Experimental studies of skin wound healing process by first intention in streptozotocin-induced diabetes mellitus rats

Various parameters were used in observing the process of wound healing in rats with streptozotocin-induced diabetes (STZ diabetes). Sections stained according to the Hematoxylin-Eosin, Van-Gieson, and Azan methods were used in observing histological changes. At the same time, wound strength during the healing was measured as a parameter for evaluating the healing process. In addition, changes in leukocytes, plasma fibrinogen, activated factor XIII (aXIII), collagen content of the incised wound, and metabolic changes were determined. Results 1. Histological studies showed that, in STZ diabetes, the inflammatory response was minimal and occurred later than in normal cases. In the incised wound, cellular infiltration of polymorpho-nuclear leukocytes and fibrin nets accumulated poorly. The fibrin net was coarse and fragile. Furthermore, epithelialization of the wound was late: it did not occur until 5 days after the operation. In cases of STZ diabetes, patterns of hyperplasia and fibroblast arrangements were abnormal. Collagen regeneration and proliferation processes were remarkably retarded. 2. In normal, wound strength increased from the 5 postoperative day. After 10 days had passed, it increased remarkably until, after 30 days, it had returned to the preoperative level. In STZ diabetes, however, no increase in wound strength occurred for the first 14 days after the operation. There after strength increased slowly; but, 40 days after the operation, 80% of the preoperative level still had not been reached. 3. Changes in leukocytes were much later occurring in STZ diabetes than in normal. Recovery took longer than in normal. 4. In STZ diabetes, increases in plasma fibrinogen and decreases of the aXIII factor were slower than in normal. The a XIII factor decreased remarkably, and recovery was slow. 5. In terms of collagen content in the wound incision, in STZ diabetes, tropocollagen increase occurred later than in normal. But, from the 5 to the 14 postoperative days, its level was higher than that in normal. Maturation-process collagen and mature collagen increased still more slowly. In normal, mature collagen had reached preoperative level 20 days after the operation, in STZ diabetes, 80% of preoperative level still had not been reached 30 days after the operation. 6. These studies showed that the following factors hinder wound healing in cases of diabetes mellitus: minimal inflammatory response, incomplete formation of the fibrin nets, retardation of epithelialization, retarded action of plasma fibrinogen and the a XIII factor, reduced fibroblast activity, and slow increase in collagen content.     

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.     

Enhanced production of mineralized nodules and collagenous proteins in vitro by calcium ascorbate supplemented with vitamin C metabolites.

BACKGROUND: Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate). METHODS: Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence. RESULTS: In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate. CONCLUSIONS: In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.     

Collagen membranes: a review

Collagen materials have been utilized in medicine and dentistry because of their proven biocompatability and capability of promoting wound healing. For guided tissue regeneration (GTR) procedures, collagen membranes have been shown to be comparable to non-absorbable membranes with regard to probing depth reduction, clinical attachment gain, and percent of bone fill. Although these membranes are absorbable, collagen membranes have been demonstrated to prevent epithelial down-growth along the root surfaces during the early phase of wound healing. The use of grafting material in combination with collagen membranes seems to improve clinical outcomes for furcation, but not intrabony, defects when compared to the use of membranes alone. Recently, collagen materials have also been applied in guided bone regeneration (GBR) and root coverage procedures with comparable success rates to non-absorbable expanded polytetrafluoroethylene (ePTFE) membranes and conventional subepithelial connective tissue grafts, respectively. Long-term clinical trials are still needed to further evaluate the benefits of collagen membranes in periodontal and peri-implant defects. This article will review the rationale for each indication and its related literature, both in vitro and in vivo studies. The properties that make collagen membranes attractive for use in regenerative therapy will be addressed. In addition, varieties of cross-linking techniques utilized to retard the degradation rate of collagen membranes will be discussed.     

Collagen deposition during wound repair in rat gingiva

Abstract – The aim of this study was to examine day-by-day changes at incisional wound edge in rat molar interdental tissue with special reference to early interactions of new and pre-existing collagen fibrils. Twenty rats aged 50 days were divided into experimental groups each comprising two animals. Following incision and post-injury observation periods of 1–10 days, the rats were given an overdose of sodium pentothal and specimens prepared for light and electron microscopy. In 1-3-day post-injury specimens fibrin was replaced by inflammatory cells. Fibroblasts were found along the cut edge in 4-day post-injury specimens. Fibrillogenesis had started at 5 days post-injury. During the period of 5–10 days post-injury increasing amounts of new collagen fibrils were laid down at the wound edge. No granular material was identified at the interface of new and preexisting collagen. In standardized mesiodistal sections, bundles of newly formed fibrils regularly appeared cross-cut in relation to the tangentially cut transseptal fiber system, suggestive of a buccolingual fibril orientation at the wound edge. The ultrastructural observations indicate that during early stages of wound repair, connective tissue continuity is generally established without direct splicing of severed fibers.     

Myofibroblasts and matrix components in healing palatal wounds in the rat

In order to identify wound contraction and scar formation during palatal mucoperiosteal wound healing in growing rats, the temporal and spatial distribution of myofibroblasts and matrix components were determined immunohistochemically. Myofibroblasts were found in the mucosal part of the palatal wound tissue between 4 and 22 days, with the highest density at 8 days post-wounding. The number of collagen type I and type III fibers gradually increased until about 8 days postwounding, and thereafter the staining intensity of collagen type III decreased. At 60 days post-wounding there were more transversely oriented collagen type I fibers and less type III fibers and elastin present in the submucosa than in normal tissue. The results suggest that in this model wound contraction mainly takes place in the mucosa between 4 and 22 days postwounding. Furthermore, palatal wounds made in young rats heal with distinct scar tissue formation. Therefore, this model is useful to test the effects of therapies that aim to reduce wound contraction and scarring after cleft palate surgery.     

Evaluation of the effect of heterologous type I collagen on healing of bone defects.

PURPOSE: The purpose of this investigation was to evaluate the effect of type I collagen on the healing of bone defects both experimentally and clinically. MATERIALS AND METHODS: In the experimental study, 16 adult male rabbits were divided into 2 groups: a collagen group and a control group. After the induction of general anesthesia with intraperitoneal ketamine, the anterior surfaces of tibias of the rabbits were surgically exposed, and a hole 4 mm in diameter was made in each tibia. In the collagen group, the defects were filled with type I collagen. The unfilled defects of the other animals were used as controls. During the study, the serum alkaline phosphatase activity of the rabbits, and radiopacity changes in the radiographs of the tibias of the rabbits were evaluated. The rabbits were killed on the 35th day, and histologic sections of the tibias were prepared. The clinical study was carried out on periapical defects in a total of 15 patients who underwent apicoectomy. After the surgical procedure, the osseous defects in periapical regions of 7 patients were filled with type I collagen. The unfilled cavities of the other patients were used for control purposes. The patients were evaluated clinically and radiographically in the postoperative period. The data were analyzed by Student's t-test and Mann-Whitney U test. RESULTS: In the experimental study, there was an increase in radiopacity corresponding with the serum alkaline phosphatase activity, and there were statistically significant differences between the control and collagen groups both radiologically and biochemically on the 14th and 28th days of the study. In the clinical study, the control cavities filled with a tissue of normal bone density in about 5 months, but the collagen cavities filled in 3 months. CONCLUSIONS: It was determined that heterologous type I collagen provides a more rapid regeneration of bone defects.     

Application of collagen hydrogel/sponge scaffold facilitates periodontal wound healing in class II furcation defects in beagle dogs

Kosen Y, Miyaji H, Kato A, Sugaya T, Kawanami M. Application of collagen hydrogel/sponge scaffold facilitates periodontal wound healing in class II furcation defects in beagle dogs. J Periodont Res 2012; 47: 626–634. © 2012 John Wiley & Sons A/S Background and Objective: A three‐dimensional scaffold may play an important role in periodontal tissue engineering. We prepared bio‐safe collagen hydrogel, which exhibits properties similar to those of native extracellular matrix. The aim of this study was to examine the effect of implantation of collagen hydrogel/sponge scaffold on periodontal wound healing in class II furcation defects in dogs. Material and Methods: The collagen hydrogel/sponge scaffold was prepared by injecting collagen hydrogel, cross‐linked to the ascorbate‐copper ion system, into a collagen sponge. Class II furcation defects (of 5 mm depth and 3 mm width) were surgically created in beagle dogs. The exposed root surface was planed and demineralized with EDTA. In the experimental group, the defect was filled with collagen hydrogel/sponge scaffold. In the control group, no implantation was performed. Histometric parameters were evaluated 2 and 4 wk after surgery. Results: At 2 wk, the collagen hydrogel/sponge scaffold displayed high biocompatibility and biodegradability with numerous cells infiltrating the scaffold. In the experimental group, reconstruction of alveolar bone and cementum was frequently observed 4 wk after surgery. Periodontal ligament tissue was also re‐established between alveolar bone and cementum. Volumes of new bone, new cementum and new periodontal ligament were significantly greater in the experimental group than in the control group. In addition, epithelial down‐growth was suppressed by application of collagen hydrogel. Conclusion: The collagen hydrogel/sponge scaffold possessed high tissue compatibility and degradability. Implantation of the scaffold facilitated periodontal wound healing in class II furcation defects in beagle dogs.     

Glycated Collagen Stimulates Differentiation of Gingival Myofibroblasts

Background: Glucose‐derived metabolites may alter the structure and biologic properties of important proteins in periodontium, such as collagens. As a consequence, it is possible that collagen‐binding cells may change their phenotypic traits. Although the glucose‐derived product methylglyoxal (MGO) has been detected in periodontal lesions, the precise effect of collagen glycation on gingival connective tissue biology is not fully understood. The present study evaluates whether collagen glycation by MGO may affect phenotypic properties and remodeling capacity of human gingival fibroblasts (HGFs). Methods: Primary cultures of HGFs were grown on Type I collagen matrices previously treated with MGO. Cell cultures were tested for cell viability, apoptosis, α‐smooth muscle actin (SMA), fibronectin (FN) production, and collagen remodeling. Mechanical properties and morphology of MGO‐treated collagen gels were evaluated using rheometry and atomic force microscopy. Statistical analysis was performed by Kruskal‐Wallis and Mann‐Whitney U tests. Results: MGO‐treated collagen did not affect cell viability or apoptosis. In addition, MGO did not induce significant changes in morphology or mechanical properties of the collagen matrix. However, MGO‐treated collagen stimulated an increase in the myofibroblast marker α‐SMA, production and assembly of FN, and contraction of collagen matrices. Moreover, use of a triple‐helical peptide that reconstitutes the collagen‐binding domain for integrins GFOGER reverted the assembly of FN induced by MGO‐treated collagen. Conclusions: The present study shows that collagen glycation by MGO stimulates differentiation of myofibroblasts and production and assembly of FN. These responses may alter the homeostatic balance and wound‐healing response of gingival connective tissues affected by diabetes mellitus or aging.     

Collagen sponge as a topical hemostatic agent in mucogingival surgery

Hemorrhage from palatal donor sites during periodontal mucogingival surgery was controlled by application of Collastat absorbable hemostatic sponges. The use of collagen sponges was evaluated in 20 free-graft cases. In contrast to microfibrillar collagen, which is normally delivered as clumps of material, a Collastat sponge was applied as a single piece that maintained structural integrity even when wet and was easy to maneuver into place. The highly porous sponges conformed to the wound, absorbed fluid and produced consistently reliable hemostasis with no secondary bleeding. By stemming the seepage of blood into the throat, patient safety and comfort were improved. The sponges were left on the wounds and removed at the 1-week postoperative visit, revealing well-formed granulation tissue. Healing proceeded normally with no evidence of infection, tissue reaction, or other adverse effects.     

Bleeding disorders: characterization, dental considerations and management

Hemostasis is a finely balanced process in which an insult to a blood vessel wall, either by injury or surgical intervention, stimulates a pair of parallel, yet associated, pathways that lead to the termination of blood loss. The coagulation cascade is initiated by the interaction between exposed subendothelial tissue factor and circulating blood and includes a series of amplification steps that result in thrombin generation. Concurrently, exposed subendothelial collagen stimulates platelets, which, in the presence of thrombin, are consolidated by fibrin to form a blood clot, thus terminating blood loss. Multiple inherited and acquired abnormalities in these pathways can seriously compromise hemostasis. Furthermore, several drugs, including over-the-counter preparations, also adversely affect hemostasis. These present significant concerns to the dentist conducting invasive procedures as they can prolong postoperative bleeding, impair wound healing and increase risk of infection. In this article, we review the current knowledge of bleeding abnormalities and discuss preoperative systemic precautions and intraoperative hemostatic measures.     

The use of in vitro models for investigating the response of fibrous joints to tensile mechanical stress

To clarify the role of mechanical deformation in the remodeling of fibrous joints, organ culture systems have been developed to apply mechanical stress to cranial sutures under controlled experimental conditions. Tensile mechanical stress applied to cranial sutures from newborn rabbits produces a two- to threefold increase in protein synthesis and a twofold increase in collagen synthesis that can be detected within 6 hours. There is also a threefold increase in the DNA content of the sutures after 48 hours. Under normal conditions sutural fibroblasts synthesize type I collagen but respond to tensile deformation by synthesizing significant amounts of type III collagen. This suggests that the biomechanical environment of a connective tissue cell is an important determinant of the collagen type synthesized. However, the effect is likely to be an indirect one by virtue of its influence on the metabolic activity of the cells. Mechanically activated cells do not preferentially synthesize structural proteins, since mechanical stress stimulates the synthesis not only of structural macromolecules but also of the enzymes responsible for their specific hydrolysis. This is not accompanied by increased degradation, however, perhaps because the metalloproteinase inhibitor TIMP synthesized by the tissues is also increased. Confluent rabbit and mouse periosteal fibroblasts synthesize and release into the culture medium factors that can inhibit bone cell proliferation and stimulate bone resorption in vitro. It seems likely that further investigation of the interaction between fibroblasts and osteoblasts at the bone--fibrous tissue interface will require a reassessment of current thinking concerning the mechanisms regulating sutural osteogenesis.     

Effect of two delivery systems for recombinant human bone morphogenetic protein-2 on periodontal regeneration in vivo

Resorbable collagen membranes for guided tissue regeneration in periodontal therapy have shown promise but are not osteoinductive. As recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to have an affinity for collagen, the use of this osteoinductive agent incorporated into a collagen vehicle may act as a suitable carrier to promote periodontal regeneration. The aim of this study was to investigate the effects of two different collagen delivery systems for rhBMP-2 in rat periodontal fenestration defects. Using the collagen membrane delivery system, 3 groups of adult Wistar rats which had surgical defects created on the right side of the mandible involving the removal of bone and exposure of the molar roots were treated with either rhBMP-2 in collagen membrane (BMPm) (n = 12 animals), or collagen membrane only (COLm) (n = 12), or were left untreated (UN) (n = 14). Using the collagen gel delivery system, surgical defects were treated with either rhBMP-2 incorporated in a collagen gel carrier (BMPg) (n = 5) or had collagen gel only (COLg) (n = 6). Animals were killed 10 d postoperatively and tissues processed for histology. New bone formation was significantly greater in BMPg compared with both BMPm and controls (p<0.05). However, new cementum formation was significantly greater in BMPm (721 ± 166 μm², mean ± SE) compared with COLm, COLg and UN (p<0.02) (190 ± 44 μm², 327±114 μm² and 172 ± 33 μm², respectively) and more than 1.5 times BMPg (451 ± 158 μm²). In conclusion, both carrier systems for rhBMP-2 significantly increased new bone formation compared with controls during the early stages of periodontal wound healing. However, the more slowly dissolving collagen membrane carrier system for rhBMP-2 produced significantly greater new cementum compared with the collagen gel carrier, suggesting that a more prolonged exposure of rhBMP-2 is required to increase cementogenesis.     

Effect of two delivery systems for recombinant human bone morphogenetic protein-2 on periodontal regeneration in vivo

Resorbable collagen membranes for guided tissue regeneration in periodontal therapy have shown promise but are not osteoinductive. As recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to have an affinity for collagen, the use of this osteoinductive agent incorporated into a collagen vehicle may act as a suitable carrier to promote periodontal regeneration. The aim of this study was to investigate the effects of two different collagen delivery systems for rhBMP-2 in rat periodontal fenestration defects. Using the collagen membrane delivery system, 3 groups of adult Wistar rats which had surgical defects created on the right side of the mandible involving the removal of bone and exposure of the molar roots were treated with either rhBMP-2 in collagen membrane (BMPm) (n= 12 animals), or collagen membrane only (COLm) (n = 12), or were left untreated (UN) (n= 14). Using the collagen gel delivery system, surgical defects were treated with either rhBMP-2 incorporated in a collagen gel carrier (BMPg) (n = 5) or had collagen gel only (COLg) (n = 6). Animals were killed 10 d postoperatively and tissues processed for histology. New bone formation was significantly greater in BMPg compared with both BMPm and controls (p<0.05). However, new cementum formation was significantly greater in BMPm (721 ± 166 μm², mean ± SE) compared with COLm, COLg and UN (p<0.02) (190±44μm², 327±114 μm² and 172 ± 33 μm², respectively) and more than 1.5 times BMPg (451 ± 158 μm²). In conclusion, both carrier systems for rhBMP-2 significantly increased new bone formation compared with controls during the early stages of periodontal wound healing. However, the more slowly dissolving collagen membrane carrier system for rhBMP-2 produced significantly greater new cementum compared with the collagen gel carrier, suggesting that a more prolonged exposure of rhBMP-2 is required to increase cementogenesis.     

TGF-beta 3 decreases type I collagen and scarring after labioplasty

Cleft lip is a common congenital malformation, and labioplasty performed on infants to repair such defects often results in severe scar formation. Since TGF-beta 3 has been implicated in wound healing, we therefore hypothesized that TGF-beta 3 functions to reduce scarring after cleft lip repair. In this investigation, we demonstrated that exogenous TGF-beta 3 reduced scar formation in an incised and sutured mouse lip in vivo. During labioplasty, endogenous TGF-beta 3 expression was also elevated. In vitro experiments showed that exogenous TGF-beta 3 reduced type I collagen accumulation. Furthermore, TGF-beta 3 inhibited alpha-smooth-muscle actin expression, a marker for myofibroblasts. In tandem, TGF-beta 3 induced the expression and activity of MMP-9. Analysis of our data suggests that TGF-beta 3 is normally secreted following labioplastic wound healing. An elevated level of TGF-beta 3 reduces type I collagen deposition by restricting myofibroblast differentiation and thereby collagen synthesis, and by promoting collagen degradation by MMP-9. In combination, these events lead to TGF-beta 3-mediated reduced scar formation.     

壳聚糖及其复合物对小鼠成骨细胞增殖及分化的影响

目的：用含壳聚糖(chitosan,CS)、Ⅰ型胶原蛋白和重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2)的培养液体外培养成骨细胞MC3T3-E1,评价3种因素对成骨细胞增殖及分化的影响。方法：实验分为4组,实验组A：CSα-MEM培养基;实验组B：CS+Ⅰ型胶原蛋白溶液的α-MEM培养基;实验组C：CS +Ⅰ型胶原蛋白溶液+rhBMP-2溶液的α-MEM培养基。对照组为含1%FBS的α-MEM培养基。采用MTT法检测加入处理因素后1、3、5、7 d的吸光度(OD)值,并绘制细胞生长曲线,观察成骨细胞的增殖情况。采用碱性磷酸酶活性测定、碱性磷酸酶染色和茜素红钙结节染色观察成骨细胞的分化作用：检测加入处理因素后1、3、5、7 d的碱性磷酸酶活性,并在细胞培养的第7天进行碱性磷酸酶染色,第14天进行茜素红钙结节染色。采用SPSS13.0软件包对所得数据进行单因素方差分析,2组之间比较采用Post Hoc检验。结果：MTT检测结果显示,实验组C的OD值高于其他组,实验组C与其他组间的两两比较具有显著差异(P<0.05)。碱性磷酸酶活性测定结果显示,实验组C的活性高于其他组,实验组C与实验组A、对照组间两两比较具有显著差异(P<0.05),与实验组B两两比较无显著差异(P>0.05)。茜素红染色和碱性磷酸酶染色结果显示,实验组C可见更多的钙盐沉积,且蓝染颗粒多于其他组。结论：壳聚糖、Ⅰ型胶原蛋白和rhBMP-2共同作用,更能促进成骨细胞的增殖与分化。     

Effect of rhPDGF-BB on bone turnover during periodontal repair

PURPOSE: Growth factors such as platelet-derived growth factor (PDGF) exert potent effects on wound healing including the regeneration of periodontia. Pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) is a well-known biomarker of bone turnover, and as such is a potential indicator of osseous metabolic activity. The objective of this study was to evaluate the release of the ICTP into the periodontal wound fluid (WF) following periodontal reconstructive surgery using local delivery of highly purified recombinant human PDGF (rhPDGF)-BB. METHODS: Forty-seven human subjects at five treatment centres possessing chronic severe periodontal disease were monitored longitudinally for 24 weeks following PDGF regenerative surgical treatment. Severe periodontal osseous defects were divided into one of three groups and treated at the time of surgery with either: beta-tricalcium phosphate (TCP) osteoconductive scaffold alone (active control), beta-TCP+0.3 mg/ml of rhPDGF-BB, or beta-TCP+1.0 mg/ml of rhPDGF-BB. WF was harvested and analysed for local ICTP levels by radioimmunoassay. Statistical analysis was performed using analysis of variance and an area under the curve analysis (AUC). RESULTS: The 0.3 and 1.0 mg/ml PDGF-BB treatment groups demonstrated increases in the amount of ICTP released locally for up to 6 weeks. There were statistically significant differences at the week 6 time point between beta-TCP carrier alone group versus 0.3 mg/ml PDGF-BB group (p<0.05) and between beta-TCP alone versus the 1.0 mg/ml PDGF-BB-treated lesions (p<0.03). The AUC analysis revealed no statistical differences amongst groups. CONCLUSION: This study corroborates the release of ICTP as a measure of active bone turnover following local delivery of PDGF-BB to periodontal osseous defects. The amount of ICTP released from the WF revealed an early increase for all treatment groups. Data from this study suggests that when PDGF-BB is delivered to promote periodontal tissue engineering of tooth-supporting osseous defects, there is a direct effect on ICTP released from the wound.     

Growth and hydrolytic enzyme production of Capnocytophaga gingivalis on different protein substrates

Capnocytophaga gingivalis was grown with proteins (albumin, collagen, mucin and hemoglobin) as carbon and energy sources in chemostat culture. The mu max (0.34 h-1) and biomass yield (0.96 g.l-1) were as high with hemoglobin (3 g.l-1) as with glucose (3 g.l-1) (20). Albumin, collagen and mucin also supported an increased mu max, or yield or both, in comparison with basal (tryptone/thiamine) medium. In steady-state, trypsin-like protease specific activity increased 3- to 5-fold in the presence of albumin, collagen and hemoglobin: whereas the greatest increase (21-fold) in alpha-glucoside activity was in the presence of mucin. There were significant, but less substantial changes in other hydrolytic enzymes (aminopeptidase, acid and alkaline phosphatases). The bulk of the detected hydrolytic activity (> 66%) was associated with the cells. The data indicate that C. gingivalis regulates its production of hydrolytic enzymes in response to environmental conditions.     

Development of an injectable composite as a carrier for growth factor-enhanced periodontal regeneration

Aim: Biomaterials are often applied in periodontal therapy; however, not always well adapted for tissue regeneration. The objective of this study was to evaluate the physico‐chemical properties and biocompatibility of an injectable, in situ setting composite for growth factor‐enhanced periodontal regeneration. Material and Methods: The composite constitutes bioresorbable poly(lactic‐co‐glycolic acid) (PLGA) and additives forming in situ a matrix designed as a carrier for recombinant human growth/differentiation factor‐5 (rhGDF‐5). In vitro characterization included the porosity, biointeraction, biodegradation, injectability, and biological activity of released rhGDF‐5. Biocompatibility was compared with granular β‐tricalcium phosphate and an absorbable collagen sponge using a canine periodontal defect model. Results: The PLGA composite showed a highly porous (500–1000 μm) space‐providing structure. It effectively induced coagulation exhibiting an intimate interaction with the fibrin clot. The biphasic biodegradation was complete within 4 weeks. The composite was conveniently injectable (90.4±3.6 N) for ease of use. It exhibited a sustained rhGDF‐5 release over 4 weeks (40.8%) after initial burst (3.4%) detected by ALP activity. Sites receiving the composite showed limited, if any, residuals and had no appreciable negative effect on periodontal wound healing. There were no noteworthy inflammatory lesions in sites receiving the PLGA composite. Conclusion: Characteristics of the PLGA composite makes it an attractive matrix to support native wound healing and rhGDF‐5‐enhanced periodontal regeneration.