SYNERGISTIC EFFECT OF HONEY AND LEMON JUICE-ENRICHED MULBERRY DIETS ON THE GROWTH OF SILKWORM, BOMBYX MORI DURING METAMORPHOSIS

Synergistic impact of honey and lemon juice-enriched mulberry diets on the growth of Bombyx mori was studied. The body size progressively increased during larval stage, but declined during pupal and adult stages. The nutrient diets promoted larval growth and positively modulated pupal and adult growth. The larval growth curves are typical Gompertz trajectories that re?ected the growth promoting nature of honey and lemon juice. The log-based growth curves were used to derive critical larval body size determinants that control molting and metamorphosis. The nutrient diets improved critical body size determinants without affecting their time schedules. The compound periodical growth rates showed instar-speci?c and stage-speci?c variations. The size speci?c growth rates in body mass, length and perimeter dimensions indicated the prevalence of an effective mass management mechanism as de?ned in the Hutchinson's investment principle. The silkworm recorded either higher or lower growth ratios indicating deviation from the Dyar's constancy rule.

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.

Comparative Evaluation of Fibrin for Bone Regeneration in Critical Size Calvarial Defects

Natural biopolymers such as collagen and fibrin have been widely used in bone regenerative applications. Despite the frequent use, their comparative biological propertiesis are largely unknown. In a previous study, we found the superiority of fibrin to collagen in the adsorption of serum proteins and the proliferation and differentiation of cultured osteoblasts. In this study, we used an in vivo model to evaluate how effectively fibrin supports bone regeneration, as compared with collagen. Collagen and fibrin were placed in critical size defects made on rat calvarial bones. Compared with collagen, fibrin supported substantially more new bone tissue formation, which was confirmed by micro-CT measurement and histological analyses. The cells in the regenerative tissues of the fibrin-filled defects were immunostained strongly for Runx2, while collagen-placed defects were stained weakly. These in vivo results demonstrate that fibrin is superior to collagen in supporting bone regeneration.

Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects

It is known that current trends on bone bioengineering seek ideal scaffolds and explore innovative methods to restore tissue function. In this way, the objective of this study was to evaluate the behavior of anorganic bovine bone as osteoblast carrier in critical-size calvarial defects. MC3T3-E1 osteoblast cells (1x10(5) cells/well) were cultured on granules of anorganic bovine bone in 24-well plates and after 24 h these granules were implanted into rat critical-size calvarial defects (group Biomaterial + Cells). In addition, other groups were established with different fillings of the defect: Blood Clot (negative control); Autogenous Bone (positive control); Biomaterial (only granules) and Cells (only MC3T3-E1 cells). After 30 days, the animals were euthanized and the calvaria were technically processed in order to allow histological and morphometric analysis. It was possible to detect blood vessels, connective tissue and newly formed bone in all groups. Particularly in the Biomaterial + Cells group, it was possible to observe a profile of biological events between the positive control group (autogenous bone) and the group in which only anorganic bovine granules were implanted. Altogether, the results of the present study showed that granules of anorganic bovine bone can be used as carrier to osteoblasts and that adding growth factors at the moment of implantation should maximize these results.

Sabe-se que uma das atuais tendências na bioengenharia óssea é procurar um carreador ideal e explorar métodos inovadores para restaurar a função do tecido. Desta forma, nosso objetivo foi avaliar o comportamento do osso bovino inorgânico como carreador de osteoblastos em defeitos ósseos de tamanho crítico em calvária de ratos. Osteoblastos da linhagem MC3T3-E1 (1x10(5) células/poço) foram cultivadas em grânulos de osso bovino inorgânico sob placas de 24 poços e após 24 h esses grânulos foram implantados em defeitos ósseos de tamanho crítico em calvária de ratos. Além deste grupo experimental (Biomaterial + Células), foram estabelecidos outros grupos com diferentes preenchimentos do defeito crítico: coágulo sanguíneo (controle negativo); osso autógeno (controle positivo); Biomaterial (apenas grânulos) e Células (apenas células MC3T3-E1). Após 30 dias, os animais foram eutanasiados e as calvárias foram processadas histotecnicamente, a fim de permitir a análise histológica e morfometria. Nossos resultados mostraram que em todos os grupos avaliados foi possível detectar vasos sanguíneos, tecido conjuntivo e osso neoformado. Em especial para o grupo tratado com Biomaterial + Células, foi possível observar um perfil de eventos biológicos intermediário ao grupo controle positivo (osso autógeno) e o grupo de biomaterial (apenas grânulos inorgânico bovino). Ao todo, nossos resultados mostraram que os grânulos de osso bovino inorgânico podem ser usados como carreador de osteoblastos e que a adição de fatores de crescimento no momento em que ocorre o implante deve maximizar os resultados.

Repair of critical-size defects with autogenous periosteum-derived cells combined with bovine anorganic apatite/collagen: an experimental study in rat calvaria

The aim of this study was to evaluate the bone repair using autogenous periosteum-derived cells (PDC) and bovine anorganic apatite and collagen (HA-COL). PDC from Wistar rats (n=10) were seeded on HA-COL discs and subjected to osteoinduction during 6 days. Critical-size defects in rat calvarias were treated with blood clot (G1), autogenous bone (G2), HA-COL (G3) and HA-COL combined with PDC (G4) (n=40), and then analyzed 1 and 3 months after surgeries. Radiographic analysis exhibited no significant temporal change. G1 and G2 had discrete new marginal bone, but the radiopacity of graft materials in G2, G3 and G4 impaired the detection of osteogenesis. At 3 months, histopathological analysis showed the presence of ossification islets in G1, which was more evident in G2, homogeneous new bone around HA-COL in G3 and heterogeneous new bone around HA-COL in G4 in addition to moderate presence of foreign body cells in G3 and G4. Histomorphometric analysis showed no change in the volume density of xenograft (p>0.05) and bone volume density in G2 was twice greater than in G1 and G4 after 3 months (p<0.05), but similar to G3. The PDC did not increase bone formation in vivo, although the biomaterial alone showed biocompatibility and osteoconduction capacity.

O objetivo deste estudo foi avaliar o reparo ósseo usando células derivadas de periósteo (PDC) e apatita inorgânica e colágeno bovinos (HA-COL). PDC de ratos Wistar (n=10) foram semeadas sobre discos de HA-COL e osteoinduzidas por 6 dias. Defeitos de tamanho crítico em calvárias de ratos foram tratados com coágulo sanguíneo (G1), osso autógeno (G2), HA-COL (G3) ou HA-COL associado a PDC (G4) (n=40) e analisados em 1 e 3 meses após as cirurgias. Análise radiográfica não exibiu mudança temporal significante, G1 e G2 tiveram aumento discreto de novo osso marginal, entretanto a radiopacidade dos materiais de enxerto em G2, G3 e G4 prejudicou a detecção de osteogênese. Análise histopatológica mostrou em 3 meses ilhotas de ossificação em G1 que foi maior em G2, novo osso homogêneo ao redor de HA-COL em G3 e novo osso heterogêneo ao redor de HA-COL em G4 além da presença moderada de células gigantes de corpo estranho em G3 e G4. Análise histomorfométrica mostrou a densidade de volume inalterada do xenoenxerto (p>0,05) e a densidade de volume de novo osso em G2 duas vezes maior que G1 e G4 após 3 meses (p<0,05), mas similar a G3. PDC não aumentaram a formação óssea in vivo apesar do biomaterial sozinho ter apresentado biocompatibilidade e capacidade osteocondutora.

Spontaneous bone regeneration after enucleation of jaw cysts: a comparative study of panoramic radiography and computed tomography

INTRODUCTION: A cyst is a closed pathologic sac containing fluid or semi-solid material in central region. The most common conventional treatment for a cyst is enucleation. It was reported that spontaneous bone healing could be accomplished without bone grafting. We are trying to evaluate bone reconstruction ability by analyzing panorama radiograph and computed tomography (CT) scan with retrograde studying after cyst enucleation. In this way we are estimating critical size defect for spontaneous healing without bone graft. MATERIALS AND METHODS: The study comprised of 45 patients who were diagnosed as cysts and implemented enucleation treatment without bone graft. After radiograph photo taking ante and post surgery for 6, 12, 18, 24 months, the healing surface and volumetric changes were calculated. RESULTS: 1. Spontaneous bone healing was accomplished clinically satisfying 12 months later after surgery. But analyzing CT scan, defect volume changes indicate 79.24% which imply incomplete bone healing of defect area. 2. Comparing volume changes of defect area of CT scan, there are statistical significance between under 5,000 mm3 and over 5,000 mm3. The defect volume of 5,000 mm3 shows 2.79x1.91 cm in panoramic view. CONCLUSION: Bone defects, which are determined by a healed section using a panoramic view, compared to CT scans which do not show up. Also we can estimate the critical size of defects for complete healing.

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