Histological Evaluation of Experimentally Induced Critical Size Defect Skin Wounds Using Exosomal Solution of Mesenchymal Stem Cells Derived Microvesicles

BACKGROUND AND OBJECTIVES: The present study investigated whether MSCs derived microvesicles (MVs) or (Exosomes) can exert therapeutic effects on an experimental model of cutaneous injury and explored the underlying involving mechanisms. METHODS AND RESULTS: Three bilateral full thickness circular wounds were created on the back of two groups of dogs using 2-cm dermal punch. The wounds were at least 2.5 cm apart. Saline was subcutaneously injected in 4 places around each wound area in group-I (control), whereas an equal volume of exosomal solution of MSCs derived MVs was similarly injected in group-II. The findings demonstrated that MSCs derived MVs had significantly promoted cutaneous wound healing, collagen synthesis, and vascularization at wound sites. The application of the exosomal solution had not only promoted the generation of newly formed vessels, but also have accelerated their development and maturation leading to a faster healing process. CONCLUSIONS: MSC-Exosomes appeared to be a superior candidate for treating cutaneous wounds than their originator cells, and may represent a promising opportunity to develop a novel cell-free therapy approach that might overcome the obstacles and risks associated with the use of native or engineered stem cells transplantation therapy.

The Rabbit Model of Femoral Bone Defects: Steps to Overcome Potential Pitfalls.

Aims: To identify the potential pitfalls and indicate procedures to prevent them, during the evaluation of biomaterials for orthopaedic and craniofacial research in the New Zealand White (NZW) rabbit animal model of femoral bone defects. Place and Duration of Study: Laboratory for Research of the Musculoskeletal System, School of Medicine, University of Athens, between June 2014 and July 2015. Materials and Methods: Pre-emptive analgesia (carprofen 2.2 mg/kg sc), chemoprophylaxis (enrofloxacin 10 mg/kg sc) and anaesthesia (ketamine/xylazine 30/5 mg/kg im) were administered to NZW rabbits (body weight 3.3±0.2 kg, mean ± SD) for the aseptic surgical creation of drilled bone defects of 6 mm diameter (“critical size defect”) in the external femoral condyle of the left limb. All rabbits recovered without post-surgical complications from the first postoperative day. Results and Discussion: Although the research group consisted of Veterinarians and Orthopaedic Surgeons with experience in this model, they were challenged with potential pitfalls which were overcome step by step. Among them is the precise localization of the defect to be drilled. Intra-operative palpation of the external femoral condyle assists in determining the site, and post-operative X-ray evaluation confirms it. Additionally the correct width and depth of the bone defect are important to adhere to, which was achieved by using a 5.5 mm diameter bone drill and observing its depth marks. Another challenge is to have the specific amount of biomaterial implanted confined to the defect. Its potential distribution in the femoral shaft, diffusion in the metaphysial trabecular bone or excessive covering of the bone surface, are also pitfalls to be avoided. Conclusions: The increased use of this animal model in the evaluation of biomaterials in orthopaedic and craniofacial research requires knowledge, skills, surgical accuracy and attention to a sequence of steps, in order to achieve homogenous results and high repeatability of the implantation technique. With the fulfillment of these conditions, the extraction of valid scientific results and reduction of the number of animals used are possible.

Investigation of a pre-clinical mandibular bone notch defect model in miniature pigs: clinical computed tomography, micro-computed tomography, and histological evaluation

OBJECTIVES: To validate a critical-size mandibular bone defect model in miniature pigs. MATERIALS AND METHODS: Bilateral notch defects were produced in the mandible of dentally mature miniature pigs. The right mandibular defect remained untreated while the left defect received an autograft. Bone healing was evaluated by computed tomography (CT) at 4 and 16 weeks, and by micro-CT and non-decalcified histology at 16 weeks. RESULTS: In both the untreated and autograft treated groups, mineralized tissue volume was reduced significantly at 4 weeks post-surgery, but was comparable to the pre-surgery levels after 16 weeks. After 16 weeks, CT analysis indicated that significantly greater bone was regenerated in the autograft treated defect than in the untreated defect (P=0.013). Regardless of the treatment, the cortical bone was superior to the defect remodeled over 16 weeks to compensate for the notch defect. CONCLUSION: The presence of considerable bone healing in both treated and untreated groups suggests that this model is inadequate as a critical-size defect. Despite healing and adaptation, the original bone geometry and quality of the pre-injured mandible was not obtained. On the other hand, this model is justified for evaluating accelerated healing and mitigating the bone remodeling response, which are both important considerations for dental implant restorations.

Experimental study of calvarial critical size defect in rats with type 2 diabetes mellitus / 实用口腔医学杂志

Objective:To explore the calvarial critical size defect (CSD)in rats with type 2 diabetes mellitus(T2DM).Methods:T2DM model of SD rats(weighted 300-320 g)was induced by high fat and high sugar diet and low dose intraperitoneal streptozotocin (STZ)injection.The rats with T2DMand the normal controls were divided into 4 groups(n=3)respectively.Defects with the diame-ter(mm)of 2,3,4 and 5 were made on the central calvaria of each rat.General observation,X-ray examination and histological study were performed 8 weeks postoperatively.Results:In the T2DM group,only the defects of 2 mm diameter were healed completely,X-ray resistance and new bone formation were observed;the defects of 3,4 and 5 mm diameter were unhealed,X-ray transmission was observed and newly formed bone was insufficient.In the control group,the defects of 2,3 and 4 mm diameter were healed completely, X-ray resistance and new bone formation were observed;the defects of 5 mm diameter were unhealed,X-ray transmission was ob-served,newly formed bone was insufficient.Conclusion:The calvarial CSD of T2DM rat model can be defined as the defect with the diameter of 3 mm.

The Effect of the Injectable Bovine Collagens (Zyderm(R)) on the Repair of Critical-Size Defect in Rat Calvaria

This study was designed to examine the capacity of injectable bovine collagen(Zyderm(R)) to mediate the repair of critical size defects of rat calvaria. Thirty-six rats were randomized into six groups(n=6) and an 8-mm circular calvarial defect was made in each rat. In Group I, defects were left untreated. Defects in Groups II and III were implanted with Zyderm I(R) and Zyderm II(R), respectively. These three groups were evaluated in the 6th week after the operation. The defects in Groups IV, V, and VI received the same treatment as in Groups I, II, and III, respectively and were evaluated at postoperative 12th week. Results were examined by gross and histologic findings, simple radiographic study, and radiodensitometric analysis for bony density quantitatively. By gross and simple radiographic findings, defects with Zyderm(R) implantation showed thick coverage and focal radioopacity within defect area, but non-treated animals showed only fibrous healing. Histologically collagen pocket and woven bone were coexisted over the defect at postoperative 6th week, but the regenerating bone appeared much robust in Group III. At postoperative 12th week, there was a well-organized lamellar pattern of new bone formation, especially on the periosteal side of the defect. In the radiodensitometric analysis, all groups of Zyderm(R) implantation showed significant bony healing. There was also statistically significant difference between Group II and Group III at 6th week. Compared with Group V, however, Group VI did not show significant bony healing at 12th week. Additionally, prolonged implantation period allowed more complete healing of the defects with the same concentration of collagen. In conclusion, Zyderm(R) appeared to be an effective mediator of bone regeneration in this critical size calvarial defect model and its concentration had an effect on bone healing in the early period.

THE BONE REGENERATIVE EFFECTS OF PARADIOXANONE ON THE CALVARIAL CRITICAL SIZE DEFECT IN SPRAGUE DAWLEY RATS / 대한치주과학회지

The major goals of periodontal therapy are the functional regeneration of periodontal supporting structures already destructed by periodontal disease as well as the reduction of signs and symptoms of progressive periodontal disease. There have been many efforts to develop materials and therapeutic methods to promote periodontal wound healing. Bone graft & guided tissue are being used for the regeneration of destroyed periodontium these days. Non-resorbable membranes were used for Guided tissue regeneration in early days, however more researches are focused on resorbable membranes these days. The aim of this study is to evaluate the osteogenesis of paradioxanone membrane on the calvarial critical size defect in Sprague Dawley rats. An 8 mm diameter surgical defect was produced with a trephine bur in the area of the midsagittal suture. The rats were divided into three groups: Untreated control group, Biomesh(R) group and paradioxanone group. The animals were sacrificed at 4, 8 and 12 weeks after surgical procedure. The specimens were examined by histologic, histomorphometric analyses. The results are as follows: 1. In histological view on Biomesh(R), no visible signs of resorption was observed at 4 weeks but progressive resorption was observed at 8 weeks through 12 weeks. Paradioxanone membrane expanded at 4 weeks, and rapid resorption was observed at 8 weeks. In both the membranes, inflammatory cells were observed around them. Inflammatory cells decreased with time but were still present at 12 weeks. More inflammatory cells were observed in paradioxanone membranes than in Biomesh(R) membrane. 2. The area of newly formed bone in the defects were 0.001+/-0.001, 0.006+/-0.005, 0.002+/-0.003 at the 4 weeks, 0.021+/-0.020, 0.133+/-0.073, 0.118+/-0.070 at the 8 weeks and 0.163+/-0.067, 0.500+/-0.197, 0.487+/-0.214 at the 12 weeks in the control group, Biomesh(R) group and experimental group respectively. Compared to the control group, Biomesh(R) group displayed significant differences at 4,8, and 12 weeks and the paradioxanone group at 8 and 12 weeks.(P<0.05) 3. The area of residual membranes were 1.143+/-0.499, 2.599+/-1.012, at the 4 weeks, 0.666+/-0.140, 0.314+/-0.131 at the 8 weeks and 0.365+/-0.110, 0.076+/-0.050 at the 12 weeks in the Biomesh(R) group and experimental group respectively. Between the two groups, significant differences were displayed at 4 weeks.(P<0.05) According to the results, when paradioxanone membrane was used in calvarial critical size defect in Sprague Dawley rat, initially the membrane expaned and regeneration of newly formed bone was small however after 8weeks new bone was formed with simultaneous resorption for the membrane. If a few problems could be solved, previously used membranes could be replaced in periodontal guided tissue regeneration.

The Repair of Critical-Size Defect with Fibrin Sealant in Rat Calvaria

This study was designed to evaluate the capacity of fibrin sealant to mediate the repair of critical size defects of rat calvaria. Twenty-four rats were randomized into four groups(n=6) and an 8-mm circular calvarial defect was made in each rat. In Group I and II, as control groups, defects were left untreated and evaluated at postoperative 6th and 12th week, respectively. In Group III and IV, defects were treated with fibrin sealant implantation and evaluated at the same point of time as in Group I and II, respectively. Results were examined by gross and histologic findings, simple radiographic study, and radiodensitometric analysis for bony density quantitatively. By gross and simple radiographic findings, animals with fibrin sealant implantation generally showed thick coverage of defects and focal radioopacity within defect area, but non-treated animals showed only fibrous healing. Histologically, small amount of new appositional bone growth was only seen at the edge of the defect at 6th week in Group III, but in Group IV, there are substantial amounts of new immature bone with well-organized patterns within the defect at 12th week postoperatively. In the radiodensitometric analysis, there was no statistically significant difference between Group I and Group III at 6th week. Compared with Group II, however, Group IV showed significant bony healing (p=0.0225) at 12th week. In conclusion, the fibrin sealant appeared to be an effective mediator in bone regeneration of this critical-sized calvarial defect model, but long-term implantation period should be needed for optimal results.

The Bone Regenerative Effects of Chitosan on the Calvarial Critical Size Defectin Sprague Dawley Rats / 대한치주과학회지

The major goals of periodontal therapy is the functional regeneration of periodontal supporting structures already destructed by periodontal disease as well as the reduction of signs and symptoms of progressive periodontal disease. There have been many efforts to develop materials and therapeutic methods to promote periodontal wound healing. There have been increasing interest on the chitosan made by chitin. Chitin is second only to cellulose as the most abundant natural biopolymer. It is a structural component of the exoskeleton of invertebrates(e.g., shrimp, crabs, lobsters), of the cell wall of fungi, and of the cuticle of insects. Chitosan is a derivative of chitin made by deacetylation of side chains. Many experiments using chitosan in various animal models have proven its beneficial effects. The aim of this study is to evaluate the osteogenesis of chitosan on the calvarial critical size defect in Sprague Dawley rats. An 8 mm surgical defect was produced with a trephine bur in the area of the midsagittal suture. The rats were divided into two groups: Untreated control group versus experimental group with 50mg of soluble chitosan gel. The animals were sacrificed at 2, 4 and 8 weeks after surgical procedure. The specimens were examined by histologic, histomorphometric and radiodensitometric analyses. The results are as follows: 1. The length of newly formed bone in the defects was 102.91+/-25.46micrometer, 219.46+/-97.81micrometer at the 2 weeks, 130.95+/-39.24micrometer, 212.39+/-89.22micrometer at the 4 weeks, 181.53+/-76.35micrometer and 257.12+/-51.22micrometer at the 8 weeks in the control group and experimental group respectively. At all periods, the means of experimental group was greater than those of control group. But, there was no statistically significant difference between the two groups. 2. The area of newly formed bone in the defects was 2962.06+/-1284.48micrometer2, 5194.88+/-1247.88micrometer2 at the 2 weeks, 5103.25+/-1375.88micrometer2, 7751.43+/-2228.20micrometer2 at the 4 weeks and 8046.02+/-818.99micrometer2, 15578.57+/-5606.55micrometer2 at the 8 weeks in the control group and experimental group respectively. At all periods, the means of experimental group was greater than those of control group. The experimental group showed statistically significant difference to the control group at the 2 and 8 weeks. 3. The density of newly formed bone in the defects was 14.26+/-6.33%, 27.91+/-6.65% at the 2 weeks, 20.06+/-9.07%, 27.86+/-8.20% at the 4 weeks and 22.99+/-3.76%, 32.17+/-6.38% at the 8 weeks in the control group and experimental group respectively. At all periods, the means of experimental group was greater than those of control group. The experimental group showed statistically significant difference to the control group at the 2 and 8 weeks. These results suggest that the use of chitosan on the calvarial defects in rats has significant effect on the regeneration of bone tissue in itself