Repair Of Critical-sized Defects Research Articles

Hybrid Mineral/Organic Material Induces Bone Bridging and Bone Volume Augmentation in Rat Calvarial Critical Size Defects.

In craniofacial bone defects, the promotion of bone volume augmentation remains a challenge. Finding strategies for bone regeneration such as combining resorbable minerals with organic polymers would contribute to solving the bone volume roadblock. Here, dicalcium phosphate dihydrate, chitosan and hyaluronic acid were used to functionalize a bone-side collagen membrane. Despite an increase in the release of inflammatory mediators by human circulating monocytes, the in vivo implantation of the functionalized membrane allowed the repair of a critical-sized defect in a calvaria rat model with de novo bone exhibiting physiological matrix composition and structural organization. Microtomography, histological and Raman analysis combined with nanoindentation testing revealed an increase in bone volume in the presence of the functionalized membrane and the formation of woven bone after eight weeks of implantation; these data showed the potential of dicalcium phosphate dihydrate, chitosan and hyaluronic acid to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in animal models.

The Effect of Photobiomodulation Therapy in Different Doses on Bone Repair of Critical Size Defects in Rats: A Histomorphometric Study.

Introduction: Photobiomodulation therapy (PBM) appears to induce osteogenesis and stimulate fracture repair; because of its capacity, it is considered a promising treatment, but the characteristics of response to different radiation doses must be investigated through in vivo studies to establish their safety and effectiveness. Thus, this paper aims to analyze the effects of the PBM at different doses on the repair of critical bone defects through histological and histomorphometric analyses. Methods: Sixty 90-day-old adult rats (Rattus norvegicus, albinus, Wistar) weighing approximately 300 g were used. Critical bone defects of 5 mm in diameter were performed in their calvaria. The animals were randomly separated into 5 groups: C-Blood clot, L15-PBM 15J/cm2, L30-PBM 30 J/ cm2, L45-PBM 45 J/cm2, L60-PBM 60 J/cm2. Each group was subdivided according to observation periods of 30 and 60 days with 6 rats in each subgroup. Low-level gallium aluminum arsenide (GaAlAs) lasers were used at a 660 nm wavelength, 30 mW and 0.04 cm2 in area. The PBM was applied over 5 points; 4 points of application were distributed on the edges while one point of application was located in the center of the bone defect. PBM occurred right after the procedure. In 30 and 60 days, the animals were euthanized by anesthesia overdose and the analyses were performed. The data were analyzed statistically by the ANOVA, together with the Tukey test, whose significance level was 5%. Results: As regards the treatment factor, the highest percentage of bone neoformation was achieved by group L45-60. The group with the highest closure, despite not having a statistically significant difference with the other doses, was 45 J with only 0.49 mm between edges. Conclusion: Thus, the present study allowed concluding that the highest percentage of bone neoformation area was achieved at 45 J/cm2 in 60 days; that is, it was significantly effective in comparison with other doses.

New Use for Old Drug: Local Delivery of Puerarin Facilitates Critical-Size Defect Repair in Rats by Promoting Angiogenesis and Osteogenesis

New Use for Old Drug: Local Delivery of Puerarin Facilitates Critical-Size Defect Repair in Rats by Promoting Angiogenesis and Osteogenesis

Icaritin Enhancing Bone Formation Initiated by Sub-Microstructured Calcium Phosphate Ceramic for Critical Size Defect Repair

Adequate bone tissue regeneration has been challenging to achieve at critical-sized bone defects caused by disease. Bone tissue engineering using a combination of scaffolds and bioactive factors provides new hope for the treatment of this extreme condition. Icaritin, a herb-derived chemical, has shown its ability to enhance bone formation both in vitro and in vivo, and it has been found that sub-micron surface structure instructs bone formation in calcium phosphate ceramics (CaPs). Here, we evaluated the possibility of using a submicron surface structured CaP ceramic as the carrier of icaritin for bone tissue regeneration in critical-sized bone defects. Icaritin, an herb-derived chemical, was loaded into a submicron surface structured porous calcium phosphate ceramic (Ø12.8 × 3 mm) to get samples with 0, 10, 50, 250, and 1,250 µg icaritin per CaP disc (M0, M10, M50, M250, M1250 groups, respectively). In vitro evaluation with the certain dosages correlated to those released from the samples showed a dose-dependent enhancement of osteogenic differentiation and mineralization of human bone marrow stromal cells with the presence of osteogenic factors in the culture medium, indicating icaritin is an osteopromotive factor. After intramuscular implantation of the samples in dogs for 8 weeks, a dose-dependent of bone formation was seen with enhanced bone formation at the dosage of 50 and 250 µg. To evaluate the in vivo osteogenic potentials of icaritin-containing CaP ceramic scaffolds in the orthopedic site, a 12.8 mm calvarial defect model in rabbits was established. Micro-computed tomography (micro-CT) and histology results at weeks 4, 8 and 12 post-surgery showed more newly formed bone in M250 group, with correspondingly more new vessel ingrowth. The results presented herein suggested that being osteopromotive, icaritin could enhance bone formation initiated by sub-microstructured CaP ceramics and the CaP ceramics scaffold incorporating icaritin is a promising biomaterial for the treatment of critical-sized defect.

Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

Doxycycline and Autogenous Bone in Repair of Critical-Size Defects.

The association of doxycycline (DOX) and autogenous bone on repair of critical-size defects was evaluated. Fifty albino rats were divided into 5 groups (n = 10). A 5-mm diameter defect was treated with: control (CO)-blood clot; DOX in Natrosol (NAT)-10% gel; NAT-gel; particulate autogenous bone (PAB); and PAB + DOX - PAB associated with 10% DOX gel. The animals were euthanized at 4 and 8 weeks postoperatively. Histomorphometric analysis was performed to assess the percentage of new bone in the defect area. Statistical analysis of the results was performed using analysis of variance and the Tukey test (P < 0.05). The results showed that new bone formation was limited to the margins of the defect. At 4 and 8 weeks, the group PAB + DOX showed higher bone formation (38.59% and 47.86%, respectively), with statistical difference in comparison with the CO (19.52%) at 4 weeks and CO (18.80%), DOX (22.05%), and NAT (15.89%) at 8 weeks (P < 0.05). The association of 10% DOX with autogenous bone significantly improved bone healing in critical-size defects.

Delivery of the improved BMP-2-Advanced plasmid DNA within a gene-activated scaffold accelerates mesenchymal stem cell osteogenesis and critical size defect repair

Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 μg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.

The influence of the protein Wnt10b as a marker of bone repair of critical size defects fille with autogenous adipose tissue graft: A study in rabbit calvaria

The proteins Wnts are considered a key regulator of the early development of the skeleton. Aim: The aim of this study was to evaluate the presence of the protein Wnt10b as a marker of bone repair in critical size defects surgically created in the calvaria of rabbits treated with fragmented autogenous adipose tissue graft. Methods: A total of 28 rabbits were divided into two groups: the Control group (C) and Adipose Tissue Graft group (ATG). A CSD measuring 15 mm in diameter was created in the calvaria of each animal. In rabbits of the C group, the defect was filled only with blood clot, and in ATG group, the defect was filled with fragmented adipose tissue graft. The two groups were divided into two subgroups (n = 7) for euthanasia 15 and 40 days after surgery. Histological and immunohistochemically analyses were performed to evaluate the neoformed bone and the presence/concentration of Wnt10b protein. The Kruskal-Wallis test was performed to compare the means and standard deviations of the number of Wnt10b + cells/mm2 in both groups in each postoperative period. It was assumed a significance level of 5%. Results: After 40 days, the mean concentration of the protein Wnt10b in ATG group was 26.26 (+-6.97) significant higher (p&lt;0,001) than the mean in C group that was 305 (37.41). Conclusion: The protein Wnt10b would play a crucial role in the signaling of bone formation in bone defects treated with fragmented autogenous adipose tissue graft.

Porcine Bone Scaffolds Adsorb Growth Factors Secreted by MSCs and Improve Bone Tissue Repair.

An ideal tissue-engineered bone graft should have both excellent pro-osteogenesis and pro-angiogenesis properties to rapidly realize the bone regeneration in vivo. To meet this goal, in this work a porcine bone scaffold was successfully used as a Trojan horse to store growth factors produced by mesenchymal stem cells (MSCs). This new scaffold showed a time-dependent release of bioactive growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in vitro. The biological effect of the growth factors-adsorbed scaffold on the in vitro commitment of MSCs into osteogenic and endothelial cell phenotypes has been evaluated. In addition, we have investigated the activity of growth factor-impregnated granules in the repair of critical-size defects in rat calvaria by means of histological, immunohistochemical, and molecular biology analyses. Based on the results of our work bone tissue formation and markers for bone and vascularization were significantly increased by the growth factor-enriched bone granules after implantation. This suggests that the controlled release of active growth factors from porcine bone granules can enhance and promote bone regeneration.

Combination of bone marrow mesenchymal stem cells and cartilage fragments contribute to enhanced repair of osteochondral defects.

Cartilage tissue engineering using stem cells and biomaterials is considered a promising approach despite poor outcomes. We hypothesise that articular cartilage fragments provides native environmental cues to enhance stem cell differentiation. As such we evaluated the chondrogenic differentiation and repair of critical size defect in a human explant osteochondral model (OD) using bone marrow derived mesenchymal stem cells (BM-MSCs) and homogenised cartilage. BM-MSCs were established from the bone-marrow plugs of patients undergoing total knee arthroplasty and characterized. Osteochondral tissue was trimmed and a central drill defect (∼2mm) was made. Chondrogenic repair was evaluated by filling the OD defect area with either BM-MSCs (Group II), homogenized cartilage (Group III) or a combination of both BM-MSCs and homogenized cartilage (Group IV). OD with no added cell or tissue served as control (Group I). Samples were maintained in chondrogenic differentiation medium for 28 days. Microscopic images showed maximal OD closure in Group IV. Partial OD closure was observed in Group II and to a lesser extent in Group III. Haematoxylin-eosin staining revealed immature cartilaginous matrix in Group II and more mature matrix in Group IV. Sircol™ Assay showed increased collagen deposition in both Group II and Group IV. Immunostaining for both groups revealed positive staining for type II collagen. Combining BM-MSCs and homogenised cartilage demonstrated enhanced cartilage formation and defect filling in a human ex-vivo osteochondral model.

Repair of critical size defects using bioactive glass seeded with adipose-derived mesenchymal stem cells.

Bioactive glass has been demonstrated as a biocompatible bone substitute. However bone healing process can be prolonged due to late resorption of the material. Adipose derived stem cells (ASC) have osteogenic differentiation potential and hence can be a cell source for bone regeneration. The aim of this study was to test whether combination of bioactive glass with ASCs would enhance bone regeneration. Following creation of critical sized defects on the calvaria of 32 Wistar rats, the animals were randomly divided into four groups: Group C (control): Defects were left untreated; Group G: Defects were covered with autologous bone graft; Group BG: Defects were filled with bioactive glass; Group BG/ASC: Defects were filled with bioactive glass seeded with ASCs. The defect size was significantly greater in Group C compared to all other groups. Bone density was significantly lower in Group C compared to Group G and Group BG/ASC. Bone regeneration score of Group C was significantly lower than other groups. Group BG/ASC demonstrated lamellar bone and havers canal formation. The results of this study demonstrated that bioactive glass implanted with ASC is a biocompatible construct stimulating radiologically and histologically evident bone regeneration similar to autologous bone grafting. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1002-1008, 2017.

Bone repair of critical size defects treated with autogenic, allogenic, or xenogenic bone grafts alone or in combination with rhBMP-2.

To evaluate the bone repair of critical size defects treated with autogenic, allogenic, or xenogenic bone grafts alone or in combination with rhBMP-2. In 112 rats, a critical bone defect of 5 mm bilaterally in the calvaria was made and filled with different bone grafts alone or combined with rhBMP-2: group autograft (AuG); group allograft (AlG); group xenograft (XeG); group AuG/BMP-2 (autograft and 5 μg rhBMP-2); group AlG/BMP-2 (allograft and 5 μg rhBMP-2); group XeG/BMP-2 (xenograft and 5 μg rhBMP-2); group BMP-2 (5 μg rhBMP-2); and control group, filled only with blood coagulum. After a period of 4 or 6 weeks, the animals were euthanized. Histological and histometric analyses were performed for new bone formation (NB), as well as the immunohistochemical detection of tartrate-resistant acid phosphatase (TRAP), and zymographic analysis (MMP) of type 2 and 9. Histological analysis showed the bone healing process was faster and favorable in AuG and AuG/BMP-2. In both periods, the grafted groups (AuG, XeG and AlG) had a greater volume of NB than the control group, which was even greater when combined with rhBMP-2. The XeG group showed a higher number of TRAP-positive multinucleated osteoclasts, and enzymatic activity revealed different levels of proMMP-2, MMP-2, and MMP-9 (6 weeks). The different types of grafts increased bone formation, mainly associated with rhBMP-2, enhancing and accelerating the repair process. These groups had higher enzymatic indices than the control group especially with XeG, which also showed higher TRAP-positive multinucleated cells similar to osteoclasts, suggesting a remodeling process.

Nonmulberry Silk Fibroin Scaffold Shows Superior Osteoconductivity Than Mulberry Silk Fibroin in Calvarial Bone Regeneration.

Recent years have witnessed the advancement of silk biomaterials in bone tissue engineering, although clinical application of the same is still in its infancy. In this study, the potential of pure nonmulberry Antheraea mylitta (Am) fibroin scaffold, without preloading with bone precursor cells, to repair calvarial bone defect in a rat model is explored and compared with its mulberry counterpart Bombyx mori (Bm) silk fibroin. After 3 months of implantation, Am scaffold culminates in a completely ossified regeneration with a progressive increase in mineralization at the implanted site. On the other hand, the Bm scaffold fails to repair the damaged bone, presumably due to its low osteoconductivity and early degradation. The deposition of bone matrix on scaffolds is evaluated by scanning electron and atomic force microscopy. These results are corroborated by in vitro studies of enzymatic degradation, colony formation, and secondary conformational features of the scaffold materials. The greater biocompatibility and mineralization in pure nonmulberry fibroin scaffolds warrants the use of these scaffolds as an "ideal bone graft" biomaterial for effective repair of critical size defects.

Evaluation of bone repair of critical size defects treated with simvastatin-loaded poly(lactic-co-glycolic acid) microspheres in rat calvaria.

Statins are hypolipemiant drugs with osteoinductive effect. We evaluated the potential of simvastatin loaded into poly(lactic-co-glycolic acid) (PLGA) microspheres to heal critical size defects in rat calvaria. PLGA scaffolds (50:50 ratio) were synthesized as pure membranes or as microspheres loaded with 2.5% simvastatin. Critical size defects (5-mm diameter) were created in the parietal bone of 3-month-old male Wistar rats; they were either left filled with blood clot (C group), covered with a PLGA membrane (M group) or with PLGA microspheres loaded with simvastatin (MSI group) or not (MM group), and then covered with the PLGA membrane. The defects were evaluated after 30 or 60 days by light and electron microscopy, immunohistochemistry for osteopontin (OPN), bone sialoprotein (BSP) and osteoadherin (OSAD), and immunocytochemistry for OPN. Scanning electron microscopy showed that the calvarial defects treated with MSI were almost completely healed after 60 days, while groups M and C presented less bone formation, whereas the bone matrix formed into the defects of MSI group was more organized and mature. The immunolabeling for OPN and BSP on the matrix in groups C and M showed typical areas of primary bone unlike the MSI that presented weak labeling at the formed area. In the MSI group, there was an intense immunostaining for OSAD in osteoid, as well as in osteocyte cytoplasm. The immunocytochemistry showed intense labeling for OPN with homogeneous distribution in the interfibrillar spaces in all groups after 30 days and after 60 days; however, while C and M groups exhibited similar aspect, the MSI specimens showed weak labeling. The ultrastructural evaluation showed the interaction between the biomaterial and the surrounding tissue where some cells established intimate contact with microspheres. The repair of critical size bone defects was accelerated and enhanced by the implantation of simvastatin-loaded PLGA microspheres.

Surfactant as a Critical Factor When Tuning the Hydrophilicity in Three-Dimensional Polyester-Based Scaffolds: Impact of Hydrophilicity on Their Mechanical Properties and the Cellular Response of Human Osteoblast-Like Cells

In tissue engineering, the hydrophilicity of porous scaffolds is essential and influences protein and cell adhesion as well as nutrient diffusion into the scaffold. The relative low hydrophilicity of degradable polyesters, which limits diffusion of nutrients, is a major drawback in large porous scaffolds of these materials when used for bone tissue engineering and repair of critical size defects. Designing porous biodegradable polymer scaffolds with improved hydrophilicity, while maintaining their mechanical, thermal, and degradation properties is therefore of clinical interest. Here, surfactants were used to tune the hydrophilicity and material properties. A total of 3-20% (w/w) of surfactant, polysorbate 80 (Tween 80), was used as an additive in poly(l-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] and poly(l-lactide)-co-(ε-caprolactone) [poly(LLA-co-CL)] scaffolds. A significantly decreased water contact angle was recorded for all the blends and the crystallinity, glass transition temperature and crystallization temperature were reduced with increased amounts of surfactant. Copolymers with the addition of 3% Tween 80 had comparable mechanical properties as the pristine copolymers. However, the E-modulus and tensile stress of copolymers decreased significantly with the addition of 10 and 20% Tween 80. Initial cell response of the material was evaluated by seeding human osteoblast-like cells (HOB) on the scaffolds. The addition of 3% Tween 80 did not significantly influence cell attachment or proliferation, while 20% Tween 80 significantly inhibited osteoblast proliferation. RT-PCR results showed that 3% Tween 80 stimulated mRNA expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), and bone morphogenetic protein-2 (BMP-2).

Effect of LLLT on autogenous bone grafts in the repair of critical size defects in the calvaria of immunosuppressed rats

The aim of this study was to evaluate the effects of low-level laser therapy (LLLT) on the bone repair of critical size defects (CSDs) filled with autogenous bone in the calvaria of immunosuppressed rats. A 5 mm-diameter CSD was created in the calvaria of 30 rats. The animals were divided into 5 groups (n = 6): Control (C) – the defect was filled with a blood clot; Dexamethasone (D) – dexamethasone treatment, and the defect was filled with a blood clot; Autogenous bone (AB) – dexamethasone treatment, and the defect was filled with autogenous bone; LLLT – dexamethasone treatment, and the defect received LLLT (660 nm; 35 mW; 24.7 J/cm2); and AB + LLLT – dexamethasone treatment, and the defect was filled with autogenous bone and received LLLT. All animals were euthanized at 30 postoperative days. Histometric and histological analyses were performed. The new bone area (NBA) was calculated as the percentage of the total area of the original defect. Data were analysed statistically (an analysis of variance and Tukey's test; P < 0.05). The AB + LLLT group showed the largest NBA of all groups (P < 0.05). The use of LLLT with AB effectively stimulated bone formation in CSDs in the calvaria of immunosuppressed rats.

Repair of critical-sized bone defects with anti-miR-31-expressing bone marrow stromal stem cells and poly(glycerol sebacate) scaffolds

The repair of critical-sized defects (CSDs) is a significant challenge in bone tissue engineering. Combining the use of progenitor cells with gene therapy represents a promising approach for bone regeneration. MicroRNAs play important roles in most gene regulatory networks, regulate the endogenous expression of multiple growth factors and simultaneously modulate stem cell differentiation. Our previous study showed that knocking down miR-31 promotes the osteogenesis of bone marrow stromal stem cells (BMSCs). To investigate the therapeutic potential of cells engineered to express anti-miR-31 for CSD repair, lentiviral vectors encoding negative control, miR-31 precursor and anti-sense sequences were constructed and transduced into osteo-inductive BMSCs. The expression of osteogenic-specific genes, alkaline phosphatase activity and Alizarin Red S staining were investigated to evaluate the effects of miR-31 on the cell fate of BMSCs over a 3-week period. In addition, miR-31-modified BMSCs seeded on poly(glycerol sebacate) (PGS) scaffolds were used to repair 8 mm critical-sized calvarial defects in rats. The results showed that miR-31 suppression significantly increased the expression of osteogenic-specific genes in vitro at the mRNA and protein levels, and that robust new bone formation with high local bone mineral density was observed in the anti-miR groups in vivo. Moreover, the PGS scaffolds carrying anti-miR-31-expressing BMSCs exhibited good biocompatibility and a high regeneration rate (~60%) within in vivo bone defects. Our results suggest that miR-31 gene delivery affects the potential of BMSCs for osteogenic differentiation and bone regeneration and that PGS is a potential substrate for genetically modified, tissue-engineered bone in the repair of large bone defects.

Bone repair of critical size defects treated with mussel powder associated or not with bovine bone graft: Histologic and histomorphometric study in rat calvaria

The objective of this study was to evaluate the bone repair of critical size defects treated with mussel powder with or without additional bovine bone. Critical size defects of 5 mm were realized in the calvaria of 70 rats, which were randomly divided in 5 groups – Control (C), Autogenous Bone (AB), Mussel Powder (MP), Mussel Powder and Bovine Bone (MP-BB) and Bovine Bone (BB). Histological and histomorphometric analysis were performed 30 and 90 days after the surgical procedures (ANOVA e Tukey p < 0.05). After 30 days, the measures of remaining particles were: 28.36% (MP-BB), 26.63% (BB) and 8.64% (MP) with a statistically significant difference between BB and MP. The percentage of osseous matrix after 30 days was, AB (55.17%), 23.31% (BB), 11.66% (MP) and 10.71% (MP-BB) with statistically significant differences among all groups. After 90 days the figures were 25.05% (BB), 21.53% (MP-BB) and 1.97% (MP) with statistically significant differences between MP-BB and MP. Percentages of new bone formation after 90 days were 89.47% (AB), 35.70% (BB), 26.48% (MP-BB) and 7.37% (MP) with statistically significant differences between AB and the other groups.Within the limits of this study, we conclude that mussel powder, with or without additional bovine bone, did not induce new bone formation and did not repair critical size defects in rat calvaria.

The role of miR-31-modified adipose tissue-derived stem cells in repairing rat critical-sized calvarial defects

With the increasing application of microRNAs (miRNAs) in the treatment and monitoring of different diseases, miRNAs have become an important tool in biological and medical research. Recent studies have proven that miRNAs are involved in the osteogenic differentiation of stem cells. However, few studies have reported the use of miRNA-modified adult stem cells to repair critical-sized defects (CSDs) using tissue engineering technology. It is known that miR-31 is a pleiotropically acting miRNA that inhibits cancer metastasis and targets special AT-rich sequence-binding protein 2 (Satb2) in fibroblasts. However, it is not clear whether the function of miR-31 is to enhance adipose tissue-derived stem cell (ASC) osteogenesis, along with its association with Satb2, during osteogenic differentiation and bone regeneration. In this study, we systematically evaluated the function of miR-31 in enhancing ASC osteogenesis and the therapeutic potential of miR-31-modified ASCs in a rat CSD model with β-tricalcium phosphate (β-TCP) scaffolds. ASCs were treated with lentivirus (Lenti)-miR-31, Lenti-as-miR-31 (antisense) or Lenti-NC (negative control). These genetically modified ASCs were then combined with β-TCP scaffolds to repair CSDs in rats. The results showed that in cultured ASCs in vitro, Lenti-as-miR-31 significantly enhanced osteogenic mRNA and protein expression when compared with the Lenti-NC group. Moreover, we firstly found that a Runt-related transcription factor 2 (Runx2), Satb2 and miR-31 regulatory loop triggered by bone morphogenetic protein-2 (BMP-2) plays an important role in ASCs' osteogenic differentiation and bone regeneration. More importantly, we found that miR-31-knockdown ASCs dramatically improved the repair of CSDs, including increased bone volume, increased bone mineral density (BMD) and decreased scaffold residue in vivo. These data confirm the essential role of miR-31-modified ASCs in osteogenesis in vitro and in vivo.

Current and Emerging Basic Science Concepts in Bone Biology

Ongoing research in bone biology has brought cutting-edge technologies into everyday use in craniofacial surgery. Nonetheless, when osseous defects of the craniomaxillofacial skeleton are encountered, autogenous bone grafting remains the criterion standard for reconstruction. Accordingly, the core principles of bone graft physiology continue to be of paramount importance. Bone grafts, however, are not a panacea; donor site morbidity and operative risk are among the limitations of autologous bone graft harvest. Bone graft survival is impaired when irradiation, contamination, and impaired vascularity are encountered. Although the dura can induce calvarial ossification in children younger than 2 years, the repair of critical-size defects in the pediatric population may be hindered by inadequate bone graft donor volume. The novel and emerging field of bone tissue engineering holds great promise as a limitless source of autogenous bone. Three core constituents of bone tissue engineering have been established: scaffolds, signals, and cells. Blood supply is the sine qua non of these components, which are used both individually and concertedly in regenerative craniofacial surgery. The discerning craniofacial surgeon must determine the proper use for these bone graft alternatives, while understanding their concomitant risks. This article presents a review of contemporary and emerging concepts in bone biology and their implications in craniofacial surgery. Current practices, areas of controversy, and near-term future applications are emphasized.