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.

Combinatorial incorporation of fluoride and cobalt ions into calcium phosphates to stimulate osteogenesis and angiogenesis.

Bone healing requires two critical mechanisms, angiogenesis and osteogenesis. In order to improve bone graft substitutes, both mechanisms should be addressed simultaneously. While the individual effects of various bioinorganics have been studied, an understanding of the combinatorial effects is lacking. Cobalt and fluoride ions, in appropriate concentrations, are known to individually favor the vascularization and mineralization processes, respectively. This study investigated the potential of using a combination of fluoride and cobalt ions to simultaneously promote osteogenesis and angiogenesis in human mesenchymal stromal cells (hMSCs). Using a two-step biomimetic method, wells of tissue culture plates were coated with a calcium phosphate (CaP) layer without or with the incorporation of cobalt, fluoride, or both. In parallel, hMSCs were cultured on uncoated well plates, and cultured with cobalt and/or fluoride ions within the media. The results revealed that cobalt ions increased the expression of angiogenic markers, with the effects being stronger when the ions were added as a dissolved salt in cell medium as compared to incorporation into CaP. Cobalt ions generally suppressed the ALP activity, the expression of osteogenic genes, and the level of mineralization, regardless of delivery method. Fluoride ions, individually or in combination with cobalt, significantly increased the expression of many of the selected osteogenic markers, as well as mineral deposition. This study demonstrates an approach to simultaneously target the two essential mechanisms in bone healing: angiogenesis and osteogenesis. The incorporation of cobalt and fluoride into CaPs is a promising method to improve the biological performance of fully synthetic bone graft substitutes.

Human mesenchymal stromal cells response to biomimetic octacalcium phosphate containing strontium.

The incorporation of bioinorganics into synthetic biomaterials is a promising approach to improve the biological performance of bone graft substitutes, while still retaining their synthetic nature. Among these bioinorganics, strontium ions (Sr(2+) ) have reported enhanced bone formation, and a reduced risk of bone fractures. While previous results have been encouraging, more detailed studies are needed to further develop specific applications. This study demonstrates the effects of Sr(2+) on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) when introduced as either a dissolved salt, or incorporated into biomimetic calcium phosphate (CaP) coatings. Upon attachment, hMSCs seeded in the presence of higher Sr(2+) concentrations presented with a more elongated shape as compared to the controls without Sr(2+) . Both Sr(2+) as a dissolved salt in the medium, or incorporated into CaP coatings, positively influenced hMSC alkaline phosphatase (ALP) activity in a dose-dependent manner. At the mRNA level, the expression of osteogenic markers ALP, bone sialoprotein, bone morphogenetic protein 2, osteopontin, and osteoclacin were increased in the presence of Sr(2+) , independent of the delivery method. Overall, this study demonstrates the positive effects of strontium on the osteogenic differentiation of human MSCs, and supports the use of strontium-incorporated CaPs for bone regeneration applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1946-1960, 2016.

Development of Highly Functional Biomaterials by Decoupling and Recombining Material Properties.

Development of functional biomaterials by a design-driven approach is described, whereby individual properties are first decoupled to investigate their sole effects on a biological process. Following this investigation, they are recombined in such a way that the overall performance and applicability of the biomaterial is improved. This is in contrast to classical, processing-driven biomaterials development where the properties of a material are mainly determined by the possibilities of the technique used to produce it.

Biological evaluation of porous aliphatic polyurethane/hydroxyapatite composite scaffolds for bone tissue engineering.

Biomaterial scaffolds meant to function as supporting structures to osteogenic cells play a pivotal role in bone tissue engineering. Recently, we synthesized an aliphatic polyurethane (PU) scaffold via a foaming method using non-toxic components. Through this procedure a uniform interconnected porous structure was created. Furthermore, hydroxyapatite (HA) particles were introduced into this process to increase the bioactivity of the PU matrix. To evaluate the biological performances of these PU-based scaffolds, their influence on in vitro cellular behavior and in vivo bone forming capacity of the engineered cell-scaffold constructs was investigated in this study. A simulated body fluid test demonstrated that the incorporation of 40 wt % HA particles significantly promoted the biomineralization ability of the PU scaffolds. Enhanced in vitro proliferation and osteogenic differentiation of the seeded mesenchymal stem cells were also observed on the PU/HA composite. Next, the cell-scaffold constructs were implanted subcutaneously in a nude mice model. After 8 weeks, a considerable amount of vascularized bone tissue with initial marrow stroma development was generated in both PU and PU/HA40 scaffold. In conclusion, the PU/HA composite is a potential scaffold for bone regeneration applications.

Calcium-mediated secondary cross-linking of bisphosphonated oligo(poly(ethylene glycol) fumarate) hydrogels.

This study demonstrates, for the first time, that synthetic PEG-based hydrogels can be cross-linked reversibly by calcium upon functionalization of the polymer backbone with bisphosphonate groups (BPs) that allow for the formation of strong coordination bonds with divalent metal ions such as Mg(2+) and Ca(2+). More specifically, it is shown that BP-functionalized hydrogels can be shaped by providing calcium ions as reversible physical cross-linkers.