Critical-sized Calvarial Bone Defects Research Articles

Biological Compatibility Profile on Biomaterials for Bone Regeneration.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of β-tricalcium phosphate (β-TCP) disks. We also tested a β-TCP/Collagen (β-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

Macroporous poly (l-lactic acid)/chitosan nanofibrous scaffolds through cloud point thermally induced phase separation for enhanced bone regeneration

A new method called cloud point thermally induced phase separation (CP-TIPS) was developed to prepare poly (l-lactic acid) (PLLA) scaffolds with macropores over 300 μm in PLLA/dioxane/H2O ternary system. The concentration of PLLA was 4% (w/v), and the solvent was dioxane with 14% of deionized water. By maintaining the clear solution of the ternary system near and above the cloud point for 2 h, followed by a large enough quenching depth (−80 °C), the crystallization of the α′-form crystal became privileged, and the microstructure of the pore wall was dominated with nanofibers. In contrast, conventional TIPS process resulted in scaffolds with similar pore size but platelet-like microstructure on the pore walls. The unique macroporous nanofibrous PLLA scaffold showed well elasticity and flexibility after swelling in 70% acetone. This property further enabled a facile “mechanical squeeze and intake” of high concentration viscous chitosan solution into the porous structure to modify the composite scaffolds. The compressive modulus of macroporous nanofibrous PLLA/CS scaffolds reached about 1 MPa. The hydrophilicity of the scaffold was greatly enhanced and the pH value remained stable in the first 3 weeks after degradation in vitro. The effect of chitosan concentration in the composite scaffolds on cell affinity and the osteogenic differentiation was evaluated. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo and the composite scaffold with 3% chitosan significantly enhanced bone regeneration.

Preparation and osteogenic properties of poly ( L-lactic acid)/lecithin porous scaffolds with open pore structure

To investigate the preparation and osteogenic properties of poly ( L-lactic acid)(PLLA)/lecithin porous scaffolds with open pore structure. PLLA/lecithin porous scaffolds with different lecithin contents (0, 5%, 10%, 20%, 30%, 40%, 50%) were prepared by thermally induced phase separation (groups A, B, C, D, E, F, and G, respectively). Scanning electron microscopy (SEM) was used to observe the surface morphology of the scaffolds. Wide-angle X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to detect the crystallinity of the scaffolds. The water uptake ability of the scaffolds was measured. The cell growth and viability of bone marrow mesenchymal stem cells (BMSCs) of mouse on each scaffold was assessed by cell counting kit 8 (CCK-8) method. The osteogenic differentiation ability of BMSCs on each scaffold was evaluated by alkaline phosphatase (ALP) activity. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo. Micro-CT was used to reconstruct the three-dimensional model of the defect area, and the bone volume and bone mineral density were quantitatively analyzed. SEM results showed that the lecithin could slightly reduce the pore size; when lecithin content was 50%, platelet-like structure could be observed on the scaffolds. Wide angle XRD and DSC showed that the crystallinity of scaffolds gradually decreased with the increase of lecithin content. The water uptake ability test showed that the hydrophilicity of scaffolds increased with the increase of lecithin content. CCK-8 assay showed that cell activity gradually increased with the increase of culture time. After 7 days of culture, the absorbance ( A) value of groups C, D, E, and F were significantly higher than that of groups A, B, and G ( P<0.05), but no significant difference was found among groups C, D, E, and F ( P>0.05). After 14 days of osteogenic induction, with the increase of lecithin content, there was a significant difference in ALP activity of each group. The ALP activity in groups D, E, F, and G were significantly higher than that in groups A, B, and C ( P<0.05). In vivo, the results of Micro-CT examination and bone volume and bone mineral density showed that the scaffolds with 30% lecithin had the best repairing effect. Prepared by thermally induced phase separation, the cytocompatibility, osteogenic differentiation, and bone repair ability of the PLLA/lecithin porous scaffold is obviously better than that of pure PLLA scaffold. PLLA/lecithin porous scaffold with suitable lecithin content is a promising scaffold material for bone tissue engineering.

Bone Regeneration Using Adipose-Derived Stem Cells in Injectable Thermo-Gelling Hydrogel Scaffold Containing Platelet-Rich Plasma and Biphasic Calcium Phosphate.

For bone regeneration, a biocompatible thermo-gelling hydrogel, hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) was used as a three-dimensional organic gel matrix for entrapping rabbit adipose-derived stem cells (rASCs). Biphasic calcium phosphate (BCP) ceramic microparticles were embedded within the gel matrix as a mineralized bone matrix, which was further fortified with platelet-rich plasma (PRP) with osteo-inductive properties. In vitro culture of rASCs in HA-CPN and HA-CPN/PRP/BCP was compared for cell proliferation and osteogenic differentiation. Overall, HA-CPN/PRP/BCP was a better injectable cell carrier for osteogenesis of rASCs with increased cell proliferation rate and alkaline phosphatase activity, enhanced calcium deposition and mineralization of extracellular matrix, and up-regulated expression of genetic markers of osteogenesis. By implanting HA-CPN/PRP/BCP/rASCs constructs in rabbit critical size calvarial bone defects, new bone formation at the defect site was successfully demonstrated from computed tomography, and histological and immunohistochemical analysis. Taken together, by combining PRP and BCP as the osteo-inductive and osteo-conductive factor with HA-CPN, we successfully demonstrated the thermo-gelling composite hydrogel scaffold could promote the osteogenesis of rASCs for bone tissue engineering applications.

Low power laser irradiation and human adipose-derived stem cell treatments promote bone regeneration of critical-sized calvarial defects in rats

The treatments of large bone defects are often accompanied by various limitations and complications. Stem cell therapy and physical treatments have both been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli on large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low power laser irradiation (LPLI) and human ADSC treatments during fracture repair using a comparative rat calvarial defect model. The viability of human ADSCs was cultured on a porous PLGA scaffold using an MTS assay. The critical-sized (7 mm) calvarial bone defect Sprague–Dawley (SD) rats were divided into 4 groups: a control group, an LPLI group, an ADSC group, and an ADSC + LPLI group. Bone formation was evaluated using micro-CT at 4, 8, 12, and 16 weeks after surgery. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses showed that both the LPLI and ADSC groups improved calvarial bone defect healing compared to the control group. In addition, the ADSC + LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC + LPLI group. There were significant differences between the groups. The ADSC and ADSC + LPLI groups showed strong signals of vWF expression in week 16. ADSC and LPLI treatments improve fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and further enhances the bone healing process.

In vitro and in vivo investigation of PLA/PCL scaffold coated with metformin-loaded gelatin nanocarriers in regeneration of critical-sized bone defects

Large bone defects constitute a major challenge in bone tissue engineering and usually fail to heal due to the incomplete differentiation of recruited mesenchymal stem cells (MSCs) into osteogenic precursor cells. As previously proposed, metformin (MET) induces differentiation of MSCs into osteoblastic lineages in vitro. We fabricated a Poly (lactic acid) and Polycaprolactone (PLA/PCL) scaffold to deliver metformin loaded gelatin nanocarriers (MET/GNs) to critical-sized calvarial bone defects in a rat model. The scaffolds were evaluated regarding their morphology, porosity, contact angle, degradation rate, blood compatibility, biomechanical, cell viability and their osteogenic differentiation. In animal study, the defects were filled with autograft, scaffolds and a group was left empty. qRT-PCR analyses showed the expression level of osteogenic and angiogenic markers considerably increased in MET/GNs-PLA/PCL. The in vivo results showed that MET/GNs-PLA/PCL improved bone ingrowth, angiogenesis and defect reconstruction. Our results represent the applicability of MET/GNs-PLA/PCL for successful bone regeneration.

A novel Lipidoid-MicroRNA formulation promotes calvarial bone regeneration

Specific microRNAs (miRs) and the Wnt signaling pathway play critical roles in regulating bone development and homeostasis. Our previous studies revealed the ability of miR-335-5p to promote osteogenic differentiation by downregulating Wnt antagonist Dickkopf-1 (DKK1). The purpose of this study was to use nano-materials to efficiently deliver miR-335-5p into osteogenic cells for tissue engineering applications. We synthesized and screened a library of 12 candidate nano-lipidoids，of which L8 was identified as the preferred biodegradable lipidoid for miRNA molecule delivery into cells. We then investigated whether a lipidoid-miRNA formulation of miR-335-5-p (LMF-335) could successfully deliver miR-335-5-p into cells to promote osteogenesis in vitro and calvarial bone healing in vivo. Transfection of C3H10T1/2 cells and bone marrow stromal cells (BMSCs) with LMF-335 led to decreased expression of DKK1 and increased expression of the key osteogenic genes. LMF-335 and LMF-335-transfected BMSCs were then used in combination with silk scaffolds to evaluate healing of critical-size calvarial bone defects in mice. The results revealed significant new bone formation in the defects in LMF-335 groups as compared with control groups. In conclusion, this first report supports the notion that lipidoid delivery of miRNA can be used to induce osteogenic differentiation of stem cells and bone regeneration.

Enhanced Repairing of Critical-Sized Calvarial Bone Defects by Mussel-Inspired Calcium Phosphate Cement.

The goal of this study is to investigate the biological response of mussel-inspired calcium phosphate cement (CPC) in vivo. Polydopamine (PDA), which is analogous to that of mussel adhesive proteins, was added in CPC. PDA-CPC was implanted into the femur, muscle, and critical-sized calvarial bone defects of rabbits. Histomorphometry of the sequential fluorescence sections showed that PDA-CPC was capable of forming more newborn bone than the control-CPC. More new bone, bone marrow cavity, and blood vessel were observed in PDA-CPC than in the control-CPC in decalcified and undecalcified histological sections. Necrosis bone was not observed in PDA-CPC, whereas it appeared in the control-CPC after 2 weeks. The histological sections in muscle witnessed that there was more ingrowth of collagen in PDA-CPC than that in the control-CPC. There were no significantly difference in the number of leukocyte between PDA-CPC and the control-CPC in blood. It was confirmed that the addition of PDA enhanced the bone repairing ability and biocompatibility of PDA-CPC. Push-out testing indicated that PDA increased the bonding strength between PDA-CPC and host bone in the early stage. These present results indicated that PDA-CPC might be one potential bone graft with gratifying biocompatibility and enhanced bone repairing.

Biomimetic mineralization of carboxymethyl chitosan nanofibers with improved osteogenic activity in vitro and in vivo

Inspired by the natural extracellular matrix, the organic-inorganic composite nanofibers are promising scaffolds for bone tissue engineering. Chitosan-based nanofibers are widely used as bone tissue engineering scaffolds with good biocompatibility but pungent solvents are frequently used for its processing. Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, has better biodegradability and bioactivity which allows CMCS to chelate Ca2+ and induce the deposition of apatite. Moreover, with water as solvent, CMCS nanofibers avoid the acidic salt removal comparing to electrospun-chitosan. In this study, we successfully prepared uniform CMCS nanofibers with the aid of polyethylene oxide (PEO) and obtained the optimized conditions with a voltage of 25 kV and PEO of molecular weight 1000 kDa. We further prepared hydroxyapatite (HA) coated electrospun CMCS nanofibers by biomimetic mineralization using 5 times simulated body fluid. The promotion of osteogenic differentiation of mouse bone marrow stromal cells (mBMSCs) in vitro was evaluated on the nanofibers scaffolds. Cell experiments revealed that CMCS-HA composite nanofibers increased the ALP activity. The gene expression level of Runx2 and ALP were about 1.6 and 4.3 folds at the 7 days, and 5.1 and 10 folds at the 14 days on CMCS-HA nanofibrous membranes than that on CMCS alone samples. The level of OCN increased by 24 and 1.5 times on the CMCS-HA scaffolds than CMCS scaffolds at the 14 and 21 days. In vivo new bone formation by nanofiber scaffolds was investigated in a critical-size rat calvarial bone defect model. Micro-CT results showed that the whole defect was covered by new bone after CMCS-HA filling the defect for 12 weeks. The results of H&E staining and Masson’s trichrome staining on histological sections further confirmed that composite nanofibers promoted new bone formation and maturation.

Low power laser irradiation and human adipose-derived stem cell treatments promote bone regeneration in critical-sized calvarial defects in rats.

Both stem cell therapy and physical treatments have been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli for large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low-power laser irradiation (LPLI) and human adipose-derived stem cell (ADSC) treatments during fracture repair using a comparative rat calvarial defect model. We evaluated the viability of human ADSCs, which were cultured on a porous PLGA scaffold using an MTS assay. The critical-sized calvarial bone defect rats were divided into 4 groups: control group, LPLI group, ADSC group, and ADSC+LPLI group. Bone formation was evaluated using micro-CT. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. Our data showed that PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses revealed that both the LPLI and ADSC groups showed improved calvarial bone defect healing compared to the control group. In addition, the ADSC+LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC+LPLI group. There were significant differences between the groups. In addition, both ADSC and ADSC+LPLI groups showed strong signals of vWF expression. ADSC and LPLI treatments improved fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and LPLI further enhances the bone healing process.

Porous Polyethylene Coated with Functionalized Hydroxyapatite Particles as a Bone Reconstruction Material.

In this study, porous polyethylene scaffolds were examined as bone substitutes in vitro and in vivo in critical-sized calvarial bone defects in transgenic Sprague-Dawley rats. A microscopic examination revealed that the pores appeared to be interconnected across the material, making them suitable for cell growth. The creep recovery behavior of porous polyethylene at different loads indicated that the creep strain had two main portions. In both portions, strain increased with increased applied load and temperature. In terms of the thermographic behavior of the material, remarkable changes in melting temperature and heat fusion were revealed with increased the heating rates. The tensile strength results showed that the material was sensitive to the strain rate and that there was adequate mechanical strength to support cell growth. The in vitro cell culture results showed that human bone marrow mesenchymal stem cells attached to the porous polyethylene scaffold. Calcium sulfate–hydroxyapatite (CS–HA) coating of the scaffold not only improved attachment but also increased the proliferation of human bone marrow mesenchymal stem cells. In vivo, histological analysis showed that the study groups had active bone remodeling at the border of the defect. Bone regeneration at the border was also evident, which confirmed that the polyethylene acted as an osteoconductive bone graft. Furthermore, bone formation inside the pores of the coated polyethylene was also noted, which would enhance the process of osteointegration.

Parathyroid hormone gene-activated matrix with DFDBA/collagen composite matrix enhances bone regeneration in rat calvarial bone defects

BackgroundGene-activated matrix (GAM) induces sustained local production of growth factors to promote tissue regeneration. GAM contains a plasmid DNA (pDNA) encoding target proteins that is physically entrapped within a biodegradable matrix carrier. GAM with a pDNA encoding the first 34 amino acids of parathyroid hormone (PTH 1–34) and a collagen matrix enhances bone regeneration in long bone defects. Demineralized freeze-dried bone allograft (DFDBA) is a widely used osteoinductive bone graft. The present study determined the osteogenic effects of PTH-GAM with a collagen or DFDBA/collagen composite (D/C) matrix for treating craniofacial bone defects. MethodsWe constructed a pDNA encoding human PTH 1–34 and performed cyclic AMP ELISA to verify the bioactivity of PTH 1–34. Next, we generated a D/C matrix and PTH-GAMs containing a collagen matrix (PTH-C-GAM) or D/C matrix (PTH-D/C-GAM). Rats with critical-sized calvarial bone defects were divided into four groups, namely, untreated rats (sham group) and rats grafted with D/C matrix, PTH-C-GAM, or PTH-D/C-GAM (D/C, PTH-C-GAM, or PTH-D/C-GAM groups, respectively). PTH expression was determined by performing immunohistochemical staining after 4 and 8 weeks. New bone formation was evaluated by performing radiography, dual-energy X-ray absorptiometry, microcomputed tomography, and histological examination. ResultsPTH pDNA-transfected cells secreted bioactive PTH 1–34. Moreover, PTH was expressed at 4 and 8 weeks after the surgery in rats in the PTH-C-GAM group but not in rats in the D/C group. New bone formation in the calvarial bone defects, from more to less, was in the order of PTH-D/C-GAM, PTH-C-GAM, D/C, and sham groups. ConclusionOur results indicate that PTH-GAM with a collagen matrix promotes local PTH production for at least 8 weeks and bone regeneration in craniofacial bone defect. Moreover, our results indicate that replacement of the collagen matrix with the D/C matrix improves the osteogenic effects of PTH-GAM.

In Situ Bone Tissue Engineering With an Endogenous Stem Cell Mobilizer and Osteoinductive Nanofibrous Polymeric Scaffolds.

Classical bone tissue engineering involves the use of culture-expanded cells and scaffolds to produce tissue constructs for transplantation. Despite promising results, clinical adoption of these constructs has been limited due to various drawbacks, including extensive cell expansion steps, low cell survival rate upon transplantation, and the possibility of immuno-rejection. To bypass the ex vivo cell culture and transplantation process, the regenerative capacity of the host is exploited by mobilizing endogenous stem cells to the site of injury. Systemic injection of substance P (SP) induce mobilization of CD29+ CD105+ CD45- cells from bone marrow and enhance bone tissue regeneration in a critical-sized calvarial bone defect model. To provide an appropriate environment for endogenous stem cells to survive and differentiate into osteogenic lineage cells, electrospun nanofibrous polycaprolactone (PCL) scaffolds are functionalized with hydroxyapatite (HA) particles via a polydopamine (PDA) coating to create highly osteoinductive PCL-PDA-HA scaffolds that are implanted in defects. The combination of the PCL-PDA-HA scaffold and SP treatment enhance in situ bone tissue formation in defects. Thus, this in situ bone regeneration strategy, which combines recruitment of endogenous stem cells from the bone marrow to defective sites and implantation of a highly biocompatible and osteoinductive cell-free scaffold system, has potential as an effective therapeutic in regenerative medicine.

CXCL12 overexpression promotes the angiogenesis potential of periodontal ligament stem cells

Periodontal ligament stem cells (PDLSCs) are a major source of mesenchymal stem cells (MSCs) in adults and are effective for tissue engineering, like promoting angiogenesis and bone regeneration. CXCL12 has been reported to be involved in the recruitment and engraftment of MSCs in wound sites. However, whether CXCL12 potentiates the angiogenesis of PDLSCs is not clear. In this experiment, we transduced PDLSCs with CXCL12, and evaluated the angiogenesis potential of CXCL12-modified PDLSCs through in vitro and in vivo studies. The results showed that CXCL12 overexpression significantly stimulated the gene and protein expressions of bFGF, VEGF, SCF and PLGF in PDLSCs; CXCL12 gene modified PDLSCs formed longer capillary‐like structure; Moreover, in vivo transplanted PDLSCs transduced with CXCL12 could significantly promote bone tissue repair and angiogenesis in a rat critical-sized calvarial bone defect model. Taken together, our study confirmed that CXCL12 can enhance the angiogenesis potential of PDLSCs, which are crucial in the repair and regeneration of bone tissue.

The combination of nano-calcium sulfate/platelet rich plasma gel scaffold with BMP2 gene-modified mesenchymal stem cells promotes bone regeneration in rat critical-sized calvarial defects

BackgroundMesenchymal stem cells (MSCs) can be differentiated into an osteoblastic lineage in the presence of growth factors (GFs). Platelet-rich plasma (PRP), which can be easily isolated from whole blood, contains a large amount of GFs, and, therefore, promotes bone growth and regeneration. The main goal of this work was to develop and investigate the effect of a new sandwich-like bone scaffold which combines a nano-calcium sulfate (nCS) disc along with PRP fibrin gel (nCS/PRP) with BMP2-modified MSCs on bone repair and regeneration in rat critical-sized calvarial defects.MethodsWe evaluated the cytotoxicity, osteogenic differentiation and mineralization effect of PRP extract on BMP2-modified MSCs and constructed a sandwich-like nCS/PRP scaffold (mimicking the nano-calcium matrix of bone and carrying multi GFs in the PRP) containing BMP2-modified MSCs. The capacity of this multifunctional bone regeneration system in promoting bone repair was assessed in vivo in a rat critical-sized (8 mm) calvarial bone defect model.ResultsWe developed an optimized nCS/PRP sandwich-like scaffold. Scanning electron microscopy (SEM) results showed that nCS/PRP are polyporous with an average pore diameter of 70–80 μm and the cells can survive in the nCS/PRP scaffold. PRP extract dramatically stimulated proliferation and differentiation of BMP2-modified MSCs in vitro. Our in vivo results showed that the combination of BMP2-modified MSCs and nCS/PRP scaffold dramatically increased new bone regeneration compared with the groups without PRP and/or BMP2.ConclusionsnCS/PRP scaffolds containing BMP2-modified MSCs successfully promotes bone regeneration in critical-sized bone defects. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for critical-sized bone defects.

Electrospun Silk Fibroin Nanofibrous Scaffolds with Two-Stage Hydroxyapatite Functionalization for Enhancing the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.

Effect of Hydrogen Dioxide Treatment on the Osteogenic Potential of Duck-beak Bone-derived Natural Bioceramic Microparticles.

As an alternative material to the autogenous bone, duck-beak bone particle for bone substitute have been attracting great attention due to their biological properties. To deliver the most favorable outcome of medical treatment, it is essential to study the effect of various processing methods of the duck-beak bone. In this study, we compared the two deproteinizing agents for manufacturing duck-beak bone. Group 1 was treated by a conventional chemical agent (ethylenediamine) and Group 2 by hydrogen dioxide (H2O2). In vitro and in vivo experiments were conducted in parallel to compare the cytocompatibility and osteogenic capability between two processing methods. For in vitro tests, human adipose-derived mesenchymal stem cells (hAD-MSCs) were planted onto each sample and their attachment and growing were evaluated. For in vivo biocompatibility and osteogenic properties, the samples were applied on the critical-sized calvarial bone defect of rats. Group 2 showed significantly higher cell attachment but Group1 showed slightly higher cell proliferation. In in vivo tests, all groups have shown biocompatibility and increased level of osteogenic potential. However, Group 2 had significantly higher bone regeneration (p<0.05). This experiment confirmed that H2O2 can be an optimal processing method for duck-beak bone particle.

Chemical inhibitors of c-Met receptor tyrosine kinase stimulate osteoblast differentiation and bone regeneration

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been recently introduced to negatively regulate bone morphogenetic protein (BMP)-induced osteogenesis. However, the effect of chemical inhibitors of c-Met receptor on osteoblast differentiation process has not been examined, especially the applicability of c-Met chemical inhibitors on in vivo bone regeneration. In this study, we demonstrated that chemical inhibitors of c-Met receptor tyrosine kinase, SYN1143 and SGX523, could potentiate the differentiation of precursor cells to osteoblasts and stimulate regeneration in calvarial bone defects of mice. Treatment with SYN1143 or SGX523 inhibited HGF-induced c-Met phosphorylation in MC3T3-E1 and C3H10T1/2 cells. Cell proliferation of MC3T3-E1 or C3H10T1/2 was not significantly affected by the concentrations of these inhibitors. Co-treatment with chemical inhibitor of c-Met and osteogenic inducing media enhanced osteoblast-specific genes expression and calcium nodule formation accompanied by increased Runx2 expression via c-Met receptor-dependent but Erk-Smad signaling independent pathway. Notably, the administration of these c-Met inhibitors significantly repaired critical-sized calvarial bone defects. Collectively, our results suggest that chemical inhibitors of c-Met receptor tyrosine kinase might be used as novel therapeutics to induce bone regeneration.

Enhanced critical-size calvarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus.

Calvarial bone repair remains challenging for adults. Although adipose-derived stem cells (ASCs) hold promise to heal bone defects, use of ASCs for critical-size calvarial bone repair is ineffective. Stromal cell-derived factor 1 (SDF-1) is a chemokine capable of triggering stem cell migration. Although recombinant SDF-1 protein is co-delivered with other molecules including BMP-2 to facilitate calvarial bone repair, these approaches did not yield satisfactory healing. This study aimed to exploit a newly developed Cre/loxP-based hybrid baculovirus for efficient gene delivery and prolonged transgene expression in ASCs. We demonstrated that transduction of rat ASCs with the hybrid Cre/loxP-based baculovirus enabled robust and sustained expression of functional BMP-2 and SDF-1. Expression of BMP-2 or SDF-1 alone failed to effectively induce rat ASCs osteogenesis and healing of critical-size calvarial bone defects. Nonetheless, prolonged BMP-2/SDF-1 co-expression in ASCs synergistically activated both Smad and ERK1/2 pathways and hence potentiated the osteogenesis. Consequently, transplantation of the hybrid baculovirus-engineered, BMP-2/SDF-1-expressing ASCs/scaffold constructs potently healed the critical-size (6mm) calvarial bone defects (filling ≈70% of defect volume), which considerably outperformed the calvarial bone repair using BMP-2/SDF-1 delivered with biomaterial-based scaffolds. These data implicated the potential of Cre/loxP-based hybrid baculovirus vector for ASCs engineering and calvarial bone healing.

Regenerative capacity of augmented bone in rat calvarial guided bone augmentation model

PurposeGuided bone regeneration (GBR) is the most widely used technique to regenerate and augment bones. Even though augmented bones (ABs) have been examined histologically in many studies, few studies have been conducted to examine the biological potential of these bones and the healing dynamics following their use. Moreover, whether the bone obtained from the GBR procedure possesses the same functions as the existing autogenous bone is uncertain. In particular, little attention has been paid to the regenerative ability of GBR bone. Therefore, the present study histologically evaluated the regenerative capacity of AB in the occlusive space of a rat guided bone augmentation (GBA) model.MethodsThe calvaria of 30 rats were exposed, and plastic caps were placed on the right of the calvaria in 10 of the 30 rats. After a 12-week healing phase, critical-sized calvarial bone defects (diameter: 5.0 mm) were trephined into the dorsal parietal bone on the left of the calvaria. Bone particles were harvested from the AB or the cortical bone (CB) using a bone scraper and transplanted into the critical defects.ResultsThe newly generated bone at the defects' edge was evaluated using micro-computed tomography (micro-CT) and histological sections. In the micro-CT analysis, the radiopacity in both the augmented and the CB groups remained high throughout the observational period. In the histological analysis, the closure rate of the CB was significantly higher than in the AB group. The numbers of cells positive for runt-related transcription factor 2 (Runx2) and tartrate-resistant acid phosphatase (TRAP) in the AB group were larger than in the CB group.ConclusionsThe regenerative capacity of AB in the occlusive space of the rat GBA model was confirmed. Within the limitations of this study, the regenerative ability of the AB particulate transplant was inferior to that of the CB particulate transplant.