Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitro

Bertrand‐Duchesne M‐P, Grenier D, Gagnon G. Epidermal growth factor released from platelet‐rich plasma promotes endothelial cell proliferation in vitro. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01205.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: The therapeutic benefits of platelet‐rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. Material and Methods: The levels of vascular endothelial growth factor (VEGF), platelet‐derived growth factor BB (PDGF‐BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium‐ and thrombin‐activated PRP samples from five donors were quantified by enzyme‐linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody‐coated beads on HUVEC proliferation was also tested. Results: Average concentrations of VEGF, PDGF‐BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody‐coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose‐dependent manner. Conclusion: This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.     

Acceleration of rat salivary gland tissue repair by basic fibroblast growth factor

A model of atrophic rat submandibular gland was used to examine the ability of basic fibroblast growth factor (bFGF) to accelerate tissue repair. The gland duct was separated carefully from associated blood vessels and nerve, and ligated with a 8-0 suture under a surgical microscope. Two weeks after ligation, the glandular tissue showed severe atrophy and weight loss (to 26% of that in a sham-operated group). Thereafter, the ligature was removed and various amounts of bFGF, isoproterenol or saline were instilled retrogradely through the duct. Both isoproterenol and bFGF increased cell proliferation significantly. bFGF accelerated the proliferation of various cell types, including both acinar and ductal. The proliferative effects of bFGF peaked at a dose of 1 ng/gland. When bFGF (1 ng/gland) was administered to the atrophic gland, its weight increased to 125% of the glands in saline-treated control animals after 2 weeks. The effects of bFGF were also examined in normal submandibular glands: bFGF stimulated cell proliferation, but the effective concentration was at least 50 times higher than that required in the atrophic gland. The results from immunohistochemical tests against anti-FGF receptor-type 1 antibody demonstrated increased immunoreactivity in the damaged gland, which might be involved in the difference in the response to bFGF between damaged and normal glands. Overall, the results indicate that bFGF can accelerate tissue repair in salivary gland.     

Accelerating effects of basic fibroblast growth factor on wound healing of rat palatal mucosa.

PURPOSE: In this study, basic fibroblast growth factor (bFGF) was examined for its ability to accelerate tissue repair in a rat oral mucosal wound. MATERIALS AND METHODS: A 4-mm mucosal defect was surgically made to the depth of the periosteum in a rat palate. bFGF was injected along the edge of the mucosal defect immediately after surgery. A control group received only phosphate-buffered saline vehicle. RESULTS: bFGF significantly accelerated granular tissue formation and reepithelialization. From the histologic analysis, the bFGF-treated group showed relatively faster collagen maturation. Starting 3 days after surgery, fibroblast growth factor receptor 1 (FGFR1)-positive cells appeared in the granular and spinous cell layers of the reepithelializing mucosa in the bFGF-treated group, whereas almost none was observed in the intact oral mucosa. By day 5, FGFR1-positive cells were seen below the stratum corneum, even in the control group. However, the number and intensity of FGFR1-positive cells in the bFGF-treated group were greater than in the control group. Results of immunostaining against proliferating cell nuclear antigen showed that bFGF stimulated cell proliferation of the basal cell layer in the regenerating epithelium. At a higher dose of bFGF, proliferating cell nuclear antigen-positive cells were also observed in the submucosal connective tissue. CONCLUSION: By the induction of its ligand protein concomitant with direct effects such as increased granular tissue formation and reepithelialization, a single topical application of bFGF facilitated wound healing in rat oral mucosa. The results of this study support the consideration for bFGF application for patients with impaired healing of oral mucosal injury.     

Heparan sulfate interacting protein (HIP/L29) negatively regulates growth responses to basic fibroblast growth factor in gingival fibroblasts

Basic fibroblast growth factor (bFGF) modulates gingival growth, and its release from heparan sulfate (HS) in the extracellular matrix (ECM) governs local tissue bioavailability. We identified a heparin/HS interacting protein (HIP/L29) that recognizes specific HS sequences. We hypothesize that HIP/L29, by modulating the interactions of bFGF with HS chains on proteoglycans, could regulate bFGF bioavailability. To investigate interactions between bFGF and HIP/L29, we isolated and cultured fibroblasts from normal gingiva and overgrown gingiva from patients on cyclosporine (CSA). bFGF significantly stimulated gingival fibroblast proliferation with or without heparin. Recombinant human HIP/L29 dramatically decreased bFGF-induced proliferation, but did not alter responses to insulin-like growth factor-1 (IGF-1). Analysis of mitogen-activated protein kinase (MAPK) phosphorylation patterns showed that bFGF stimulation of p44 (Erk-1), but not p42 (Erk-2), also was inhibited by HIP/L29 in a dose-dependent manner. Together, these results support our hypothesis that HIP/L29 modulates the bioavailability and action of bFGF.     

Epidermal growth factor in the submandibular salivary glands of congenitally athymic mice.

The submandibular salivary glands of a group of congenitally athymic (“nude”) mice were assayed for their epidermal growth factor (EGF) content and their histology was examined by light microscopy. The ability of the submandibular salivary glands from athymic mice to respond to an androgenic agent was assessed. The histology of the submandibular salivary glands resembled that of normal mice. Athymic mice had concentrations of EGF in their submandibular salivary glands which were similar to those reported previously for normal mice As in normal mice, male athymic mice had more prominent granular convoluted tubules than female mice, but, as in normal mice, testosterone treatment of female athymic mice resulted in an increase in both the EGF content and in the number of granules within the cells of the granular tubules of the duct system of the submandibular salivary gland.These results provide no evidence for a relationship between the level of submandibular salivary gland EGF and the immunological deficiency of nude mice, and show that the abnormalities in development of nude mice do not extend to their submandibular salivary glands.     

Epidermal growth factor concentration in hyperplastic and hypertrophic submandibular salivary glands of mice.

Submandibular salivary glands of mice contain high concentrations of epidermal growth factor (EGF). The EGF content of mouse submandibular salivary glands undergoing hyperplastic and hypertrophic changes was measured and compared to that in the glands of control mice. Salivary gland hyperplasia was induced by giving mice a single injection of isoprenaline and hypertrophy was produced either by repeated, daily injections of isoprenaline, repeated amputation of a lower incisor tooth or by removing the right submandibular salivary gland and thus producing a compensatory hypertrophy of the left submandibular gland. The EGF content of the hyperplastic submandibular salivary glands was not different from that of the control glands. While the EGF content of the hypertrophied glands resulting from either repeated isoprenaline injections or partial sialoadenectomy did not differ from that of the control glands, the concentration of EGF was significantly lower. This reduced concentration is probably a reflection of acinar hypertrophy with a resultant smaller proportionate contribution of the granular tubules to the mass of the gland. Incisor-amputation-induced hypertrophy did not result in a reduced concentration of EGF in the submandibular salivary glands, but the reason for the different response is unknown.The findings provide no evidence for the involvement of EGF in the induced changes of submandibular salivary gland hyperplasia or hypertrophy resulting from either isoprenaline treatment or partial sialoadenectomy. The reason for the higher concentration of EGF in hypertrophied submandibular salivary glands resulting from incisor amputation compared to that measured in hypertrophied glands resulting from the other stimuli used remains unresolved.     

bFGF及其受体FGFRS在成年大鼠唾液腺中的免疫组化定位和mRNA表达

目的 本文研究碱性成纤维细胞生长因子[basic fibroblast growth factor(bFGF)]和成纤维细胞生长因子受体1、2、3和4(FGFR1、FGFR2、FGFR3和FGFR4)在大鼠唾液腺中分布及表达特点。方法 应用免疫组化和RT-PCR的方法研究bFGF和FGFR1，FGFR2，FGFR3和FGFR4在成年大鼠腮腺和颌下腺中的免疫组化定位和mRNA表达。结果 bFGF和FGFR1，FGFR2，FGFR3和FGFR4在成年大鼠腮腺和颌下腺的各级腺管上皮细胞均有较强的特异性免疫染色。在成年大鼠腮腺和颌下腺可以检测到bFGF和FGFR1，FGFR2，FGFR3mRNA表达。结论 成年大鼠腮腺和颌下腺是合成、分泌bFGF的重要源泉。     

Dynamic distribution of basic fibroblast growth factor during epulis formation: an immunohistochemical study in an enhanced healing process of the gingiva

Basic fibroblast growth factor (bFGF) is thought to play an important role in wound healing. However, its histological localization, both in normal and pathological conditions in the oral mucosa, has not been well documented. We have studied the immunolocalization of bFGF in normal gingiva and gingivaf epulis specimens corresponding to different organizing stages. In normal gingiva. bFGF was detected in subpopulations of macrophages. mast cells and most endothelial cells in the lamina propna. Granulation tissue in epulides was histopathologically classified into six organizing stages. In stages 1 and 2. a small number of bFGF-positive macrophages was seen at the periphery of ulcer bases. In stages 3 and 4. histologically characterized by prominent capillary proliferation, large numbers of bFGF-positive macrophages and mast celis were located within granulation tissue. A positive reaction for bFGF was also found in some endothelia! cells and in myxoedematous stroma that was rich in heparan sulfate proteoglycan. In stages 5 and 6, when fibrosis was accelerated. bFGF-positive macrophages and mast cells decreased in number and were localized only at the periphery of the fibrous tissue. These findings suggest that maximum amounts of bFGF are synthesized and released from some macrophages and mast cells into the extracellular matrix during neovascularization of granulation tissue.     

Regeneration of periodontal tissues by basic fibroblast growth factor

Several growth factors (or cytokines) have recently received attention because of their ability to actively regulate various cellular functions of periodontal ligament (PDL) cells and the effects of topical application of such factor(s) on periodontal tissue regeneration has been evaluated. In this study, we examined the role of basic fibroblast growth factor (bFGF) in the wound healing and regeneration of periodontal tissues. Alveolar bone defects (such as 2-wall, 3-wall and furcation class II bone defects) were created surgically in beagle dogs and primates. Recombinant bFGF was topically applied to the artificial bony defects. Six or 8 wk after application, the periodontal regeneration was morphologically and histomorphometrically analyzed. In all sites where bFGF was applied, significant periodontal ligament formation with new cementum deposits and new bone formation was observed in amounts greater than in the control sites. We found it noteworthy that no instances of epithelial down growth, ankylosis or root resorption were observed in the bFGF sites. In vitro studies demonstrated that bFGF enhances the proliferative responses of human PDL cells, which express FGF receptor-1 and -2, but inhibits the induction of alkaline phosphatase activity and mineralized nodule formation by PDL cells. Interestingly, we observed that the mRNA level of laminin in PDL cells, which plays an important role in angiogenesis, was specifically upregulated by bFGF stimulation, but that of type I collagen was downregulated. The present study demonstrates that bFGF can be applied as one of the therapeutic modalities which actively induce periodontal tissue regeneration. The results of in vitro studies suggest that by suppressing the cytodifferentiation of PDL cells into mineralized tissue forming cells, bFGF may play important roles in wound healing by promoting angiogenesis and inducing the growth of immature PDL cells, and may in turn accelerate periodontal regeneration.     

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.     

神经生长因子及其受体在下颌骨骨折愈合中的表达及意义

目的:探讨神经生长因子(nerve growth factor,NGF)及其受体(p75和TrkA)在骨折愈合过程的表达及其对骨折愈合的影响。方法:将32只白兔随机分为8组(每组4只),4只无骨折作为健康对照组2,8只制作右下颌骨骨折模型,立即行钛板固定。术后实验组动物分别在1、3、5、7、14、21、28 d各取4只处死。切取下颌骨脱钙后制作骨组织切片,行HE染色、NGF及受体的免疫组化染色。结果:NGF在骨折愈合过程中表达阳性3,～5 d达到高峰,在未骨折组仅有轻度阳性表达。NGFR-TrkA在骨折愈合过程中表达强阳性7,d达到高峰,到21 d时软骨细胞强阳性。NGFR-p75在骨折愈合过程中表达阳性,5～7 d达到高峰,到21 d时仍为阳性表达。结论:在骨折愈合过程中NGF、p75和TrkA共同调节骨细胞活性,对骨的修复重建起到重要作用。     

Basic fibroblast growth factor up-regulates the expression of vascular endothelial growth factor during healing of allogeneic bone graft

Recently we reported that basic fibroblast growth factor (bFGF) improved the healing of allogeneic bone grafts. However, the mechanism of action of the bFGF was not known. Therefore, the present study was designed to identify the expression pattern of vascular endothelial growth factor (VEGF) in the presence of bFGF reconstituted in demineralized intramembranous bone matrix (DBM_IM) during the healing of allogeneic bone grafts. Eighteen critical size (15 mm × 10 mm) defects were created on rabbit mandibles bilaterally. Three groups of six defects each were grafted with allogeneic bone alone, allogeneic bone and DBM_IM, and allogeneic bone and bFGF reconstituted in DBM_IM. Three weeks later, the defects were retrieved for immunohistochemistry and in situ hybridization for VEGF. The percentage of positive staining area was quantified by using image analyzer. The increase (517%) in the expression of VEGF mRNA was accompanied by an increase (492%) of immunoreactive VEGF protein in allogeneic bone graft augmented by bFGF reconstituted in DBM_IM. A close correlation existed between levels of VEGF production and the amount of newly formed bone. The results show that bFGF reconstituted in DBM_IM markedly up-regulated the expression of VEGF in the grafted area. Basic FGF augments the healing of allogeneic bone grafts by enhancing vascularization through the up-regulation of VEGF.     

明胶海绵复合生长因子加速颌骨骨折愈合的实验研究

目的 探索明胶海绵复合生长因子缓释系统加速颌骨骨折愈合的作用和机制,为临床加速颌骨骨折愈合提供新的方法。方法 按每100 μg基因重组人骨形态发生蛋白（BMP）-2用1 mL重组牛碱性成纤维细胞生长因子（bFGF）液完全溶解后,取40 μL滴加到明胶海绵（0.5 cm×0.5 cm×1.0 cm）组织块中,冻干后制成bFGF/BMP/明胶海绵缓释系统。在12只新西兰大白兔两侧下颌制造线样骨折,左侧为对照组,只用钛板固定;右侧为实验组,钛板下放置bFGF/BMP/明胶海绵。术后2、4、12周行大体观察、X线检查、组织学检查。结果 术后2周,实验组较对照组在骨折断端处可见更多纤维组织长入;术后4周,实验组骨折间隙可见到纤维性骨痂,对照组可见纤维组织和血管长入;12周后实验组和对照组骨折均已完全愈合。结论 bFGF/BMP/明胶海绵能加速骨折愈合,提高骨折愈合效果。     

Effects of basic fibroblast growth factor on cultured rat and human submandibular salivary gland cells

Basic fibroblast growth factor (bFGF) is a strong mitogen for most mesoderm- and ectoderm-derived cells. Although bFGF exists in rat and human salivary glands, its physiological role in those glands is unknown. In this study, the effects of bFGF were investigated in monolayer culture of normal rat and human submandibular gland cells. Epithelial cells from rat and human submandibular glands were cultivated with the aid of 3T3 cells as a feeder layer. The effects of different concentrations of bFGF on the second passage of these cultured cells were examined. In both the rat and human cells, the percentage of bromodeoxyuridine (BrdU)-positive cells gradually increased up to 50 ng/ml, and then increased sharply at 100 ng/ml. However, at concentrations higher than 100 ng/ml, the percentages of BrdU-positive cells reached a plateau. In both rat and human cells, total cell numbers at 100 ng/ml bFGF were significantly higher than those of the control group from culture day 4. On the other hand, the morphology of the cultured cells showed no difference either with or without bFGF. These results indicate that a major effect of bFGF on salivary gland epithelial cells is to act as a mitogenic stimulus.     

Zoledronic acid decreases gene expression of vascular endothelial growth factor and basic fibroblast growth factor by human epithelial cells

Delayed wound healing in patients taking bisphosphonates could result from decreased expression of growth factors, which are directly related to cell proliferation and migration. In this study, we evaluated the gene expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by epithelial cells exposed to zoledronic acid 5 μmol for 48 h using real-time polymerase chain reaction. The gene expression of VEGF and bFGF by epithelial cells exposed to zoledronic acid decreased by 34% and 51%, respectively (p = 0.0001 and p = 0.0001). We conclude that zoledronic acid can decrease the expression of growth factors by epithelial cells.     

Histomorphometric characteristics and expression of epidermal growth factor and its receptor by epithelial cells of normal gingiva and hereditary gingival fibromatosis

OBJECTIVE: The objective of this study was to examine the histomorphometric features and evaluate the expression of epidermal growth factor (EGF) and transmembranic receptor (EGFr) and the proliferative potential of epithelial cells from normal and hereditary gingival fibromatosis (HGF) gingival tissues. BACKGROUND: EGF is a multifunctional cytokine with a variety of biological effects including stimulation of cell proliferation by binding to its specific EGFr. METHODS: Immunohistochemistry was performed to measure EGF and EGFr expression and the epithelial cell proliferation was determined by measuring proliferating cell nuclear antigen (PCNA). RESULTS: Histomorphometric evaluation indicated that in HGF the mean height of the epithelial papillae was higher compared to the normal gingiva (NG), whereas mean epithelial area and number of epithelial papillae were quite similar in both groups. The EGF and EGFr positive cells were observed in the basal, spinous and granular cell layers of both normal and HGF tissues, with a gradual reduction from the basal layer. Although the expressions of EGF and EGFr in the control group were significantly higher than those from HGF, in HGF the epithelial papilla tips showed increased number of proliferating cells and elevated expression of EGF and EGFr. There was a correlation between the proliferative potential of epithelial cells and the expression of EGF or EGFr only in the epithelial papilla tips of HGF gingiva. CONCLUSION: Our data suggest that EGF and EGFr in the oral epithelium of HGF gingiva may stimulate epithelial cell proliferation, with the resultant apical migration of the oral epithelium and formation of the slender deep epithelial papillae; however, without hyperplastic alterations.     

聚乳酸和骨生长因子复合植入块修复骨缺损的实验研究

目的 探讨碱性成纤维细胞生长因子对重组人骨形成蛋白２诱导骨形成的影响,评价多孔聚乳酸复合人工骨修复骨缺损的作用。方法 在５６例下颌骨缺损的兔模型中分别植入ＰＬＡ／ｒｈＢＭＰ－２／ｂＦＧＦ、ＰＬＡ＝ｒｈＢＭＰ－２、ＰＬＡ／ｂＦＧＦ、ＰＬＡ并设空白对照。植入后２、４、８周行Ｘ线摄片和组织学观察骨缺损区骨形成的情况。结果 ３种材料复合物较两赤道昨合物成骨更好,而后者又 单一的ＰＬＡ成骨好,所有含骨生长因     

表皮生长因子与碱性成纤维细胞生长因子对人牙髓干细胞分化的影响

目的探讨表皮生长因子(epidermal growth factor,EGF)和碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)碱性磷酸酶(alkaline phosphatase,ALP)活性的影响。方法确定EGF与bFGF最大效应浓度后,对细胞分组,根据EGF和bFGF单独或联合应用,分为4组:EGF组、bFGF组、EGF联合bFGF组、不加任何生长因子的对照组。作用1、3、5、7、14 d后,采用ALP活性检测法(酶动力法)检测hDPSCs细胞ALP活性。结果 1～14 d bFGF组ALP活性与对照组相比差异无统计学意义(P>0.05);在5、7、14 d,EGF组和EGF联合bFGF组ALP活性显著高于对照组(P<0.05);EGF联合bFGF组的ALP活性明显高于bFGF组(P<0.05),但EGF联合bFGF组ALP活性与EGF组相比差异无统计学意义(P>0.05)。结论 bFGF单独应用不能诱导hDPSCs分化,EGF在hDPSCs的分化中发挥作用,EGF和bFGF无明显协同作用。     

种植体周围骨愈合及骨缺损愈合中神经生长因子作用的实验研究

目的观察神经生长因子(NGF)在种植体周围骨愈合及骨缺损愈合中的表达,探讨NGF在骨愈合中的作用。方法选用29只SD大鼠,按观察时间不同随机分4组,每组大鼠随机选取4只,左侧后大腿股骨植入种植体设为种植组;另外3只左侧后大腿股骨备洞设为骨缺损组。右侧后大腿均无骨损伤,设为对照组。术后取股骨标本拍X线片,去种植体制作切片行HE染色、NGF免疫组化染色。剩余1只大鼠取颌下腺作为NGF染色阳性对照。结果种植体无松动,种植体-骨结合良好。免疫组化染色结果显示,种植组及骨缺损组在愈合过程中可见骨髓基质细胞、骨母细胞、骨细胞、成纤维细胞及成骨细胞有NGF阳性染色。对照组相同位置骨组织中,未见NGF阳性染色。统计学结果显示,1周时种植组和骨缺损组NGF阳性染色率的比较有统计学差异;2、3、4周时2组NGF阳性染色率比较无统计学差异。结论 NGF可能参与了种植体周围骨愈合及骨缺损愈合过程。在种植早期,植体周围的NGF分泌情况与骨缺损愈合不同,较晚时期二者相同。