Critical-sized Rat Calvarial Defects Research Articles

Preparation and evaluation of osteogenic nano-MgO/PMMA bone cement for bone healing in a rat critical size calvarial defect.

The clinical outcomes of polymethylmethacrylate (PMMA) bone cement used to fill gaps or marrow cavities of bones and bone defects are limited due to poor handling properties, mismatched mechanical properties with natural bone and lack of osteogenesis for bone healing. In this study, a series of PMMA bone cements containing active nano-MgO particles (nano-MgO/PMMA) were prepared. The handling and mechanical properties were systemically evaluated according to an International Standardization Organization standard (ISO 5833:2002). The biocompatibility and osteogenic activity of nano-MgO/PMMA were also analysed in vitro. The osteogenic effects of nano-MgO/PMMA were assessed in a rat calvarial critical bone defect model. The addition of less than 15 wt% nano-MgO to PMMA improved the handling properties of PMMA. Compared with PMMA, the compression modulus and strength of 20MP (20 wt% nano-MgO to PMMA) decreased to 0.725 ± 0.023 GPa and 25.38 ± 2.82 MPa, respectively. In vitro studies with MC3T3-E1 showed that nano-MgO/PMMA had better biocompatibility than the PMMA group after 7 days of culture. The nano-MgO/PMMA groups showed more calcium nodules and higher osteogenic gene expression levels than PMMA after 12 days of osteogenic induction of the rat BMSCs. The in vivo studies analysed by micro-CT and histomorphology results proved that nano-MgO/PMMA could significantly enhance new bone formation. The mean new bone mineral density in the nano-MgO/PMMA group was 50% greater than that in the PMMA group. In addition, biomechanical tests showed that nano-MgO/PMMA was superior to PMMA in bone-bonding strength after 12 weeks implantation. Therefore, the nano-MgO/PMMA bone cement has good potential in joint fixation and bone defect filling applications.

In vivo study of polyurethane and tannin-modified hydroxyapatite composites for calvarial regeneration

Biomaterial mediated bone regeneration is an attractive strategy for bone defect treatment. Organic/inorganic composites have been well established as effective bone graft. Here, the bone regenerative effect of the composites made from tannic acid (TA) modified hydroxyapatite (HA) (THA) or TA & silver nanoparticles (Ag NPs) modified HA (Ag-THA) and polyurethane (PU) was evaluated on critical-sized calvarial defects in rats. The in vivo study indicates that PU/THA and PU/Ag-THA scaffolds exhibited acceptable biocompatibility and induced significantly enhanced bone mineral densities comparing with the blank control (CON) group as well as PU/HA group. The inclusion of TA on HA brought the composites with enhanced osteogenesis and angiogenesis, evidenced by osteocalcin (OCN) and vascular endothelial growth factor (VEGF) immunohistochemical staining. Tartrate resistant acid phosphatase (TRAP) staining showed high osteoclast activity along with osteogenesis, especially in PU/THA and PU/Ag-THA groups. However, further introduction of Ag NPs on HA depressed the angiogenesis of the composites, leading to even lower VEGF expression than that of CON group. This study once more proved that THA can serve as a better bone composite component that pure HA and can promote osteogenesis and angiogenesis. While, the introduction of antimicrobial Ag NPs on HA need to be controlled in some extent not to affect the angiogenesis of the composites.

Bioinspired nanocomposite fibrous scaffold mediated delivery of ONO-1301 and BMP2 enhance bone regeneration in critical sized defect.

Treatment aiming to enhance bone tissue regeneration can benefit from multiple growth factor or small molecule delivery. In the present study, the objective was to evaluate the feasibility of using nanocomposite fibrous scaffold to deliver prostacyclin I2 agonist ONO-1301 in combination with BMP2 for treating critical sized bone defect. For this, ONO-1301 at three different concentrations (1.67μg, 5μg, 15μg) and a fixed dose of BMP2 (5μg) was loaded on the scaffold via physical adsorption. The results showed fast release of ONO-1301 for two weeks, whereas BMP2 exhibited slow and sustained release for four weeks. The scaffold with dual factors promoted the migration and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro when compared to the scaffold with BMP2 alone. It also augmented bone tissue regeneration in critical sized rat calvarial defect at 12weeks; mainly with lower dose of ONO-1301. However, synergistic effect on osteogenic differentiation and bone regeneration were not obtained through the concurrent release of BMP-2 and ONO-1301.

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis invitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

Periosteal matrix-derived hydrogel promotes bone repair through an early immune regulation coupled with enhanced angio- and osteogenesis

Bone healing is a complex physiological process initiated by early regulation of the inflammatory immunity and entails multiple events including angiogenesis, osteogenic differentiation, and biomineralization. Here, we fabricated an injectable periosteal extracellular matrix (PEM) hydrogel that dynamically integrates multiple biological functions and, therefore, acts at different stages of the fracture healing process. PEM hydrogels were fully characterized compared with a collagen I hydrogel. The effects of PEM hydrogels on the different phases of the healing process were assessed in vitro. PEM hydrogels induced the recruitment and M2-polarization of macrophages, promoted the differentiation of MSCs into endothelial-like cells, HUVEC tube formation, osteogenic differentiation of primary calvarial osteoblasts and MSCs, and mineralization after being immersed in simulated body fluid. The dynamic and multiphase effects of the hydrogels were evaluated using a rat critical-sized calvarial defect model in vivo. During the early phase of repair, PEM hydrogels facilitated the M1-to-M2 transition of macrophages. As bone repair progressed, PEM hydrogels promoted blood vessel migration, the development of relative larger blood vessels, and functional vascularization. These effects were also verified in a subcutaneous embedding model. Eventually, PEM hydrogels promoted mature bone formation in large bone defects to a greater extent than collagen I hydrogels. These biological effects coordinated well with the natural process of bone regeneration. Thus, PEM hydrogels may serve as promising biomaterials in bone tissue engineering.

Silicone resin derived larnite/C scaffolds via 3D printing for potential tumor therapy and bone regeneration

Three dimensional (3D) printing has been used to fabricate bioceramic scaffolds for treating the tumor-related defects in recent years, but the fabrication process and the introduction of anti-tumor agents are still challenging. In this study, porous free carbon-embedding larnite (larnite/C) scaffolds have been successfully fabricated by 3D printing of the silicone resin loaded with CaCO3 filler and high temperature treatment under an inert atmosphere. The fabricated larnite/C scaffolds had uniform interconnected macropores (ca. 400 μm), and exhibited excellent photothermal effect, which was able to kill human osteosarcoma cells (MNNG/HOS) and inhibit the tumor growth in nude mice. Moreover, the larnite/C scaffolds could stimulate the expression of osteogenesis-related gene (ALP, OCN and Runx-2) in rat bone mesenchymal stem cells (rBMSCs), and also promoted new bone formation in critical-sized rat calvarial defects. Therefore, the combination of 3D printing with polymer-derived ceramics strategy could fabricate multifunctional bioceramic scaffolds, which would be promising for potential application in treating tumor-related bone defects.

Platelet-rich plasma incorporated electrospun PVA-chitosan-HA nanofibers accelerates osteogenic differentiation and bone reconstruction

Biologically active materials and polymeric materials used in tissue engineering have been one of the most attractive research areas in the past decades, especially the use of easily accessible materials from the patients that reduces or eliminates any patient's immune response. In this study, electrospun nanofibrous scaffolds were fabricated by using polyvinyl-alcohol (PVA), chitosan and hydroxyapatite (HA) polymers and platelet-rich plasma (PRP) as a bioactive substance isolated from human blood. Fabricated scaffold's structure and cytotoxicity were evaluated using scanning electron microscope and MTT assays. Scaffolds osteoinductivity was investigated by osteogenic differentiation of the mesenchymal stem cells (MSCs) at the in vitro level and then its osteoconductivity was examined by implanting at the critical-sized rat calvarial defect. The in vitro results showed that scaffolds have a good structure and good biocompatibility. Alkaline phosphatase activity, calcium content and gene expression assays were also demonstrated that their highest amount was detected in MSCs-seeded PVA-chitosan-HA(PRP) scaffold. For this reason, this scaffold alone and along with the MSCs was implanted to the animal defects. The in vivo results demonstrated that in the animals implanted with PVA-chitosan-HA(PRP), the defect was repaired to a good extent, but in those animals that received MSCs-seeded PVA-chitosan-HA(PRP), the defects was almost filled. It can be concluded that, PVA-chitosan-HA(PRP) alone or when stem cells cultured on them, has a great potential to use as an effective bone implant.

Collagen-infilled 3D printed scaffolds loaded with miR-148b-transfected bone marrow stem cells improve calvarial bone regeneration in rats.

Differentiation of progenitors in a controlled environment improves the repair of critical-sized calvarial bone defects; however, integrating micro RNA (miRNA) therapy with 3D printed scaffolds still remains a challenge for craniofacial reconstruction. In this study, we aimed to engineer three-dimensional (3D) printed hybrid scaffolds as a new ex situ miR-148b expressing delivery system for osteogenic induction of rat bone marrow stem cells (rBMSCs) in vitro, and also in vivo in critical-sized rat calvarial defects. miR-148b-transfected rBMSCs underwent early differentiation in collagen-infilled 3D printed hybrid scaffolds, expressing significant levels of osteogenic markers compared to non-transfected rBMSCs, as confirmed by gene expression and immunohistochemical staining. Furthermore, after eight weeks of implantation, micro-computed tomography, histology and immunohistochemical staining results indicated that scaffolds loaded with miR-148b-transfected rBMSCs improved bone regeneration considerably compared to the scaffolds loaded with non-transfected rBMSCs and facilitated near-complete repair of critical-sized calvarial defects. In conclusion, our results demonstrate that collagen-infilled 3D printed scaffolds serve as an effective system for miRNA transfected progenitor cells, which has a promising potential for stimulating osteogenesis and calvarial bone repair.

Fabrication of vascularized and scaffold-free bone tissue using endothelial and osteogenic cells differentiated from bone marrow derived mesenchymal stem cells

Over-dependence on existing synthetic scaffolds and insufficient vascularization limit the development of tissue engineered bone (TEB). The purpose of this study is to fabricate vascularized and scaffold-free bone tissue using cell sheet technology and to assess its feasibility to repair critical-sized calvarial defects in rats. Firstly, the pre-vascularized cell sheet was formed by seeding BMSC-derived endothelial cells (ECs) on an undifferentiated BMSCs cell sheet layer in vitro. After 3 days of co-culture, ECs migrated and rearranged to form lumens on the BMSC sheet. Secondly, osteogenic cell sheet was formed by inducing osteogenic differentiation of high density BMSCs. Then, the pre-vascularized cell sheet was stacked on BMSC-derived osteogenic cell sheet to fabricate a scaffold-free construct for bone regeneration. Finally, the scaffold-free construct with both angiogenic and osteogenic potential was implanted into critical-sized calvarial defects in adult Wistar rats. Results showed that more functional perfused blood vessels and new bone tissue formed in the pre-vascularized group than that in the controls (both empty and non-pre-vascularized cell sheet group). This study indicates a new promising strategy for bone tissue regeneration.

Rapid Regeneration of Vascularized Bone by Nanofabricated Amorphous Silicon Oxynitrophosphide (SiONP) Overlays.

Fracture healing is a complex biological process. Severe bone loss and ischemia from traumatic fractures lead to inflammation and accumulation of damaging reactive oxygen species (ROS). Fixative devices that not only provide mechanical support but also stimulate antioxidants such as superoxide dismutase (SOD1) and influence signaling pathways for extracellular matrix (ECM) mineralization, are critical for normal healing of such fractures. In this study, we report a novel biomaterial, silicon oxynitrophosphide (SiONP) that provides sustained release of ionic silicon (Si+4) and phosphorous (P) over few weeks under physiological conditions. Anti-oxidant role of Si+4 and augmented ECM mineralization by P ions lead to enhanced osteogenesis coupled with quick revascularization for rapid bone regeneration. Plasma enhanced chemical vapor deposition (PECVD) provided a conformal, well adherent and highly reproducible surface chemistry overlaid onto nanofabricated bioinspired surfaces. The Nitrogen to P and O content ratio was observed to change the dissolution rate and the release kinetics of the overlaid film. The SiONP films with optimal release kinetics promoted anti-oxidant expression via enhanced SOD1, which downstream upregulated other osteogenic markers with MC3T3-E1 cells. These surfaces also promoted angiogenesis evident by formation of thicker tubules by Human umbilical vein endothelial cells (HUVEC). In-vivo evaluation using a rat critical-sized calvarial defect model showed rapid bone-regeneration for these nanofabricated biomaterials as compared to control groups, and opens new horizon for future clinical trials of new antioxidant materials on biomedical devices that can reduce healing time, lower medical care cost, and increase the quality of newly formed bone in critical size defects.

Supercritical CO2 foamed composite scaffolds incorporating bioactive lipids promote vascularized bone regeneration via Hif-1α upregulation and enhanced type H vessel formation

Bone tissue engineering has substantial potential for the treatment of massive bone defects; however, efficient vascularization coupled with bone regeneration still remains a challenge in this field. In the current study, supercritical carbon dioxide (scCO2) foaming technique was adopted to fabricate mesoporous bioactive glasses (MBGs) particle-poly (lactic-co-glycolic acid) (PLGA) composite scaffolds with appropriate mechanical and degradation properties as well as in vitro bioactivity. The MBG-PLGA scaffolds incorporating the bioactive lipid FTY720 (designated as FTY/MBG-PLGA) exhibited simultaneously sustained release of the bioactive lipid and ions. In addition to providing a favorable microenvironment for cellular adhesion and proliferation, FTY/MBG-PLGA scaffolds significantly facilitated the in vitro osteogenic differentiation of rBMSCs and also markedly stimulated the upregulation of Hif-1α expression via the activation of the Erk1/2 pathway, which mediated the osteogenic and pro-angiogenic effects on rBMSCs. Furthermore, FTY/MBG-PLGA extracts induced superior in vitro angiogenic performance of HUVECs. In vivo evaluation of critical-sized rat calvarial bone defects indicated that FTY/MBG-PLGA scaffolds potently promoted vascularized bone regeneration. Notably, the significantly enhanced formation of type H vessels (CD31hiEmcnhi neo-vessels) was observed in newly formed bone tissue in FTY/MBG-PLGA group, strongly suggesting that FTY720 and therapeutic ions released from the scaffolds synergistically induced more type H vessel formation, which indicated the coupling of angiogenesis and osteogenesis to achieve efficiently vascularized bone regeneration. Overall, the results indicated that the foamed porous MBG-PLGA scaffolds incorporating bioactive lipids achieved desirable vascularization-coupled bone formation and could be a promising strategy for bone regenerative medicine. Statement of SignificanceEfficacious coupling of vascularizationandbone formation is critical for the restoration of large bone defects. Anoveltechnique was used to fabricate composite scaffolds incorporating bioactive lipids which possessedsynergistic cues of bioactive lipids and therapeutic ions to potently promotebone regenerationas well as vascularization. The underlying molecular mechanism for the osteogenic and pro-angiogenic effects of the compositescaffolds was unveiled. Interestingly, the scaffolds were furtherfoundto enhance the formation oftype H capillarieswithin the bone healing microenvironment to couple angiogenesis to osteogenesis to achieve satisfyingvascularizedbone regeneration.These findings provide a novel strategy to develop efficiently vascularized engineering constructs to treat massive bone defects.

Imaging of nano-hydroxyapatite/chitosan scaffolds using a cone beam computed tomography device on rat calvarial defects with histological verification.

Τhis study aims at determining the ability of cone beam computed tomography (CBCT) to visualize critical-size defects (CSD) created at rat calvaria and filled with 75/25 w/w nano-hydroxyapatite/chitosan (nHAp/CS) scaffolds, prior to their histological investigation. Thirty adult Sprague Dawley rats, 15 males and 15 females, were used. Two CSD, 5mm in diameter, were bilaterally trephined in the parietal bone. The right CSD was filled with nHAp/CS scaffold, while the left CSD remained empty, as the control group. Two female rats died post-operatively. Rats were euthanized at 2, 4, and 8weeks post-surgery. Twenty-eight specimens (15 × 2 × 10mm) were resected-containing both CSDs-and then scanned using a NewTom VGi CBCT imaging unit (Verona, Italy). The manufacturer's software trace region profile tool (NNT v6.2, Verona, Italy) was used in selected axial slices. The greyscale value (in VGiHU) and the traced/selected region of interest (ROI, in mm2) of those areas were automatically calculated. Subsequently, all specimens were histologically examined. An increased VGiHU (P = 0.000), was observed in the experimental group relative to the control group. The ROI of CSD (in mm2) was significantly reduced (P = 0.001) from the fourth to the eighth week in both groups. No statistically significant difference between male and female rats (P = 0.188) was observed with respect to VGiHU. The nHAp/CS scaffolds are easily visualized using a particular high-resolution CBCT device. Both the CBCT measurements and also the histological results suggest that the nHAp/CS scaffold presence contributes to new bone formation in rat calvarial CSD.

Thermally triggered injectable chitosan/silk fibroin/bioactive glass nanoparticle hydrogels for in-situ bone formation in rat calvarial bone defects

Copper-containing bioactive glass nanoparticles (Cu-BG NPs) with designed compositions and sizes were synthesized and incorporated into chitosan (CH)/silk fibroin (SF)/glycerophosphate (GP) composites to prepare injectable hydrogels for cell-free bone repair. The resulting Cu-BG/CH/SF/GP gels were found to exhibit well-defined injectability and to undergo rapid gelation at physiological temperature and pH. They were highly porous and showed the ability to administer Si, Ca and Cu ions at their respective safe doses in a sustained and controlled manner. In vitro studies revealed that the gels supported the growth of seeded MC3T3-E1 and human umbilical vein endothelial cells, and effectively induced them toward osteogenesis and angiogenesis, respectively. In vivo bone repair based on a critical-size rat calvarial bone defect model demonstrated that the optimal Cu-BG/CH/SF/GP gel was able to fully restore the bone defect with formation of vascularized bone tissue and mineralized collagen deposition during a treatment period of 8 weeks without utilization of any cells and/or growth factors. The results suggest that the presently developed Cu-BG/CH/SF/GP composite hydrogels have great potential and translation ability for bone regeneration owing to their thermo-sensitive properties, cell-free bioactivity, and cost-effectiveness. Statement of SignificanceHydrogels loaded with cells and/or growth factors exhibit potential in bone repair. However, they have been facing obstacles related to the clinic translation. Here, a novel type of hydrogel system consisting of copper-containing bioactive glass nanoparticles and chitosan/silk fibroin composite was developed. These gels showed injectability and thermally triggered in situ gelation properties and were able to administer the release of ions at safe but effective doses in a controlled manner while inducing the seeded cells toward osteogenesis and angiogenesis. The optimal gel showed the ability to fully repair critical-size rat calvarial bone defects without involving time consuming cell processing and/or the use of expensive growth factors, confirming that this novel hydrogel system has great potential for translation to the clinic.

Adenoviral mediated mono delivery of BMP2 is superior to the combined delivery of BMP2 and VEGFA in bone regeneration in a critical-sized rat calvarial bone defect

Apart from osteogenesis, neovascularization of the defect area is an important determinant for successful bone healing. Accordingly, several studies have employed the combined delivery of VEGFA and BMP2 for bone regeneration. Nevertheless, the outcomes of these studies are highly variable. The aim of our study was to compare the effectiveness of adenoviral mediated delivery of BMP2 alone and in combination with VEGFA in rat bone marrow stromal cells (rBMSC) seeded on a poly(LLA-co-CL) scaffold in angiogenesis and osteogenesis using a critical-sized rat calvarial defect model. Both mono delivery of BMP2 and the combined delivery of a lower ratio of VEGFA and BMP2 (1:4) led to up-regulation of osteogenic genes (Alpl and Runx2) and increased calcium deposition in vitro, compared with the GFP control. Micro computed tomography (microCT) analysis of the rat calvarial defect at 8 weeks showed that the mono delivery of BMP2 (43.37 ± 3.55% defect closure) was the most effective in healing the bone defect, followed by the combined delivery of BMP2 and VEGFA (27.86 ± 2.89%) and other controls. Histological and molecular analyses supported the microCT findings. Analysis of the angiogenesis, however, showed that both mono delivery of BMP2 and combined delivery of BMP2 and VEGFA had similar angiogenic effect in the calvarial defects. Examination of the key genes related to host response against the adenoviral vectors showed that the current model system was not associated with adverse immune response. Overall, the results show that the mono delivery of BMP2 was superior to the combined delivery of BMP2 and VEGFA in healing the critical-sized rat calvarial bone defect. These findings underscore the importance of appropriate growth factor combination for the successful outcome in bone regeneration.

Developing a Strontium-Releasing Graphene Oxide-/Collagen-Based Organic-Inorganic Nanobiocomposite for Large Bone Defect Regeneration via MAPK Signaling Pathway.

Significant efforts have been dedicated to fabricating favorable biomaterial-based bone substitutes for the repair of large bone defects. However, the development of bone biomaterials with suitable physiochemical and osteoinductive properties remains a challenge. Here, novel strontium-graphene oxide (Sr-GO) nanocomposites that allow long-term release of Sr ions are fabricated, which are used to reinforce collagen (Col) scaffolds through covalent cross-linking. The prepared Sr-GO-Col scaffold demonstrates significantly high water retention rates and excellent mechanical properties compared with unmodified Col scaffolds. The Sr-GO-modified Col scaffolds display a strong effect on adipose-derived stem cells by facilitating cell adhesion and osteogenic differentiation and by promoting the secretion of angiogenic factors to stimulate the in vitro tube formation of endothelial cells. Additionally, the secretion of angiogenic VEGF and osteogenic BMP-2 proteins is increased, which may be attributed to the synergistic effects of GO and Sr on the activation of the MAPK signaling pathway. The Sr-GO-Col constructs were then transplanted into rat critical-size calvarial bone defects, which showed the best bone regeneration and angiogenesis outcome at 12 weeks. Moreover, histological staining results show that the Sr-GO-Col group achieved complete defect bridging with the newly formed bone tissue and the residual Sr-GO nanoparticles are phagocytosed and degraded by multinucleated giant cells. These findings reveal that the incorporation of inorganic Sr-GO nanocomposites into biocompatible Col scaffolds is a viable strategy for fabricating favorable substitutes that enhance the regeneration of large bone defects.

Sinapic acid-loaded chitosan nanoparticles in polycaprolactone electrospun fibers for bone regeneration in vitro and in vivo

Sinapic acid (SA) is a plant-derived phenolic compound known for its multiple biological properties, but its role in the promotion of bone formation is not yet well-studied. Moreover, the delivery of SA is hindered by its complex hydrophobic nature, limiting its bioavailability. In this study, we fabricated a drug delivery system using chitosan nanoparticles (nCS) loaded with SA at different concentrations. These were incorporated into polycaprolactone (PCL) fibers via an electrospinning method. nCS loaded with 50 μM SA in PCL fibers promoted osteoblast differentiation. Furthermore, SA treatment activated the osteogenesis signaling pathways in mouse mesenchymal stem cells. A critical-sized rat calvarial bone defect model system identified that the inclusion of SA into PCL/nCS fibers accelerated bone formation. Collectively, these data suggest that SA promoted osteoblast differentiation in vitro and bone formation in vivo, possibly by activating the TGF-β1/BMP/Smads/Runx2 signaling pathways, suggesting SA might have therapeutic benefits in bone regeneration.

Bovine bone particulates containing bone anabolic factors as a potential xenogenic bone graft substitute

BackgroundAlternative grafts are needed to improve the healing of bone non-union. Here, we assessed a bovine bone product which retains the inorganic and organic components of bone, as an alternative bone graft.MethodsBovine bone matrix proteins (BBMPs) were isolated from bovine bone particulates (BBPs) and tested in vitro. Primary rat osteoblast viability, differentiation, and mineralisation were assessed with alamarBlue®, real-time PCR, and von Kossa staining assays, respectively. Osteoclast formation was assessed in primary murine bone marrow cultures with TRAP staining.Human osteoblast growth and differentiation in the presence of BBPs was evaluated in 3D collagen gels in vitro using alamarBlue® and real-time PCR, respectively. The efficacy of BBPs as an alternative bone graft was tested in a rat critical-size calvarial defect model, with histology scored at 4 and 12 weeks post-surgery.ResultsIn vitro, the highest concentration of BBMPs increased mineral deposition five-fold compared to the untreated control group (P < 0.05); enhanced the expression of key osteoblast genes encoding for RUNX2, alkaline phosphatase, and osteocalcin (P < 0.05); and decreased osteoclast formation three-fold, compared to the untreated control group (P < 0.05).However, the BBPs had no effect on primary human osteoblasts in vitro, and in vivo, no difference was found in healing between the BBP-treated group and the untreated control group.ConclusionsOverall, despite the positive effects of the BBMPs on the cells of the bone, the bovine bone product as a whole did not enhance bone healing. Finding a way to harness the positive effect of these BBMPs would provide a clear benefit for healing bone non-union.

Catalpol promotes the osteogenic differentiation of bone marrow mesenchymal stem cells via the Wnt/\u03b2-catenin pathway

BackgroundRehmanniae Radix is a traditional herbal medicine in East Asia that has been widely used to treat patients with osteoporosis. However, the effect of catalpol, the primary active principle component of Rehmanniae Radix, on the function of bone marrow mesenchymal stem cells (BMSCs) and the underlying molecular mechanisms associated with its activity remain poorly understood.MethodsThe effect of catalpol on the proliferation of BMSCs was evaluated using a Cell Counting Kit-8 assay. Alkaline phosphatase (ALP) staining, ALP activity and Alizarin Red staining were performed to elucidate the effect of catalpol on the osteogenesis of BMSCs. qRT-PCR, Western blotting and immunofluorescence were performed to evaluate the expression of osteo-specific markers and the Wnt/β-catenin signalling-related genes and proteins. Moreover, a rat critical-sized calvarial defect model and a rat ovariectomy model were used to assess the effect of catalpol on bone regeneration in vivo.ResultsCatalpol significantly enhanced osteoblast-specific gene expression, alkaline phosphatase activity and calcium deposition in BMSCs in vitro. This phenomenon was accompanied by an upregulation of Wnt/β-catenin signalling. In addition, the enhanced osteogenesis due to catalpol treatment was partially reversed by a Wnt/β-catenin antagonist. Furthermore, catalpol increased the bone healing capacity of BMSCs in a rat critical-sized calvarial defect model and attenuated bone loss in a rat ovariectomy model.ConclusionsThese data suggest that catalpol enhances the osteogenic differentiation of BMSCs, partly via activation of the Wnt/β-catenin pathway. Catalpol may provide a new strategy for bone tissue engineering and can be a potential agent for the treatment of postmenopausal osteoporosis.

ScCO &lt;sub&gt;2&lt;/sub&gt; Foamed Composite Scaffolds Incorporating Bioactive Lipids Promote Vascularized Bone Regeneration Via Hif-1α Upregulation and Enhanced Type H Vessel Formation

Bone tissue engineering has substantial potential for the treatment of massive bone defects; however, efficient vascularization coupled with bone regeneration still remains a challenge in this field. In the current study,supercritical carbon dioxide (scCO2) foaming technique was adopted to fabricate mesoporous bioactive glasses (MBGs) particle-poly (lactic-co-glycolic acid) (PLGA) composite scaffolds with appropriate mechanical and degradation properties as well as in vitro bioactivity. The MBG-PLGA scaffolds incorporating the bioactive lipid FTY720 (designated as FTY/MBG-PLGA) exhibited simultaneously sustained release of thebioactive lipid and ions. In addition to providing a favorable microenvironment for cellular adhesion and proliferation, FTY/MBG-PLGA scaffolds significantly facilitated the in vitro osteogenic differentiation of rBMSCs and also markedly stimulated the up regulation of Hif-1α expression via the activation of the Erk1/2 pathway, which mediated the osteogenic and pro-angiogenic effects on rBMSCs. Furthermore, FTY/MBG-PLGA extracts induced superior in vitro angiogenic performance of HUVECs. In vivo evaluation of critical-sized rat calvarial bone defects indicated that FTY/MBG-PLGA scaffolds potently promoted vascularized bone regeneration. Notably, the significantly enhanced formation of type Hvessels (CD31hiEmcnhineo-vessels) was observed in newly formed bone tissue in FTY/MBG-PLGA group, strongly suggesting that FTY720 and therapeutic ions released from the scaffolds synergistically induced moretype H vessel formation, which indicated the coupling of angiogenesis and osteogenesis to achieve efficiently vascularized bone regeneration. Overall,the results indicated that the foamed porous MBG-PLGA scaffolds incorporating bioactive lipids achieved desirable vascularization-coupled bone formation and could be a promising strategy for bone regenerative medicine.

Lanthanum phosphate/chitosan scaffolds enhance cytocompatibility and osteogenic efficiency via the Wnt/\u03b2-catenin pathway

BackgroundFabrication of porous scaffolds with great biocompatibility and osteoinductivity to promote bone defect healing has attracted extensive attention.MethodsIn a previous study, novel lanthanum phosphate (LaPO4)/chitosan (CS) scaffolds were prepared by distributing 40- to 60-nm LaPO4 nanoparticles throughout plate-like CS films.ResultsInterconnected three dimensional (3D) macropores within the scaffolds increased the scaffold osteoconductivity, thereby promoting cell adhesion and bone tissue in-growth. The LaPO4/CS scaffolds showed no obvious toxicity and accelerated bone generation in a rat cranial defect model. Notably, the element La in the scaffolds was found to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the Wnt/β-catenin signalling pathway and induced high expression of the osteogenesis-related genes alkaline phosphatase, osteocalcin and Collagen I (Col-I). Moreover, the LaPO4/CS scaffolds enhanced bone regeneration and collagen fibre deposition in rat critical-sized calvarial defect sites.ConclusionThe novel LaPO4/CS scaffolds provide an admirable and promising platform for the repair of bone defects.