The aim of the current study was to reduce the clinically used supra-physiological dose of bone morphogenetic protein-2 (BMP-2) (usually 1.5 mg/mL), which carries the risk of adverse events, by using a more effective release system. A slow release system, based on an injectable hydrogel composed of BMP-2-loaded recombinant collagen-based microspheres and alginate, was previously developed. Time- and dose-dependent subcutaneous ectopic bone formation within this system and bone regeneration capacity in a calvarial defect model were investigated. BMP-2 doses of 10 µg, 3 µg and 1 µg per implant (50 µg/mL, 15 µg/mL and 5 µg/mL, respectively) successfully induced ectopic bone formation subcutaneously in rats in a time- and dose-dependent manner, as shown by micro-computed tomography (µCT) and histology. In addition, the spatio-temporal control of BMP-2 retention was shown for 4 weeks in vivo by imaging of fluorescently-labelled BMP-2. In the subcritical calvarial defect model, µCT revealed a higher bone volume for the 2 µg of BMP-2 per implant condition (50 µg/mL) as compared to the lower dose used (0.2 µg per implant, 5 µg/mL). Complete defect bridging was obtained with 50 µg/mL BMP-2 after 8 weeks. The BMP-2 concentration of 5 µg/mL was not sufficient to heal a calvarial defect faster than the empty defect or biomaterial control without BMP-2. Overall, this injectable BMP-2 delivery system showed promising results with 50 µg/mL BMP-2 in both the ectopic and calvarial rat defect models, underling the potential of this composite hydrogel for bone regeneration therapies.

Currently, autografts still represent the gold standard treatment for the repair of large bone defects. However, these are associated with donor-site morbidity and increased pain, cost, and recovery time. The ideal therapy would use biomaterials combined with bone growth factors to induce and instruct bone defect repair without the need to harvest patient tissue. In this line, bone morphogenetic proteins (BMPs) have been the most extensively used agents for clinical bone repair, but at supraphysiological doses that are not without risk. Because of the need to eliminate the risks of BMP2 use in vivo, we assessed the ability of three putative osteogenic factors, nel-like molecule type 1 (NELL-1), high mobility group box 1 (HMGB1), and CCN2, to enhance the essential processes for bone defect repair in vitro and compared them to BMP2. Although it has been reported that NELL-1, HMGB1, and CCN2 play a role in bone formation, less is known about the contribution of these proteins to the different events involved, such as cell migration, osteogenesis, and vasculogenesis. In this study, we investigated the effects of different doses of NELL-1, HMGB, CCN2, and BMP2 on these three processes as a model for the recruitment and differentiation of resident cells in the in vivo bone defect repair situation, using cells of human origin. Our data demonstrated that NELL-1, HMGB1, and CCN2 significantly induced mesenchymal stem cell migration (from 1.58-fold increase compared to control), but BMP2 did not. Interestingly, only BMP2 increased osteogenesis in marrow stromal cells, whereas it inhibited osteogenesis in preosteoblasts. Moreover, the four proteins studied promoted significantly endothelial cell migration, reaching a maximum of 2.4-fold increase compared to control, and induced formation of tube-like structures. NELL-1, HMGB1, and CCN2 had these effects at relatively low doses compared to BMP2. This work indicates that NELL-1, HMGB1, and CCN2 might enhance bone defect healing via the recruitment of endogenous cells and induction of vascularization and act via different processes than BMP2.

The use of biomaterials and signaling molecules to induce bone formation is a promising approach in the field of bone tissue engineering. Follistatin (FST) is a glycoprotein able to bind irreversibly to activin A, a protein that has been reported to inhibit bone formation. We investigated the effect of FST in critical processes for bone repair, such as cell recruitment, osteogenesis and vascularization, and ultimately its use for bone tissue engineering. In vitro , FST promoted mesenchymal stem cell (MSC) and endothelial cell (EC) migration as well as essential steps in the formation and expansion of the vasculature such as EC tube-formation and sprouting. FST did not enhance osteogenic differentiation of MSCs, but increased committed osteoblast mineralization. In vivo , FST was loaded in an in situ gelling formulation made by alginate and recombinant collagen-based peptide microspheres and implanted in a rat calvarial defect model. Two FST variants (FST288 and FST315) with major differences in their affinity to cell-surface proteoglycans, which may influence their effect upon in vivo bone repair, were tested. In vitro , most of the loaded FST315 was released over 4 weeks, contrary to FST288, which was mostly retained in the biomaterial. However, none of the FST variants improved in vivo bone healing compared to control. These results demonstrate that FST enhances crucial processes needed for bone repair. Further studies need to investigate the optimal FST carrier for bone regeneration.

New solutions for large bone defect repair are needed. Here, in situ gelling slow release systems for bone induction are assessed. Collagen-I based Recombinant Peptide (RCP) microspheres (MSs) are produced and used as a carrier for bone morphogenetic protein 2 (BMP-2). The RCP-MSs are dispersed in three hydrogels: high mannuronate (SLM) alginate, high guluronate (SLG) alginate, and thermoresponsive hyaluronan derivative (HApN). HApN+RCP-MS forms a gel structure at 32 ºC or above, while SLM+RCP-MS and SLG+RCP-MS respond to shear stress displaying thixotropic behavior. Alginate formulations show sustained release of BMP-2, while there is minimal release from HApN. These formulations are injected subcutaneously in rats. SLM+RCP-MS and SLG+RCP-MS loaded with BMP-2 induce ectopic bone formation as revealed by X-ray tomography and histology, whereas HApN+RCP-MS do not. Vascularization occurs within all the formulations studied and is significantly higher in SLG+MS and HApN+RCP-MS than in SLM+RCP-MS. Inflammation (based on macrophage subset staining) decreases over time in both alginate groups, but increases in the HApN+RCP-MS condition. It is shown that a balance between inflammatory cell infiltration, BMP-2 release, and vascularization, achieved in the SLG+RCP-MS alginate condition, is optimal for the induction of de novo bone formation.

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Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.

For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induced the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.

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