Human osteoclast formation and resorptive function on biomineralized collagen.

Biomineralized collagen composite materials pose an intriguing alternative to current synthetic bone graft substitutes by offering a biomimetic composition that closely resembles native bone. We hypothesize that this composite can undergo cellular resorption and remodeling similar to natural bone. We investigate the formation and activity of human osteoclasts cultured on biomineralized collagen and pure collagen membranes in comparison to cortical bone slices. Human monocytes/macrophages from peripheral blood differentiate into multinucleated, tartrate-resistant alkaline phosphatase (TRAP)-positive osteoclast-like cells on all substrates. These cells form clear actin rings on cortical bone, but not on biomineralized collagen or pure collagen membranes. Osteoclasts form resorption pits in cortical bone, resulting in higher calcium ion concentration in cell culture medium; however, osteoclast resorption of biomineralized collagen and collagen membranes does not measurably occur. Activity of osteoclast enzymes - TRAP, carbonic anhydrase II (CA-II), and cathepsin-K (CTS-K) - is similar on all substrates, despite phenotypic differences in actin ring formation and resorption. The mesh-like structure, relatively low stiffness, and lack of RGD-containing binding domains are likely the factors responsible for preventing formation of stable actin rings on and resorption of (biomineralized) collagen membranes. This insight helps to guide further research toward the optimized design of biomineralized collagen composites as a more biomimetic bone-graft substitute.

Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts.

BACKGROUND: Biomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts. METHODS: To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium. RESULTS: The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded. CONCLUSIONS: This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Proliferation and Osteogenic Differentiation of hMSCs on Biomineralized Collagen.

Biomineralized collagen with intrafibrillar calcium phosphate mineral provides an excellent mimic of the composition and structure of the extracellular matrix of bone, from nano- to micro-scale. Scaffolds prepared from this material have the potential to become the next-generation of synthetic bone graft substitutes, as their unique properties make them closer to the native tissue than synthetic alternatives currently available to clinicians. To understand the interaction between biomineralized collagen and cells that are relevant in the context of bone regeneration, we studied the growth and osteogenic differentiation of bone marrow derived human mesenchymal stromal cells (hMSCs) cultured on biomineralized collagen membranes, and compared it to the cell behavior on collagen membranes without mineral. Cells proliferated normally on both biomimetic membranes, and were more triggered to differentiate toward the osteogenic lineage by the biomineralized collagen. This was shown by the elevated mRNA levels of RUNX2, SPP1, ENPP1, and OCN after 3 days of culture, and COL1A1 after 14 days of culture on mineralized collagen. The mRNA levels of the tested markers of osteogenesis were lower on collagen membranes without mineral, with the exception of OCN, which was more highly expressed on collagen than on biomineralized collagen membranes. Expression by hMSCs of OPG, a gene involved in inhibition of osteoclastogenesis, was higher on biomineralized collagen at day 3, while M-CSF, involved in osteoblast-osteoclast communication, was upregulated on both membranes at day 3 and 14 of culture. Alkaline phosphatase activity of hMSCs was high on both biomimetic membranes when compared with cells cultured on tissue culture plastic. Cell-induced mineralization was observed on collagen membranes, while the high mineral content of the biomineralized membranes prohibited a reliable analysis of cell-induced mineralization on these membranes. In conclusion, we have identified that both collagen and biomineralized collagen support proliferation, osteogenic differentiation and mineralization of hMSCs, with biomineralized membranes having a more pronounced positive effect. These findings support the existing evidence that biomineralized collagen is a promising material in the field of bone regeneration.

Biomineralization-Inspired Material Design for Bone Regeneration.

Synthetic substitutes of bone grafts, such as calcium phosphate-based ceramics, have shown some good clinical successes in the regeneration of large bone defects and are currently extensively used. In the past decade, the field of biomineralization has delivered important new fundamental knowledge and techniques to better understand this fascinating phenomenon. This knowledge is also applied in the field of biomaterials, with the aim of bringing the composition and structure, and hence the performance, of synthetic bone graft substitutes even closer to those of the extracellular matrix of bone. The purpose of this progress report is to critically review advances in mimicking the extracellular matrix of bone as a strategy for development of new materials for bone regeneration. Lab-made biomimicking or bioinspired materials are discussed against the background of the natural extracellular matrix, starting from basic organic and inorganic components, and progressing into the building block of bone, the mineralized collagen fibril, and finally larger, 2D and 3D constructs. Moreover, bioactivity studies on state-of-the-art biomimicking materials are discussed. By addressing these different topics, an overview is given of how far the field has advanced toward a true bone-mimicking material, and some suggestions are offered for bridging current knowledge and technical gaps.