledgements vii Chapter one- Introduction 1 1 - N o r m a l g ing iva 1-1- M a c r o s c o p i c anatomy 1 1- 2 - M i c r o s c o p i c anatomy 1 2- G i n g i v a l overgrowth 2 2 -1 - De f in i t i on 2 2-2- Prevalence and c l i n i c a l features o f cyclospor ine- induced g ing iva l overgrowth 2 2- 3- C l i n i c a l compl ica t ions 4 3- C y c l o s p o r i n e - A 5 3-1- H i s to ry 5 3-2- M e c h a n i s m o f A c t i o n 5 3-3- D i s p o s i t i o n and pharmacokinet ics 6 3-4- Therapeutic uses 7 3-5- S ide effects 7 4- Pathogenesis o f cyc lospor ine- induced g ing iva l overgrowth 8 4 -1 - C e l l s 8 -Macrophages 8 - Fibroblasts 10 - M a s t cel ls 11 - Kerat inocytes 12 4-2- G r o w t h factors 12 Platelet der ived growth factor 12 Connec t ive tissue growth factor 14 - Transforming growth factor-P 14 B a s i c fibroblast g rowth factor 17 Kera t inocyte growth factor 18 4-3- Cy tok ines 18 - Interleukin-1 18 Interleukin-6 19 4-4- Integrins 21 i v 4-5- M a t r i x Metal loproteinases 21 4- 6- Proteoglycans and g lycosaminoglycans 24 5- H i s t o l o g i c a l features o f cyc lospor ine- induced g ing iva l overgrowth 25 5- 1- E p i t h e l i u m 25 5-2- Connec t ive Tissue 25 6- Prevent ion, Treatment and Maintenance o f cyclospor ine- induced g ing iva l O v e r g r o w t h 25 Chapter 2- The objective of the Study 28 Chapter 3- Materials and Methods 29 1 - G i n g i v a l overgrowth group 29 2- C o n t r o l group 30 3- C l i n i c a l examinat ions 31 4- Su rg i ca l treatment 31 5- Tissue co l l ec t ion 32 6- H i s tochemica l staining 33 7- Immunohis tochemica l staining 34 8- A n t i b o d y staining 35 9- Considera t ions to a l l o w semi-quantitative analysis o f immunohis tochemica l staining 35 10- M o r p h o m e t r i c analysis 36 10-1- Ep i the l i a l changes 3 6 10-2- In f lammat ion 36 10- 3 - C o n n e c t i v e tissue changes 37 11 - Immunohis tochemica l analysis 40 11 -1 - C D 4 4 (The Hya lu ronan receptor) 40 11 -2- 2 B 1 -Ver s i can staining 42 11- 3- L C 2 - V e r s i c a n staining 43 11 -4- 1 A 4 - a-smooth muscle actin 43 11-5- P C N A - Prol i fera t ing ce l l s 43 12- Statist ical analysis 43 v Chapter 4- Results 44 1 - C l i n i c a l F ind ings 44 2- F ind ings i n ep i the l ium 52 2 - 1 - H & E staining 52 2-2- P C N A staining 54 3- F i n d i n g s i n Connec t ive tissue 56 3 -1 - H & E staining 5 6 3-2- G o m o r i t r ichrome/ aldehyde fuschin staining 56 3-3- A l c i a n blue/ P ic ros i r ius red staining 57 3-4- a -Smooth muscle actin 62 3-2- C o r e o f vers ican antibody 62 3-3- C T e r m i n a l o f V e r s i c a n 65 3.4. C D 4 4 Sta in ing 66 4- C o - l o c a l i z a t i o n 69 Chapter 5- Discussion 73 Limitations of the present study 81 References 82 Appendix 1 90 Appendix 2 91 v i ACKNOWLEDGEMENTS I w o u l d l ike express m y gratitude and appreciat ion to D r . C l i v e R . Roberts for h i s valuable guidance and supervis ion that made this project possible. M a n y thanks to m y colleagues i n Tehran: D r . B . E s l a m i , D r . S. Seyedein, D r . J . E g h b a l , D r . B . E i n o l l a h i , D r . M . Nafar , D r . K . N i l fo rooshan , and D r . R . K h o s r a v i for their sincere cont r ibut ion i n the c l i n i c a l aspect o f the pro jec t . I w o u l d l i ke to acknowledge the genuine cooperat ion o f a l l the patients w h o participated i n this project. I w o u l d l i ke to thank A n d r e a Connor , Sa loumeh Pourmalek , Sean M a u r i c e and Charlotte M o r r i s o n for their help i n the technical aspects o f the project. M a n y thanks to the members o f m y committee D r . E d w a r d Putnins and D r . D o u g Water f ie ld F i n a l l y , I deeply want to thank D r . Saeed A h m a d i , D r . Hassan N o u r i , D r . Sed i N o u r i , and other members o f m y f a m i l y and friends for a l l their love and support. v i i " L o v e is the roo t o f a l l . " ( O s t a d E l a h i 1895-1974) Chapter One- Introduction 1- Normal Gingiva; 1-1 Macroscopic Anatomy: T h e g i n g i v a is that part o f the masticatory mucosa w h i c h covers the a lveolar process and surrounds the ce rv ica l por t ion o f the teeth (Lindhe et al 2003). The g ing iva is d iv ided anatomica l ly into marginal , attached and interdental areas ( N e w m a n et al 2002). 1-2 Microscopic Anatomy: The g i n g i v a consists o f a central core or connect ive tissue covered by stratified squamous epi thel ium. T h e ep i the l ium is d i v i d e d into four ce l l layers: basal , p r ick le ce l l or spinous, granular and kerat inized ce l l layer. E p i t h e l i u m can be orthokeratinized, parakeratinized or non-kerat inized. Orthokerat inized ep i the l ium has no nucle i i n the kerat inized ce l l layer and the granular ce l l layer is wel l -def ined. In parakerat inized epi the l ium, the kerat inized ce l l layer contains pyknot ic nuc le i . Non-kera t in ized ep i the l ium has neither granular nor kerat in ized ce l l layer. The superficial cel ls i n this type o f ep i the l ium have viable nuc le i . The p r inc ipa l c e l l type o f the g ing iva l ep i the l ium is the keratinocyte. The keratinocytes undergo ce l l d i v i s i o n i n the basal ce l l layer and then migrates across the epi the l ium to reach the superficial layers. Other cel ls found in the epi thel ium are melanocytes, langerhans cel ls , merck le ' s cel ls and inf lammatory cel ls . These cel ls are also cal led clear ce l ls . The boundary between the ep i the l ium and the connective tissue has a wavy course. The connect ive tissue portions w h i c h project into the epi the l ium are ca l led connect ive tissue papil lae and are separated from each other by retepegs. The epi the l ium is jo ined to the connective tissue by a basal l amina consists o f l amina densa and l am ina l uc ida by electron microscopy . The major components o f the connect ive tissue are co l lagen fibers, fibroblasts, vessels and nerves w h i c h are embedded i n an amorphous ground substance. C o l l a g e n type I forms the bulk o f the connective tissue. Type I V col lagen is found i n the basement 1 membranes. E las t ic fibers are also distributed among col lagen fibers. Fibroblasts synthesize col lagen and elastic fibers as w e l l as the glycoproteins and g lycosaminoglycan-conta in ing proteoglycans o f the amorphous intercel lular matrix. Besides fibroblasts, the connective tissue also harbors mast cel ls and macrophages as w e l l as inf lammatory cel ls ( N e w m a n et al 2002, L i n d h e et al 2003) . 2 - G i n g i v a l o v e r g r o w t h : 2-1 Definition: G i n g i v a l overgrowth or g ing iva l enlargement is the increase i n size o f the g ing iva , w h i c h is a c o m m o n feature o f g ing iva l disease. These are both c l i n i c a l descriptive terms that a v o i d the erroneous pathologica l connotations o f terms used i n the past such as g ing iva l hyperplasia . Different types o f g ing iva l enlargement are classif ied accord ing to e t io logic factors and pa thologic changes such as; inf lammatory enlargement, drug-induced enlargement, enlargements associated w i t h systemic diseases and condi t ions and neoplastic enlargements (Carranza & H o g a n 2002). In addi t ion to cyclospor ine A , phenytoin and nifedipine are the two other major medicat ions w h i c h can cause g ing iva l overgrowth. A l l o f these drugs function by inh ib i t ing the cel lu lar uptake o f c a l c ium (L indhe et al 1998). 2-2 Prevalence and clinical features of cyclosporine-induced gingival overgrowth: G i n g i v a l overgrowth is observed in i t i a l ly as a papi l lary enlargement w h i c h is prominent on the labia l aspects. The enlargement coalesces, result ing in a lobulated appearance (Ty ldes ley et al 1984). G i n g i v a has been reported to be erythematous, edematous, mobi le , tender and tending toward spontaneous hemorrhage i n some cases (Ros tock et al 1986). Enlarged tissues associated w i t h C S A treatment are general ly more hyperemic than the other medicat ions- induced g ing iva l overgrowth. The first cases o f g ing iva l overgrowth caused by C S A medicat ion were reported by Ratei tschak et al i n 1983. These authors studied 50 k idney transplant patients, most o f w h o m developed g ing iva l 2 enlargement after 4-6 weeks o f C S A treatment. Howeve r , it has also been reported by several authors that g ing iva l overgrowth starts over several months, usual ly between the first and third month after transplantation and often reaches a plateau after 1 year o f C S A therapy ( H a l l m o n & R o s s m a n n 1999, A f o n s o et al 2003). C S A overgrowth se ldom occurs at sites distant from teeth or i n edentulous patients ( M a r s h a l l & B a r t o l d 1999). Because o f this f inding , F u et al (2001) carr ied out an an imal study to examine overgrowth occurrence at the edentulous ridge after C S A therapy i n rats. T h e y extracted a l l r ight m a x i l l a r y molars i n 16 rats and separated animals into 2 groups, a control and a CSA- t r ea t ed group. T h e edentulous morphology , i nc lud ing the bucco- l ingual w i d t h and the vert ical height was measured after sacr i f ic ing the animals. Results showed that C S A therapy produced a significant increase o f the r idge w i d t h and height, w h e n compared to controls. U n d e r histometry, C S A treatment resulted i n a significant increase o f the epi the l ium, connective tissue and total soft tissue areas. Based o n the results o f this study, the authors questioned the hypothesis that tooth or P D L is an essential component for the overgrowth development. The reported incidence for C S A - i n d u c e d g ing iva l overgrowth ranges f rom 1 3 % to 8 1 % (Afonso et al 2003). ). In other studies the prevalence was determined to range f rom 2 5 % to 5 0 % ( H a l l m a n & Rossmann 1999). The reasons for this extensive range are the nature o f the disease be ing treated, the age o f the patient, the method o f measuring g ing iva l overgrowth and addit ional medicat ions being taken by the patient ( M a r s h a l l & Bar to ld 1999, A f o n s o et al 2003). Some authors found a posi t ive correlat ion between C S A b l o o d concentration and incidence o f overgrowth (Marsha l & Bar to ld 1999). Heft i et al (1994) i n a double b l i n d c l i n i ca l trial reported that 17% o f those on C S A w i t h b lood levels be low 400 ng /ml developed g ing iva l overgrowth, whereas 5 9 % tak ing C S A w i t h b lood levels above 400 ng /ml developed enlarged g ing iva . M a n y studies have reported no significant correlat ion between the occurrence o f g ing iva l overgrowth and durat ion o f C S A treatment ( M c G a w et al 1987, Pernu et al 1992, C e b e c i et al 1996, C a k i r et al 3 1998, A f o n s o et al 2003). There are also some addit ive effects reported w h e n C S A is administered concurrent ly w i t h nifedipine (NIF) .Thomason and Seymour (1993) compared 32 renal transplant patients w h o were medicated w i t h C S A for at least 3 months w i t h 23 s imi la r patients w h o were medicated w i t h both C S A and N I F . Patients on combined treatment demonstrated a s ignif icant ly higher g ing iva l overgrowth score when compared to the patients on C S A . The prevalence o f g ing iva l overgrowth was 4 8 % for the combined group and 3 7 % for the C S A group. The same authors i n another study assessed the g ing iva l health o f cardiac transplant patients w h o were o n cyc lospor ine or a combina t ion o f C S A and N I F . 6 2 % o f those taking combined treatment and 26% o f the patients on ly on C S A exhibi ted g ing iva l overgrowth. T h e y found that there was a 25- fo ld increased r isk o f requir ing surgery i f a patient was tak ing both medicat ions. They also reported that young patients are more susceptible to drug induced g ing iva l overgrowth as w e l l as noting tissue changes to be more prevalent i n males (Thomason & Seymour 1995). 2-3 C l i n i c a l c o m p l i c a t i o n s : A s the overgrowth becomes more pronounced, there are increased esthetic concerns. In addi t ion to esthetic problems, the overgrowth may result in uncleansible areas that are more prone to infect ion, caries, and the development o f periodonti t is ( W i l l i a m s o n et al 1994). G i n g i v a l overgrowth also may adversely affect the abi l i ty to masticate (Rostock et al 1986). One recent study (Bora tynska et al, 2003) demonstrated a higher frequency o f loss o f graft function due to chronic graft nephropathy among patients treated w i t h C S A w h o suffer f rom g ing iva l overgrowth. N o research on a possible correlat ion between g ing iva l hyperplas ia and chronic graft nephropathy has been performed to date. H o w e v e r , based on the above ment ioned study, there are c o m m o n pathologica l mechanisms for both k idney fibrosis and g ing iva l overgrowth . The authors 4 stated that TGF-(3 may be one factor responsible for this pathogenesis. Based upon the findings o f this study, w e can conclude that g ing iva l overgrowth is o f c l in i ca l s ignif icance, not on ly for the oral compl ica t ions it causes, but also it can be a s ign showing a higher r isk o f nephropathy. 3 - C y c l o s p o r i n e A ; 3-1 H i s t o r y : The d i scovery o f cyc lospor ine A ( C S A ) is attributed to Jean B o r e l ( A l a g i l l e 1994). Its first reported use i n renal transplant was by Ca lne et al (1978). A l t h o u g h it was first isolated i n Swi tze r land i n 1970 as a metabolite o f a fungus species, it p roved to have little value as an antifungal antibiotic. However , as a potent immunosuppress ive agent, it prolongs surv iva l o f al logeneic transplants ( H a l l m o n & R o s s m a n n 1999). 3-2 M e c h a n i s m o f A c t i o n : C s A suppresses some humoral immuni ty but is more effective against T-cel l-dependent immune mechanisms such as those under ly ing transplant rejection and some forms o f auto immuni ty . It preferentially inhibi ts antigen-triggered s ignal transduction in T lymphocytes , b lunt ing expression o f many lymphokines , inc lud ing I L - 2 , as w e l l as expression o f anti-apoptotic proteins. Studies on the b io log i ca l mechanisms o f C S A have shown that the C S A - c y c l o p h i l i n c o m p l e x binds and inhibi ts the act ivat ion o f ca lc ineur in , a c a l c i u m and camodo l in dependent serine threonine phosphatase. Th i s inact ivat ion prevents dephosphoryla t ion o f nuclear factor o f activated T cel ls ( N F - A T ) , and thereby the nuclear import o f N F - A T and the formation o f a t ranscr ipt ional ly active N F - A T complex . T h e net consequence, i nh ib i t i on o f I L - 2 gene expression at the transcript ional leve l , is considered to be the pr imary mechanism for the immunosuppress ive 5 act iv i ty o f C S A (shin et al 1997). C S A can inhibi t some in vitro B cell responses to T ce l l independent antigens but the c l i n i c a l s ignificance o f this is unclear ( O ' G a r r a et al 1986). 3-3 D i s p o s i t i o n a n d P h a r m a c o k i n e t i c s C S A is var iab ly absorbed i n the gut; peak p lasma concentration is reached 3-4 h after administrat ion, it also has a var iable serum half- l i fe (17-40 hours). H o w e v e r there is a h igh var iab i l i ty i n the data reported o n the terminal half-l ife o f C S A depending on the assay appl ied and on the target populat ion ( C P S 2001) . B i o a v a i l a b i l i t y and t ime unt i l the serum concentration peaks vary greatly between ind iv idua l s ( M a r s h a l l & B e r t o l d 1998). The drug is most ly bound to both cel ls (erythrocytes 5 0 % , lymphocytes 5%) and l ipoproteins (40%), w i t h approximately 5% free i n the p lasma (Hasse l l & Hef t i 1991). C S A accumulates i n the pancreas, spleen, l iver , fat, k idney, lung, bone mar row, heart and whole b lood ( A t k i n s o n et al 1982). The total concentration o f the drug per weight in tissue can be up to 20- fo ld greater than i n p lasma and varies considerably between organs and ind iv idua ls . C S A is metabol ized i n the l iver microsomes ; i n particular by members o f the cytochrome P -450- I I IA gene fami ly , w h i c h is also responsible for the metabol i sm o f hydrophobic compounds such as n i fedipine (Ya t s co f f et al 1991). Ano the r cytochrome, P-450 h P C N 3 , has also been shown to be i n v o l v e d i n metabol iz ing C S A ( A o y a m a et al 1989). These monooxygenase enzymes produce at least 15 metabolites, w h i c h are not as immunosuppress ive as the parent compound . C S A is excreted ma in ly v i a the bi le , through the faeces. There is on ly 6% excreted i n the urine wi th only 0 .1% excreted in urine as unchanged drug (Massha l l & Bar to ld 1998, C P S 2001). 6 3-4 T h e r a p e u t i c Uses The success achieved w i t h C S A treatment i n transplant medic ine and a w ide variety o f autoimmune diseases a l lows us to estimate that one b i l l i o n persons w o r l d wide w i l l be tak ing C S A i n the next decade (Iacopino et a l , 1996). The m a i n indica t ion for this medicat ion is so l id organ transplantation. C S A i n fo rm o f N e o r a l capsules and oral so lu t ion and Sandimmune I . V . are indicated i n the prevent ion o f graft rejection f o l l o w i n g transplantation ( H a l l m o n , Rossmann 1999 and C P S 2001). Other indicat ions include bone mar row transplantation, psoriasis, rheumatoid arthritis, nephri t ic syndrome ( M a r s h a l & Bar to ld 1998, H a l l m o n & Rossmann 1999, C S P 2001), Behce t ' s syndrome (Pisanty et al 1987), insulin-dependent diabetes mel l i tus and systemic lupus erythematosus (Ros tock et al 1986). 3-5 S i d e Ef fec t s : Severa l side effects o f C S A treatment have been reported, among w h i c h are chronic nephrotoxici ty , hepatotoxici ty and neurotoxici ty, lymphoprol i fera t ive neoplasms, hypertension, th romboembol ic compl ica t ions , b i l i a ry calculus disease, diabetes, altered bone metabol ism, h i rsu t i sm, cutaneaous disorders, h igh r isk o f opportunistic infections and f inal ly g ing iva l overgrowth (Mar sha l l & Bar to ld 1998, Ros tock et al 1986, Rateitschak et al 1983). Nephro tox ic i ty is a c o m m o n compl i ca t ion o f C S A therapy. It occurs as C S A causes increases i n serum creatinine and urea levels as a result o f reduced glomerular filtration rate, even at recommended doses. Cyc lospor ine also might cause a reversible increase i n serum b i l i r ub in and l iver enzymes, leading to hepatotoxicity. W i t h regard to these side effects, close moni to r ing o f renal and hepatic funct ion is required for patients treated w i t h C S A . C S A also has the potential to induce tremor, convu l s ion and paresthesia. M a l i g n a n c i e s and lymphoprol i fera t ive disorders can develop as w e l l , but their incidence and dis t r ibut ion are s imi la r to 7 those i n patients treated w i t h convent ional immunosuppress ive therapy ( C P S 2001). G i n g i v a l overgrowth is a side effect o f C S A that is less damaging to the patient 's health. H o w e v e r , consider ing its h igh incidence and the c l i n i ca l compl ica t ions that it causes, it is a significant side effect for the patients ' qual i ty o f l i fe (Afonso et al 2003). Studies addressing the mechan i sm o f g ing iva l overgrowth i n a rat m o d e l , demonstrated that C S A immunosuppress ion inhibi ts the act ivi ty o f matr ix metalloproteinase 2 and 9 i n the early phase o f granulat ion tissue in heal ing dental socket ( S i l v a et al 2001) Based on these f indings, the authors suggested that C S A may interfere w i t h the w o u n d heal ing f o l l o w i n g dental extractions. However , more c l i n i c a l studies are needed to determine whether C S A interferes w i t h the tissue heal ing. 4-Pathogenesis of CSA-induced gingival overgrowth: Studies o f the pathogenesis o f C S A - i n d u c e d g ing iva l overgrowth o n the ce l lu lar and extracellular components o f periodontal tissues have produced considerable data. It seems that complex interactions between var ious mediators o f inf lammat ion and tissue mode l ing are i n v o l v e d in the g ing iva l overgrowth. 4-1 C e l l s : - M a c r o p h a g e s : There have been a number o f studies o f macrophage subpopulations i n g ing iva l overgrowth induced by cyc lospor ine and n i f id ip ine , N u r m e n n i e m i et al (2002) studied g ing iva l samples taken from ind iv idua l s o n C S A and nifedipine treatment and compared those to samples taken f rom healthy ind iv idua l s . An t ibod ie s used for immunohi s tochemica l staining were 2 7 E 1 0 , R M 3 / 1 and 2 5 F 9 . 2 7 E 1 0 antibody identifies a type o f macrophages found in cases o f acute in f lammat ion . R M 3 / 1 antigen is 8 expressed by a reparative/proliferative macrophage phenotype, w h i l e antibody 2 5 F 9 stains cel ls i n the differentiation pathway from monocytes to macrophages. The total numbers o f labeled cel ls were determined i n connect ive tissue beneath sulcular ep i the l ium, connect ive tissue beneath oral epi thel ium and midd le connect ive tissue. The investigators reported a s ignif icantly higher incidence o f specimens expressing 2 7 E 1 0 posi t ive cells throughout the oral epi thel ium i n C S A treated group when compared to control and ni f id ip ine groups. In addi t ion, the number o f R M 3 / 1 - p o s i t i v e macrophages was s ignif icant ly greater i n the C S A group i n connect ive tissue beneath oral ep i the l ium than i n the control group. Ano the r study done by Pernu and K n u u t t i l a (2001) was a imed at evaluat ing the differences i n macrophage ( C D 6 8 ) and lymphocyte subpopulations ( C D 4 , C D 8 and C D 2 0 ) i n n i f id ip ine and cyc lospor ine associated g ing iva l overgrowth. T o test this objective, b iopsy samples o f overgrown g ing iva l tissues f rom patients rece iv ing either o f the two medications were taken and compared to samples taken f rom healthy g ing iva . The authors d iv ided the connective tissue area i n each section into three different zones: 1- the connective tissue beneath the.sulcular ep i the l ium, 2- the midd le connect ive tissue and 3- the connective tissue beneath the oral epi the l ium. T h e y observed that in the n i f id ip ine group, the proport ion o f C D 8 labeled cel ls was signif icantly higher i n the connective tissue beneath the sulcular epi thel ium when compared to control group. In both medicated groups the propor t ion o f C D 6 8 labeled cel ls was higher i n a l l three zones than in control group. O n the other hand, there were no significant differences between medicated and control groups regarding C D 4 and C D 2 0 labeled cel ls . In another study, Iacopino et al (1997) observed that the inf lammatory subset ( 2 7 E 1 0 - the macrophage), w h i c h produces s ignif icant ly greater amounts o f I L - i p , is found i n severely inf lamed g ing iva l tissue but not in drug-induced g ing iva l overgrowth. T h i s f ind ing contrasts the study o f N u r m e n n i e m i et al (2002). The reparative/proliferative subset ( R M 3 / 1 macrophage) produces 9 s igni f icant ly greater amounts o f P D G F - B and is found i n P H T - i n d u c e d hyperplast ic tissue and not i n severely in f lamed tissue. These data appear to support the hypothesis that macrophage phenotype may determine whether tissues enter into a state o f breakdown, repair/maintenance, or prol i ferat ion. - F i b r o b l a s t s : In order to examine the cel lu lar mechanisms pertinent to C S A - i n d u c e d g ing iva l overgrowth, Barber et al (1992) performed a study to observe the effect o f C S A on fibroblasts. The fibroblasts that were used i n this research were taken from 3 different sources: 1- normal healthy human g ing iva , 2- cyc lospor ine- induced overgrown g ing iva and 3- human fetal lung. In addi t ion , they also analyzed the effect o f Escherichia coli L P S on the same cel ls . The results demonstrated that cel ls taken from the g ing iva l overgrowth , when exposed to different concentrations o f C S A , were not stimulated to synthesize D N A at a higher level compared to controls. C e l l s f rom normal g i n g i v a and fetal lung d isp layed an increased proliferat ive act ivi ty i n response to C S A . It was conc luded that C S A has the capacity to stimulate fibroblast prol iferat ion direct ly. The fact that cel ls f rom overgrown g ing iva l d i d not demonstrate a significant response to C S A support this f inding that C S A - i n d u c e d g ing iva l overgrowth does not represent a ce l lu lar hyperplast ic response. There was also a synergist ic effect noted w h e n the proteoglycan output o f normal g ing iva l cel ls was assessed in response to co- incubat ion w i t h both C S A and L P S (Barber et al 1992). So , based on this latter f ind ing , it might be concluded that bacterial L P S m a y be an important co-factor i n the pathogenesis o f C S A - i n d u c e d g ing iva l over growth . C o t r i m et al (2003) performed a study to analyze the effect o f C S A and TGF-(31 o n the prol i ferat ion o f the normal g ing iva l ( N G ) fibroblasts. They observed that C S A at the concentrations o f 50 -200 n g / m l increased N G fibroblasts prol iferat ion, w i t h the m a x i m a l s t imulat ion observed at 200 n g / m l . W i t h increasing concentration o f C S A (400 & 800 ng/ml) N G fibroblasts prol i ferat ion was 10 inhibi ted . T h e y also reported that the prol iferat ing ce l l nuclear antigen ( P C N A ) levels i n N G fibroblasts that were incubated wi th 200 ng /ml o f C S A were also s ignif icant ly higher than those o f controls . In addi t ion, the authors studied the effect o f incubat ion wi th TGF-(31 for 24 hours on these cel ls and observed a significant increase i n the proliferative rate o f N G fibroblasts at a T G F - p i concentrat ion o f 0.1-1 ng /ml . However , at a concentration o f 10 ng /ml T G F - p i decreased N G ce l l prol i ferat ion. C o r t i m and colleagues conc luded that; 1) C S A stimulates N G fibroblast prol i ferat ion at the same concentrations found i n the serum o f patients undergoing C S A treatment (human serum leve l range: 100-250 ng/ml) , and 2) C S A stimulates N G fibroblast proliferat ion through autocrine s t imula t ion o f T G F - P 1 . -Mast cells: Pisanty et al (1990) used both l ight mic roscopyand S E M to study g ing iva l samples o f patients suffering f rom Behce t ' s syndrome who were treated w i t h C S A for up to 20 months. They demonstrated the presence o f mast cel ls , both intact and degranulated, i n the g ing iva l epi thel ium. In another study, to observe the ultrastructural and his tochemical aspects o f cyc lospor ine- induced g ing iva l overgrowth M a r i a n i et al (1996) obtained tissue samples f rom k idney transplant patients. They observed a h igh numbers o f mast cel ls i n overgrown tissues. Based o n this f ind ing , they stated that mast cel ls presence cou ld result i n a h igh release o f histamine, favor ing the transfer o f f luids f rom b lood to the amorphous ground substance by means o f the mic ropynocy to t i c vesic les o f the capi l lar ies . The authors conc luded that the mast cel ls m a y contribute s ignif icant ly to the accumula t ion o f abnormal ly h igh quantities o f intercellular l i qu id . O n the other hand, Asaha ra et al (2000) used mast c e l l deficient m ice to determine the role o f mast cel ls i n C S A - i n d u c e d g ing iva l overgrowth. They fed this group o f mice and a control group w i t h different concentrations o f cyc lospor ine da i ly . Af ter 30 11 days the m i c e were sacrif iced to get the g ing iva l sections. B o t h groups, fed w i t h 600 mg/kg/day o f cyc lospor ine , demonstrated g ing iva l hyperplasia . They concluded that the mast cel ls are not necessary i n the development o f the overgrowth and that the increased number o f mast cel ls i n the enlarged g i n g i v a observed by M a r i a n i et al (1996) may be a secondary effect o f g ing iva l hyperplasia . -Keratinocytes: In order to compare the mitot ic activit ies o f keratinocytes i n sulcular, oral sulcular , and oral g ing iva l ep i the l ium i n nifedipine and C S A - i n d u c e d g ing iva l overgrowth tissue and in normal g ing iva , N u r m e n n i e m i et al (2001) obtained g ing iva l samples from the above groups. Cryostat sections were stained w i t h monoc lona l antibody for Ki-67 and the mitot ic act ivi ty o f epi thel ia l cel ls was determined as a percentage o f the labeled cells in relat ion to total numbers o f epi thel ial cel ls i n the basal layers o f 3 different zones. It was observed that mi to t ic activit ies were s ignif icant ly higher i n medicated groups when compared to controls. The difference was most c lear ly seen i n the oral ep i the l ium. T h i s study conc luded that the epi thel ial th ickening i n ni fedipine- and C S A - i n d u c e d g ing iva l overgrowth is associated w i t h increased mitot ic act ivi ty. Differences in mitot ic act ivi ty between the oral epi thel ium, oral sulcular and sulcular epi thel ium suggest that loca l g ing iva l in f lammat ion affects the drug-induced changes. 4-2 Growth Factors: -Platelet-Derived Growth Factor (PDGF): P D G F is a d imer ic polypept ide, consis t ing o f A and B chains, in homodimer ( A A , B B ) or hetrodimer ( A B ) combinat ions . The A cha in is be l ieved to p lay a minor role i n ce l l prol i ferat ion and tissue repair. The B chain , however , is a potent mi togen for cel ls o f mesenchymal o r ig in and has been demonstrated 12 to promote g rowth and heal ing o f connect ive tissues. A d d i t i o n a l l y , P D G F - B is a major chemoattractant for fibroblasts, s t imulat ing fibroblast prol iferat ion and synthesis o f g lycosaminog lycans , proteoglycans, f ibronect in, and col lagen. P D G F is released f rom platelets, macrophages, endothelial cel ls , smooth muscle cel ls and fibroblasts. In terms o f proliferat ive act ivi ty , the release o f P D G F - B f rom macrophages is bel ieved to be responsible for connect ive tissue prol i ferat ion associated w i t h inf lammat ion. Thus , the increased levels o f P D G F - B m a y promote fibroblast prol i fera t ion and/or fibroblast product ion o f extracellular matr ix constituents i n hyperplastic tissues (Nares et al 1996). In situ hybr id iza t ion studies performed on tissues from control and C S A - treated patients exhib i t ing g ing iva l overgrowth have shown that adminis t ra t ion o f C S A increases macrophage P D G F - B gene expression (P lemmons et al 1996). Nares and colleagues (1996) performed a study to quantify P D G F - B gene express ion i n hyperplastic tissues i n patients rece iv ing C S A therapy. They extracted the total R N A f rom human g ing iva l samples obtained f rom C S A - t r e a t e d and control patients. In addi t ion, dual fluorescence immunohis tochemis t ry for mature macrophage marker antigen ( C D 5 1 ) and intracellular P D G F - B was performed. The study demonstrated a significant increase i n P D G F - B m R N A i n hyperplastic tissues f rom CSA- t r ea t ed patients. S ince C S A caused a 48- fo ld increase i n P D G F - B m R N A compared to 8-fold increase i n the amount o f this growth factor caused by inf lammat ion , the authors conc luded that C S A - i n d u c e d increase was inflammation-independent. P D G F - B produc ing cel ls were also ident i f ied as mature macrophages w i t h a non-uni form dis t r ibut ion l imi ted to the papi l lary connect ive tissue. They hypothesized that the macrophages may play a pr imary role in this process through C S A - s t i m u l a t e d up-regulat ion o f var ious growth factors/cytokines. S i m i l a r l y , i n an an imal study, Iacopino et al (1997) tested rat macrophages for P D G F - A / P D G F - B synthesis in response to var ious levels o f C S A , us ing an 13 ELISA assay. The results demonstrated that CSA produce a significant increase of PDGF-A/B levels in conditioned media in all concentrations. -Connective tissue growth factor (CTGF): CTGF was first identified as a cysteine-rich polypeptide secreted by vascular endothelial cells in culture. CTGF functions as both an autocrine and paracrine-signaling molecule to maintain and perhaps to amplify/synchronize the response of fibroblastic cell proliferation and extracellular matrix synthesis. Elevated levels of CTGF occur in skin sclerosis, renal fibrosis and lung fibrosis. The close association of CTGF with vascular endothelial cells led to the concept that CTGF could also play an important role in angiogenesis. It has been suggested that CTGF could facilitate the growth of fibroblasts in part by a mechanism involving increased angiogenesis during development and wound repair. CTGF is strongly and rapidly induced by TGF-(3 in vivo and in vitro (Uzel et al 2001). These authors investigated the presence and distribution of CTGF protein by immunohistochemistry in tissue samples taken from patients undergoing therapy with phenytoin, nifidipine and cyclosporine. The data from their study showed significantly higher CTGF staining in phenytoin-induced gingival overgrowth tissues compared to controls, CSA, or nifidipine-induced gingival overgrowth. -Transforming growth factor-P (TGF-P): In mammals there are three TGF-P isoforms; p i , P2 and P3.These can be distinguished by their effects on cell growth, cytokine expression and receptor binding characteristics. They are multifunctional cytokines known to be important in both healing and fibrosis The TGF-P isoforms can be expressed by most cells, including gingival inflammatory cells, endothelial cells and fibroblasts (Border & 14 Rouslahti 1992). Increased TGF-P expression may be associated with cyclosporine-induced renal fibrosis (Wondimu et al 1997). James et al (1998) investigated the potential role for TGF-pl in pathogenesis of CSA-induced gingival overgrowth. In their in-vitro study they determined that the cellular activity of gingival fibroblasts is dependent on culture conditions and that fibroblasts derived from overgrown gingival tissue are more responsive to TGF-P 1 than normal gingival fibroblasts when cultured in type I collagen gel. Coletta et al (1999) believed that TGF-P 1 is a key mediator of tissue fibrosis causing extracellular matrix accumulation in pathologic states such as hereditary gingival fibromatosis. Wright et al (2001) performed a study to determine whether there is any altered expression of TGF-P isoforms or their receptors in tissue from patients with drug induced (PHT, CSA, NIF) gingival overgrowth or hereditary gingival fibromatosis compared to the controls. They observed that fibroblasts from both drug-induced gingival overgrowth and hereditary gingival fibromatosis expressed more TGF-P 1 compared to controls. Cells expressing TGF-P2 were present at control levels in DIG but were significantly reduced in hereditary gingival fibromatosis. In contrast the number of TGF-P3 positive cells was the same in overgrowth tissues and controls. They concluded that qualitative differences in TGF-P isoforms and receptor expression by fibroblasts in gingival overgrowth may contribute to disease pathogenesis. However, one critique of this study is that they had both healthy periodontium and periodontitis samples in their control group. Since we expect to see alterations in growth factors in association with periodontitis, this group is not the most suitable one to use for comparison. 15 In the study of Saito et al 1996, it was noted that there were increased numbers of fibroblast-like cells that were positive for TGF-P 1 in the lamina propria of nifidipine and phenytoin-induced gingival overgrowth tissues compared to control gingiva. Budneli et al (2001) investigated the level of T G F - p l i n gingival crevicular fluid (GCF) samples of CSA-treated patients and compared the results with control groups. A l l the patients in C S A group exhibited severe gingival overgrowth. Control groups included both patients with gingivitis and subjects with healthy periodontium. G C F samples were harvested from sites exhibiting gingival overgrowth (GO+) and sites not exhibiting gingival overgrowth (GO-). The TGF-P level was analyzed by E L I S A . The results demonstrated that the concentration of TGF-P 1 in sites with and without gingival overgrowth were similar and significantly higher than that of healthy sites. They also showed an increased amount of TGF-P 1 in gingival overgrowth group compared to gingivitis group, although the differences were non-significant. The authors supported the theory that C S A increases the synthesis of TGF-P 1 in G C F . However, they concluded that due to lack of statistically significant differences between C S A GO+ and C S A G O - , it seems unlikely that G C F T G F - p l is the sole factor responsible for the gingival overgrowth. The authors did not mention whether the patients in C S A group or even control patients were taking any other type of medications. In an in-vitro study (Cortim et al 2002) fibroblasts from normal human gingiva were admixed with different concentrations of C S A (0, 100 or 200 ng/ml of C S A ) and cultured for 24 hours. Using reverse transcriptase-polymerase chain reaction (RT-PCR) , the expression and production of TGF-P 1 was then determined. M M P and T I M P expression levels were also analyzed by the same technique. In addition, in order to determine the effect of TGF-P 1 on the expression of M M P and T I M P by human gingival fibroblasts incubated with C S A , the cells were treated with sense oligonucleotides (SON) or antisense oligonucleotides ( A O N ) . The result of this study demonstrated that C S A stimulates 16 expression and production of TGF-P 1, while it inhibits expression of MMP-1 and MMP-2 by gingival fibroblasts. TIMP expression was not statistically significant. Furthermore, the authors observed that blocking TGF-pl synthesis with AON resulted in an increased effect of CSA on the MMP-1 and MMP-2 expression by gingival fibroblasts, whereas TIMP-1 and TIMP-2 were not affected. In conclusion, the authors stated that TGF-pl, acting in an autocrine fashion, may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CSA-induced gingival overgrowth, which favors the accumulation of extracellular matrix. -Basic fibroblast growth factor (bFGF): Saito et al (1996) demonstrated that the increased synthesis of bFGF as well as TGF-p and their respective receptors may be related to the pathogenesis of drug-induced gingival over growth. Hong and Trackman (2002) performed a study to determine whether cytokines reported to be present at elevated levels in drug-induced gingival overgrowth regulate lysyl oxidase, elastin or a-l type I collagen when administered to human gingival fibroblasts. They obtained samples from systemically healthy individuals. They treated the fibroblasts with different concentrations of IL-1P, bFGF and PDGF-BB. The results demonstrated that bFGF significantly reduced fibroblast lysyl oxidase and a-l type I collagen mRNA levels in a dose- and time-dependent manner. Controversially, no significant changes were observed using IL-1 (3 and PDGF-BB. The authors concluded that the strong downregulation of lysyl oxidase and collagen type I by bFGF suggested the role for this cytokine might be regulatory. As such it could limit stimulation of connective tissue synthesis induced by other fibrogenic factors such as TGF-pl and CTGF that are known to be present in gingival tissues. Alternatively, as bFGF is mitogenic and angiogenic, a possible role for bFGF may be to stimulate cell proliferation and neovascularization that could ultimately contribute to gingival overgrowth. 17 - K e r a t i n o c y t e g r o w t h fac to r : Kera t inocyte g rowth factor stimulates the growth and act ivi ty o f epi thel ial cel ls v i a the keratinocyte growth factor receptor (Das et al 2002). 4-3 C y t o k i n e s : It has been postulated that C S A - i n d u c e d alterations o f cytokine levels i n g ing iva l tissues might p lay a role i n the drug- induced g ing iva l overgrowth. - Inter luekin -1 (IL-1): IL-1 is the term for two polypeptides, IL -1 a and IL-1 p. They possess a w i d e spectrum o f inf lammatory , metabol ic , phys io log ic , and i m m u n o l o g i c properties. A l t h o u g h both forms o f IL-1 are produced by different genes, they recognize the same ce l l surface receptors and share the s imi lar b io log ic act ivi t ies. IL-1 (3 belongs to a group o f cytokines (pro-inf lammatory cytokines) w i t h over lapping b io log ica l properties. IL-1 P is produced predominant ly by macrophages, but it can also be released f rom platelets, fibroblasts, endothelial ce l l s , and keratinocytes. In macrophages, significant amount o f IL-1 p are not observed in health. H o w e v e r , there is a dramatic increase in IL-1 p in response to infect ion, m i c r o b i a l toxins , inf lammatory agents, products o f activated lymphocytes and complement ( Job l ing et a l 1988- Iacopino et al 1997). M a n y studies have demonstrated that the concentrat ion o f IL-1 p appears to be a major determinant in the progression and extent o f tissue degradation i n chronic in f lammat ion (Stashenko et al 1991, Heasman et al 1993). IL-P has also been demonstrated at increased levels i n inf lamed g ing iva l tissue and g ing iva l c revicular f l u id harvested from per iodonta l ly diseased sites. It is also among the most potent inducers o f bone resorption ( H o w e l l s 1995). In terms o f extracellular matr ix turnover IL-1 p induces connect ive tissue degradation 18 by i nduc ing increased expression o f the matr ix metalloproteinases collagenase and s t romelysin , w h i l e reducing levels o f tissue inhibi tor o f metalloproteinases ( T I M P ) (B i rkeda l 1993). T o investigate the effects o f C S A on the product ion o f some cytokines , i nc lud ing IL-1 p, M y r i l l a s et al (1999) obtained human g ing iva l fibroblasts from biopsies o f normal g ing iva l tissue f rom healthy volunteers and f rom biopsies o f patients w i t h cyc lospor ine induced g ing iva l overgrowth . The results o f this study demonstrated that C S A induced a statistically significant inh ib i t ion o f IL-1 P product ion by peripheral mononuclear cel ls . In the study o f Iacopino et al (1997), no increase i n amount o f IL-1 p was found when macrophages were treated w i t h C S A , i n contrast to the increase i n the level o f P D G F - B as noted above. Several studies have shown that there is a posi t ive correlat ion between the degree o f in f lammat ion and g ing iva l c revicular f l u id ( G C F ) cytokine levels (Masada et al 1990). In order to determine the levels o f IL -1 P i n G C F , A t i l l a et al (1998) col lec ted G C F samples f rom renal transplant patients rece iv ing C S A w i t h and without g ing iva l overgrowth and compared them to samples taken from unmedicated ind iv idua l s w i t h g ingiv i t i s as w e l l as to samples from subjects w i t h a c l i n i c a l l y healthy per iodont ium. They conc luded that C S A therapy does not alter G C F I L - i p levels , w h i l e g ing iva l in f l ammat ion plays a significant role i n the e levat ion o f G C F IL-1 p levels . -Interleukin-6 (IL-6): I L - 6 is produced by a variety o f cel ls , i nc lud ing T-ce l l s , B - c e l l s , monocytes , keratinocytes, endothel ial ce l l s , some tumor cells and fibroblasts. W h i l e I L - 6 produces many signals for these cel ls , one proposed act ion is to inhibi t fibroblast growth ( K i s h i m o t o 1989). In terms o f connect ive tissue turnover, IL -1 p and I L - 6 have oppos ing effects w i t h IL-1 p induc ing tissue b reakdown through the induc t ion o f M M P s secretion and I L - 6 reducing tissue breakdown through increased T I M P expression. 19 In addi t ion, I L - 6 has been shown to stimulate the proliferat ion o f human g ing iva l keratinocytes, an effect w h i c h cou ld account for the epi thel ial hyperplasia observed i n ove rg rown g ing iva l tissues (B i rkeda l -Hansen 1993). In the study o f M y r i l l a s et al (1999), the product ion o f I L - 6 by g ing iva l fibroblasts and peripheral b lood mononuclear cel ls ( P B M C ) under the effect o f C S A was investigated. Resul ts revealed that at a concentration o f 2000 ng /ml , C S A stimulated I L - 6 product ion by P B M C . They also observed that fibroblasts der ived from overgrown g ing iva produced s ignif icant ly higher levels o f I L - 6 than their normal counterparts. The authors concluded that C S A - i n d u c e d g ing iva l overgrowth tissue contained s ignif icant ly higher levels o f I L - 6 than inf lamed or normal g ing iva . H o w e v e r , the results o f this study contrast those o f a study done by Y o s h i m u r a et al (1991), w h i c h demonstrated that C S A signif icant ly reduced serum levels o f I L - 6 . The study was performed on serum and I L - 6 product ion o f human peripheral b lood mononuclear cel ls . W i l l i a m s o n et al (1994) also performed a study to determine whether C S A therapy affects I L - 6 gene expression in g ing iva l tissues. They obtained samples from indiv iduals w h o received k idney or l iver transplant and were on C S A therapy and compared those to samples taken f rom healthy indiv iduals . In a l l test subjects, g ing iva l overgrowth was determined to be moderate. Rad io immunoassay and in-s i tu hybr id iza t ion demonstrated s ignif icant ly elevated I L - 6 levels i n CSA- t r ea t ed tissues. The authors conc luded that C S A causes an upregulat ion o f I L - 6 gene expression i n human g ing iva . The f ind ing suggests a mechan i sm for induc ing g ing iva l overgrowth. S tudying G C F samples o f patients rece iv ing C S A w i t h and wi thout g ing iva l overgrowth, A t i l l a et al (1999) stated that C S A therapy does not alter G C F I L - 6 levels di rect ly , and that in f lammat ion probably plays an important role i n the elevat ion o f these cytokines . 20 4-4- Integrins: Integrins are the pr inc ip le mediators o f the molecula r dialogue between a ce l l and its extracellular matr ix environment . The unique combinat ions o f integrins subunits determine w h i c h extracellular matr ix molecules w i l l be recognized by a ce l l (Gumbine r et al 1996). B o t h a l p i and a 2 p i integrins are c e l l surface receptors for type I co l lagen i n fibroblasts. Based on the study o f L e e et al (1996), the in i t ia l b ind ing step o f col lagen phagocytosis relies on adhesive interaction between fibroblasts and col lagen, and that a 2 integrin plays a cr i t ica l role in the phagocyt ic regulat ion o f co l lagen internal izat ion. K a t a o k a et al (2003) studied the expression o f a 2 integrin i n fibroblasts der ived f rom g ing iva o f rats fed a diet w i t h or without C S A , and investigated whether the difference i n expression o f a 2 integr in is i n v o l v e d i n the regulat ion o f co l lagen phagocytosis . The f indings o f their study revealed that fibroblasts f rom CSA- t r ea t ed g ing iva showed less phagocytosis . It was also conf i rmed that C S A inhibi ted the expression o f a 2 integrin i n fibroblasts. The level o f a 2 integrin m R N A i n the fibroblasts isolated f rom C S A treated rat g ing iva was apparently lower than that o f the control fibroblasts. Further studies were suggested to clar i fy the role o f a 2 integrin i n the induct ion o f C S A induced G O . 4-5-Matrix Metalloproteinases (MMPs): The matr ix metalloproteinase ( M M P ) fami ly consists o f at least 13 members w i t h c lose ly related doma in structures and discrete functions. A l l have a 21 -kd catalytic doma in that contains a Zn++ b ind ing site. M M P fami ly is responsible for the remodel ing o f extracellular matr ix i n both phys io log ica l and pathological condi t ions. M M P - 1 or collagenase is produced by a variety o f ce l l types i n c l u d i n g keratinocytes, fibroblasts, and macrophages. Th i s enzyme is an important regulator o f connect ive tissue remodel ing and is present i n h igh concentration i n inf lamed g ing iva l regions, 21 i nc lud ing those affected w i t h periodontal disease; whereas in hyperplast ic tissue and fibrosis we expect to f ind a lower l eve l . M M P - 1 can hydro lyze type I, II, III, V I , V I I I and X collagens. The gelatinase group o f M M P s has 2 prominent members, M M P - 2 and M M P - 9 . B o t h o f these enzymes have a h i g h affinity for gelatin. St romelysins have a broad specif ici ty w i t h the abi l i ty to degrade proteoglycans, basement membrane, l amin in , f ibronectin, in addi t ion to col lagens. Three s tromelysins have been described so far; stromelysin-1 ( M M P - 3 ) , s tromelysin-2 ( M M P - 1 0 ) and stromelysin-3 ( M M P - 1 1 ) (Bar to ld & Narayanan 1998, Tuter et al 2002). M M P s are a l l spec i f ica l ly inhibi ted by tissue inhib i tor o f metalloproteinase-1 ( T I M P - 1 ) . T I M P s are expressed by many cel ls i nc lud ing fibroblasts, keratinocytes, monocytes/macrophages, endothelial cel ls and osteoblasts ( M e i k l e et al 1994). B a l z a n i et al (2000) investigated the product ion and act ivi ty o f M M P s i n C S A - i n d u c e d g ing iva l overgrowth. T h e y u t i l i zed both animal models and in vi t ro ce l l cultures to study this issue. Thi r ty s ix male rats received injections o f C S A da i ly for 60 days. F o r the i n vi tro study g ing iva l fibroblasts, obtained f rom seven healthy volunteers were incubated w i t h cyclospor ine . The results demonstrated that the g ing iva l overgrowth tissue had lower gelat inolyt ic activit ies o f M M P - 2 than the control g ing iva l tissues i n rat. In addi t ion it was observed that C S A signif icant ly inhibi ts , at the protein leve l , the product ion o f both M M P - 1 and M M P - 3 by g ing iva l fibroblasts. The authors noted that inh ib i t ion o f M M P s occured at C S A concentration near those found i n the serum o f C S A immunosuppressed patients. T h e y conc luded that there is a co-operative role o f M M P - 1 and M M P - 3 i n the pathogenesis o f C S A - i n d u c e d g ing iva l overgrowth ( inh ib i t ion o f these M M P s may contribute to an abnormal accumula t ion o f f ibronect in and proteoglycans). In another study done by Y a m a d a et al (2000) normal human g ing iva l fibroblasts were cultured w i t h or without phenytoin or cyc lospor ine A and the total R N A and ce l lu lar proteins were col lected da i ly for R T - P C R analyses and measurement o f lysosomal 22 enzyme act iv i ty . The results revealed that these medicat ions suppressed the expression o f M M P - 1 and T I M P - 1 and cathepsin L , but not the cathepsin B i n a time-dependent manner. T h e y concluded that the decreased ab i l i ty o f protein degradation by lysosomal enzymes is, at least, one o f the factors i n the pathogenesis. In another study, C o t r i m et al (2002) demonstrated that C S A inhib i ted expression o f M M P - 1 and M M P - 2 by human g ing iva l fibroblasts taken from normal tissue, whereas it has little effect o n T I M P - 1 and T I M P - 2 expression. Tuter et al in 2002 reported conf l i c t ing f indings when compar ing M M P - 1 and T I M P - 1 levels i n ce l l cultures o f g ing iva l tissue der ived f rom both renal transplant patients rece iv ing C S A and exhib i t ing g ing iva l overgrowth and per iodonta l ly healthy control subjects. T h e y also investigated the effects o f C S A on M M P - 1 and T I M P - 1 levels in C S A - treated g ing iva l fibroblast cultures der ived f rom periodontal ly healthy subjects. T h e y classif ied g ing iva l fibroblast cultures as: 1- C S A G O (g ing iva l overgrowth) 2- H + C S A ( C S A treated healthy g ing iva l fibroblast culture) and 3- H (healthy g ing iva l fibroblasts). The results showed the levels o f T I M P - 1 were s ignif icant ly lower i n C S A G O than H . There was no statistically significant difference i n the levels o f M M P - 1 between H and C S A G O . The authors conc luded that C S A therapy does not have a s ignif icant effect on M M P - 1 levels; however , l o w T I M P - 1 levels can be an important factor i n the pathogenesis o f C S A G O . S i l v a et al (2001) used a rat molar extraction mode l to observe the influence o f C S A - i n d u c e d immunosuppress ion on M M P s act ivi ty i n dental socket granulat ion tissue. In their study it was observed that C S A inhibi ts the act ivi ty o f M M P - 2 and M M P - 9 i n the early phase o f granulat ion tissue i n the heal ing dental sockets. However , some enhanced act ivi t ies o f M M P - 1 was observed, w h i c h is i n agreement wi th the study o f Tuter et al (2002). 23 4-6 -Pro teoglycans a n d G l y c o s a m i n o g l y c a n s : Pro teoglycan is a f ami ly o f macromolecules composed o f one or more o f g lycosaminog lycans ( G A G s ) covalent ly bound to a protein core (Balazs 1967, Roberts 2003). G A G s are the pr inc ip le carbohydrate components o f proteoglycans and they include hyaluronan, chondroit in-4-sulfate, dermatan sulfate, heparin sulfate, heparin and keratan sulfate. Excep t keratan sulfate, a l l o f the G A G s are composed o f repeating disaccharide units o f uronic ac id and hexosamine. Ke ra t i n sulfate contains D-galactose i n the place o f uronic ac id . In addi t ion to the g lycosaminog lycan chains, smaller ol igosaccharides have been identif ied i n most proteoglycans (Bar to ld & Narayanan 1998). Proteoglycans are large, h igh ly anionic glycoprote ins o f connective tissues, w h i c h are located w i t h i n the matr ix as integral components o f the mat r ix structure as w e l l as on ce l l surfaces and w i t h i n ce l l organelles. The i r functions include tissue hydrat ion, regulat ion o f col lagen fiber formation, g rowth factor b ind ing , ce l l adhesion, and c e l l growth. Proteoglycans are c lass i f ied into at least three separate groups; matr ix organizers and space fillers, ce l l surface proteoglycans, and intracel lular proteoglycans o f the hematopoiet ic cel ls (Bar to ld & Narayanan 1998). Ve r s i can , an important member o f the first group, was o r ig ina l ly isolated f rom fibroblasts. S ince then it has been found to be present in a w ide variety o f tissues i nc lud ing aorta, brain, cartilage, placenta as w e l l as sk in (Ruosla t i 1989). The ultrastructural study o f M a r i a n i et al (1993) demonstrated a part icular abundance o f amorphous ground substance i n connect ive tissue o f C S A - i n d u c e d g ing iva l overgrowth. Bensada et al (1995, 1997) have demonstrated that vers ican is consistently associated w i t h prol i ferat ing myofibroblasts in early lesions o f pu lmonary f ibrosis , but absent f rom areas o f dense col lagen deposits consistent w i t h a transient role for vers ican i n the ce l l b i o l o g y o f proliferative lung disease and its degradation concomitant w i t h myofibroblasts apoptosis. 24 5- Histological features of CSA- induced gingival overgrowth: 5 - 1 - E p i t h e I i u m : In a his tologic study o f C S A induced g ing iva l overgrowth M i r i a n i et al (1996) found that the basal and spinous layers o f epi the l ium show distinct di latat ion o f the intercel lular spaces. T h e y found the d imens iona l increase i n g ing iva l overgrowth to be due to an increased product ion o f amorphous ground substance by fibroblasts. Other his tologic features being described for C S A - induced g ing iva l overgrowth are epithelial ridges penetrating deep into C T and h igh ly vascular ized C T w i t h focal accumula t ion o f infi l t rat ing inf lammatory cel ls . The predominant inf lammatory ce l l type is reported to be the p lasma ce l l , w i t h lymphocytes seen to a lesser degree. Acan thos i s and parakerat inizat ion o f the epi the l ium w i t h psuedoepithelioumatous prol i ferat ion can be observed. Immunohis tochemica l examinat ion o f C S A induced g ing iva l overgrowth exhibi ts active protein synthesis and reduced cyto toxic or degenerative changes. 5- 2 -Connect ive T i s s u e : A c c o r d i n g to Ratei tschak et al (1983), the connect ive tissue o f C S A - i n d u c e d g ing iva l overgrowth exhibi ted i r regular ly arranged col lagen bundles and h igh vascular izat ion. F o c a l accumulat ions o f inf i l t rat ing inf lammatory cel ls were also observed. M a r i a n i et al (1993) also report the presence o f amorphous ground substance i n the connective tissue. A c c o r d i n g to the authors, this substance passes through the spaces between adjacent basal ce l l s , w h i c h at some points assumed the features o f intercel lular canals. 6- Prevention, Treatment and Maintenance of gingival overgrowth: The general consensus is that a r igorous oral hygiene program is extremely important in the preventive and therapeutic management o f drug induced g ing iva l overgrowth and should be instituted pr ior to 25 starting such therapy whenever possible ( H a l l m o n & Rossmann 1999). Ph i l s t rom et al (1980) studied the effectiveness o f a preventive dental p rogram i n 13 patients who were taking phenytoin ( P H T ) . A l l patients rece ived O H I and S R P w i t h i n 30 days o f ini t ia t ing P H T therapy. R e c a l l appointments were repeated every 3 months. They observed a smal l increase i n g ing iva l enlargement anteriorly dur ing the first 6 months w i t h no further progression. F u et al (1997) examined the role o f plaque retention on C S A induced g ing iva l overgrowth i n rats. Ligatures were placed around the first molar unilateral ly and the rats adminis tered C S A in different dosages. G i n g i v a l overgrowth was s ignif icant ly increased i n sites w i t h higher C S A dosage and longer treatment duration. G i n g i v a l overgrowth was enhanced i n ligated sites regardless o f C S A dose. T h e y suggested that plaque retention magnifies C S A - i n d u c e d g ing iva l overgrowth. Ingel i et al (1999) studied 38 patients who were on C S A or N I F . A l l patients received in i t ia l per iodontal therapy, after w h i c h anterior segments i n each patient were surgica l ly treated. Surg ica l therapy consisted o f the flap technique w i t h a 90 degree g ing ivec tomy inc i s ion . Patients were placed on a maintenance therapy for 18 months. Recurrence was observed i n 34%. A g e , g ing iva l inf lammat ion , and attendance at recal l appointments were significant determinants for the recurrence o f severe g ing iva l overgrowth. Pi la t t i and Sampaio (1997) h is to logica l ly assessed the influence o f da i ly appl ied 0 .12% C H X on the severity o f C S A - i n d u c e d g ing iva l overgrowth i n rats. T h e y concluded that the use o f C H X m a y lead to an i m p r o v e d plaque and g ingiv i t i s control , w h i c h may help reduce the severity o f g ing iva l overgrowth. Camagro et al (2001) in their article mentioned that considerat ion should be g iven to the poss ib i l i ty o f changing medicat ions i n consultat ion w i t h the patient 's phys ic ian . Al te rna t ive medicat ions to phenytoin inc lude carbamazepine and va lpro ic ac id , both o f w h i c h have been reported to have a lesser impact i n i nduc ing g ing iva l overgrowth. D i l t i a z e m and verapami l are other c a l c i u m channel b lockers exh ib i t ing a lesser prevalence o f g ing iva l enlargement (20% and 4 % respect ively in compare to 4 4 % 26 reported for N I F ) . It also has been shown that C S A induced g ing iva l enlargement can spontaneously resolve i f the drug is substituted by t racol imus. There is also pre l iminary evidence that the antibiot ic az i t h romyc in m a y a id i n decreasing the severity o f C S A induced g ing iva l overgrowth . T h e y recommended that whenever the drug substitution is attempted, it is important to a l l o w for 6-12 months to elapse between discont inuat ion o f the offending drug and the poss ible resolut ion o f g ing iva l enlargement. W h e n g ing iva l enlargement persists, despite drug substitution and good plaque control , these cases need to receive surgical treatment, w h i c h cou ld be either g ing ivec tomy or periodontal f lap therapy. The advantages o f g ing ivec tomy are s impl i c i ty and quickness, but un l ike the periodontal flap therapy, it w i l l not a l l o w for osseous recontouring and may sacrifice kerat in ized tissue. In addit ion, g ing ivec tomy results i n hea l ing by secondary intention, w h i c h causes discomfort and an increased chance o f post-operative bleeding. In general, smal l areas (up to s ix teeth) presenting w i t h drug- induced g ing iva l enlargement where there is no evidence o f attachment loss can be effectively treated w i t h g ing ivec tomy. H o w e v e r , the amount o f kerat in ized tissue should be considered i n these cases. It is recommended that at least 3 m m o f kerat inized tissue in the apicocoronal d i rec t ion remain after the surgery is concluded . A n y situation i n w h i c h the g ing ivec tomy technique may result i n the e l imina t ion o f a l l kerat inized tissue and consequent creation o f mucog ing iva l problems should be treated wi th the periodontal flap surgery (Camarego et al 2001). Recurrence o f drug-induced g ing iva l enlargement is c o m m o n i n surgical ly treated cases (Rees et al 1995). Proper oral hygiene care, ch lorhexid ine rinse, and regular scal ing and root p lann ing can decrease the rate and the degree o f recurrence (Saravia el al 1990). Recurrence may occur as early as 3-6 months after the surgical treatment, but i n general, surgical results mainta ined for at least 12 months ( P i l l o n i et al 1998). 27 Chapter 2- The objectives of the study: The a i m o f this study is to characterize the tissue changes that occur in g ing iva l overgrowth associated w i t h cyc lospor ine A therapy in k idney transplant patients. In order to do this, ce l l and matr ix populat ions in h is to logica l sections o f g ing iva l overgrowth biopsies w i l l be compared w i t h those o f sys temica l ly healthy controls, w h i c h are obtained i n the procedure o f c r o w n lengthening. The objective o f this study: • Identify and select matched disease and control patients • Sys temat ica l ly sample and process tissues to a l l o w quantitative and semi-quantitative h is to logica l analysis o f tissue changes associated w i t h g ing iva l overgrowth • Conduc t and analysis o f tissue changes i n the epi thel ium and connect ive tissue o f the g ing iva l overgrowth samples and compare it to the ones in the control group The hypothesis is that immunosuppress ive drugs may have a direct effect on growth factor and cytokine synthesis and release i n the oral cavi ty result ing in a proteoglycan-myofibroblas t complex that is responsible for g ing iva l overgrowth. The nature o f the matr ix molecules and ce l l populat ions i n v o l v e d w i l l be determined. 28 Chapter 3- Materials and Methods: The co l l ec t ion o f c l i n i c a l information and tissue samples took part i n Shahid Behesht i ( S H B ) S c h o o l o f Dent is t ry , private practice, and the k idney transplant center o f Labaf i hospi tal in Tehran, Iran. It i nc luded patient selection, oral examinat ion, periodontal surgery and co l lec t ion o f tissue samples at the t ime o f surgery. T h i s study was approved by the research committee o f S H B S c h o o l o f Dentis t ry. The procedure and reasons for the study were expla ined to the patients i n both groups and patients a l l gave their in formed consent to use o f surgical samples for research. F o r those patients under the age o f eighteen, the consent forms were signed by one o f the parents. ( A p p e n d i x 1 shows the consent form both i n Pers ian and Eng l i sh ) . The study a imed to compare the f o l l o w i n g two groups o f patients: 1- G i n g i v a l overgrowth group and 2- Con t ro l group. 1- Gingival overgrowth group (The GO group): The g ing iva l overgrowth group consisted o f 18 ind iv idua ls (6 females and 12 males wi th an age range o f 16-50 years) w h o had al l undergone k idney transplant surgery at least one year pr ior to this study and were tak ing cyc lo spo r ine -A . A l l o f these patients demonstrated g ing iva l overgrowth o f degree II or III based o n M c G a w index ( M c G a w et al 1988). A c c o r d i n g to this index, the g ing iva l overgrowth is c lass i f ied as fo l lows : • Grade 0: N o overgrowth or feather-edged g ing iva l margin . • Grade I: B l u n t i n g o f g ing iva l marg in . • Grade II: Modera te g ing iva l overgrowth, < 1/3 o f c r o w n length. • Grade III: G i n g i v a l overgrowth, > l / 3 o f c r o w n length. 29 These patients were selected from those referred to Labaf i hospital i n Tehran, Iran for their regular b l o o d pressure and renal function post-transplant check-up. Th i s selection was conducted by a Per iodont ics resident and a trained dental student. The steps for patient select ion were as fo l lows : • R e v i e w i n g o f charts o f patients w h o had an appointment on the same day. • Ini t ia l ora l examinat ion on those w h o had had k idney transplant for at least one year and were prescribed C S A . • Se lec t ion o f indiv iduals w i t h Grade II or III G O from the above group and explanat ion o f the study to them; informed consent. • C l i n i c a l examinat ions and treatment. 2- Control Group: C o n t r o l group consisted o f 12 systemical ly healthy ind iv idua ls , sex and age-matched w i t h the G O group (4 females and 8 males w i t h an age range o f 24-56 years). These ind iv idua l s were referred to the Per iodont ics department o f S H B dental school to receive a c r o w n lengthening procedure for restorative purposes. The i r c l i n i c a l diagnosis was g ing iv i t i s and, based on the informat ion obtained f rom their charts, they d i d not appear to have periodonti t is . The purpose o f this study was expla ined to them and they were asked to read and s ign the consent form to give us pe rmiss ion to use the excised tissue for research purposes. Tissue exc i s ion is generally a part o f the c r o w n lengthening procedure. T h i s tissue was saved instead o f be ing discarded as w o u l d normal ly be the case. 30 3- Clinical Examinations: Genera l informat ion inc lud ing demographic data, general health concerns other than k idney problems, medicat ions taken, and history o f s m o k i n g was recorded for a l l patients in both groups. In the G O group, details regarding their k idney transplant were also col lected based o n informat ion p rov ided by the patients and/or f rom their medica l charts i n Labaf i Hosp i t a l . These data inc luded the reason for k idney failure, the date o f transplant, the dosage o f C S A and other medicat ions taken, and the patients ' serum levels o f C S A . In the oral examinat ion o f this group G i n g i v a l Index ( G I , L o e & Silness 1964), oral hygiene effectiveness, probing depth ( P D ) and g ing iva l overgrowth index were recorded pr ior to any treatment. Consul t s w i th physicians were done for the g ing iva l overgrowth patients regarding their systemic health and i n order to k n o w whether they required any antibiotic p rophy lax i s or a change in the amount o f cor t icosteroid taken. ( A p p e n d i x 2 shows the fo rm that was used to col lect the c l i n i c a l information) . 4- Surgical Treatment: Pr io r to surgical treatment, oral hygiene instruction and scaling/root p lann ing were performed in both groups. A l l g ing iva l overgrowth patients received periodontal flap surgery for accessing the bone surface and per forming osseous corrections i f required. C r o w n lengthening patients also received periodontal flap surgery. F o l l o w i n g reverse bevel inc i s ion , the excised tissues were careful ly removed and saved for the next step. After comple t ion o f the surgery, periodontal dressing (CoePak) was used i n both groups and patients received post-op instructions. Ch lo rhex id ine 0 .12% (1 minute o f r ins ing, b id) was also prescribed. D u r i n g the f o l l o w i n g vis i t , one week later, the per iodontal dressing and sutures were r emoved and the patients were taught h o w to perform their oral hygiene. It was also expla ined to the G O patients that there is a poss ib i l i ty o f recurrence o f overgrowth , and i n order to prevent this, they needed to be meticulous w i t h their dai ly oral hygiene. Further, they were advised to 31 receive scaling/root p lanning ( S R P ) on a regular basis. Photographs were taken pr ior to and f o l l o w i n g surgery i n the G O group. Per iapical radiographs were also obtained to study the poss ib i l i ty o f bone loss and also to ensure the absence o f any pathologic condi t ion i n the surgical site. Surgeries in the G O and control groups occurred in private practice and at the dental school , respectively. 5- Tissue Collection: Tissue samples were harvested from interdental papil lae as w e l l as margina l g i n g i v a i n both groups and they were immedia te ly rinsed w i t h normal saline and soaked in fo rmal in 10% for 24 hours. That same day, the exc ised tissues were transferred to the pathology laboratory at the S H B dental school . The next day, the tissues were embedded i n paraffin by the technicians o f the pathology lab. A t this point , they were safe and ready to be carr ied to Vancouve r . Paraffin processing was done as fo l lows : 1. 7 0 % a lcohol 45 minutes. 2. 7 0 % a lcohol 45 minutes. 3. 95%) a lcohol 45 minutes. 4. 95%> a lcohol 45 minutes. 5. 100% a lcoho l 45 minutes. 6. 100% a lcoho l 45 minutes. 7. 100% a lcohol 45 minutes. 8. X y l e n e 45 minutes. 9. X y l e n e 45 minutes. 10, X y l e n e 45 minutes. 11. Paraff in w a x 1 hour 12. Paraffin w a x 1 hour 32 6- Histochemical Staining: Tissue sect ioning and his tochemical stainings were done i n Department o f Pa thology at the U B C hospi ta l . In i t ia l ly , samples were cut to sections o f 3 microns thickness. De-para f f in iz ing was then performed us ing the f o l l o w i n g technique: 1. X y l e n e #1 for 3 minutes. 2. X y l e n e #2 for 3 minutes. 3. X y l e n e #3 for 3 minutes. 4. 100% a lcoho l #1 for 20 dips 5. 100% a lcohol #2 for 20 dips 6. 100% a lcohol #3 for 20 dips 7. 9 5 % a lcoho l for 20 dips 8. R inse slides i n tap water for 1 minute. 9. D r a i n w e l l before staining. Three different techniques o f staining were done inc lud ing hematoxyl in and eos in ( H & E ) , G o m o r i ' s Tr ichrome-a ldehyde fuschin stain ( G o m o r i ) , and a lc ian blue p H 2.5- picrosirus red ( A B / P S R ) . H & E staining was done to study general features o f both epi thel ium and connect ive tissue, i nc lud ing changes i n retepegs number, length and thickness, and inf lammatory changes i n connect ive tissue. G o m o r i staining was used to v i sua l ize co l lagen fibers, elastin and g lycosaminog lycans i n connective tissue. A n d f ina l ly , A B / P S R cou ld detect the presence o f proteoglycans and g lycosaminoglycans ( G A G ) i n connect ive tissue (blue) and col lagen fibers (red). 33 7- Immunohistochemical Staining: Immunohis tochemica l staining was performed i n D r . Rober ts ' laboratory. Sect ions were de-waxed i n X y l e n e and brought through a graded series o f a lcohol to water, us ing the method described above. De ta i l ed procedures were developed as needed for different antibodies. F o r staining, V e c t o r system and Vectas ta in A B C kits were u t i l ized . The technique is described i n be low: 1 - R i n s i n g for 5 minutes i n tap water. 2- Incubat ing the sections w i t h 0 .3% H2O2 for 30 minutes 3- W a s h i n g slides i n Tr i s buffered saline ( T B S ) for 5 minutes 4- Incubating sections for 20 minutes w i th di luted normal b l o c k i n g serum. 5- B l o t t i n g the excess serum from sections. 6- Incubat ing sections for 30 minutes w i t h pr imary antiserum, no pr imary antiserum, or control antiserum di lu ted in T B S w i t h 3 % bovine serum a lbumin ( T B S - B S A ) . 7- W a s h i n g slides i n buffer for 5 minutes. 8- Incubating sections for 30 minutes w i t h secondary antibody solut ion or no secondary ant ibody as control . 9- W a s h i n g slides i n buffer for 5 minutes. 10- Incubating sections for 30 minutes w i t h Vectas ta in A B C reagent. 11- W a s h i n g slides i n buffer for 5 minutes. 12- Incubating sections i n peroxide substrate solut ion to get the desired stain intensity (between 7- 10 minutes) . 13- R i n s i n g sections i n tap water. 14- Counters ta ining, c lear ing and mount ing . 34 8- Antibody Staining: Pr imary antibodies used i n this study and their concentrations are col lected i n table 1. Table 1: Primary antibodies, the concentrations and their targets. Pr imary A n t i b o d y Concentrat ion Target 1 A 4 1/1000 a - smooth musc le act in P C N A 1/100 Prol i ferat ing cel ls L C 2 1/500, 1/1000, 1/5000 C terminal o f V e r s i c a n 2 B 1 1/200 Core o f V e r s i c a n C D 4 4 1/200 Hya lu ronan receptors ( H A ) (Predominantly on macrophages) 9- Considerations to allow semi-quantitative analysis of immunohistochemical staining The intensity o f immunohis tochemica l ( I H C ) staining is inf luenced by many factors inc lud ing : • Concent ra t ion o f antibody • T i m e and temperature o f incubat ion • Sec t ion thickness • T i m e and temperature o f co lor development In order to a l l o w semi-quantitative compar i son o f tissue staining between the control and G O groups, we took the f o l l o w i n g precautions: • Standardized tissue processing up to paraffin embedding • Tissues to be compared were stored, sectioned and processed together. • Sections to be compared were stained together i n the same staining procedure. 35 10- Morphometric Analysis: Afte r r e v i e w i n g the stained slides, we developed a number o f h i s to logica l grading schemes, to a l l o w semi-quantitative analysis o f h is tochemical and immunohis tochemica l data. In h is tochemical features, epi thel ia l and connective tissue changes were studied us ing the f o l l o w i n g cr i ter ion: 10-1 E p i t h e l i a l changes : Ep i the l i a l changes inc lud ing presence or absence o f acanthosis, hyperkeratosis, and parakeratosis as w e l l as length and thickness changes o f retepegs were recorded. 10-2 I n f l a m m a t i o n : T o assess the inf lammatory infi l t rat ion, a rating scheme for the degree o f in f lammat ion was developed. E x a m p l e s o f each category are shown i n the next page (see Figure 1): • Degree 0- M i n i m a l infi l t rat ion • Degree 1 - M i l d infi l trat ion • Degree 2- Moderate inf i l t rat ion • Degree 3- Severe inf i l t rat ion 36 F i g 1-C F i g 1-D F i g u r e 1- Different degrees o f inf lammatory inf i l t ra t ion i n tissue samples. A - M i n i m a l inf i l t rat ion o f inf lammatory cel ls , B - M i l d inf i l t rat ion o f inf lammatory ce l ls , C - Moderate inf i l t rat ion o f inf lammatory ce l ls , D - Severe inf i l t rat ion o f inf lammatory cel ls . B l a c k arrow shows the inf lammatory inf i l t ra t ion and y e l l o w arrow points the epi thel ium, (or ig inal magnif icat ions o f a l l slides x 100) Degree o f inf lammat ion i n h i s to logica l sections was assessed and recorded w i t h reference to the scheme developed and standard images. 10-3 C o n n e c t i v e t issue changes : Connec t ive tissue changes inc lud ing G A G accumula t ion and the amount o f co l lagen and elastin fibers were assessed. In the h is to logica l features stained w i t h A B / P S R , the blue staining areas were representative o f G A G s accumulat ion. A s for the in f lammat ion scheme, a semi-quantitative grading scheme was developed as presented i n the next page: 37 • Degree 0= M i n i m a l amount o f G A G accumula t ion • Degree 1= Modera te amount o f G A G accumulat ion • Degree 2= Severe amount o f G A G accumula t ion The tissue sections were assessed for G A G accumula t ion according to this scheme. W e also can observe the co l lagen bundles as red areas i n these slides. E x a m p l e s o f different degrees are shown i n Figure 2. Fig2-C Figure 2. Different degrees o f A l c i a n blue/ P ic ros i r ius red staining i n tissue samples. A - M i n i m a l or loss o f G A G s accumula t ion and dense col lagen bundles, B - Modera te accumula t ion o f G A G s , C - Severe accumula t ion o f G A G s . B and C both demonstrate the hydrated col lagen. Y e l l o w and b lack arrows show G A G accumula t ion and col lagen bundles, respectively (or ig inal magni f ica t ion o f a l l slides x 100) 38 In G o m o r i ' s t r ichrome stained slides, w h i c h show the accumula t ion o f col lagen bundles, the cr i ter ion was based o n presence or absence o f hydrated or dense col lagen (Figure3) . Dense and hydrated co l lagen cou ld also be v i sua l i zed i n A l c i a n b l u e / P S R stained slides. In G o m o r i sl ides elastin fibers c o u l d also be detected. They were v i sua l i zed as th in greenish fibers between the bundles o f col lagen. F i g 3-A F i g 3 -B F i g u r e 3- G o m o r i ' s t r ichrome staining- A - (magnif icat ion x 20) G o m o r i staining i n g ing iva l overgrowth group showing co l lagen bundles ( Y e l l o w arrow), retepeg ( B l a c k arrow) and elastin fibers (Orange arrow), B - (or ig inal magnif ica t ion x 100) the same section. In the above slides, the ep i the l ium is exhib i t ing the purple staining. Fibroblasts and inf lammatory ce l l s are v i s ib le i n the same color . C o l l a g e n bundles appear to be condensed/ hydrated i n a blue color . E las t in fibers are detected as t iny dark green fibers. W e can also observe red b lood cel ls i n the vessels. 39 11-Immunohistochemical Analysis: 11-1 CD44 (The hyaluronan receptor): The f o l l o w i n g scores were used as a cr i ter ion i n evaluat ion o f tissue sections stained w i t h C D 4 4 antibody, w h i c h detects hyaluronan receptors that are predominant ly present on macrophages. • Degree 0= N o stained cel ls detected or the m i n i m u m amount • Degree 1= S m a l l numbers o f stained cel ls • Degree 2= Modera te numbers o f stained cel ls • Degree 3= M a n y stained cel ls F igure 4 shows the above scores used to describe the number o f stained cel ls . 40 Fig 4- A Fig 4- B Fig 4- C Fig 4- D Figure 4 - Tissue sections stained w i t h C D 4 4 antibody. A - Absence o f stained macrophages, B - M i l d accumula t ion o f macrophages, C - Modera te accumula t ion o f macrophages, D - M a n y C D 4 4 + macrophages. B l a c k ar row points the stained cel ls . In these slides ep i the l ium shows the s imi la r antigenici ty and demonstrates staining i n a l l slides (ye l l ow arrow) or ig ina l magnif ica t ion o f a l l sl ides x l O O . 41 1 1 - 2 2 B 1 - V e r s i c a n S t a i n i n g A s imi la r index was made to classify staining for the core protein o f vers ican (using monoc lona l ant ibody 2 B 1 ) as fo l lows : • Degree 0= N o stained areas are detected • Degree 1= M i l d accumula t ion o f vers ican • Degree 2= Modera te accumula t ion o f vers ican • Degree 3= Severe accumula t ion o f vers ican Figure 5 shows different degrees o f staining: F i g 5 -C F i g 5- D F i g u r e 5 - 2 B 1 staining o f tissue samples demonstrating different degrees o f accumula t ion o f vers ican i n connective tissue. A - Absence o f versican, B - M i l d accumula t ion o f versican, C - moderate accumulat ion o f vers ican, D - Severe accumula t ion o f versican. Y e l l o w arrow points Stained areas (or iginal magnif ica t ion x 100) 42 11-3 LC2- Versican Staining: In these slides the C- te rmina l o f vers ican proteins was subjected to s taining. The result o f this s taining d i d not lead us to make any scoring, since it was not as prominent as what w e observed w i t h 2 B 1 . 11-4 1A4- a-Smooth muscle actin: T o analyze the slides stained w i t h this antibody, i n addi t ion to the degree o f vascular iza t ion, presence or absence o f myofibroblas ts were determined. 11-5 PCNA- Proliferating cells: Instead o f us ing any scores to analyze these sl ides, the number o f stained cel ls , w h i c h were m a i n l y located i n the basal layer, was quantif ied us ing a c e l l counter. The count ing procedure was performed twice for each sl ide, and i n case o f any discrepancy the mean was calculated. Resul ts expressed as numbers o f pos i t ive stained cel ls / 200 cel ls ( i n a r andom field) . 12-Statistical Analysis: M e a n and S D were calculated for variables such as age, dose and serum l eve l o f C S A , P D , O H I and G I . T-student test performed to reveal the degree o f s imilar i t ies between the variables i n control and D G O group. T o compare the f indings i n 2 groups, Student 's t-test was used to compare the 2 groups; statistically significant differences were considered i f p-values o f 0.05 or less were calculated. 43 Chapter 4- Results: 1- Clinical Findings: In the g ing iva l overgrowth group there were 8 w o m e n and 12 m e n w i t h an age range o f 16 to 50 years. The c l i n i c a l measurements made i n this group inc lud ing p rob ing depth ( P D ) , ora l hygiene ( O H ) , g ing iva l index (GI) i n addi t ion to the dose and serum leve l o f cyclospor ine and number o f years after transplant are s h o w n i n table 2. The e t io logy o f renal failure and subsequent transplant i n most o f the cases was u n k n o w n . In 3 cases some k i n d o f congeni ta l deficiencies were ment ioned (usual ly i n younger patients) and i n one case renal failure had happened secondary to hypertension but for the other 14 patients, even i n the med ica l chart there was no certain e t io logy specif ied. M o s t o f ind iv idua l s i n g ing iva l overgrowth group were tak ing some other medicat ions i n addi t ion to C S A , such as prednisone 5-10 mg/day, and azathioprine (Imuran) 50-100 mg/day. Aza th iop r ine produces immunosuppress ion by inh ib i t ing purine synthesis i n cel ls , thereby prevent ing R N A and D N A synthesis. It is prescr ibed i n renal and bone m a r r o w transplants, and autoimmune diseases such as pemphigus (Gage & Picket t 2001) . T w o patients were also tak ing nifedipine for their hypertension (patients # 2 & 5). C l i n i c a l oral examinat ion i n this group also revealed changes i n g ing iva l co lor , consistency, texture, contour and fo rm. The changes were l oca l i zed to facial aspects o f m a x i l l a r y and mandibular anterior segments i n most o f the cases. In a few cases some changes were observed o n the bucca l surfaces o f posterior teeth, w h i c h were not as severe as the anterior ones. Ove rg rowth generally occurred i n areas, where teeth were present. W e d i d not recognize any tissue changes i n those edentulous patients wear ing complete dentures. 44 Photographs were taken to show the c l i n i c a l changes (Figs 6-11) and the manifestations are discussed i n be low: Changes in Color: A l m o s t a l l o f the patients showed erythematous changes i n g ing iva . In some instances a b lu i sh red co lor was observed. Changes in consistency: The ove rg rown tissues were soft, spongy and extremely fragile i n most o f patients. In addi t ion, the tissue b l ed easi ly upon prob ing . These patients were also c o m p l a i n i n g about spontaneous b leeding or b leeding upon brushing. In a few cases, f ibrotic changes were observed (Figure 10) and the tissue seemed to be f i r m and resil ient. Changes in texture: L o s s o f s t ippl ing was observed i n a l l cases. The g ing iva l surface was generally shiny and nodular. Changes in contour and form: In most o f the cases both papi l la ry and marg ina l g i n g i v a were i nvo lved . In many instances the papi l la ry and the marg ina l g i n g i v a were uni ted and developed into a massive tissue fo ld cover ing a considerable por t ion o f the crowns. The first post-operation images are shown i n Figures 12 and 13. 45 Table 2- Demographic and clinical findings in gingival overgrowth patients V Data PatientsX Sex Age Dose of CSA mg/ day CSA serum level ng/ml Years of kidney transplant Dose of Prednisone Mg/day Other drugs P D O H G I 1 M 16 225 135 2 years 5 - 3.9 40% 1.8 2 F 21 200 85 9 years 10 Imuran-Nif 3.9 65% 1.8 3 M 35 275 144 2 years 10 - 5.1 60% 1.6 4 M 36 225 125 2 years 10 - 4.8 50% 2.0 5 M 17 250 150 4 years 5 Imuran-Nif 4.3 66% 2.6 6 F 25 200 125 4 years 5 - 3.9 75% 1.9 7 M 30 250 135 4 years 5 - 5.2 29% 2.6 8 F 22 250 135 3 years 10 - 3.6 55% 1.7 9 F 25 200 170 9 years 5 - 4.6 66% 2.0 10 M 32 150 106 6 years 10 Imuran 3.9 74% 1.9 11 M 17 200 125 4 years 5 - 3.9 55% 2.5 12 M 30 200 128 2 years 10 - 5.3 50% 2.5 13 M 16 150 183 2 years 5 Imuran 4.9 45% 2.7 14 M 21 300 162 8 years 10 Imuran 4.8 37% 2.5 15 F 24 250 136 1 year 5 Imuran 3.9 50% 2.3 16 M 34 275 155 8 years 5 Atenolol 5.9 62% 2.5 17 M 50 225 148 4 years 10 - 4.8 40% 2.2 18 F 42 200 N M 3 years 5 - 5.3 50% 2.2 Range L F = 6 EM=12 16-50 150-300 85-183 1-9 5-10 6 Imuran 1 Atenolol 2 Nifedipine 3.6- 5.9 29-75 1.6-2.7 M e a n ± S D 2 7 . 4 ± 9.5 2 2 3 . 6 ± 40.46 1 3 8 ± 23.27 4 . 3 ± 2.6 6 . 6 ± 2.4 4 . 5 ± 0.6 5 4 ± 12.8 2 . 2 ± 0.35 N M = N o t ment ioned P D = Prob ing depth G I = G i n g i v a l index O H = O r a l hygiene effectiveness 46 Figure 6- Patient # 12, GOIII Figure 7- Patient # 5, GOI II Figure 8- Patient # 17 GOI II 47 Figure 9- Patient # 7, GOI III Figure 10- Patient # 16, GOI III In a l l above pictures we can observe dramatic changes i n g ing iva . Papi l lae are lobulated and cover a part o f the c rown . G i n g i v a l margins do not exhibi t the knife-edge shape. The tendency toward bleeding (on probing or spontaneously) can be v i sua l ized . A l l o f the pictures except f ig 10 demonstrate erythematous changes. In patient #16 (fig 10) the tissue is more fibrotic compared to the other cases and patient reported o f hav ing g ing iva l surgery once and recurrence o f the overgrowth i n less than one year but i n a less degree o f severity. W e can see spacing between the teeth i n some o f the cases. It was asked o f the patients whether they were aware about the onset o f the spacing, w h i c h i n some cases was attributed to g ing iva l overgrowth. 48 Fig 12- A Fig 12- B Figure 12- Patient #12- A- Before operation, B- One week post-op Fig 13- A Fig 13- B Figure 13- Patient # 13- A- Before operation, B- one week post-op In one week post-op we s t i l l can observe some degrees o f in f lammat ion , w h i c h is not unusual i n periodontal surgeries. The excess tissue is removed and we can see the normal length o f the c l i n i c a l c r o w n . Patients were instructed to perform oral hygiene at this stage again. Regard ing m u c o g i n g i v a l problems, patient # 12 was referred to S H B dental school , Per iodont ics Department to receive other necessary treatments. In none o f the cases d i d we have any prob lem w i t h heal ing process. W e d i d not observe any case w i t h infect ion or delay i n heal ing. 49 Demograph ic f indings i n control group inc lud ing sex and age i n addi t ion to c l i n i c a l f indings are shown i n table 3. A s prev ious ly stated, control group was sys temical ly healthy and the ind iv idua l s were not taking any k i n d o f medicat ions. There was o n l y 1 smoker i n each group and because o f this l o w number it is not s h o w n i n the tables. Table 3- Demographic and clinical findings in control group " ^Data Patients^^^ Sex Age PD mm O H % GI 1 F 22 2.3 70% 1.2 2 F 38 2.1 50% 1.4 3 M 36 3.2 65% 1.3 4 F 24 3.1 50% 1.9 5 M 25 3.7 45% 2.1 6 M 36 2.9 75% 1.8 7 M 22 2.6 60% 2.0 8 M 52 2.2 70% 1.5 9 F 39 3.4 85% 1.4 10 M 45 3.1 75% 1.9 11 M 32 3.1 45% 2.2 12 M 30 3.5 50% 1.7 Range EF=4 EM=8 22-52 2.1-3.7 50-85% 1.2-2.2 MeaniSD 33.4±9.4 2.9±0.52 61.6±13.5 1.7±0.33 50 A s we can observe i n tables 2 and 3, the propor t ion o f female/male i n both groups is the same. The groups were age-matched and t-student test d i d not show any statistically significant difference between them. W i t h O H effectiveness also w e d i d not detect any signif icant difference between two groups. Regard ing p rob ing depth and g ing iva l index, the overgrowth group exhib i ted a statist ically significant higher scores compared to the control group, w h i c h is expected. Table 4 shows the statistical analysis regarding the demographic and c l i n i c a l f indings between 2 groups. Table 4- Statistical analysis of the demographic and clinical findings between DGO and control groups. Variables Groups Sex Age Mean± SD PD mm Mean± SD O H % Mean± SD GI Mean± SD Control group DGO group F=4 33.4± 9.4 27.4± 9.5 2.9± 0.52 4.5± 0.6 61.6 ± 13.5 54 ± 12.8 1.7± 0.33 2.2± 0.35 M=8 F=6 M= 12 T-student test P-value F/M= 1/2 T=1.7 P<0.1 T=8 P < 0.001 T= 1.6 P<0.1 T= 3.5 P < 0.01 51 2-Findings in Epithelium 2-1: H&E staining: W e compared g ing iva l overgrowth to control samples. In the g ing iva l overgrowth group, mos t ly a th ick stratified squamous ep i the l ium was cover ing the under ly ing connect ive tissue. The retepegs were elongated, either th in or th ick and were extended deeply into the connect ive tissue. Based o n the compar i son w i t h the control group and normal g i n g i v a h is to logic pictures, w e determined the retepegs d imens iona l changes. T h e number o f retepegs was also increased i n the G O group and we observed the prol i ferat ion o f the basal layer. In some areas, confluent retepegs were v i sua l i zed . W e can observe confluent retepegs w h e n f o l l o w i n g elongat ion, they j o i n each other. S igns o f hyperkeratosis, parakeratosis and acanthosis were observed i n most o f the cases i n this group ( F i g 14 and table 5). These terms are defined as fo l lowings : • Hyperkera tos is : E x c e s s i v e l y th ickened layer o f the stratum corneum. • Parakeratosis: Presence o f residual nuc le i i n kerat in layer • Acan thos i s : Excess ive th ickening o f the spinous layer o f squamous epi the l ium, result ing i n broadening and elongat ion o f the retepegs (Sapp et a l 2004) . Table 5- Summary of histological findings in epithelium of GO and control groups. " ^ ^ - ^ ^ Groups H i s t o l o g i c a l ^ " " " - - ^ ^ ^ Features ^ " " " ^ - ^ C o n t r o l n=12 G i n g i v a l Ove rg rowth n=18 Ep i the l i a l Changes Retepegs longer 0 / 1 2 12/18 Th icke r 4/ 12 9118 Acanthos i s 2/12 10/18 Hyperkeratos is 1/12 5/18 Parakeratosis 1/12 12/18 52 Fig 14- A Fig 14- B Fig 14- C Fig 14- D Fig 14- E Fig 14- F Figure 14 - H e m a t o x y l i n and E o s i n staining- A , B (or iginal magnif ica t ion x 10) -Long and th in retepegs penetrated deeply into connect ive tissue i n g ing iva l overgrown tissue (white ar row points elongated retepegs) C , D (or ig inal magni f ica t ion x 100)-Normal g ing iva l specimens demonstrating normal retepegs (black ar row shows the retepegs), E (magnif icat ion x 300)- Elongated retepegs i n g ing iva l overgrowth tissue(black arrow), F (magnif ica t ion x 200)- No te acanthosis (black arrow) 53 2-2 PCNA staining P C N A ant ibody was used to stain the prol i ferat ing cel ls . E igh teen overgrowth and 11 normal samples were stained w i t h concentrations o f 1/100, 1/200, 1/500 and 1/1000. Three slides remained as controls. In a l l the slides one area was randomly selected and the number o f pos i t ive stained cel ls out o f 200 cel ls was counted. The differences between 2 groups were then compared. S ta in ing as expected was more prevalent i n the basal layer cel ls i n both groups. C o m p a r i s o n between 2 groups revealed that g ing iva l overgrowth samples demonstrate more stained cel ls . T h i s f ind ing is i n agreement w i t h the changes w e observed i n H & E stained sections, w h i c h the latter also demonstrated prol iferat ive changes i n retepegs o f G O group. A l t h o u g h most o f the stained cel ls are observed i n the basal layer, w e c o u l d detect some stained cel ls i n the spinous layer, as w e l l . The presence o f staining i n this layer can be a result o f qu ick turnover, w h i c h is probably because o f prol iferat ion. F i g 15 and table 6 show the differences i n the number o f stained cel ls i n both groups. Table 6- Statistical analysis of PCNA stained cells in the control and GO group ^\Steined cells/200 cells Groups Mean SD Control 61 37.5 GO 126 31.5 Student-1 1= 5.3 P-value P < 0.01 54 Fig 15-A Fig 15-B Fig 15- E Fig 15- F Fig 15- S ta in ing w i t h P C N A i n the basal layer o f ep i the l ium i n both groups. A , B (or iginal magnif ica t ion x 100) - no rmal tissue samples demonstrate a lesser number o f stained cel ls . C , D (or iginal magnif ica t ion x 100) Increased number o f stained cel ls i n overgrowth tissue samples can be observed. Orange ar row show the stained cel ls i n basal layer and y e l l o w ar row points the ones i n spinous layer. E (magnif icat ion x 150) less number o f stained cel ls can be observed i n one o f the control samples compared to G O samples. F (magnif ica t ion x 200) more stained cel ls i n G O samples compared to F i g E 55 3- Findings in Connective Tissue 3-1- H & E S t a i n i n g : A dense col lagenous fibrous connect ive tissue was observed i n most o f overgrowth g ing iva l specimens. W e observed increased co l l agen bundles compared to controls . The tissue was expanded i n g ing iva l overgrowth group compared to healthy group. Increased number o f fibroblasts and inf lammatory cel ls cou ld also be observed i n this group. Inf lammatory cel ls were m a i n l y representative o f chronic inf lammatory inf i l t rat ion. The degree o f vascular iza t ion also seemed to be higher compare to the control group ( F i g 16). It was also noted that some o f vessels exhib i ted th ickened wa l l s , and apparent vascul i t i s . 3-2 - G o m o r i T r i c h r o m e / A l d e h y d e f u c h i n S t a i n i n g : In these slides, the most prominent feature was the bundles o f co l lagen , w h i c h demonstrated a deep b lu i sh green co lo r and a w a v y course. In some o f the areas the co l lagen fibers appeared dense and i n the other areas some empty spaces were observed i n between. The other observations inc luded keratinocytes, w h i c h appeared purple, vessels w i t h red b l o o d cel ls i n the l u m e n and elast in fibers l o o k i n g l ike t iny purple fibers surrounded b y co l lagen bundles. W e also observed per ivascular inf i l t ra t ion i n some o f the features ( F i g 17). Since the areas representing the proteoglycans also demonstrate the purple co lor , it was diff icul t to differentiate them f rom other features w i t h the same staining reaction, so, the a lc ian blue/ p icros i r ius red staining was performed to get a more precise observat ion regarding proteoglycans and g lycosaminoglycans . C o l l a g e n and elast in fibers appeared s imi la r i n content and appearance between the diseased and the control groups. 56 3-3- Alcian blue/ Picrosirius red Staining: In the slides stained w i t h A B / P red, the red areas are representative o f co l lagen bundles w i t h i n the connect ive tissue, w h i c h can be either dense or hydrated ( in the case latter w e can observe whi te spaces i n between). E p i t h e l i u m i n these slides demonstrates the l ight p ink staining ( F i g . 18). In some o f the cases, between the co l lagen bundles we can observe some areas stained i n l ight blue w h i c h are representative o f proteoglycan and g lycosaminoglycans . These areas are usua l ly found i n deeper parts o f connect ive tissue. A s it was ment ioned i n materials and methods w e used a grading scheme, based on that, G A G accumula t ion was class i f ied as m i n i m u m , moderate and severe. Table 7 demonstrates the h is to logica l f indings i n connect ive tissue i n both groups and table 8 shows the statistical analysis between the two groups regarding inf lammatory inf i l t ra t ion and G A G accumula t ion . 57 Fig 16- A Fig 16-B Fig 16-C Fig 16-D Figure 16- H e m a t o x y l i n and E o s i n staining- A (or iginal magni f ica t ion x 100)- Severe inf i l t ra t ion o f inf lammatory cel ls i n g ing iva l overgrowth sample, B (or iginal magnif ica t ion x 100)- Modera te inf i l t rat ion o f inf lammatory cel ls i n control g ing iva l sample, C (magnif icat ion x 150)- Inflammatory infi l t rat ion i n g ing iva l overgrowth sample, ma in ly composed o f p lasma cel ls (arrow) and lymphocytes , D (magnif icat ion x 200) C o n t r o l group sample, most o f the cel ls are fibroblasts (arrow) 58 Fig 17-A Fig 17-B Fig 17-E Fig 17-F Fig 17- G o m o r i T r i ch rome / A l d e h y d e fuchin Sta in ing: A , B - Dense bundles o f co l lagen i n control group. C , D , E - Dense co l lagen bundles i n g ing iva l overgrowth group. F - Less density o f co l lagen bundles, b l o o d vessels w i t h R B C and keratinocytes are observed i n D G O group. Per ivascular inf i l t rat ion can be observed (arrow), (or ig inal magni f ica t ion o f a l l slides x 100) 59 F i g 1 8 - E F i g u r e 18- A l c i a n b lue/ P ic ros i r ius red staining i n control and g ing iva l overgrowth groups. A , B (or iginal magni f ica t ion x 100) - m i n i m u m accumula t ion o f G A G i n control group. Y e l l o w ar row points the col lagen bundles, C (magnif icat ion x 150)- moderate accumulat ion o f G A G s i n g ing iva l overgrowth group, D (magnif icat ion x 150)- severe accumula t ion o f G A G s i n g ing iva l overgrowth group. B l a c k ar row points G A G s , E (magnif ica t ion x 200) - G A G accumula t ion i n g ing iva l overgrowth group. 60 Table 7- Summary of histochemical findings in the connective tissue of gingival overgrowth and control groups. Groups Histological ' Features * ^ Control n=12 Gingival Overgrowth n=18 Degree of Inflammation Scores 0-3 M e a n = 1.4 SD = 0.66 Range =1 -3 M e a n = 2.6 SD = 0.48 Range = 2-3 Connective Tissue Changes GAG content M e a n = 0.8 SD = 0.49 Range= 1-2 M e a n = 1.5 SD= 0.58 Range=l-2 Collagen Hydrated 4/12 15/18 Dense 9/12 4/18 A s w e can observe i n table 7, there are more cases w i t h hydrated co l lagen i n the overgrowth group, w h i c h can be attributed to G A G accumula t ion . In table 8 w e can see that both G A G content and inf lammatory inf i l t ra t ion i n the G O group demonstrate statist ically significant higher amounts compared to the control group. Table 8- Statistical analysis of histochemical findings between GO and control groups. >w Variables Groups^. GAG content Mean± SD Inflammatory infiltration Mean ± SD Control group 0.8 ± 0.49 1.4 ± 0.66 GO group 1.5 ± 0.58 2.6 ± 0.48 T- test t = 23 t = 30 P- value P < 0.01 P < 0.01 61 3-4 a-smooth muscle actin - A total o f 28 slides (10 healthy, 18 g ing iva l overgrowth) were stained w i t h 1 A 4 antibody i n 2 o f experiment. In each round o f staining 4 slides (both healthy and g ing iva l overgrowth) were kept as controls, not r ece iv ing the p r imary antibody. 1 A 4 targets the a - smooth musc le act in, w h i c h can be found i n vessel wa l l s and myofibroblas ts . In a l l specimens, overgrowth and healthy g ing iva l tissues, vessel wa l l s were stained (brown color) . The staining demonstrated more vascular iza t ion i n overgrowth tissues was more prominent i n this group. In addi t ion, some areas representative o f myofibroblas ts c o u l d be observed i n overgrowth group. These cel ls appeared to be i n the matr ix rather than b l o o d vessel wa l l s and their cytoplasms were stained. W e c o u l d not detect myofibroblas ts i n the control group. In F igure 19 we can see the differences between the number and the size o f b l o o d vessels i n ove rg rown and normal tissues i n addi t ion to presence o f myofibroblas ts . 3-5 Core of versican antibody: 2 B 1 ant ibody was used to stain the core o f vers ican. A total o f 30 slides (18 overgrowth samples, 12 normal samples) were stained w i t h this ant ibody and 6 slides were served as controls . W e observed that a l l o f the overgrowth tissue samples demonstrate a higher degree o f s taining (brown color) , w h i c h was distributed almost evenly throughout the connect ive tissue. In no rma l tissue samples, w e observed some degree o f staining, w h i c h was s ignif icant ly less compared to the g ing iva l overgrowth group (figure 20). Based o n this f ind ing , w e conc luded that vers ican is i n a part responsible for the d imens iona l changes, w h i c h happens i n C S A - i n d u c e d G O . 62 F i g 1 9 - E F i g 19- Sta in ing w i t h 1 A 4 to demonstrating a -smooth muscle act in ( in b l o o d vessels and myofibroblasts) . F i g A , B (or ig inal magni f ica t ion x 100) - B l o o d vessels demonstrate the staining i n normal tissues. F i g C , D (or ig inal magnif ica t ion x 100) - Increased i n number and size o f b l o o d vessels i n g ing iva l overgrowth tissues compared to no rma l ones. E (magnif icat ion x 150) - Presence o f myofibroblasts i n g ing iva l overgrowth tissue (orange arrow). 63 F i g 20- E F i g u r e 20- S ta in ing w i t h 2B1 ant ibody to reveal core o f vers ican. F i g A , B (or iginal magni f ica t ion x 100) - m i l d accumula t ion o f vers ican is seen i n normal tissues. F i g C , D (or iginal magni f ica t ion x 100) - Severe accumula t ion o f vers ican i n ove rg rown tissues. F i g E (magnif icat ion x 200) Severe stained areas representative o f vers ican accumula t ion i n g ing iva l overgrowth tissue 64 3-4 C terminal of Versican: L C 2 antibody was used to stain the C terminal o f versican. A total o f 36 (20 g ing iva l overgrown and 16 normal samples) slides were treated w i t h this antibody at concentrations o f 1/500, 1/1000, 1/5000. 8 slides remained as controls. A l t h o u g h we d i d not get the same strong staining as what we got w i t h 2 B 1 , we s t i l l can notice the differences between two groups, w i t h more accumula t ion o f vers ican i n the overgrowth samples. U s i n g higher concentrations o f this antibody d i d not make a major difference i n the amount o f staining. F igure 21 shows the differences between 2 groups. F i g 2 1 - A F i g 2 1 - B Figure 2 1 - A (or iginal magni f ica t ion x 100) - L C 2 staining i n normal tissue. B (or ig inal magni f ica t ion x 100) - L C 2 staining i n ove rg rown tissue. A l t h o u g h w i t h this antibody a strong staining reaction was not observed, we s t i l l can see the difference between the control and diseased tissue. 65 3-7 CD 44 Staining: C D 4 4 ant ibody was used to stain the hyaluronan receptors, w h i c h are predominant ly o n macrophages. To ta l ly , 30 slides (18 diseased and 12 normal samples) were stained w i t h different concentrations o f ant ibody (1/200, 1/500, and 1/1000). 6 slides served as controls and received no p r imary ant ibody. A l l overgrowth tissue samples exhibi ted more stained cel ls i n connect ive tissue compared to the controls , w h i c h were more prominent i n sub epi thel ia l connect ive tissue. W i t h this antibody, ep i the l ium exhibi ted the same staining. Be tween the stained cel ls , w e c o u l d differentiate macrophages cons ider ing their prominent nuc le i . W e c o u l d also observe some p lasma cel ls . The different degrees o f s taining i n 2 groups are s h o w n i n F i g 22 . Table 9 demonstrates the statistical analysis. 66 F i g 22- E F i g 22- S ta in ing w i t h C D 4 4 i n both overgrowth and normal g ing iva l samples. A , B (or iginal magni f ica t ion x 100) - m i l d accumula t ion o f the stained cel ls i n the connective tissue o f normal samples. C , D (or iginal magni f ica t ion x 100) - moderate and severe accumula t ion o f staining i n overgrowth g ing iva l samples. E (magnif icat ion x 200) - moderate staining i n overgrowth g ing iva l sample. A r r o w points the macrophages. E p i t h e l i u m exhibits the s imi la r antigenici ty i n a l l the slides. 67 Table 9- Statistical analysis of immunohistochemical findings in the GO and control groups. ^^^^ Groups Scores CD44 Mean± SD 2B1 Mean ± SD Control group 1.08 ± 0.5 1.25 ± 0.45 GO group 1.88 ± 0.67 2.27± 0.66 T- student T= 3.8 T= 25 P-value P < 0.01 P < 0.01 A c c o r d i n g to the above table, w e have statistically significant higher amounts o f s taining w i t h CD44 and 2 B 1 i n the D G O group compared to the control group. W e can see that the greatest difference happens w i t h 2 B 1 . 68 Co-Localization: T o compare different stainings i n the same slides and see h o w the changes are related together, figures 23 , 24 and 25 show the co- local iza t ions o f various stainings i n 3 patients w i t h g ing iva l overgrowth. In F igure 23 , we can observe that the same areas i n A l c i a n / b l u e stained slides demonstrate the staining i n those slides stained w i t h 2 B 1 , both indica t ing the presence o f G A G s . In F igure 24, the same areas show the staining reaction w i t h L C 2 , 2 B 1 and A l c i a n / b l u e , w h i c h again demonstrate correspondence between the staining for g lycosaminoglycans and staining for vers ican. In figure 25 , we see that same areas i n the slides stained w i t h A l c i a n / b l u e and 2 B 1 exhibi t the accumula t ion o f G A G s , w h i l e i n the sl ide stained w i t h L C 2 w e see less amount o f s taining i n that area. A s L C 2 is raised against C- te rmina l o f versican, that m a y means vers ican i n tissues has been pro teo ly t ica l ly processed and lacks the C- te rmina l domain . 69 Fig 23- E Fig 23- F Fig 23- C o - L o c a l i z a t i o n o f different stainings i n patient # 6- A - Sta ining w i t h 1 A 4 , B - S ta in ing w i t h A l c i a n / b l u e , C - C o n t r o l specimen, D - S ta in ing w i t h 2B1, E - C D 4 4 staining, F - G o m o r i / t r ichomorious staining (or iginal magnif ica t ion x 100 i n a l l the slides). 70 Fig24-C Fig 24-D Fig 24-E Fig 24-F Fig 24- Co -Local iza t ion o f the same sections w i t h different stainings i n patient # 10. A - 2 B 1 staining B - A l c i a n / B l u e staining, C - C D 4 4 staining, D - G o m o r i staining, E - 1 A 4 staining, F - L C 2 staining, (or ig inal magnif ica t ion x 100 i n a l l slides). 7 1 Fig 25-A Fig 25-B Fig 25-F Figure 25- C o - l o c a l i z a t i o n o f the same slides and different stainings. A - 1 A 4 staining, B - 2 B 1 staining, C - A l c i a n / b l u e staining, D - C D 4 4 staining, E - G o m o r i staining, F - L C 2 staining (or ig inal magnif ica t ion x 100 i n a l l slides) 72 Chapter 5- Discussion: The major n e w f indings o f this study are as fo l l ows : • Observa t ion o f Myof ib rob las t s i n connect ive tissue o f G O group • Detec t ion o f vers ican accumula t ion i n Connec t ive tissue o f G O group • Increased number o f prol i ferat ing cel ls i n the basal layer o f the ep i the l ium, i n G O group 1-Comparison of the findings in the epithelium of overgrowth gingival samples to the previous findings in literature: H & E staining: H & E stained slides o f D G O group revealed significant differences compared to those o f control group. W e observed a th ick stratified squamous ep i the l ium w i t h different degrees o f acanthosis and parakeratosis, w h i c h penetrates connect ive tissue w i t h long , th in/ thick and irregular retepegs. These observations are i n concordance w i t h the f indings o f Ratei tschak et al (1983) and Ros tock et al (1986), w h o reported an irregular, mul t i layered parakerat inized ep i the l ium w i t h va ry ing thickness. O n the other hand, Ros tock et al (1986) also ment ioned the presence o f spongiosis i n epi the l ium, w h i c h was not observed i n our samples. H o w e v e r , that study was a case report and the biopsies were taken o f one A f r i c a n - A m e r i c a n male patient. T h i s rac ia l d ivers i ty might exp l a in the different features. The S E M study done by Pisanty et al (1988) o n patients suffering f rom Behcet syndrome and rece iv ing C S A revealed epi thel ia l acanthosis and the presence o f needle l ike crystal l i tes i n epi the l ium, w h i c h were regarded as representing the accumulated and deposited drug. Acan thos i s was one o f the changes that we observed i n most o f our cases, however , no crystal l i te structure was found i n the samples, w h i c h might be expla ined by a l imi ta t ion o f l ight mic roscopy . O ' v a l l e et al (1994) observed va ry ing degrees o f papi lomatosis , acanthosis and epi thel ia l spongiosis i n a l l o f their specimens. M e l l e r and colleagues (2002) also report a th ickened ep i the l ium and hypertrophy o f epi thel ial cel ls i n rats that received a dose o f 40 m g / k g for eight weeks. 73 Proliferating Cell Nuclear Antibody: W i t h P C N A antibody we observed increased staining i n the basal layer o f C S A - i n d u c e d G O group compared to the controls, w h i c h indicates active prol i ferat ion in that ce l lu lar layer ( 1 2 6 ± 31.5 i n G O group versus 6 1 ± 31.5 in control group). T h i s f ind ing is in accordance w i t h other studies. Saito et al (1999) s tudying the percentage o f K i - 6 7 to demonstrate the mi to t ic act ivi ty i n basal layer o f n i fedipine- induced overgrown g ing iva and compare w i t h the controls , found about 2- fo ld increases o f k i -67 staining percentage in the diseased group. N u r m e n n i e m i et al (2001) also compared the mitot ic act ivi ty o f the basal ce l l layer i n drug- induced g ing iva l ove rg rown samples by us ing monoc lona l antibody for k i -67 , and determined the mi to t ic act ivi t ies o f epi thel ia l cel ls as percentage o f k i -67 labeled cel ls i n relat ion to total numbers o f epi thel ia l cel ls i n the basal layer o f ora l , oral sulcular and sulcular epi the l ium. They concluded that the increased epithelial thickness is associated w i t h increased mi to t ic act ivi ty especial ly i n the oral ep i the l ium. O n the other hand, one recent study from B u l u t et al 2004 revealed controversial f indings. U s i n g K i - 6 7 , they reported s imi l a r proliferat ive act ivi ty i n the basal layer cel ls o f the C S A - i n d u c e d g ing iva l overgrowth and control groups. Based on the periodontal parameters o f their study popula t ion , the control group has more g ing iva l in f lammat ion as revealed by G I (g ingiva l index). A c c o r d i n g to Schroeder et al (1973) a prol i ferat ion o f retepegs can be observed i n early and established les ion o f dentogingival ep i the l ium. Thus , more inf lammat ion i n the control group can be responsible for observ ing the same degree o f prol i ferat ion i n basal layer o f two groups. Based o n the f indings w i t h P C N A staining w h i c h show us more prol iferat ion o f the basal layer, we can exp la in the reason we see d imensional changes i n epi thel ial retepegs o f the g ing iva l overgrowth samples. 2-Comparison of the findings in connective tissue of overgrowth gingival samples to previous reports: The changes that we not iced i n connect ive tissue inc luded a h igh l eve l o f vascular izat ion, 74 i r regular ly arranged col lagen fiber bundles, a significant infi l t rat ion o f inf lammatory cel ls ma in ly composed o f mononuclear cells and accumulat ion o f amorphous ground substances. Other investigations (Rateitschak et al 1983, Ros tock et al 1986, O ' v a l l e et al (1994), Seymour & T h o m s o n 1996, H a l l m o n & Rossmann 1999) conf i rm these f indings. Howeve r , there is a controversy regarding the number o f fibroblasts. W h i l e many authors bel ieve that there is no increase i n the number o f fibroblasts (Ros tock et al 1986, Pisanty et al 1988), other investigators ( M a r i a n i et al 1996) report observing a h igh number o f fibroblasts and c l a i m that fibroblasts constituted the predominat ing ce l lu lar elements i n the connective tissues. Despi te this f inding , the authors suggested that the term "hyperp las ia" is an inappropriate name for this condi t ion and since the increase i n v o l u m e o f g ing iva is brought by abnormal quantity o f amorphous substance o f the connective tissue, the term should be changed to "d imens iona l increase o f g ing iva l tissue". In the present study, we observed that inf lammatory cel ls constitute the highest number o f ce l l populations and we c o u l d not f ind a drastic difference i n the number o f fibroblasts between control and D G O groups. In those slides stained wi th A l c i a n blue/picrosi r ius red we observed that most o f D G O group exhibi ted a s ignif icant amount o f G A G accumula t ion i n connective tissue. Th i s observat ion is i n agreement w i t h many previous studies. It has been reported by But le r & But le r (1974) that the non-col lagenous matr ix composed 2 0 % o f the dry weight i n g ing iva l tissue from phenytoin- induced g ing iva l overgrowth and on ly 7%> i n normal tissue. Kan tour & Hassa l (1983) studying the cultures o f g ing iva l fibroblasts f rom patients tak ing phenytoin observed that these cel ls synthesize increased amounts o f sulphated g lycosaminog lycans ( G A G ) . Pisanty et al (1988) also reported the accumula t ion o f non-collagenous extracel lular matr ix . Suresh et al (1992) extracted G A G s from human normal , in f lamed and phenytoin- induced g ing iva l overgrowth tissue by proteolysis and a lcohol precipi tat ion. They observed that G A G s were decreased in inf lammat ion , w h i l e it was increased i n the overgrowth cases. M a r i a n i et 75 al (1993) observed a particular abundance of amorphous substance when compared to fibrous material in tissue samples obtained from kidney transplant patients. Their histochemical data showed that overgrowth gingival samples contains increased amounts of both sulphated and non-sulphated GAGs. These authors also noted that the increased number of GAGs in the ground substance leads naturally to a greater amount of bound water, and correspondingly to a higher volume and an increased osmotic pressure in the extracellular matrix, which would produce a water-logged connective tissue, thereby accounting for the gingival edema. Saito et al (1996) observed heparan sulfate G A G in the lamina propria of hyperplastic gingival tissue while less immunostaining was observed in the control group. Newell and Erwin (1997) compared G A G and hyaluronan synthesis by fibroblasts derived from normal and CSA-induced overgrown gingival tissue and suggested that a direct promotion of G A G synthesis by gingival fibroblasts in response to CSA may play a role in the pathogenesis of gingival overgrowth. On the other hand, Rocha et al (2000) observed that there were no differences on the total and relative amounts of G A G between CSA-induced overgrown gingiva and control group. They analyzed the purified GAGs by agarose gel electrophoresis and observed the presence of chondroitin 4- and 6- sulfate, dermatan sulfate, heparan sulfate and hyaloronic acid to be the same in both normal gingiva and CSA- induced overgrowth. From the same group of authors Martins et al (2003), observed no significant difference in the molecular size distribution of hyaluronic acid and sulfated glycosaminoglycans among healthy and drug-induced overgrown gingival tissues. The results we obtained from our study, all indicated that there was a marked difference on G A G accumulation between diseased and control group, as most of the other studies observed the same finding. The reason for the controversy could be due to the different techniques which were applied. The set of slides were stained with Gomori Trichrom/Aldehyde fuchin demonstrated collagen bundles, which were dense or hydrated. 76 Versican: To study more specifically about G A G accumulation we aimed to determine the amount of versican in connective tissue of the gingival overgrowth group and to compare it to the control group. Using 2B1 and LC2 antibodies we were able to observe versican deposition in tissue samples, with 2B1 giving a better staining response. In most of gingival overgrowth samples there was a higher accumulation of versican compared to those of controls. To our knowledge, this is the first report of versican presence in cyclosporine-induced gingival overgrowth. In the study of Gnoatto et al (2003), mRNA expression of the proteoglycans perlecan, decorin, biglycan and versican was analyzed by reverse transverse polymerase chain reaction in CSA-induced gingival overgrown samples. The results revealed an increase in perlecan expression compared to control group. However, other proteoglycans including versican did not show any significant difference with controls. a-Smooth Muscle Actin: Antibody 4A1 was used to identify the ct-smooth muscle actin, which is present in both vessel walls and also myofibroblasts. Because of high vascularization in DGO group, there was a marked staining in vessel walls in that group. As mentioned previously, many studies mention this increase in vascularization and its effects on the general picture of the pathological changes. According to Mariani et al (1996), the importance of endothelial cells is that they have a high number of micropynocitotic vesicles in overgrown gingiva, which appear to derive from saccular invaginations of the plasma membrane and have openings on the side of the capillary lumen and the opposite side. The vesicles allow the endothelial cells to actively intervene in the exchange of materials through the capillary walls. Some of the other features of this staining were that the stained areas indicated the presence of myofibroblasts (cells, which could not be detected in control group). Yamasaki et al (1987) were the first to report the presence of myofibroblasts in tissue samples taken from CSA-induced gingival overgrowth. They reported the presence of cells containing microfilament 77 bands w i t h semiper iodic dense nodes and nuclear indentations. D i l l & Iacopino (1997) performed a t ransmiss ion electron mic roscopy study o f phenytoin- induced g ing iva l overgrowth and demonstrated the presence o f cel ls w i t h ultrastructure characteristics o f myofibroblasts w h i c h inc lude; microf i laments , electron-dense bodies associated w i t h fi lament bundles, ves icular structure i n close associat ion w i t h surface membrane, presence o f desmosomes and hemidesmosomes and a w e l l developed rough endoplasmic re t iculum. The latest study discussing the presence o f these cel ls is the one done by B u l l o n et al (2003), i n w h i c h they observed myofibroblasts i n tissue samples taken from patients on c a l c i u m channel blockers . They described myofibroblasts as cel ls w i t h a very fusiform shape, elongated nucle i and w e l l presented cytoplasmic organelles par t icular ly endoplasmic re t iculum. The observat ion o f myofibroblasts is significant regarding the association o f these cel ls w i t h the process o f tissue turnover/repair and fibrosis. In a series o f studies performed by Roberts and colleagues (1996, 1997, 2001) presence o f these cel ls was noticed i n pu lmonary fibrosis. A c c o r d i n g to these studies, myofibroblas ts express cc-smooth muscle actin and resemble smooth ce l l muscles ultrastructually. They both synthesize the structural elements and contract the granulat ion tissue, w h i c h the later be ing an important step i n c los ing the wound . -CD44: C D 4 4 ant ibody also demonstrated significant numbers o f stained ce l l s i n overgrown tissues. These ce l l s were ma in ly macrophages. The reason that we used this antibody is because o f the important potential role that macrophages have i n the pathogenesis o f g ing iva l overgrowth. A c c o r d i n g to studies o f Iacopino et al 1997 and N u r m e n n i e m i et al 2002, macrophages are a determinant i n pathogenesis o f drug-induced g ing iva l overgrowth. Schematic picture i n figure 26 shows the potential role o f macrophages i n drug-induced g ing iva l overgrowth. W e observed accumula t ion o f proteoglycans i n connect ive tissue o f g ing iva l overgrowth tissue, w h i c h i n turn can be a result o f 78 act ivat ion o f myofibroblas ts . A c c o r d i n g to the literature, C S A can have this effect on fibroblast through prol i ferat ion o f more macrophages and product ion o f more growth factors ( P D G F - B , TGF -P) . C D 4 4 according to K a j i t a et al (2001) is a mult is tructural and mul t i funct ional ce l l adhesion molecule that is i n v o l v e d i n ce l l - ce l l and ce l l -matr ix interactions. It has been shown to play roles i n many important phys io log ica l and pathological processes such as lymphocyte homing , T c e l l act ivat ion, w o u n d heal ing, angiogenesis and metastatic spread o f cancer cel ls . Discussion C S A \ . I )in-cr effect S\ nrhcsis of Vcrsit.m I l l \ Fig 26- Poss ible role o f macrophages i n pathogenesis o f D G O It is clear that g ing iva l overgrowth as a side effect o f cyc lospor ine therapy is d r iven by both the direct effects o f cyclospor ine o n macrophages and/ or fibroblasts and an effect o f altered g ing iva l microf lora . A s we k n o w that oral hygiene plays a very important part i n determining the severity o f g ing iva l 79 overgrowth , it seems probable that there is a posi t ive feedback loop that dr ives g ing iva l overgrowth i n these patients. I propose that C S A has effects on macrophages and fibroblasts that may cause increased matr ix synthesis. A t the same t ime, immunosuppress ive effects o f C S A may either alter g ing iva l mic ro f lo ra or alter the tissue react ion to the b i o f i l m present i n these patients. It is possible that C S A causes both increased matr ix synthesis and increased b i o f i l m that drives matr ix synthesis leading to a posi t ive feedback loop. H o w e v e r , despite numerous investigations, w h i c h have been done i n the f i e ld o f drug-induced g ing iva l overgrowth, there s t i l l are many controversies and many questions to answer regarding issues such as suscept ibi l i ty , histopathology, pathogenesis, treatment and prevention. Cons ide r ing the increased number o f organ transplants, w h i c h are taking place, there is a need to overcome the side effects o f these drugs. A s a consequence, we s t i l l need more studies to increase our knowledge and understand the mechanisms that contribute to tissue overgrowth as a side effect o f cyc lospor ine therapy. 80 Limitations of the present study: 1- The differences between the overgrown and normal g ing iva l samples were so obvious and clear under the l ight microscopy , that there was no way to perform this study in a b l i n d way . W h e r e possible , ce l l counts p rov ided an objective approach to data co l lec t ion . The scor ing systems were conf i rmed by independent analysis by 2 observers. H o w e v e r , as both observers c o u l d clear ly see that each sample belonged to one o f 2 obvious groups, the poss ib i l i ty o f observer bias cannot be discounted extremely. 2- It was not possible to make quantitative measurements. W e had to make our o w n grading schemes for some features, compare the two groups. It is obvious that this qualitative scor ing is not a very precise and exact method to describe a l l the changes. 3- W e compared the g ing iva l overgrowth samples w i t h c l i n i c a l l y healthy g ing iva l samples. The G O patients exhibi ted some changes that might be secondry to periodonti t is , and that might confound analysis . It w o u l d have improved the study i f w e cou ld have inc lude groups o f patients w i th various degrees o f periodonti t is to see whether we c o u l d separate changes due to in f lammat ion from those associated w i t h g ing iva l overgrowth. T h i s w o u l d o n l y have been possible w i t h a m u c h larger group o f control and G O patients, stratified based on periodontit is score. 4- Ano the r possible design for this study is a cohort design. W i t h this type o f study we can overcome many o f confounding variables. Da ta co l lec t ion i n this study shou ld be done before patients receive C S A . 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J Per iodontol 2001 ; 72:921-31 W i l l i a m s o n M S , M i l l e r E K , P lemons J , Rees T , Iacopino A M : Cyc lospo r ine A upregulates inter leukin-6 gene expression i n human g ing iva : possible mechanism for g ing iva l overgrowth. J Per iodonto l 1994; 65:895-903 W r i g h t H J , Chapp ie I L , Mat thews J B : T G F - P isoforms and T G F - p receptors i n drug- induced and hereditary g ing iva l overgrowth. J O r a l Pathol M e d 2001; 30: 281-289 88 W y s o c k i G P , Gretz inger H A , Laupac is A et a l : Side effect o f cyc lospor in A therapy. O r a l Surg O r a l M e d O r a l Pa th 1983; 274-278 Y a m a d a H , N i s h i m u r a F , Na ru i sh i K , C h o u H , Takash iba S, A l b r i g h t G M , Nares S, Iacopino A M , M u r a y a m a Y : Phenyto in and cyclospor ine A suppress the expression o f M M P - 1 , T I M P - 1 , and cathepsin L , but not Cathepsin B in cultured g ing iva l fibroblasts J Per iodonto l 2000; 71 : 955-960 Y a m a s a k i A , Rose G G , Pinero G J , M a h a n C J : U l t r a structure o f fibroblasts in c y c l o s p o r i n e - A induced g ing iva l hyperplasia . J Ora l Pathol 1978; 16: 129-134 Y a t s c o f f R , Rosano T , Bower s L . The c l i n i c a l s ignif icance o f cyclospor ine metabolites. C l i n b iochem 1991;24 :23-35 Y o s h i m u r a N , K a h a n B D , O k a t. The i n v i v o effect o f cyclospor ine on inter leukin-6 gene expression i n renal transplant recipients. Transplant Proc 1991; 23: 985-960 Z e b r o w s k i E J , Py lpas S P , O d i u m O , Johnson R B : Compara t ive metabo l i sm o f H 3 - g lucosamine by fibroblast popula t ion exposed to cyclospor ine . J Per iodonto l 1994; 65: 565-56 89 Appendix I: 4j y~-« j j S j J a m j J AJ] ( j ^ l J > LUC- U Ij . J j i . C J j L j a j jLui . j j j i o . J J j j 3 I. ''d ? 'jl jl-JJ-o I j ^Liil* ftjUU 4JK .AJJJJ Jlj j l ̂ jjljla. <_jl J J A £ jJjjJ^uijlSoLui < _ ? J J ^ j l cs-"Lj 4-Jajlc jUjJS jjiaio Cjij lAlji. jl£ 4j i-ilaiâ ." «1 i il jjjalo 4j OAJJJ AJjuSljjJ 4JJ A J J A J ŷ̂ l ja. 1̂5\JA jJ <\£ ^IUIA sl£l i_JJLaJjl I express m y consent regarding periodontal flap surgery by D r . M a s o u m e h N o u r i for treating m y g ing iva l overgrowth. I was been expla ined that g ing iva l overgrowth is a side effect o f c y c l o s p o r i n usage. I a m aware that the discarded g u m sample w i l l be used for research purposes. Signature 90 Appendix 2: • U N I V E R S I T Y O F B R I T I S H C O L U M B I A D e p a r t m e n t o f O r a l B i o l o g i c a l a n d M e d i c a l Sc iencesJ . B . M a c d o n a l d B u i l d i n g , R m 3982199 Wesbrook M a l l V a n c o u v e r B C V 6 T l Z 3 C a n a d a T e l : l - (604) 822-6819; Fax : l - (604) 822-3562 • I D N O : N A M E O F T H E C E N T E R : • P A T I E N T ' S N A M E : • P A T I E N T A D D R E S S : • Date and year o f birth • S E X : F E M A L E M A L E • G i n g i v a l Index: O H I : P D : G O I : • S M O K I N G H A B I T : Y E S N O • IF Y E S : D U R A T I O N : N O O F C I G / D A Y • O T H E R S Y S T E M I C D I S E A S E S : Y E S N O • E x p l a i n , us ing another sheet i f necessary • T Y P E O F M E D I C A T I O N : N I F C S A P H T D O S A G E : • D U R A T I O N : Serum level o f C S A • Dura t ion o f g ing iva l overgrowth ( i f known) • The patient has read and understands the insti tutional consent fo rm • S igned (by the patient) G i n g i v a l I n d e x o r G I ( L o e & Si lness ) : • 0 = N o r m a l g ing iva • 1 = M i l d inf lammat ion , slight change i n the color , sl ight edema, no b leed ing o n palpat ion • 2 = Modera te inf lammat ion, redness, edema, and g laz ing ; b leeding on p rob ing • 3 = Severe inf lammat ion , marked redness and edema, tendency to spontaneous bleeding The tissues surrounding each tooth are d i v i d e d into four g ing iva l scor ing units: the distofacial pap i l l a , the facial margin , the mesiofacia l pap i l l a and the entire l ingual marg in . E a c h surface is g iven one score, the scores are totaled and d iv ided by four; the G I score for each tooth is obtained. 17 16 15 14 13 12 11 21 22 23 24 25 26 27 47 46 45 44 43 42 41 31 32 33 34 35 36 37 91 Probing Depth: • The distance between the margin o f the g ing iva and the bot tom o f the pocket in m m . The prob ing depth is measured for three surfaces (mesial , middle , distal) i n facial and l ingual surfaces o f each tooth. 17 16 15 14 13 12 11 21 22 23 24 25 26 27 47 46 45 44 43 42 41 31 32 33 34 35 36 37 Gingival Overgrowth Index or GOI (McGaw et al): • 0 = N o overgrowth, feather-edged g ing iva l marg in • 1 = B l u n t i n g o f g ing iva l marg in • 2 = Modera te g ing iva l overgrowth, < 1/3 c r o w n length • 3 = > 1/3 c r o w n length 12 22 23 43 42 41 31 32 33 Oral Hygiene effectiveness (OH %): Absence o f dental plaque on four surfaces o f the Ramfjord teeth 92

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