Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair.

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100-150 µm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo.

Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9's mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.

Effectiveness of BMP-2 and PDGF-BB Adsorption onto a Collagen/Collagen-Magnesium-Hydroxyapatite Scaffold in Weight-Bearing and Non-Weight-Bearing Osteochondral Defect Bone Repair: In Vitro, Ex Vivo and In Vivo Evaluation.

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.

Comparison of two carbonated apatite ceramics in vivo.

Carbonated apatite ceramics, with a composition similar to that of bone mineral, are potentially interesting synthetic bone graft substitutes. In the present study, two porous carbonated apatite ceramics were developed, characterized and tested for their bone repair capacity and osteoinductive potential in a goat model. Although the two ceramics were prepared from a similar starting powder, their physico-chemical and structural characteristics differed as a consequence of different preparation methods. Both ceramics had an open and interconnected porous structure with a porosity of about 80%. The morphology of the surface of CA-A and CA-B at the submicron level differed significantly: CA-A consisted of irregular grains with a size of 5-20microm, with 1-10microm large micropores among the grains, whereas CA-B surface consisted of much smaller and regular shaped grains (0.05-0.5microm), with most micropores smaller than 1microm. The specific surface area of CA-B was about 10 times larger than that of CA-A due to its significantly smaller grain size. CA-A and CA-B ceramics contained 3 and 5 wt.% of B-type carbonated apatite, respectively. Although neither ceramic succeeded in completely bridging the 17mm iliac wing defect with new bone after 12weeks of implantation, CA-A showed significantly more bone formation in the pores of the implant than CA-B. The total area percentage of new bone in the total defect area was 12.7+/-1.81 and 5.51+/-1.37 (mean+/-SEM) for CA-A and CA-B, respectively. Intramuscular implantation of the ceramics led to ectopic bone formation by CA-A in all three implanted specimens, in contrast to CA-B, where no new bone was observed in any of the 11 animals. CA-A showed a more pronounced degradation than CA-B both in vitro and in vivo at both implantation sites, which was unexpected based on the physico-chemical and structural properties of the two ceramics. Both physico-chemical and structural properties of the ceramics could, dependently or independently, have affected their in vivo behaviour, emphasizing the importance to control individual parameters for successful bone repair.

Structural characterization and mechanical performance of calcium phosphate scaffolds and natural bones: a comparative study.

PURPOSE: The knowledge of the mechanical response of bones and their substitutes is pertinent to numerous medical problems. Understanding the effects of mechanical influence on the body is the first step toward developing innovative treatment and rehabilitation concepts for orthopedic disorders. METHODS: This was a comparative study of 5 synthetic scaffolds based on porous calcium phosphates and natural bones, with regard to their microstructural, chemical, and mechanical characterizations. The structural and chemical characterizations of the scaffolds were examined by means of X-ray diffraction, scanning electron microscopy, and X-ray spectroscopy analysis. The mechanical characterization of bones and bone graft biomaterials was carried out through compression tests using samples with noncomplex geometry. RESULTS: Analysis of the chemical composition, surface features, porosity, and compressive strength indicates that hydroxyapatite-based materials and trabecular bone have similar properties.

Comparative in vivo study of six hydroxyapatite-based bone graft substitutes.

Improvement of synthetic bone graft substitutes as suitable alternatives to a patient's own bone graft remains a challenge in biomaterials research. Our goal was to answer the question of whether improved osteoinductivity of a material would also translate to better bone-healing orthotopically. Three porous biphasic calcium phosphate (BCP) ceramics (BCPA, BCPB, and BCPC), consisting of hydroxyapatite and beta-tricalcium phosphate, a porous biphasic calcium phosphate ceramic reinforced with a bioresorbable polylactic acid to improve its mechanical properties (BCPC+), a pure hydroxyapatite ceramic (HA), and a carbonated apatite ceramic (CA) were implanted intramuscularly and orthotopically by using a transverse process model in 11 goats for 12 weeks. BCPA and BCPB had similar chemical composition but differed in their microstructure. BCPB was not osteoinductive at all, but BCPA induced ectopic bone formation in 9 of 11 animals. Orthotopically, BCPA performed better than BCPB in both the amount and rate of bone formation. BCPC, similar to BCPA structurally and physicochemically, showed comparable results ectopically and orthotopically. Addition of resorbable polymer to BCPC made the material less osteoinductive (4 of 11 animals) and delayed bone formation orthotopically. Neither HA nor CA were osteoinductive, and their orthotopic performance was inferior to the osteoinductive ceramics. The results of the present study showed that material-derived osteoinduction significantly enhanced bone healing orthotopically, and that this material property appeared more sensitive for predicting orthotopic performance than physicochemical and structural characteristics.

Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics.

Purpose of this study was the analysis of the role of density and pore interconnection pathway in scaffolds to be used as bone substitutes. We have considered 2 hydroxyapatite bioceramics with identical microstructure and different macro-porosity, pore size distribution and pore interconnection pathway. The scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold A), and (b) foaming (scaffold B). Bone ingrowth within the two bioceramics was obtained using an established model of in vivo bone formation by exogenously added osteoprogenitor cells. The histological analysis of specimens at different time after in vivo implantation revealed in both materials a significant extent of bone matrix deposition. Interestingly enough, scaffold B allowed a faster occurrence of bone tissue, reaching a steady state as soon as 4 weeks. Scaffold A on the other hand reached a comparable level of bone formation only after 8 weeks of in vivo implantation. Both scaffolds were well vascularised, but larger blood vessels were observed in scaffold A. Here we show that porosity and pore interconnection of osteoconductive scaffolds can influence the overall amount of bone deposition, the pattern of blood vessels invasion and finally the kinetics of the bone neoformation process.