950 D3000 new imprint Word template Vol 13, No 1 (2025) ISSN 2167-8677 (online) DOI 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu Expression of TGFB3 in Pyogenic Granuloma Asmaa Ali Hussein College of Medicine, University of Fallujah, Anbar, Iraq Abstract Objec0ve: The aim of the study was to determine the expression of TGFB3 in pyogenic gran- uloma. Material and Methods: Pathologist used a double-headed light microscope to calcu- late cutoff values for all the anTbodies employed in the invesTgaTon. Stained slides were thoroughly washed for 4 minutes with Gill's Hematoxylin SoluTon, dehydrated, and mounted using DPX mounTng soluTon. Results: Transforming growth factor beta-3 protein expression was measured using immunohistochemistry. In pyogenic granuloma Tssues expression, neg- aTve scores (0) were found in 4.5 of 16 samples (18.1%), whereas posiTve values (+) were found in 10 of 16 samples (33.06%). Conclusion: TGFB3 is expressed in most pyogenic gran- ulomas. Open Access Cita%on: Hussein AA. (2025) Expression of TGFB3 in Pyo- genic Granuloma. Den%stry 3000. 1:a001 doi:10.5195/d3000.2025.950 Received: May 25, 2025 Accepted: June 27, 2025 Published: August 13, 2025 Copyright: ©2025 Hussein AA. This is an open access ar- %cle licensed under a Crea%ve Commons ATribu%on Work 4.0 United States License. Email: mmss2006@uofallujah.edu.iq Introduc)on Pyogenic granuloma is a benign tumor that grows in the mouth or skin tissues. Because of the vascular effects of female hormones, it mostly affects young girls in their second decade of life. Proliferation of granulation tissue with inflammatory infiltration and high angiogenic potential is the most preva- lent form of hyperplasia in the mouth. This uncommon disease necessitates accurate di- agnosis and treatment, particularly during pregnancy [1,2]. Pyogenic granuloma is a le- sion that responds to a variety of low-level stimuli, including recurrent trauma, aggres- sions, hormonal variables, and some medica- tions. High levels of estrogen and progester- one during pregnancy are linked to a higher occurrence of this lesion. Lesions are more prevalent in the upper jaw, anterior regions, and gingiva's vestibular zone [3,4]. The gums are the most common site (75% of cases), although it can also occur on the lips, tongue, oral mucosa, and palate. In diverse cells and tissues throughout the body, the TGF-beta superfamily of proteins has a vari- ety of often conflicting actions. TGF-3 (trans- forming growth factor beta-3) is a protein produced by the TGFB3 gene. This protein may be found across the body and is neces- sary for development both before and after birth. To fulfill its responsibilities, it con- nects (binds) to cell surface receptor pro- teins. This binding initiates signal transmis- sion inside the cell, controlling a variety of cellular functions [5,6]. TGF-beta-3 is a protein that controls cell dif- ferentiation and embryogenesis. Different stages are involved in maturation into ma- ture form: Latency-associated peptide (LAP) and Transforming growth factor beta-3 (TGF -beta) chains remain non-covalently con- nected after cleavage of the proprotein in the Golgi apparatus [7,8]. The effects of TGF- on bone resorption vary depending on the tar- get cell and the settings of the experiment. TGF stimulates the replication of osteo- blastic cells and bone production in mice, but it does not promote osteoblastosis [9,10]. The primary objective of this research was to investigate the expression levels of trans- forming growth factor (TGF) protein in pyo- genic granuloma, aiming to better under- stand its potential role in the lesion's patho- genesis. Material and Methods After a Yirst wash with washing buffer, all the slides were coated with Peroxidase-Blocking Reagent and incubated for 10 minutes. After allowing the slides to cool for 20 minutes at room temperature, the margins around the samples were marked with a liquid blocker pap pen to prevent the contents from leaking out during the IHC run. The ideal primary antibody dilution (the di- lution of each Ab and the volume were calcu- lated and adjusted earlier) was applied to the slides, incubated for 60 minutes, and then thoroughly washed with washing buffers. Negative controls included incubations with- out the particular antibodies [11,12]. The stained slides were thoroughly washed for 4 minutes with Gill's Hematoxylin Solution, dehydrated, and mounted using DPX mount- ing solution. The stained slides were kept in the fume hood for at least one hour to dry at room temperature [13,14]. A pathologist uti- lized a double-headed light microscope to calculate cutoff values for all the antibodies employed in the investigation. Expression of TGFB3 in Pyogenic Granuloma Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu 2 Overexpression of TGFB3 was deYined as positive nuclei staining in at least 10% of the cells, whereas normal expression was de- Yined as fewer than 10% positive cells. Stain- ing was carried out according to the instruc- tions of the Dako detection system, with the following software and protocol: "pouring the solutions (on the slide for each location 100 l maximum 300 l per slide)". The slides were then de parafYin zed by run- ning them through xylene (twice) and graded dilutions of alcohol (100 percent twice, 95 percent, 70 percent, and 50 per- cent) before incubating them in distilled wa- ter for 10 minutes. The slides were incubated for demasking (Antigen Retrieval). Solution for retrieval, Citrate Buffer; pH9 for (TGFB3) in a water bath for 60 minutes at 97°C. Fol- lowing many experiments utilizing various incubation durations (20, 30, 45, 60, and 90 minutes) for Ag recovery of samples, the 60- minute optimum incubation time was estab- lished. The slides were allowed to cool for 20 minutes at room temperature before the liq- uid blocker pap pen was used to mark the edges around the samples to prevent the contents from leaking out during the IHC run. After a Yirst wash with washing buffer, all slides were coated with Peroxidase- Blocking Reagent and incubated for 10 minutes. Because some cells or tissues have endogenous peroxidase, it includes H2O2, which is a peroxidase substrate. The washing buffer was used to clean the slides for 30 minutes, blocking reagent (Ab diluent) was administered to block the excess site of an- other nonspeciYic protein and reduce back- ground generation. Washing buffer was used on the slides. The optimal dilution of primary antibody was applied to the slides (the dilu- tion of each Ab and the volume were previ- ously calculated and adjusted), then force- fully rinsed with washing buffer after incu- bation for 60 minutes. Negative controls were incubations that did not include antibodies previously adjusted to minimize the creation of a strong back- ground in the dyed slides that would inter- fere with the results) and rinsed two times thoroughly. The slides were rinsed thor- oughly with water after being counter- stained with Gill's Hematoxylin Solution for 4 minutes. The slides were dehydrated before being mounted using DPX mounting media. For at least one hour in the fume hood, the stained slides were permitted to cure at room tem- perature [15,16]. With the help of a trained pathologist and a double-headed light micro- scope, the stained slides were scored to- gether. With the aid of a pathologist, cut off values for all the antibodies employed in the study were determined. Cut-off values for the DAKO procedure are as follows: According to Sophia et al., TGFB3 scoring was utilized. TGFB3 oveexpression was de- Yined as positive nuclei staining in at least 10% of the cell nuclei overexpression, Nor- mal expression was deYined as those with fewer than 10% positive cell nuclei. (Nega- tive, score 0; weak or mild staining (5–10% score 1); moderate staining (10–25% score 2); severe staining (25–50% score 3) and ex- tremely strong staining (25–50% score 4) (over 50 percent score 4). Statistical Analysis As a cutoff criterion, we utilized the median IRS=4 score. Patients with higher IRS scores (>4) were categorized as having strong TGFB3 expression. p=0.05 was used as the statistical signiYicance cutoff. To examine the pT and Clark level variables, Cochran– Armitage statistics were used. Results In this work, immunohistochemistry meth- ods were used to investigate the protein Transforming growth factor beta-3, which is generated by the human TGFB3 gene, in pa- tients from Iraq who developed pyogenic granulomas. Negative results (-0) were seen in 4.5 of 16 samples (18.1%) when TGFB3 expression in pyogenic granuloma tissues was conYirmed by immunohistochemistry. Ten of the 16 samples, or 33.06 percent, re- ceived positive ratings (+), one out of 16, or 2 percent, had positive ratings (++), and one out of 16, or 4.5 percent, had positive ratings (+++). The control had lesions (1) and a Neg- ative score (0), as shown in Table. The ex- pression of TGFB3 in tissues from pyogenic granulomas was markedly different from control samples (P = 0.001) in comparison. Figure 1 displays the expression of TGFB3 with the nucleus stained with Brown to demonstrate positive TGFB3 expression and without nucleus staining to show negative TGFB3 expression. Figure 2 displays the im- munohistochemical staining of TGFB3 in py- ogenic granuloma slices with counterstained blue hematoxyline and brown peroxi- dase/DAB (400X) (400X). Discussion A driving force behind growth Among other ways, beta-3 can stop development, cause cell death, alter the cell matrix on the out- side, and lessen the body's reaction to sick- ness [17]. TGFB3 has also been demon- strated to enhance the production of endo- thelial growth factors (TGF-I), as well as hav- ing a variety of additional anti-swelling properties. TGFB3, a novel host factor that can't be weakened by infection, is another essential cytokine, organize tissue development, proliferation, and growth (us- ing different expressions). When individuals with pyogenic granuloma are followed with obvious health values, TGFB3 is commonly accessible [18]. Patients with erosive form had substantially greater TGFB3 tissue expression levels than those with retinal form. Regardless, there is no re- current disparity in numbers, suggesting that TGFB3 levels differ signiYicantly be- tween patients with pyogenic granuloma and healthy controls. Another study found a strong link between TGFB3 levels and vari- ous kinds of pyogenic granuloma, as well as an increase in saliva levels for patients with TGFB3 in pyogenic granuloma. In compari- son to the retinal form, the corrosive and de- structive forms included more TGFB3 [19]. In addition, 8 of 11 individuals with pyogenic granuloma had an unaffected response to TGFB3. Despite this, pyogenic granuloma was shown to be negative in recent tissue samples from healthy people; also, the levels of TGFB3 and different types of pyogenic granuloma were shown to have a signiYicant relationship. The reticular type had lower TGFB3 levels than the atrophic and erosive forms. In addition, in 8 out of 11 individuals with pyogenic granuloma, an unable to harm response was shown against TGFB3 [20]. Conclusions According to the results of the study, TGFB3 expression is high in pyogenic granuloma. Conflict of Interested Statement None to declare. References 1. Tobouti PL, Olegário I, de Sousa SC. Benign vascular lesions of the lips:Diagnostic approach. J Cutan Pathol. 2017;44(5):451–5. 2. daSilva FC, Piazzetta CM, Torres-Pereira CC, Schussel JL, Amenábar JM. Gingival proliferative lesions in children and adolescents in Brazil:A 15- year-period cross-sectional study. J Indian Soc Per- iodontol. 2016;20(1):63–6. 3. Wollina U. Subungual telangiectatic granu- loma. Dtsch Arztebl Int. 2017;114(8):136. 4. Silva de Araujo Figueiredo C, Gonçalves Car- valho Rosalem C, Costa Cantanhede AL, Abreu Fonseca Thomaz ÊB, Fontoura Nogueira da Cruz MC. Systemic alterations and their oral manifesta- tions in pregnant women. J Obstet Gynaecol Res. 2017;43(1):16–22. 5. Abreu-Dos-Santos F, Câmara S, Reis F, Freitas T, Gaspar H, Cordeiro M. 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Misawa S, Sakamoto H, Kurogochi A, Kirii Y, Nakamura S, Misawa T, Yoneda S, Hirano M, Owa O, Takagi H, Ota H. Rare cause of severe anemia due to pyogenic granuloma in the jejunum. BMC Gastroenterol. 2015;15:126. 11. Matsuzaki K, Imamura Y, Ozawa M, Nakajima T, Ikeda A, Konishi T, Jikuya T. Intrave- nous lobular capillary hemangioma in the subcla- vian vein. Ann Thorac Surg. 2016;102(5):e427–9. 12. Brightman LA, Geronemus RG, Reddy KK. Laser treatment of port-wine stains. Clin Cosmet Investig Dermatol. 2015;8:27–33. 13. Wollina U, Zielinski M, Knopf B, Hipler C. [Eruptive capillary hemangioma following argon laser therapy of naevus nlammeus] Hau- tarzt. 1989;40(4):212–4. 14. Rana R, Ramachandra SS, Prasad UC, Ag- garwal P, Dayakara JK. Recurrent pyogenic granu- loma with a satellite lesion. Cu- tis. 2015;96(2):E27–30. 15. Gupta V, Mridha AR, Sharma VK. Pediatric Dermatology Photoquiz:Multiple erythematous papules on the back. Recurrent pyogenic granulo- mas with satellitosis. Pediatr Derma- tol. 2016;33(1):97–8. 16. Finnson KW, McLean S, Di Guglielmo GM, Philip A. Dynamics of transforming growth factor beta signaling in wound healing and scarring. Adv Skin Wound Care. 2013;2(5):195–214. 17. Penn JW, Grobbelaar AO, Rolfe KJ. The role of the TGF-β family in wound healing, burns and scarring: a review. Int J Burns Trauma. 2012;2(1):18–28. 18. Fleisch MC, Maxwell CA, Barcellos-Hoff MH. The pleiotropic roles of transforming growth fac- tor beta in homeostasis and carcinogenesis of endocrine organs. Endocr Relat Cancer. 2006;13(2):379–400. 19. Devescovi V, Leonardi E, Ciapetti G, Cenni E. Growth factors in bone repair. Chir Organi Mov. 2008;92(3):161–8. 20. Lehnert SA, Akhurst RJ. Embryonic expres- sion pattern of TGF beta type-1 RNA suggests both paracrine and autocrine mechanisms of action. De- velopment. 1988;104(2):263–73. Table 1. TGFB3 expression in pyogenic granuloma was investigated using immunohistochemistry. Score group Negative scored + ++ +++ Pyogenic granuloma 4.5 (18.1%) A 10(33.06%) B 1 (2%) B 1 (4.5%) C Control Negative scored (0) *** P <0.005 Figure 1. Immunohistochemical staining of TGFB3 in pyogenic granuloma sections with peroxidase/DAB (brown) counterstained with blue hematoxyline (400X). Expression of TGFB3 in Pyogenic Granuloma Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu 4 Figure 2. Immunohistochemical staining of TGFB3 in pyogenic granuloma slices with peroxidase/DAB (brown) and blue hematoxylin coun- terstained (400X).