Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction

Background and Objective: Platelet‐derived growth factor‐BB is a potent mediator of tooth‐supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet‐derived growth factor‐BB application is its short half‐life in vivo. Gene therapy has shown strong promise for the long‐term delivery of platelet‐derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet‐derived growth factor‐B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad‐PDGF‐B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell‐surface proteins associated with cellular signaling. Material and Methods: HGFs from human subjects were treated by adenoviral PDGF‐B, PDGF‐1308 (a dominant negative mutant of PDGF) and recombinant human platelet‐derived growth factor‐BB, and then incubated in serum‐free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF‐B was measured by RT‐PCR and Western blot. Cell proliferation was evaluated by [methyl‐³H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF‐B gene transfer. The expression of α and β PDGFR and Akt were measured by Western blot analysis. Results: Sustained in vitro expression of PDGF‐B in HGFs by Ad‐PDGF‐B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF‐BB and Ad‐PDGF‐1308, Ad‐PDGF‐B maintained cell growth in serum‐free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF‐B also prolonged downregulation of the growth arrest specific gene (gas) PDGFαR. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long‐term cell signaling effect as a result of the over‐expression of Ad‐PDGF‐B, compared with pulse recombinant human platelet‐derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad‐PDGF‐B, for at least up to 96 h. Conclusion: These findings further demonstrate that gene delivery of PDGF‐B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet‐derived growth factor‐BB protein. These data on platelet‐derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.     

Effects of epidermal growth factor and/or nerve growth factor on Malassez’s epithelial rest cells in vitro: expression of mRNA for osteopontin,bone morphogenetic protein 2 and vascular endothelial growth factor

Yamawaki K, Matsuzaka K, Kokubu E, Inoue T. Effects of epidermal growth factor and/or nerve growth factor on Malassez’s epithelial rest cells in vitro: expression of mRNA for osteopontin, bone morphogenetic protein 2 and vascular endothelial growth factor. J Periodont Res 2010; 45: 421–427. © 2010 John Wiley & Sons A/S Background and Objective: Malassez’s epithelial rest (MER) cells are involved in the maintenance and homeostasis of the periodontal ligament (PDL). The purpose of this study was to determine the effects of epidermal growth factor (EGF) and/or nerve growth factor (NGF) in vitro on these functions of MER cells. Material and Methods: MER cells from porcine PDL were incubated for 3 or 9 h after the addition of EGF and/or NGF to final concentrations of 10 ng/mL. Cells cultured without those growth factors were used as controls. The expression of mRNA for osteopontin, bone morphogenetic protein 2 (BMP‐2) and vascular endothelial growth factor (VEGF) was analyzed using quantitative RT‐PCR. Results: There was a decrease in the expression of osteopontin mRNA by MER cells treated for 9 h with NGF and the level of mRNA expressed was lower than that of the control and EGF‐treated groups. The expression of BMP‐2 mRNA by MER cells treated with NGF for 9 h also decreased, and was lower than that of the control and EGF‐treated groups. The expression of VEGF mRNA by MER cells treated with EGF for 3 or 9 h was higher than in the control and NGF‐treated groups. The expression of VEGF mRNA was lower in MER cells treated with NGF for 3 and 9 h than in the control and EGF‐treated groups, and decreased from 3 to 9 h of treatment. EGF stimulated MER cells to secrete VEGF, which suggests that EGF plays an important role in maintaining the homeostasis of the PDL. NGF acts on MER cells to inhibit calcification in the PDL. Furthermore, in the EGF+NGF‐treated MER cells, expression of mRNA for BMP‐2 and VEGF was similar to that of the NGF‐treated group, but cell proliferation and expression of osteopontin mRNA were similar to that of the EGF‐treated group. Conclusion: EGF and NGF play important roles in maintaining the PDL.     

Activation of human platelet-rich plasmas: effect on growth factors release, cell division and in vivo bone formation

OBJECTIVES: Aims of this controlled study were to determine the effects of activated human platelet-rich plasmas (PRPs) on early and mature bone formation in vivo, and to characterize the effect of PRP activation on growth factors release and endothelial cell division in vitro. MATERIAL AND METHODS: PRPs were prepared from four volunteers with the platelet concentrate collector system (PCCS) system and activated with three concentrations of calcium and thrombin. Platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and interleukin-1beta (IL-1beta) levels released in supernatants were measured by ELISA, at time 0, 1h, 24h and 6 days following PRP activation. Mitogenic potential of PRP supernatants were tested on endothelial cells in vitro, and the effects of activated human PRPs on bone formation in vivo were measured in athymic rats by micro-CT analyses. RESULTS: Activation of PRPs with calcium and thrombin triggered an immediate release of VEGF, PDGF-BB and TGF-beta and a delayed release of IL-1beta in PRP supernatants. Higher endothelial cell division was observed with supernatants from activated PRPs than from non-activated PRPs. Positive correlations were observed between VEGF levels and endothelial cell division and bone formation. A negative correlation was also found between PDGF-BB concentration and bone formation. However, early and mature bone formations with activated PRPs did not significantly differ from the ones obtained in the control group. CONCLUSIONS: Activation of PRPs with calcium and thrombin regulates growth factors release and endothelial cell division in vitro. However, activated PRPs does not improve the early or mature bone formations in vivo in this athymic rat model.     

Effects of HB-EGF and epiregulin on wound healing of gingival cells in vitro

Oral Diseases (2011) 17 , 785–793 Objective: Gingival wound healing is important to periodontal disease and surgery. This in vitro study was conducted to assess the manner in which heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and epiregulin cooperatively participate in the wound‐healing process in the gingival epithelial and fibroblast cells of the oral mucosa. Material and Methods: Gingival epithelium and fibroblast were separated from gingival tissue biopsies and prepared to primary cultures. The changes in the mRNA expression were evaluated via real‐time PCR. The effects on cell proliferation, migration, and repopulation were evaluated in vitro. Results: The different regulation of expressions of HB‐EGF, epiregulin, and epidermal growth factor receptors was observed over time and with different gingival cell types. HB‐EGF exerted a cell migration‐inducing effect on both epithelial and fibroblast cells, whereas epiregulin did not. Both growth factors functioned as mitogens for epithelial cell proliferation, but not for fibroblast proliferation. HB‐EGF strongly promoted epithelial cell repopulation and mildly promoted fibroblast repopulation, whereas epiregulin promoted only fibroblast repopulation. Conclusion: These results indicated that both growth factors might function importantly in the wound‐healing process of human gingival tissue via the different regulation of the expression, cell migration, proliferation, and repopulation.     

Effect of autologous and allogenic platelet-rich plasma on human gingival fibroblast function

Oral Diseases (2012) 18 , 494–500 Objective: Platelet‐rich plasma (PRP) has been proposed as a method of delivering growth factors to enhance regeneration. The aim of this study was to investigate the use of autogenous and allogenic PRP and platelet‐poor plasma (PPP) on migration and proliferation of human gingival fibroblasts in vitro. Methods: Various concentrations of PRP, as well as PPP, were prepared from autologous and allogenic sources and applied to primary gingival fibroblasts. Migration was determined by assessing the fibroblast response to a concentration gradient. ³H‐thymidine incorporation and crystal violet colorimetric assays were utilized to assess DNA synthesis and proliferation. Results: Platelet‐rich plasma provides a significant migratory stimulus to gingival fibroblasts. Furthermore, the various concentrations of PRP (50%, 20% and 10%) do not promote DNA synthesis in the short term (24 h), but over the longer term (5 days) they stimulate an increase in cell proliferation. Compared with PPP, PRP was superior in terms of encouraging migration, but was inferior in terms of promoting DNA synthesis and cell proliferation. No difference was noted between the autologous and allogenic PRP preparations on cell function. Conclusion: Both PPP and PRP promote gingival fibroblast migration and proliferation in vitro, without differences between preparations obtained from autologous and allogenic sources.     

富血小板血浆对人真皮细胞增殖及PDGF、TGF-β和VEGF表达的影响

目的:研究富血小板血浆(PRP)对人真皮成纤维细胞(hFbs)增殖、分化及胞质中PDGF、TGF-β和VEGF表达量的影响。方法:2次离心法制备PRP,成骨诱导条件下hFbs培养液中加入不同浓度PRP于第12天、21天钙-钴法染色,第21天茜素红染色;免疫细胞化学法检测加不同浓度PRP后第4天细胞PDGF、TGF-β和VEGF的表达;碱性磷酸酶染色(ALP)检测细胞活性;CCK-8法检测有和无成骨诱导条件下不同浓度PRP对细胞增殖的影响。结果:茜素红染色显示,2.8%PRP组钙结节形成最明显;钙-钴法染色显示,2.8%PRP组矿化作用最强;免疫细胞化学结果表明PDGF表达存在剂量依赖性,TGF-β和VEGF表达虽无剂量依赖性,但有适宜浓度的要求;ALP染色表明3%PRP组细胞增殖明显,活性最强。CCK-8检测表明各浓度PRP对hFbs均有促增殖作用,PRP加成骨诱导组比单纯PRP组表现出更明显的促增殖作用,其中4.8%PRP促增殖作用最强。结论:适宜浓度的PRP可促进hFbs增殖,但相关因子的表达存在适宜浓度的要求,PRP通过促进细胞增殖,增加相关因子表达促进创伤愈合。     

Platelet concentrates: effects of calcium and thrombin on endothelial cell proliferation and growth factor release

BACKGROUND: Clinical evidence suggests that platelet concentrate (PC) could have beneficial therapeutic effects on hard and soft tissue healing, due to the contents of growth factors (GFs) stored in the platelets. The objectives of this study were: 1) to determine the concentrations of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) released from PCs and whole blood (WB), before and after the addition of various concentrations of calcium and thrombin, and 2) to assess the physiological importance of the released GFs on angiogenesis. METHODS: WB and PCs were harvested and prepared from three healthy volunteers. Enzyme-linked immunosorbent assay tests, specific for PDGF-BB, TGF-beta1, VEGF, and bFGF, were performed on WB and PC supernatants, collected before and 30 minutes after the addition of various concentrations of calcium and thrombin. The supernatants were also added to human umbilical vein endothelial cell (HUVEC) cultures in order to measure their effects on endothelial cell proliferation. RESULTS: Growth factor concentrations detected in PC supernatants were significantly greater (280% to 800% increase) than concentrations present in WB supernatants. Calcium and thrombin induced immediate GF release from PCs in a dose-dependent fashion. Furthermore, PC supernatants led to greater HUVEC proliferation rates than WB supernatants. However, there was no correlation between the concentrations of specific GFs and HUVEC proliferation rates. CONCLUSION: These results suggest that PCs could stimulate blood vessel formation. They also reinforce the relevance for using PCs in regenerative therapies.     

Platelet‐Poor and Platelet‐Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells

Background: Plasma‐derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). Methods: The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme‐linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. Results: Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet‐derived growth factor (PDGF) isoforms (PDGF‐AA, ‐BB, and ‐AB), insulin‐like growth factor binding protein (IGFBP)‐2, and IGFBP‐6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. Conclusions: PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.     

Platelet‐Derived Growth Factor Promotes Periodontal Regeneration in Localized Osseous Defects: 36‐Month Extension Results From a Randomized,Controlled, Double‐Masked Clinical Trial

Background: Recombinant human platelet‐derived growth factor (rhPDGF) is safe and effective for the treatment of periodontal defects in short‐term studies up to 6 months in duration. We now provide results from a 36‐month extension study of a multicenter, randomized, controlled clinical trial evaluating the effect and long‐term stability of PDGF‐BB treatment in patients with localized severe periodontal osseous defects. Methods: A total of 135 participants were enrolled from six clinical centers for an extension trial. Eighty‐three individuals completed the study at 36 months and were included in the analysis. The study investigated the local application of β‐tricalcium phosphate scaffold matrix with or without two different dose levels of PDGF (0.3 or 1.0 mg/mL PDGF‐BB) in patients possessing one localized periodontal osseous defect. Composite analysis for clinical and radiographic evidence of treatment success was defined as percentage of cases with clinical attachment level (CAL) ≥2.7 mm and linear bone growth (LBG) ≥1.1 mm. Results: The participants exceeding this composite outcome benchmark in the 0.3 mg/mL rhPDGF‐BB group went from 62.2% at 12 months, 75.9% at 24 months, to 87.0% at 36 months compared with 39.5%, 48.3%, and 53.8%, respectively, in the scaffold control group at these same time points (P <0.05). Although there were no significant increases in CAL and LBG at 36 months among all groups, there were continued increases in CAL gain, LBG, and percentage bone fill over time, suggesting overall stability of the regenerative response. Conclusion: PDGF‐BB in a synthetic scaffold matrix promotes long‐term stable clinical and radiographic improvements as measured by composite outcomes for CAL gain and LBG for patients possessing localized periodontal defects ( ClinicalTrials.gov no. CT01530126).     

Analysis of a rapid, simple, and inexpensive technique used to obtain platelet-rich plasma for use in clinical practice

The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.     

Migration induced by epidermal and hepatocyte growth factors in oral squamous carcinoma cells in vitro: role of MEK/ERK, p38 and PI-3 kinase/Akt

J Oral Pathol Med (2012) 41 : 547–558 Background: Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti‐tumour signal inhibitors on the migratory activity. Methods: Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time‐lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting. Results: In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose‐dependent activation of cell migration. Addition of the EGFR‐specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF‐induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI‐3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF‐induced migration was particularly sensitive to PI‐3 K‐inhibition, while in C12 cells, both HGF‐ and EGF‐induced migration were highly sensitive to p38‐blockade. Conclusion: The results demonstrate that the MEK/ERK, p38 and PI‐3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.     

Comparison of platelet,leukocyte, and growth factor levels in point-of-care platelet-enriched plasma,prepared using a modified Curasan kit,with preparations received from a local blood bank

The potential use of autologous thrombocytic growth factors to accelerate bone regeneration requires improved methods of isolating platelet-rich plasma (PRP). In addition to discontinuous cell separation, a second method by which PRP is produced at the point-of-care has now become available. In this study, growth factor levels in PRP from these two sources were compared. Whole blood was drawn from 115 healthy donors (73 males, 42 females) aged 21 - 62 years (mean 36, SD 10). The PRP was separated by the blood bank (BB) using the discontinuous cell separation method or at the 'point-of-care' by the so-called 'buffy coat' method (analogous to the Curasan PRP Kit). Growth factor content differed significantly for TGF-beta1 (BB 268.65+/-70.77 ng/ml, Curasan 95.02+/-60.67 ng/ml (sign test P<0.001)) and PDGF-AB (BB 133.59+/-46.26 ng/ml, Curasan 233.70+/-111.86 ng/ml (P<0.001)), while the content of IGF-I (BB 85.37+/-25.58 ng/ml, Curasan 101.72+/-47.7 ng/ml (P<0.160)) showed no significant difference. The higher thrombocyte count in the BB PRP (BB 1434300+/-351960/ microl, Curasan 908.500+/-492.30/microl) seems to result in higher TGF-beta1 levels, while the higher leukocyte count in the Curasan PRP (BB 160+/-320/ microl, Curasan 30130+/-12500/microl) seems to result in higher PDGF-AB levels. The similar IGF-I levels in the two preparations might merely reflect similar amounts of plasma in the PRP produced by each approach.     

Platelet‐Derived Growth Factor‐BB Release Profile in Gingival Crevicular Fluid After Use of Marginal Periosteal Pedicle Graft as an Autogenous Guided Tissue Membrane to Treat Localized Intrabony Defects

Background: The aim of this study is to evaluate levels of platelet‐derived growth factor‐BB (PDGF‐BB) in gingival crevicular fluid (GCF) during the early stages of healing for sites treated by marginal periosteal pedicle (MPP) graft as an autogenous guided tissue membrane compared to that of the control open flap debridement (OFD). Methods: Fifteen non‐smoking patients (13 males and 2 females) with severe chronic periodontitis participated in this prospective, controlled, masked trial. Each subject contributed matched pairs of 2‐ or 3‐walled intrabony interproximal defects in premolar or molar teeth. Interproximal contralateral defects were randomly assigned to either the MPP group 1 or control OFD group 2. GCF samples were collected at 1, 3, 7, 14, and 30 days after surgery. PDGF‐BB in the GCF samples was measured using a human PDGF‐BB enzyme‐linked immunosorbent assay kit. Results: In both MPP and OFD, PDGF‐BB concentrations peaked in the samples obtained during the early postoperative days (days 2 and 3) and decreased sharply in the samples obtained 7, 14, and 30 days post‐surgery. Conclusion: Periosteal coverage of periodontal defects is not associated with a significant increase in PDGF‐BB levels.     

Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation

INTRODUCTION: Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. MATERIAL AND METHODS: Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. RESULTS: The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. CONCLUSION: Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.     

Platelet-rich plasmas: growth factor content and roles in wound healing

Platelet-rich plasmas (PRPs) are used in a variety of clinical applications, based on the premise that higher growth factor content should promote better healing. In this study, we have determined the effects of calcium and thrombin on the release of EGF, TGF-alpha, IGF-1, Ang-2 and IL-1beta from PRPs, and assessed the mitogenic potential of PRP supernatants on osteoblast and endothelial cell division. ELISA assays indicate that (i) mean growth factor concentrations vary from traces (TGF-alpha) to 5.5 ng/mL (IGF-1), (ii) there are significant variations in growth factor concentrations between individuals, and (iii) calcium and thrombin regulate growth factor release, synthesis, and/or degradation in stereotyped patterns that are specific to each growth factor. PRP supernatants promote strong osteoblast and endothelial cell divisions, supporting the concept that PRPs may be beneficial in wound healing. Abbreviations: PRPs, platelet-rich plasmas; GFs, growth factors; EGF, epidermal growth factor; TGF-alpha, transforming growth factor-alpha; IGF-1, insulin-like growth factor-1; Ang-2, angiopoietin-2; IL-1beta, interleukin-1 beta; HUVECs, human umbilical vein endothelial cells; hFOB 1.19, human fetal osteoblasts; and FBS, fetal bovine serum.     

Crevicular Fluid Growth Factors Release Profile Following the Use of Platelet‐Rich Fibrin and Plasma Rich Growth Factors in Treating Periodontal Intrabony Defects: A Randomized Clinical Trial

Background: The open, usually contaminated nature of periodontal defects could negatively affect availability and activity of platelet concentrate–suggested growth factors (GF). The aim of this study is to test this hypothesis and investigate concentrations of: 1) vascular endothelial growth factor (VEGF) and 2) platelet‐derived growth factor (PDGF‐BB) in gingival crevicular fluid (GCF) from localized intrabony defects treated with platelets rich in growth factors (PRGF) or platelet‐rich fibrin (PRF) compared with a control xenograft defect filling. Methods: Thirty non‐smoking patients suffering severe chronic periodontitis were allocated to this randomized, prospective, single‐masked trial. Each patient had one interproximal defect randomly distributed to: 1) group 1: bone‐substitute grafting control (n = 10); 2) group 2: experimental PRGF (n = 10); or 3) group 3: PRF (n = 10). Clinical parameters were measured at baseline and 6 and 9 months following therapy. GCF samples were obtained on days 1, 3, 7, 14, 21, and 30 after therapy for evaluation of VEGF and PDGF‐BB levels. Results: On days 1, 3, and 7 following surgery, mean levels of VEGF and PDGF‐BB at sites treated with PRGF and PRF were not significantly different versus the control. Levels of PDGF‐BB and VEGF were higher in the PRGF‐treated group, but differences were not significant. Growth factor levels decreased significantly in samples collected on days 14, 21, and 30 with non‐significant differences among the three groups. No significant clinical differences were reported among the three groups during the two observation periods (early period: days 1, 3, and 7; and later period: days 14, 21, and 30). Conclusions: Within the limits of the present study, it can be concluded that PRF and PRGF platelet concentrate failed to augment clinical effects achieved with the xenograft alone in treating intrabony defects. Periodontal defects could not retain extraphysiologic levels of GF suggested to be associated with platelet concentrate.     

Effect of platelet-derived growth factor-BB on root resorption after reimplantation of partially denuded tooth in dog

Abstract – The prognosis for a reimplanted tooth depends largely on the condition of the root. Platelet‐derived growth factor (PDGF)‐BB has been shown to regenerate periodontal tissue in animal and human clinical studies. However, information regarding the effect of PDGF‐BB on tooth reimplantation is limited. The objective of this study was to investigate the effect of PDGF‐BB on root resorption after reimplantation of a partially denuded tooth in dog. A total of 15 healthy female beagle dogs were used. Mandibular third and fourth premolars were endodontically treated and then extracted as atraumatically as possible. The coronal portion of each root was carefully scaled and planed. The roots on the right side of the mandible were treated with PDGF‐BB and reimplanted, while the roots on the left side served as controls. After 2, 4, or 8 weeks, specimens were collected and processed for histopathological examination. By the 4th week after reimplantation, new periodontal ligament (PDL)‐like tissue had formed around the PDGF‐BB‐treated root surfaces and new bone. By the 8th week, healing of the PDGF‐BB‐treated roots was characterized by newly formed PDL with inserting attachment formation. In contrast, control roots showed multiple areas of replacement resorption. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) performed at 2 weeks after reimplantation showed that the number of PCNA‐positive cells in the connective tissue area was statistically significantly greater in the PDGF‐BB‐treated group than in the control group (P < 0.001). The application of PDGF‐BB resulted in a significantly lower occurrence and extent of root resorption and ankylosis. These results suggest that the use of PDGF‐BB reduces occurrence of ankylosis and root resorption in tooth reimplantation.