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.     

The effects of length of surgery on healing of full and partial thickness flaps.

Partial and full thickness flaps which were were made in the facial gingiva of 10 dogs were reflected for 15 or 90 min. Specimens were examined histologically and biochemically from 3 to 28 days after initial surgery. The results indicate that flaps refelected for 15 min exhibited faster epithelial closure than those reflected for 90 min. Partial thickness flaps reflected for both 15 and 90 min showed less inflammation than full thickness flaps at 3 and 7 days post surgery and healed faster in respect to epithelial closure, mesenchymal healing, and bone regeneration. Biochemical analysis of the collagen concentration of the tissure flaps revealed similar collagen content in full and partial thickness flaps reflected for 15 min; however, the collagen concentration of partial thickness flaps opened for 90 min was higher than that in full thickness flaps.     

The effects of length of surgery on healing of full and partial thickness flaps

Partial and full thickness flaps which were made in the facial gingiva of 10 dogs were reflected for 15 or 90 min. Specimens were examined histologically and biochemically from 3 to 28 days after initial surgery, The results indicate that flaps reflected for 15 min exhibited faster epithelial closure than those reflected for 90 min. Partial thickness flaps reflected for both 15 and 90 min showed less inflammation than full thickness flaps at 3 and 7 days post surgery and healed faster in respect to epithelial closure, mesenchymal healing, and bone regeneration. Biochemical analysis of the collagen concentration of the tissue flaps revealed similar collagen content in full and partial thickness flaps reflected for 15 min; however, the collagen concentration of partial thickness flaps opened for 90 min was higher than that in full thickness flaps.     

Response of periradicular tissues to growth factors introduced into the surgical site in the root-end filling material

AIM: The objective of this study was to evaluate the healing of the periradicular tissues when exogenous growth factors were delivered to the respected root-end. The healing response was compared with that when Diaket was used as a control. METHODOLOGY: Non-surgical root canal treatment was performed on mandibular teeth in mongrel dogs. Surgical treatment followed and included root-end resection and root-end cavity preparation. Insulin-like growth factor in combination with platelet-derived growth factor, or fibroblast growth factor alone, were then placed in the root-end preparations on a polylactic acid carrier (Atrisorb) with or without the incorporation of the carrier tetracalcium phosphate. The healing was evaluated at 60 days with regard to presence of inflammatory response, bone regeneration, periodontal ligament formation and cementum formation. RESULTS: Osseous regeneration in the excisional would and periodontal formation were significantly greater when Diaket was used as the root-end filling material. Likewise, cementum deposition occurred significantly more frequently in the Diaket group (P < 0.05). The polylactic carrier Atrisorb remained in the surgical sites for the duration of the study. CONCLUSIONS: The use of specific growth factors, FGF and a combination of IGF/PDGF, delivered to the prepared root end in a collagen carrier did not initiate the desired periradicular tissue response of regeneration. Diaket, as used in this study, did stimulate a periradicular tissue response compatible with regeneration.     

Human deciduous teeth dental pulp cells with basic fibroblast growth factor enhance wound healing of skin defect

In this research, we examined the effect on wound healing applying basic fibroblast growth factor (b-FGF) that is approved for clinical use to enhance wound healing and human deciduous teeth dental pulp cells (hDPCs) in clinics, but that have been attracting attention as a novel stem cell source in recent years. Human deciduous teeth were harvested from healthy volunteers, and hDPCs were isolated. We used a nude mouse full-thickness skin defect model and evaluated wound healing by macroscopic view and histologic and histomorphometric analysis. The mice were randomly divided into 4 groups: phosphate-buffered saline-treated group (control group), b-FGF-treated group (b-FGF group), hDPC-treated group (hDPC group), and hDPC and b-FGF-treated group (hDPC/b-FGF group). Basic fibroblast growth factor and hDPC groups accelerated wound healing compared with the control group. There was no statistically significant difference in wound healing observed between the hDPC and b-FGF groups. The hDPC/b-FGF group demonstrated accelerated wound healing compared with other groups. At day 14, PKH26-positive cells were surrounded by human type I collagen in hDPC and hDPC/b-FGF groups in immunohistologic evaluation. Significantly increased collagen fibril areas in wound tissues were observed in b-FGF, hDPC, and hDPC/b-FGF groups as compared with the control group at days 7 and 14. Our results showed that the hDPC/b-FGF group significantly promotes wound healing compared with other groups. This study implies that deciduous teeth that are currently considered as medical spare parts might offer a unique stem cell resource for potential of new cell therapies for wound healing in combination with b-FGF.     

Controlled local application of basic fibroblast growth factor (FGF-2) accelerates the healing of GBR. An experimental study in beagle dogs

This animal study was performed to ascertain whether the regeneration of membrane-protected bone defects can be accelerated by the controlled application of basic fibroblast growth factor (FGF-2) using a new drug delivery system. Standardized alveolar bone defects were made surgically in 9 beagle dogs, and FGF-2 was administered using specially made collagen minipellets. A minipellet containing either 0.15 microgram FGF-2 (FGF) or 0 microgram FGF-2 (placebo) was placed in the defect or no minipellet was used (control), and bone regeneration was evaluated radiologically, histologically, and histometrically 8 weeks after the operation. Radiographs showed a surprisingly large radiopaque region in FGF sites compared with placebo or control sites. Histologically, mature bone filled the majority of the inner space of the membrane-protected defect in FGF sites. New bone formation was also seen in the control and the placebo sites, however, it filled less than half the area of the defect. Histometrically, the area of regenerated bone in FGF sites was significantly higher than in the other sites (P < 0.01). These results demonstrate that the controlled application of FGF-2 accelerates bone regeneration in membrane-protected bone defects in the canine model.     

碱性成纤维细胞生长因子/壳聚糖衍生物/胶原温敏型复合水凝胶促大鼠牙周组织再生的研究

目的 探讨不同种壳聚糖衍生物温敏型复合水凝胶作为支架材料应用于牙周组织工程的可行性。方法制备磺化壳聚糖（SCS）、磷酸化壳聚糖（PCS）及磷酸化磺化壳聚糖（PSCS） 3种具有不同生物学特性的壳聚糖衍生物,构建3种碱性成纤维细胞生长因子（bFGF）/壳聚糖衍生物/胶原温敏型复合水凝胶。选取20只雄性wistar大鼠,大鼠双侧上颌第一磨牙近中建立三壁骨袋,随机分为空白对照组、空白凝胶组、bFGF/SCS/胶原温敏型复合水凝胶组、bFGF/PCS/胶原温敏型复合水凝胶组、bFGF/PSCS/胶原温敏型复合水凝胶组,术后6周处死大鼠,采集标本,进行大体、苏木精-伊红染色、Masson染色观察。结果 术后6周,3种bFGF/壳聚糖衍生物/胶原温敏型复合水凝胶组与空白对照组在相对牙槽骨高度比值、相对上皮根向下移比及牙周组织再生分级计数等方面的差异均具有统计学意义（P<0.05）。结论 bFGF/壳聚糖衍生物/胶原温敏型复合水凝胶在牙周组织工程邻域具有良好的应用前景。     

Does root surface conditioning with citric acid delay healing?

Abstract Effects of topical citric acid application on tissue maturation was studied in standardized periodontal defects in 6 beagle dogs. Following elevation of facial mucoperiosteal flaps, fenestration defects, 3 mm in diameter, were made through the cortical bone and recessed 0.5 mm into the dentin of maxillary canines. 1 defect in each dog was conditioned with a saturated solution of citric acid for 3 min and then rinsed with saline. Control defects in contralateral teeth were treated with saline only for the same length of time. The defects were covered with an expanded polytetrafluoroethylene membrane and the flaps re-positioned and sutured. 14 days postsurgery, healing appeared more advanced along the defect walls and floor than in the center of the defect in all instances. Histometrically, citric acid-conditioned defects exhibited a higher density of collagen fibers along the defect walls and floor and adjacent to the barrier membrane as well as more advanced resolution of the residual blood clot than the surgical controls. Differences in fibroblast density within specimen pairs were non-significant. All control defects but none of the acid-conditioned defects showed an artifactual split between the dentin walls and the granulation tissue. This study failed to support the contention that topical application of citric acid to root surfaces may delay healing following periodontal surgery.     

Controlled release of fibroblast growth factor 2 stimulates bone healing in an animal model of diabetes mellitus

PURPOSE: Bone formation and the healing of calvarial defects in mice is diminished in chemically induced type 1 diabetes. The present study investigated whether controlled local release of fibroblast growth factor 2 (FGF-2) stimulates bone defect healing in this model of diabetes. MATERIALS AND METHODS: First, in vitro release kinetics of different doses of recombinant human FGF-2 (rhFGF-2) from polyglycolate:polylactide membranes was determined over a 14-day period by incubating loaded membranes in PBS with constant shaking. The amount of FGF-2 was measured by enzyme-linked immunosorbent assay. Then, the effects of rhFGF-2-loaded and control membranes on calvarial defect healing over a 14-day healing period were determined in diabetic and nondiabetic mice. The degree of healing was determined by histomorphometric analyses of bone area percentage and by area measurements. The significance of the data was determined by statistical analyses, including analysis of variance. RESULTS: Kinetic release data in vitro showed that membranes loaded with 5 microg FGF-2 released measurable levels of growth factor for more than 14 days. Data from the in vivo study supported the previous finding that diabetes inhibits bone formation. Membranes containing rhFGF-2 significantly (P < .05) stimulated bone formation in diabetic animals to near normal levels during the healing period. CONCLUSION: FGF-2-loaded membranes may be useful in further studies aimed at developing therapeutic strategies for correcting deficient bone healing in patients with diabetes.     

Guided tissue regeneration may modulate gene expression in periodontal intrabony defects: a human study

Background and Objective: Guided tissue regeneration has been shown to lead to periodontal regeneration; however, the mechanisms involved remain to be clarified. The present study was carried out to assess the expression of genes involved in the healing process of periodontal tissues in membrane‐protected vs. nonprotected intrabony defects in humans. Material and Methods: Thirty patients with deep intrabony defects (≥ 5 mm, two or three walls) around teeth that were scheduled for extraction were selected and randomly assigned to receive one of the following treatments: flap surgery alone (control group) or flap surgery plus guided tissue regeneration (expanded polytetrafluorethylene (e‐PTFE) membrane) (test group). Twenty‐one days later, the newly formed tissue was harvested and quantitatively assessed using the polymerase chain reaction assay for the expression of the following genes: alkaline phosphatase, receptor activator of nuclear factor‐κB ligand, osteoprotegerin, osteopontin, osteocalcin, bone sialoprotein, basic fibroblast growth factor, interleukin‐1, interleukin4, interleukin‐6, matrix metalloproteinase2 and matrix metalloproteinase9. Results: Data analysis demonstrated that mRNA levels for alkaline phosphatase, receptor activator of nuclear factor‐κB ligand, osteoprotegerin, osteopontin, bone sialoprotein, basic fibroblast growth factor, interleukin‐1, interleukin‐6, matrix metalloproteinase‐2 and matrix metalloproteinase ‐9 were higher in the sites where guided tissue regeneration was applied compared with the control sites (p < 0.05), whereas osteocalcin mRNA levels were lower (p < 0.05). No difference was observed in interleukin‐4 mRNA levels between control and test groups. Conclusion: Within the limits of this study, it can be concluded that genes are differentially expressed in membrane barrier‐led periodontal healing when compared with flap surgery alone, and this may account for the clinical outcome achieved by guided tissue regeneration.     

Effects of basic fibroblast growth factor administration on vascular changes in wound healing of rat palates.

OBJECTIVE: The purpose of this study was to investigate the vascular changes induced by mucoperiosteal denudation of rat palate and to elucidate the effects of basic fibroblast growth factor (bFGF) administration on the palatal vascular network in wound healing. METHODS: A total of 117 male Wistar rats were used for the study on their 20th postnatal day. The animals were divided into three groups: a scar formation group, a basic fibroblast growth factor group, and a control group. The scar formation and basic fibroblast growth factor groups had lateral mucoperiosteum excised from the palate. In the basic fibroblast growth factor group, a solution of basic fibroblast growth factor was injected into the operated area 1 week after excision. At 6, 8, and 10 weeks postoperatively, palatal vascular changes were investigated by immunohistochemical staining and corrosion cast techniques. RESULTS: Throughout the experimental period, there were significantly fewer vessels in the scar formation group than in the control and basic fibroblast growth factor groups. In the basic fibroblast growth factor group, the elongation of new vessels and capillary proliferation proceeded, and after 10 weeks a highly organized vascular network was established. The scar formation group showed few Volkmann's canals that were shrunken or closed, whereas the basic fibroblast growth factor group evidenced Volkmann's canals with arterioles or venules, as seen in the control. CONCLUSIONS: The results suggested that injection of basic fibroblast growth factor into palatal wounds improves the vascular supply to the operated mucosa and underlying bone during and after palatal wound healing, which may contribute to tissue remodeling of the palate during growth.     

Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs

Abstract The effect of a collagen gel matrix as a submembranous space-maintaining material was evaluated in guided tissue regeneration procedures. In 4 dogs, contralateral surgical circular fenestration defects, 5 mm in diameter, were produced at the midbuccal aspect of the alveolar bone in 8 maxillary canines. Removal of bone, PDL and cementum was complete. Experimental sites were filled with collagen gel and covered with collagen membranes; control sites were covered with collagen membranes and the underlying space was spontaneously filled with blood. Mucogingival flaps were repositioned. Histological and histomorphometric observations, 6 weeks post-surgery, indicated that defects covered by collagen membranes presented the most impressive regeneration with almost complete coverage of the denuded root by new cementum (98.4%) and new bone (63.2%). In the experimental defects. 83.5% coverage of new cementum with only 21.9% new bone regeneration was observed. These results suggest that collagen gel. interfered with healing by PDL and bone-derived cells in the submembranous space.     

纳米羟基磷灰石材料复合碱性成纤维细胞因子促进牙周组织再生的实验研究

目的:探讨新型纳米羟基磷灰石材料—纳米羟晶/胶原仿生骨材料(nHAC)作为生长因子载体和牙周组织工程支架材料应用的可行性,观察评价其对牙周组织再生修复的影响和意义。方法:将人牙周膜细胞(HPDLCs)接种于nHAC三维支架上复合培养,体外扫描电镜观察HPDLCs在nHAC支架上的附着、生长情况,并将碱性成纤维细胞生长因子(bFGF)与nHAC复合植入动物人工牙周组织缺损,表面覆盖聚四氟乙烯(e-PTFE)膜,以空白对照组和单纯置膜组作为对照。术后8周进行组织学观察和测量,分析评价其牙周组织的再生情况。结果:扫描电镜可见nHAC具有良好的多孔网状结构,PDLCs在nHAC支架上贴附紧密,生长旺盛,伸展充分,动物实验结果显示nHAC/bFGF/膜复合植入组较空白对照组和单纯置膜组有更多新生的牙槽骨、牙周膜和牙骨质生成,结果具有显著性差异。结论:nHAC具有良好的三维空间结构和细胞相容性,与bFGF复合植入牙周缺损后可显著促进牙周组织再生,提示nHAC有望成为理想的生长因子载体和牙周组织工程支架材料。     

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.     

碱性成纤维细胞生长因子对拔牙后牙槽骨骨密度的影响

研究碱性成纤维细胞生长因子（bFGF）对拔牙后牙槽骨愈合的影响。方法：采用随机双盲对照临床研究,双能X线吸收法测量实验组、对照组拔牙后即时、1、2、3和6月时牙槽骨骨密度的变化。结果：应用bFGF的实验组牙槽骨骨密度在1、2和3月时均明显高于对照组（P＜0．01或0．05）拔牙后即时和6月时牙槽骨骨密度两组无显著差异。结论:拔牙后局部应用成纤维细胞生长因子有良好的促进拔牙创骨愈合的作用。     

Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction

OBJECTIVE: To understand bone regeneration process after tooth extraction could be a clue to develop a new strategy for alveolar bone reconstruction. Recently, accumulated evidences support that connective tissue growth factor (CTGF) is implicated in tissue repair of many tissues. In this study, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets. DESIGN: Five weeks old wild type male rats (weighing 120 g) were used for this experiment. Expression of CTGF was determined by immunohistochemistry and in situ hybridization in the rat upper molar tooth extraction sockets at 2, 4, 7, 10 and 14 days after tooth extraction. RESULTS: CTGF was expressed strongly in the endothelial cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. During the reparative process, no apparent chondrocyte-like cell appeared in the sockets, while osteoblast-like cells proliferated in the sockets with low CTGF expression at 7, 10, 14 days after extraction. As expected, no staining was observed with the preimmune rabbit IgG and CTGF sense probe. CTGF may play an important role in angiogenesis and granulation tissue formation specifically at early healing stage after tooth extraction to initiate alveolar bone repair. CONCLUSION: CTGF was expressed at early healing stage of the rat tooth extraction wound.     

碱性成纤维细胞生长因子对大鼠下颌骨缺损Bio-oss植骨的影响

目的研究外源性碱性成纤维细胞生长因子对大鼠下颌骨缺损Bio-oss植骨愈合的影响。方法选用48只体重约450g的Wistar大鼠随机分为2组,实验组24只,对照组24只。在大鼠左侧下颌角处制备5mm×4mm×2mm的骨缺损区。实验组植入含有基因重组大鼠碱性成纤维细胞生长因子的Bio-oss颗粒。对照组植入含生理盐水的Bio-oss颗粒。于术后2、4、6、8周分四批处死大鼠,取其左侧下颌骨进行X线和组织学检查。结果术后各个阶段实验组骨愈合的速度均高于对照组。结论外源性碱性成纤维细胞生长因子与Bio-oss复合后对大鼠下颌骨缺损的修复效果明显优于单纯应用Bio-oss骨。     

Human buccal plate extraction socket regeneration with recombinant human platelet-derived growth factor BB or enamel matrix derivative

The objective of this study was to assess the osseous healing of buccal plate extraction socket defects. There were four cohorts: group A (mineral collagen bone substitute [MCBS] scaffold alone), group B (MCBS with recombinant human platelet-derived growth factor BB [rhPDGF-BB; 0.3 mg/mL]), group C (MCBS with enamel matrix derivative [EMD]), and group D (combination of EMD with bone ceramic). The primary outcome of bone quality was evaluated using light microscopy, backscatter scanning electron microscopy, and histomorphometrics. Reentry surgery provided an opportunity for clinical observation of the healed ridge morphology. Sixteen patients with buccal wall extraction socket defects were randomized into four treatment groups of equal size. Grafting was provided at the time of extraction with advancement of the buccal flap for primary closure. A trephine core biopsy of the implant site preparation was performed after 5 months for implant placement. Histologic examination identified new bone healing around the biomaterial scaffolds. Statistically significant differences in new bone formation were not observed among the treatment groups. There was a histomorphometric trend toward more new bone for the rhPDGF-BB-treated group (group B). This group had the most favorable ridge morphology for optimal implant placement.