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.     

Epidermal growth factor and bone morphogenetic proteins upregulate osteoblast proliferation and osteoblastic markers and inhibit bone nodule formation

Objective

The aim of this study was to investigate the in vitro osteogenic activity of EGF in association with bone morphogenetic proteins BMP2 and BMP7.

Methods

SaOS-2 (osteoblast-like cell line from human osteosarcoma) were cultured in the presence of EGF and BMPs for various culture periods to assess (a) cell proliferation by MTT assay, (b) Runx2, alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression using quantitative RT-PCR and ELISA, and (c) bone tissue mineralization using Alizarin Red staining.

Results

EGF alone was able to stimulate osteoblast growth in a time-dependent manner. When mixed with BMP2, BMP7, and their combination, EGF greatly promoted osteoblast growth, compared to the BMP- and EGF-stimulated cells, suggesting a possible synergistic effect between EGF and BMPs on osteoblast growth. Stimulation with EGF, EGF/BMP2, and EGF/BMP2/BMP7 for 7 days upregulated Runx2 mRNA expression by the osteoblasts. EGF downregulated ALP mRNA expression, which was recovered when the BMP2/BMP7 combination was added to the osteoblast culture. Tested on OC mRNA expression, EGF had no effect and inhibited the enhancing effect of BMP2 and BMP7 on osteocalcin expression. The bone mineralization assay showed that EGF reduced both the number and size of the bone nodules. This reducing effect was observable even in the presence of BMP2 and BMP7.

Conclusion

This study demonstrated that EGF may act in the early phase to promote osteoblast growth and specific marker expression rather than the late phase involving cell differentiation/mineralization.     

表皮生长因子对口腔鳞癌细胞系血管内皮生长因子和NF-κB活性的影响

目的：探讨表皮生长因子（Epidermal growth factor,EGF）和口腔鳞癌侵袭转移的关系。方法：采用ELISA和免疫组化法测量不同浓度EGF作用于口腔鳞癌TSCCa细胞系及颈淋巴转移癌GNM细胞系后血管内皮生长因子（vascular endothelial growth factor,VEGF）活性和阳性表达变化;同时采用凝胶电泳迁移实验（EMSA）检测不同浓度EGF下核转录因子-κB（nuclear factor kappa B,NF-κB）的活性变化。结果：不同浓度的EGF（0ng/mL,10ng/mL,40ng/mL,80ng/mL）作用于两种细胞系后,随着外源性EGF浓度的增加,GNM和TSCCa细胞中VEGF活性和阳性表达有增加,NF-κB的活化明显增强,VEGF和NF-κB的活性与EGF有剂量依赖关系,与对照组相比差异有显著性（P〈0.05）;而在相同的浓度内TSCCa细胞较GNM细胞VEGF和NF-κB的活性增加较明显,两者相比差异有显著性（P〈0.05）。结论：EGF可能是通过增加VEGF的活性和阳性表达,调节NF-κB的信号传导路径来促进OSCC细胞侵袭和转移的。     

血清饥饿和低氧对口腔鳞癌细胞系血管内皮生长因子表达的影响

目的：探讨血清饥饿和低氧对体外培养的口腔鳞癌细胞系血管内皮生长因子（VEGF）表达的调节。方法：采用半定量逆转录聚合酶链反应（RT—PCR）检测血清饥饿和缺氧条件下口腔鳞癌TSCCa细胞系和颈淋巴转移癌GNM细胞系中VEGF mRNA表达情况，同时用酶联免疫吸附实验（ELISA）的方法定量测定了2细胞系中VEGF的活性变化。结果：RT—PCR结果显示GNM细胞VEGFmRNA表达水平均较TSCCa细胞高（P〈0．05），血清饥饿和低氧处理时在TSCCa细胞和GNM细胞中VEGF mRNA均有明显的增加，以TSCCa细胞系中VEGF mRNA增加的尤为明显。低氧处理4h时，VEGF的活性便显著增加，8h时达到最高。GNM细胞中VEGF增加至2倍，而TSCCa细胞中VEGF增加至6倍。血清饥饿组在TSCCa细胞中VEGF的活性增加至4倍，明显高于GNM细胞中的2倍。结论：血清饥饿和低氧能显著上调口腔鳞癌细胞VEGF的表达和活性，可能在口腔鳞癌血管形成中起重要作用。     

口腔扁平苔藓组织中表皮生长因子及其受体表达变化的研究

目的探讨表皮生长因子(EGF)、表皮生长因子受体(EGFR)及两种原癌基因c-fos、c-jun,在口腔扁平苔藓中的基因表达、蛋白含量变化及其生物学意义。方法选择新鲜组织标本31例,包括糜烂型扁平苔藓10例、网状型扁平苔藓12例和正常口腔黏膜9例,使用逆转录聚合酶链反应技术(RT-PCR)检测EGF、EGFR、c-fos和c-jun基因在不同组织中的表达;同时使用免疫组化方法比较这4种蛋白在不同组织中的定位和表达量的变化。结果糜烂型扁平苔藓EGFR基因的mRNA和蛋白含量分别为(55.9±23.1)%、(71.1±10.1)%,均明显高于网状型扁平苔藓及正常口腔黏膜组织(P<0.05)。糜烂型扁平苔藓中的c-fos和c-jun基因表达呈相同的变化规律,两种基因的mRNA含量均显著高于正常口腔黏膜。糜烂型扁平苔藓的C-fos、C-jun蛋白的阳性细胞率分别是正常黏膜的1.3和1.5倍,蛋白含量明显高于正常口腔黏膜(P<0.01)。结论糜烂型扁平苔藓的癌变率可能大于网状型扁平苔藓,其中EGF及其受体引导的信号传导通路可能起着十分重要的作用。     

The effect of combined delivery of recombinant human bone morphogenetic protein-2 and recombinant human vascular endothelial growth factor 165 from biomimetic calcium-phosphate-coated implants on osseointegration

Objectives: The delivery of growth factors for enhanced osseointegration depends on the effectiveness of the carrier systems at the bone–implant interface. This study evaluated the effect of solo and dual delivery of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and recombinant human vascular endothelial growth factor (rhVEGF₁₆₅) from biomimetically octacalcium phosphate‐coated implants on osseointegration. Materials and methods: Biomimetic implants, bearing either a single growth factor (BMP or VEGF) or their combination (BMP+VEGF), were established, and compared with acid‐etched (AE, control) and biomimetic implants without growth factor (CAP). Implants were placed into frontal skulls of nine domestic pigs. The quality of osseointegration was evaluated using microradiographic and histomorphometric analysis of bone formation inside four defined bone chambers of the experimental implant at 1, 2 and 4 weeks. Results: Biomimetic implants, either with or without growth factor, showed enhanced bone volume density (BVD) values after 2 and 4 weeks. This enhancement was significant for the BMP and BMP+VEGF group compared with the control AE group after 2 weeks (P<0.05). All biomimetic calcium‐phosphate (Ca‐P) coatings exhibited significantly enhanced bone–implant contact (BIC) rates compared with the uncoated control surface after 2 weeks (P<0.05). However, the combined delivery of BMP‐2 and VEGF did not significantly enhance BIC at the final observation period. Conclusion: It was concluded that the combined delivery of BMP‐2 and VEGF enhances BVD around implants, but not BIC. Therefore, it may be assumed that changes in the surface characteristics should be considered when designing growth factor‐delivering surfaces. To cite this article:
Ramazanoglu M, Lutz R, Ergun C, von Wilmowsky C, Nkenke E, Schlegel K A. The effect of combined delivery of recombinant human bone morphogenetic protein‐2 and recombinant human vascular endothelial growth factor 165 from biomimetic calcium‐phosphatecoated implants on osseointegration.
Clin. Oral Impl. Res. xx , 2011; 000–000.
doi: 10.1111/j.1600‐0501.2010.02133.x     

Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery

Objective: To evaluate the synergistic effect of bone morphogenetic protein 2 (BMP‐2) and vascular endothelial growth factor (VEGF) on the repair of bone defects around dental implants. Material and methods: Five groups of scaffold were fabricated by a freeze‐drying method, including pure chitosan/collagen scaffold; scaffold loaded with adenoviruses expressing BMP‐2, adenoviruses expressing VEGF, both adenoviruses expressing BMP‐2 and VEGF, VEGF protein and adenovirus expressing BMP‐2. In vitro studies examined whether bone marrow stromal cells were responsive to these scaffolds over time. Bone formation capacity, bone‐to‐implant contact, as well as removal torque values were investigated in vivo. Differences between the various groups were statistically analyzed using the one‐way analysis of variance test. Results: The in vitro study revealed a burst and rapid release of VEGF with a sustained high‐level expression of BMP‐2 in scaffold combined with VEGF protein and adenoviruses expressing BMP‐2. Histomorphometry demonstrated that scaffolds expressing BMP‐2 enhanced more bone formation compared with other groups; VEGF alone is insufficient to promote bone formation. New bone formation in the bone defects around dental implants, bone‐to‐implant contact and mean peak removal torque showed statistically significant difference for the adenoviral vector encoding human bone morphogenetic protein 2 (Ad‐BMP‐2) and VEGF protein and adenovirus expressing BMP‐2 groups. Furthermore, scaffold combined with VEGF protein and Ad‐BMP‐2 represented the best outcomes in this model. Conclusions: A combination of BMP‐2 gene and VEGF protein could have a synergistic effect in promoting bone healing. To cite this article:
Luo T, Zhang W, Shi B, Cheng X, Zhang Y. Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery.
Clin. Oral Impl. Res. 23 , 2012 467–474.
doi: 10.1111/j.1600‐0501.2011.02164.x     

BMP9联合NGF对C3H10T1/2间充质干细胞骨向分化能力影响的实验研究

目的:探讨骨形态发生蛋白9(BMP9)与神经生长因子(NGF)联合处理对间充质干细胞(MSCs)骨向分化的影响.方法:以重组腺病毒法将BMP9导入C3H10T1/2细胞,构建MSCs骨向分化实验模型,设定分组为绿色荧光(GFP)对照组、NGF单独处理组、BMP9单独处理组以及BMP9+ NGF联合处理组,分别在处理3h、12 h、24 h、48 h、3d、7d后检测早期成骨分化标志物Ⅰ型胶原、Runt相关转录因子2(RUNX2)和碱性磷酸酶(ALP)在细胞中的表达.检测方法采用ALP染色、实时定量RT-PCR检测mRNA转录水平以及Western blot检测蛋白表达.结果:BMP9+ NGF组较各单独处理组ALP活性显著增加,Ⅰ型胶原、RUNX2的mRNA水平在BMP9+ NGF组的表达明显高于其他各单独处理组,且各组在3h即出现显著差异;RUNX2蛋白在各实验组均表达,BMP9+ NGF组表达最高.结论:BMP9联合NGF在成骨诱导间充质干细胞分化早期,协同促进其骨向分化.     

Fiber system degradation,and periostin and connective tissue growth factor level reduction,in the periodontal ligament of teeth in the absence of masticatory load

Choi JW, Arai C, Ishikawa M, Shimoda S, Nakamura Y. Fiber system degradation, and periostin and connective tissue growth factor level reduction, in the periodontal ligament of teeth in the absence of masticatory load. J Periodont Res 2011; 46: 513–521.© 2011 John Wiley & Sons A/S Background and Objective: The periodontal ligament (PDL), which is interposed between the alveolar bone and roots, supports teeth against mechanical stress. Periostin and connective tissue growth factor (CTGF) might play essential roles in maintaining PDL fiber integrity under mechanical stress. However, this relationship has not been studied at the protein and gene levels. Therefore, the aim of this study was to assess the PDL fiber system without masticatory load to determine the structural changes in the PDL in the absence of mechanical stress. Material and Methods: The study included 45 Wistar male rats (12 wk of age) whose upper‐right first molars were relieved from occlusion for 24 h, 72 h, 7 d or 21 d. The PDL was examined histologically, and changes in the gene and protein levels of periostin and CTGF were investigated. Results: The PDL space width was reduced significantly. Histologically, an initial reduction in the fiber number and thinning of PDL fibers were observed, followed by disarrangement of the PDL fibers and their attachments to the alveolar bone; finally, the PDL fibers lost their meshwork structure. Real‐time RT–PCR results revealed sharp down‐regulation of the periostin and CTGF mRNA levels at 24 and 72 h, respectively, which continued throughout the experiment. Immunohistochemical analysis revealed that periostin localized to both the cellular elements and the extracellular matrix, whereas CTGF localized only to the cellular elements. Periostin and CTGF immunoreactivities became very weak without masticatory load. Conclusion: In the absence of mechanical stress, the PDL fiber system undergoes degradation concomitantly with a reduction in the periostin and CTGF levels in the PDL.     

Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro

The periodontal ligament (PDL) contains precursor cells for osteoblasts and cementoblasts. It has been shown that epidermal growth factor (EGF) inhibits dexamethasone-induced differentiation and up-regulates EGF-receptor (EGF-R) expression, whereas EGF-R is down-regulated in the course of differentiation. Thus it was suggested that EGF and its receptors act as a negative regulator of osteoblastic differentiation in PDL cells. In order to investigate further this hypothesis, human PDL cells were now used to elucidate the role of EGF and EGF-R in their proliferation and differentiation under mechanical stress-loaded conditions in vitro, as the PDL regularly receives mechanical stress from occlusal forces. As a model of mechanical stress, a cyclic stretch of 9 or 18% elongation was applied to the cells with a Flexercell cell-strain unit system. Alkaline phosphatase activity and osteocalcin mRNA expression were significantly induced by loading cyclic stretch for more than 4 days, whereas stretch slightly inhibited cell proliferation. Visualization of the actin stress fibres of the cells by rhodamine phalloidin revealed that approx. 10% of the total number of cells had become aligned perpendicularly to the direction of the stretch. The effects of stretch on alkaline phosphatase activity and cell proliferation were totally abolished by the presence of 10 ng/ml EGF. Western blotting of EGF-R protein demonstrated that stretch-induced differentiation accompanied the decreased expression of EGF-R protein in the cells. However, the amount of tyrosine-phosphorylated EGF-R upon EGF stimulation was restored to the control level in stretched cells. These results suggest that the EGF/EGF-R system acts as a negative regulator of differentiation of PDL cells regardless of the type of differentiation stimuli. Also, interaction between mechanical stress and the EGF/EGF-R system may participate in the osteoblastic differentiation of PDL cells and thereby regulate the source of cementoblasts and osteoblasts.     

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.     

Doxycycline Counteracts Bone Morphogenic Protein 2–Induced Osteogenic Mediators

Background: Microbial colonization during wound healing may exaggerate the inflammatory response and could adversely affect the outcome of periodontal regeneration. Bone morphogenic proteins (BMPs) directly augment bone regeneration. Interestingly, inhibitors of tissue collagenases, such as sub‐antimicrobial–dose doxycycline, also indirectly promote hard‐tissue regeneration. In this study, it is hypothesized that BMP2‐mediated bone regeneration would be positively affected by simultaneous treatment of sub‐antimicrobial–dose doxycycline. Methods: Human periodontal ligament (PDL) cells were stimulated with: 1) 10 ng/mL BMP2; 2) 1 μg/mL doxycycline; or 3) a combination of the two. The expressions of alkaline phosphatase, osteocalcin, osteonectin, and osteopontin were analyzed along with in vitro mineralized nodule formation and calcium accumulation. Results: BMP2 was a potent inducer of osteocalcin/osteopontin (statistically significant at P <0.01) and osteonectin in PDL cells relative to stimulation with doxycycline. However, doxycycline relative to BMP2 (statistically significant at P <0.001) upregulated the expression of alkaline phosphatase and in vitro mineralized nodule formation. Contrary to expected results, combined BMP2 and doxycycline induced a statistically significant (P <0.001) downregulation of alkaline phosphatase, osteocalcin, osteonectin/osteopontin, and in vitro mineralized nodule formation compared to stimulation with either BMP2 or doxycycline alone. Conclusions: Combined treatment of BMP2 and doxycycline in PDL cells counteracts the osteogenic mediators. Molecular interaction of growth factors should be explored before using a combination of these biologic molecules. It is important and clinically relevant to determine whether tetracycline and its other derivatives also counteract BMP functions. Animal models should be used to confirm these in vitro results.