rhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. METHODS: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. RESULTS: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. CONCLUSIONS: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Macroporous Calcium Phosphate/Chitosan Composites Prepared via Unidirectional Ice Segregation and Subsequent Freeze-Drying.

Calcium phosphate chitosan-based composites have gained much interest in recent years for biomedical purposes. In this paper, three-dimensional calcium phosphate chitosan-based composites with different mineral contents were produced using a green method called ice segregation induced self-assembly (ISISA). In this methodology, ice crystals were used as a template to produce porous structures from an aqueous solution of chitosan (CS) and hydroxyapatite (Hap) also containing acetic acid (pH = 4.5). For better characterization of the nature of the inorganic matter entrapped within the resulting composite, we performed either oxygen plasma or calcination processes to remove the organic matter. The nature of the phosphate salts was studied by XRD and NMR studies. Amorphous calcium phosphate (ACP) was identified as the mineral phase in the composites submitted to oxygen plasma, whereas crystalline Hap was obtained after calcination. SEM microscopy revealed the formation of porous structures (porosity around 80-85%) in the original composites, as well as in the inorganic matrices obtained after calcination, with porous channels of up to 50 µm in diameter in the former case and of up to 20 µm in the latter. The biocompatibility of the composites was assessed using two different cell lines: C2C12GFP premyoblastic cells and MC3T3 preosteoblastic cells.

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation.

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.

In vivo comparison of the effects of rhBMP-2 and rhBMP-4 in osteochondral tissue regeneration.

The aim of this work is to investigate the use of bone morphogenetic proteins (rhBMP-2, rhBMP-4) alone or in combination with cells delivered in a calcium alginate gel for the treatment of osteochondral defects. For this purpose, alginate gels were prepared mixing a 2% sodium alginate solution and a 200 mM calcium chloride solution (1:1). Osteochondral defects were created (4 mm wide, 5 mm deep) in the internal femoral condyle of rabbit knee and gels were directly formed into the defects. 3 months after surgery samples were harvested, gross morphology was documented and histological appearance was evaluated. The performed histological observations revealed subchondral bone regeneration in rhBMP-2 samples and moderate hyaline cartilage regeneration in rhBMP-4 samples. Thus, results indicate that alginate gel may serve as an appropriate delivery vehicle for rhBMP-2, rhBMP-4 and stromal cells. With this carrier material, differential behaviour between the evaluated proteins was observed. rhBMP-2 shows better restoration of subchondral bone in contrast to the superior efficiency of rhBMP-4 for hyaline cartilage repair.

Gene expression profile on chitosan/rhBMP-2 films: A novel osteoinductive coating for implantable materials.

This study focusses on the gene expression profile related to a new rhBMP-2 carrier material, chitosan film. This film could be suitable for use as an osteoinductive coating of commercially available titanium implants. The developed material was characterized, biocompatibility was tested and the cellular response was extensively characterized by transcriptional expression studies. Finally, in vivo studies were carried out to confirm the osteoinductivity of the developed coating. Results show good material properties for cell adhesion and proliferation. Presented data show cellular differentiation to the osteoblastic phenotype due to rhBMP-2, with a 90% common transcriptional response between the control rhBMP-2 treatment and the developed chitosan/rhBMP-2 film. The growing surface also had an influence on the observed cellular response and was quantified as 7% of the total. These results indicate that both the growth factor and the material induce a cell response, but this is mainly driven by the osteoinductor factor. In vivo, new bone formation and early vascularization was observed around chitosan/rhBMP-2 coated titanium pieces implanted in mouse muscle. In contrast, control implants did not induce this reaction. This work, therefore, shows both in vitro and in vivo that chitosan/rhBMP-2 film is a promising osteoinductive coating for titanium implantable materials.

Improvement of porous beta-TCP scaffolds with rhBMP-2 chitosan carrier film for bone tissue application.

Ceramic materials are osteoconductive matrices extensively used in bone tissue engineering approaches. The performance of these types of biomaterials can be greatly enhanced by the incorporation of bioactive agents and materials. It is previously reported that chitosan is a biocompatible, biodegradable material that enhances bone formation. In the other hand, bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive factor. In this work we coated porous beta-tricalcium phosphate (beta-TCP) scaffolds with recombinant human BMP-2 (rhBMP-2) carrier chitosan films and studied how they could modify the ceramic physicochemical properties, cellular response, and in vivo bone generation. Initial beta-TCP disks with an average diameter of 5.78 mm, 2.9 mm thickness, and 53% porosity were coated with a chitosan film. These coating properties were studied by X-ray diffraction, Fourier transform-infrared analysis, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). Treatment modified the scaffold porous distribution and increased the average hardness. The biocompatibility did not seem to be altered. In addition, adhered C2C12 cells expressed alkaline phosphatase activity, related to cell differentiation toward osteogenic lineage, due to the incorporation of rhBMP-2. On the other hand, in vivo observations showed new bone formation 3 weeks after surgery, a much shorter time than control beta-TCP ceramics. These results suggest that developed coating improved porous beta-TCP scaffold for bone tissue applications and added osteoinductive properties.

Multiwall carbon nanotube scaffolds for tissue engineering purposes.

The use of scaffolds composed of a major fraction of multiwall carbon nanotubes (MWCNT, up to 89 wt.%) and a minor one of chitosan (CHI), and with a well-defined microchannel porous structure as biocompatible and biodegradable supports for culture growth is described. Cell adhesion, viability and proliferation onto the external surface of MWCNT/CHI scaffolds with C2C12 cell line (myoblastic mouse cell), which is a multipotent cell line able to differentiate towards different phenotypes under the action of some chemical or biological factors, has been evaluated in vitro and quantified by MTT assays. The evolution of the C2C12 cell line towards an osteoblastic lineage in presence of the recombinant human bone morphogenetic protein-2 (rhBMP-2) has also been studied both in vitro (e.g., following the appearance of alkaline phosphatase activity) and in vivo (e.g., by implantation of MWCNT/CHI scaffolds adsorbed with rhBMP-2 in muscle tissue and evaluation of the ectopic formation of bone tissue).