Delivery Of BMP-2 Research Articles

Vertical bone augmentation with simultaneous implantation using deproteinized bovine bone block functionalized with a slow delivery of BMP-2.

We hypothesized that a biomimetic calcium phosphate (CaP) coating which incorporates morphogenetic protein 2 (BMP-2) on the deproteinized bovine bone (DBB) blocks could be used to enhance the vertical alveolar ridge augmentation for the one-stage onlay surgery with simultaneous implants insertion. We aimed to test this hypothesis in vivo. Beagles dogs were used for the study (n=6 specimens per group). One month after building the edentulous animal model, 4mm vertical alveolar bone loss were surgically created and four groups of blocks (W×L×H: 7mm×10mm×4mm) were randomly fixed onto the reduced alveolar ridge by implants: (a) DBB blocks alone (negative control group); (b) DBB blocks with superficial adsorption of 50μg BMP-2 (ad.BMP-2 group); (c) DBB blocks coated by biomimetic CaP coating which incorporates 50μg BMP-2 (inc.BMP-2 group); and (d) autologous bone blocks (positive control group). After 3months of healing, samples were harvested for micro-CT and histomorphometric analyses. In histomorphometry, the inc.BMP-2 group showed a significantly thicker (coronal-apically) and wider (buccal-lingually) augmented bone area, better bone-to-implant contact than the negative control group. In both the micro-CT and histomorphometry, the inc.BMP-2 group showed more mineralized tissue than the negative control group and the inc.BMP-2 group also showed significantly more newly formed bone and residual grafts than the negative control group in the upper half of the blocks. In micro-CT, the inc.BMP-2 group showed significantly more bone-to-graft contact percentage than the ad.BMP-2 group. In both micro-CT and histomorphometry, the inc.BMP-2 group showed significantly more percentage of mineralized tissue than the ad.BMP-2 group. No significant differences were found between the inc.BMP-2 group and the positive control group either in micro-CT or in histomorphometry. The DBB blocks with coating-delivered BMP-2 significantly enhanced the efficacy of vertical alveolar bone augmentation, compared with the unloaded blocks and blocks with adsorbed BMP-2, in the one-stage onlay surgery with simultaneous implant insertion.

Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration.

Supraphysiologic doses of bone morphogenetic protein-2 (BMP-2) are used clinically to promote bone formation in fracture nonunions, large bone defects, and spinal fusion. However, abnormal bone formation (i.e., heterotopic ossification) caused by rapid BMP-2 release from conventional collagen sponge scaffolds is a serious complication. We leveraged the strong affinity interactions between heparin microparticles (HMPs) and BMP-2 to improve protein delivery to bone defects. We first developed a computational model to investigate BMP-2-HMP interactions and demonstrated improved in vivo BMP-2 retention using HMPs. We then evaluated BMP-2-loaded HMPs as a treatment strategy for healing critically sized femoral defects in a rat model that displays heterotopic ossification with clinical BMP-2 doses (0.12 mg/kg body weight). HMPs increased BMP-2 retention in vivo, improving spatial localization of bone formation in large bone defects and reducing heterotopic ossification. Thus, HMPs provide a promising opportunity to improve the safety profile of scaffold-based BMP-2 delivery.

Super-assembled core/shell fibrous frameworks with dual growth factors for in situ cementum-ligament-bone complex regeneration.

The regeneration of periodontal tissue defects remains a clinical challenge due to its complex tissue structure (e.g. periodontal ligament, alveolar bone and cementum) and poor self-healing ability. In situ tissue engineering has emerged as a promising approach that combines frameworks with growth factors that are specifically chosen for the recruitment of endogenous stem cells to the site of injury and to evoke the innate regenerative potential of the body. Herein, a core/shell fibrous super-assembled framework (SAF)-based sequential growth factor delivery system is developed, in which basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) are designed to release in a sequential manner to facilitate in situ regeneration of the cementum-ligament-bone complex. The in situ tissue engineering framework (iTE-framework) shows ameliorated physicochemical properties and improved hydrophilicity, with an initial burst release of bFGF in the first few days, followed by a slow and constant release of BMP-2 up to 4 weeks. The iTE-framework shows excellent biocompatibility, significantly promoting the proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in vitro. After implantation in rat periodontal defects, the iTE-framework effectively triggers the recruitment of mesenchymal stem cells (MSCs) to the defect site, significantly promotes the formation of new bones, and facilitates the regeneration of the periodontal ligament and cementum tissue in vivo. Therefore, this sequential delivery system provides a promising therapeutic strategy for cementum-ligament-bone complex regeneration.

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.

A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair

BackgroundOsteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study.MethodIn this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair.ResultsWe observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining.ConclusionsWe conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

Bone Morphogenic Protein 2-Loaded Porous Silicon Carriers for Osteoinductive Implants.

Bone morphogenetic proteins (BMPs) are probably the most important growth factors in bone formation and healing. However, the utilization of BMPs in clinical applications is mainly limited due to the protein poor solubility at physiological pH, rapid clearance and relatively short biological half-life. Herein, we develop degradable porous silicon (PSi)-based carriers for sustained delivery of BMP-2. Two different loading approaches are examined, physical adsorption and covalent conjugation, and their effect on the protein loading and release rate is thoroughly studied. The entrapment of the protein within the PSi nanostructures preserved its bioactivity for inducing osteogenic differentiation of rabbit bone marrow mesenchymal stems cells (BM-MSCs). BM-MSCs cultured with the BMP-2 loaded PSi carriers exhibit a relatively high alkaline phosphatase (ALP) activity. We also demonstrate that exposure of MSCs to empty PSi (no protein) carriers generates some extent of differentiation due to the ability of the carrier’s degradation products to induce osteoblast differentiation. Finally, we demonstrate the integration of these promising BMP-2 carriers within a 3D-printed patient-specific implant, constructed of poly(caprolactone) (PCL), as a potential bone graft for critical size bone defects.

BMP-2 gene delivery in cell-loaded and cell-free constructs for bone regeneration.

To induce osteogenicity in bone graft substitutes, plasmid-based expression of BMP-2 (pBMP-2) has been successfully applied in gene activated matrices based on alginate polymer constructs. Here, we investigated whether cell seeding is necessary for non-viral BMP-2 gene expression in vivo. Furthermore, to gain insight in the role of BMP-producing cells, we compared inclusion of bone progenitor cells with non-osteogenic target cells in gene delivery constructs. Plasmid DNA encoding GFP (pGFP) was used to trace transfection of host tissue cells and seeded cells in a rat model. Transgene expression was followed in both cell-free alginate-ceramic constructs as well as constructs seeded with syngeneic fibroblasts or multipotent mesenchymal stromal cells (MSCs). Titration of pGFP revealed that the highest pGFP dose resulted in frequent presence of positive host cells in the constructs. Both cell-loaded groups were associated with transgene expression, most effectively in the MSC-loaded constructs. Subsequently, we investigated effectiveness of cell-free and cell-loaded alginate-ceramic constructs with pBMP-2 to induce bone formation. Local BMP-2 production was found in all groups containing BMP-2 plasmid DNA, and was most pronounced in the groups with MSCs transfected with high concentration pBMP-2. Bone formation was only apparent in the recombinant protein BMP-2 group. In conclusion, we show that non-viral gene delivery of BMP-2 is a potentially effective way to induce transgene expression in vivo, both in cell-seeded as well as cell-free conditions. However, alginate-based gene delivery of BMP-2 to host cells or seeded cells did not result in protein levels adequate for bone formation in this setting, calling for more reliable scaffold compatible transfection methods.

FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

Osteoinductive bone morphogenetic proteins (BMPs), including BMP-2, have a unique capability of mediating bone formation both in orthotopic and ectopic locations. Immunosuppresive macrolides have been shown to potentiate BMP-2 activity through FKBP12, but these have yet to translate to effective osteoinductive therapies. Herein, we show the osteogenic activity of FK506 as a stand-alone agent in direct comparison to BMP-2 both in vitro and in vivo. FK506 was capable of producing stand-alone alkaline phosphatase induction in C2C12 cells comparable to that seen with rhBMP-2. FK506 treatment activated the BMP receptor, as shown by increased pSmad1/5 levels, and produced significantly higher mRNA levels of the early response genes in BMP and TGF-β pathways. Additionally, the FK506 induction of alkaline phosphatase was shown to be resistant to Noggin treatment. In vivo osteogenic activity of FK506 was tested by local delivery on a collagen sponge in an ectopic subcutaneous implantation model in the rat. Dose responses of FK506 showed increasing levels of ectopic mineralization comparable to the mineral volume produced by BMP-2 delivery. These findings suggest that the use of FK506 can enhance osteoblastic differentiation in vitro and can induce mineralization when delivered locally in vivo.

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.

Development of CaCO3 microsphere-based composite hydrogel for dual delivery of growth factor and Ca to enhance bone regeneration.

Injectable scaffolds have attracted much attention because of their minimum surgical invasiveness. However, limited osteogenic induction property and low mechanical properties hampered their application in bone tissue engineering. CaCO3 microspheres, which possess osteoinductivity, rough surfaces and specific binding sites for BMP-2, were first fabricated; after BMP-2 uploading, microspheres were further entrapped in fibrin-glue hydrogel. CaCO3 microspheres were co-functionalized with casein and heparin. To obtain a high encapsulation of heparin and thus BMP-2 uploading, along with controlled release and simultaneous maintenance of the presence of vaterite which had osteogenic induction property, fabrication parameters were optimized and microspheres were characterized using XRD, FITR and SEM. The formed CaCO3 had a microsphere morphology of ∼1 μm. Both vaterite and calcite phases were present and the relative amount of calcite phase increased with the amount of heparin. Sample 25 mM_4-1Hep with the highest loading amount of heparin was selected as carrier for BMP-2 and BMP-2 loaded CaCO3 microspheres were further entrapped in fibrin-glue hydrogel (FC-B). For the as-prepared composite hydrogel, mechanical properties were characterized and the presence of CaCO3 significantly elevated the tensile strength; controlled release of BMP-2 was sustained until day 21. Based on ALP activity, alizarin red staining and RT-PCR, in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was found to be significantly enhanced under induction of FC-B. Rabbit tibia bone defect model was applied to evaluate its in vivo performance. After implantation for 4 weeks, presence of composite hydrogel was observed in defects. After 8 weeks, bone defects of FC-B group were nearly completely healed. Using the fact that autologous scaffolds can be derived based on fibrin-glue hydrogel, the well-designed BMP-2 loaded fibrin-glue composite hydrogel demonstrated good potential in bone tissue engineering.

Interplay of topographical and biochemical cues in regulating osteoblast cellular activity in BMP-2 eluting three-dimensional cellular titanium alloy mesh structures.

The objective of this study is to elucidate the elution response of bone morphogenetic protein (BMP-2) in tuning cellular functions on 3D-printed titanium alloy mesh scaffolds subjected to microarc/plasma electrolytic oxidation process. The microtopographical cues enabled strong interaction with BMP-2 protein, which led to controlled release. Furthermore, the interaction of BMP-2 with the surface microtopographical cue regulated osteoblast cellular activity by contributing to the early phase differentiation and mineralization of osteoblasts. The in vitro BMP-2 release kinetics showed an initial burst of BMP-2 after day 1, followed by a controlled release on plasma electrolytic oxidized mesh structure. The profile represented that the rate of release decreased with increase in time on both as-fabricated and plasma electrolytic oxidized mesh structures after 24 h, with relatively higher degree on plasma electrolytic oxidized mesh structure. Furthermore, the in vitro osteoblast cellular activity indicated enhanced osteogenic induction on BMP-2 adsorbed plasma electrolytic oxidized mesh structure. Cells penetrated into micropores through several filopodia-like cellular extensions by increasing the area of contact, leading to stronger cell adhesion on BMP-2 adsorbed plasma electrolytic oxidized mesh structure. The up-regulation of biochemical markers and quantification of the expression level of cell secreted proteins underscored the determining role of BMP-2 eluting 3D mesh structure in modulating osteoblasts functions. The study emphasizes the potential of BMP-2 in rendering plasma electrolytic oxidized 3D-printed titanium alloy mesh structure osteoinductive and controlled delivery of BMP-2. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 49-60, 2019.

Inductive effects of growth factors sequential-release scaffolds on osteogenic differentiation of bone mesenchymal stem cells

Objective To prepare scaffolds containing different growth factors, investigate the inductive effects of bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) on differentiation of bone mesenchymal stem cells (BMSCs). Methods BMP-2-Poly D, L-Lactic Acid (PLLA) microspheres were prepared with a double emulsion method. Scaffolds were prepared with supercritical CO2 foaming technique. The effects of BMP-2 and VEGF on differentiation of BMSCs were investigated. Results BMP-2-PLLA microspheres were spherical shape with diameters of 10.022 μm. The encapsulation efficiency was 64.881%. Scaffolds were obtained with the porosity of 78.706% and sequentially released rate. Cell proliferation of the scaffolds group (1.121±0.110) was significantly higher than that of blank group (0.762±0.150) and the free growth factors group (0.882±0.140) (P=0.005). ALP production and calcium nodule numbers of the scaffolds group (9.720±0.330, 0.269±0.000) were significantly higher than that of blank group (6.242±0.660, 0.149±0.020) and the free growth factors group (7.989±0.420, 0.204±0.010) (P=0.006, P=0.003). Conclusion The composite scaffolds had excellent performance, realized the sequential delivery of BMP-2 and VEGF, and could induce BMSCs osteogenic differentiation effectively. Key words: Bone morphogenetic protein-2; Vascular endothelial growth factor; Sequential release; Osteogenic differentiation

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.

Bone formation by heterodimers through non-viral gene delivery of BMP-2/6 and BMP-2/7.

Non-viral gene delivery is a safe technique to release sustained physiologic dosages of bone morphogenetic protein (BMP). Co-delivery of multiple BMPs can result in the formation of more potent BMP heterodimers. In this study, non-viral co-delivery of BMP-2/6 and BMP-2/7, as a mean to produce heterodimers, was assessed. Goat MSCs were non-virally transfected with plasmid DNA encoding BMP isoforms (pBMP) known to be relevant for osteogenesis: BMP-2, -6 or -7. As a result, BMP-2, -6 and -7 were produced and detectable for up to 14 d and their combined delivery (pBMP-2 with pBMP-6 or pBMP-7) was used to create BMP-2/6 and BM-2/7 heterodimers. Formation and secretion of the heterodimer proteins was validated by sandwich enzyme-linked immunosorbent assay (ELISA). Produced BMPs and heterodimers were biologically active, as confirmed by differentiation of reporter cells and MSCs. To assess bone formation, transfected MSCs were seeded on to ceramic scaffolds and implanted subcutaneously into nude mice. Bone formation was significantly enhanced in the pBMP-2/6 condition and a trend for more bone formation was observed in the pBMP-2/7 and pBMP-6 homodimer condition. No bone was found in the pBMP-2, pBMP-7 or control condition. In conclusion, simultaneous delivery of pBMP-2 with pBMP-6 or -7 resulted in the production of heterodimers that were beneficial for bone formation as compared to BMP homodimers. Combination of BMP sequences could reduce the need for high BMP protein dosages and might enhance prolonged availability of the growth factors.

Fibrin glue mediated delivery of bone anabolic reagents to enhance healing of tendon to bone.

Tendon graft healing in bone tunnels for the fixation of intra-articular ligament reconstructions may limit clinical outcome by delaying healing. This study assesses the effects of hydrogel-mediated delivery of bone anabolic growth factors in a validated model of tendon-to-bone tunnel healing. Forty-five Wistar rats were randomly allocated into three groups (BMP2-treated, GSK126-treated, and placebo). All animals underwent a tendon-to-bone tunnel reconstruction. Healing was evaluated at 4 weeks by biomechanical assessment, micro-computed tomography (bone mineral density, bone volume, cross sectional area of bone tunnels), and traditional histology. Adverse events associated with the hydrogel-mediated delivery of drugs were not observed. Results of our biomechanical assessment demonstrated favorable trends in animals treated with bone anabolic factors for energy absorption (P = 0.116) and elongation (P = 0.054), while results for force to failure (P = 0.691) and stiffness (P = 0.404) did not show discernible differences. Cross sectional areas for BMP2-treated animals were reduced, but neither BMP2 nor GSK126 administration altered bone mineral density (P = 0.492) or bone volume in the bone tunnel. These results suggest a novel and positive effect of bone anabolic factors on tendon-to-bone tunnel healing. Histological evaluation confirmed absence of collagen fibers crossing the soft tissue-bone interface indicating immature graft integration as expected at this time point. Our study indicates that hydrogel-mediated delivery of BMP2 and GSK126 appears to be safe and has the potential to enhance tendon-to-bone tunnel healing in ligament reconstructions.

Bioabsorbable Bone Plates Enabled With Local, Sustained Delivery of Bmp2 for Bone Regeneration

Bioabsorbable Bone Plates Enabled With Local, Sustained Delivery of Bmp2 for Bone Regeneration

3D printed microchannel networks to direct vascularisation during endochondral bone repair

Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.

MBG scaffolds containing chitosan microspheres for binary delivery of IL-8 and BMP-2 for bone regeneration.

Biomimetic delivery of chemokines and growth factors based on stem cell recruitment and endochondral ossification, as the key steps in natural regenerative process, has been an area of intense research in recent years. An inflammatory chemokine, interleukin-8 (IL-8), was recently reported with high recruitment efficiency of bone marrow stem cells, chondrogenic inductivity and immune regulatory functions. In this study, the effect of IL-8 action duration on bone morphogenetic protein-2 (BMP-2)-induced bone regeneration was studied to achieve an optimal synergism of these two proteins. Herein, a mesoporous bioactive glass (MBG)-based scaffold with BMP-2 entrapment and IL-8-loaded chitosan microspheres (CMs) was developed. The MBG scaffold with size-matched mesopores was adopted for the long-term sustained delivery of BMP-2; and CMs with different sizes, prepared using a modified ionotropic gelation method, were customized to match the optimal action time of IL-8. The results indicated that CMs of 100 μm diameter and medium crosslinking density exhibited an 85% release of IL-8 in 7 days and the MBG substrate exhibited a long-term sustained release of BMP-2. Furthermore, the binary delivery system exhibited excellent biocompatibility and synergistically enhanced osteoinductivity. In an in situ bone regeneration model of a rabbit radius large segmental defect, the system efficiently accelerated the whole regenerative process, with the highest bone formation amount from an early stage and the highest degree of regenerative completion. Since delivery systems for multiple cytokines have been in great demand due to the requirement of complicated biological processes, we believe that this new binary delivery system could be customized to design other dual delivery systems for improving bone-repairing biomaterials with higher regenerative efficiency.

Rapid initiation of guided bone regeneration driven by spatiotemporal delivery of IL-8 and BMP-2 from hierarchical MBG-based scaffold

Initiation of endogenous repair mechanisms, including key steps of stem cell recruitment and cartilage intermediate formation in endochondral ossification, is vital to regeneration of large bone defects. To biomimetically promote a rapid initiation and ensuing osteogenic stimulation, exogenous chemokine IL-8 and growth factor BMP-2 were orchestrated in a mesoporous bioactive glass (MBG)-based spatiotemporal delivery system, to achieve a rapid release of IL-8 followed by a long-term sustained release of BMP-2. The synergistic effect of IL-8 and BMP-2 on initiation stage of bone healing and underlying mechanism were thoroughly investigated in vitro and in vivo. Intriguingly, apart from its superiority in stem cell recruitment to BMP-2, IL-8 not only endowed a histological “prep-state” of endochondral ossification by up-regulating chondrogenic genes and inducing the formation of extensive cartilage tissues, facilitating rapid bone transformation by BMP-2, but also triggered a cellular “prep-state” with high expression of BMP receptors, enhancing the osteoinductivity of BMP-2. With the spatiotemporal delivery system, orchestrated signal stimuli of IL-8 and BMP-2 induced a rapid initiation including efficient stem cell recruitment and a “chondrogenic/osteogenic balance” at the first stage of endochondral ossification, and the scaffold facilitated sufficient osteoconductivity, together resulting in early extensive bone mineralization and an advanced regeneration throughout the repair of large bone defect. We believe this new idea could provide insights toward designing bone-repairing biomaterials with higher regenerative efficiency.

Evaluation of synergistic osteogenesis between icariin and BMP2 through a micro/meso hierarchical porous delivery system.

BMP2 is well known as an outstanding growth factor for inducing new bone formation. However, improvements are still required to use BMP2 effectively and expand its clinical application due to the potential side effects at high doses. In this study, icariin (IC), a type of traditional Chinese medicine, was originally proposed to be a cooperative factor for BMP2. An alkaline phosphatase (ALP) activity assay showed that IC promoted BMP2 osteogenesis in a concentration-dependent manner with significant enhancement at 38.4 µM versus that for BMP2 at 0.8 µg/mL. Furthermore, we developed a composite hierarchical porous scaffold (SF/SBA15; composed of micropores of silk fibroin [SF] scaffold and mesopores of SBA15) for the controlled delivery of BMP2 and IC. This composite scaffold was investigated by a series of physical characterizations and displayed good in vitro cell biocompatibility. In addition, the composite scaffold also showed the degradation rate of 12% dry weight loss and a slight change in the microstructures within 10 days. Moreover, BMP2 and IC were loaded into the SF and SBA15 structures, respectively, of the SF/SBA15 scaffold. This protein/drug loading system (SFBMP2/SBA15IC) provided delivery of BMP2 with an initial burst release of 60.9%±0.9% in the first 24 hours and a gradual release over the subsequent 6 days to 97.9%±0.8%, whereas IC exhibited a burst release of 64.2%±0.7% in the first 24 hours, followed by a sustained release to 92.4%±0.8% over 10 days. With the prolonged local retention and interaction duration of BMP2 and IC, the SFBMP2/SBA15IC scaffold provided better osteogenic differentiation than other groups with different loading modes of BMP2 or IC, as determined by ALP staining and quantitation and Alizarin red staining. Finally, the results of quantitative real-time polymerase chain reaction analysis indicated that the SFBMP2/SBA15IC scaffold induced a significantly higher increase in the RUNX2, ALP, COL I, and OCN expression levels of cocultured bone marrow mesenchymal stem cells than other payload composite scaffolds. This study suggests that a micro/meso hierarchical porous delivery system of BMP2 and IC ensures osteogenic synergy and demonstrates promise for bone tissue engineering.