Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in a simulated osteochondral environment is hydrogel dependent.

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.

Platelet-released supernatant induces osteoblastic differentiation of human mesenchymal stem cells: potential role of BMP-2.

Platelet-rich preparations have recently gained popularity in maxillofacial and dental surgery, but their beneficial effect is still under debate. Furthermore, very little is known about the effect of platelet preparations at the cellular level, and the underlying mechanisms. In this study, we tested the effect of platelet-released supernatant (PRS) on human mesenchymal stem cell (MSC) differentiation towards an osteoblastic phenotype in vitro. Cultures of MSC were supplemented with PRS and typical osteoblastic markers were assessed at up to 28 days post-confluence. PRS showed an osteoinductive effect on MSC, as shown by an increased expression of typical osteoblastic marker genes such as collagen Ialpha1, bone sialoprotein II, BMP-2 and MMP-13, as well as by increased 45Ca²+ incorporation. Our results suggest that the effect of PRS on human MSC could be at least partially mediated by BMP-2. Activated autologous PRS could therefore provide an alternative to agents like recombinant bone growth factors by increasing osteoblastic differentiation of bone precursor cells at bone repair sites, although further studies are needed to fully support our observations.

Stimulatory effects of creatine on metabolic activity, differentiation and mineralization of primary osteoblast-like cells in monolayer and micromass cell cultures.

The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically. The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.

In serum-free culture thyroid hormones can induce full expression of chondrocyte hypertrophy leading to matrix calcification.

A serum-free culture system has been developed to examine the biologic factors involved in the regulation of cellular maturation, extracellular matrix assembly, and calcification in the physis of the bovine fetal growth plate. Isolated prehypertrophic chondrocytes in high density culture undergo a process of cellular maturation whereby full expression of the hypertrophic phenotype is characterized first by type X collagen synthesis followed by matrix calcification. Using this culture system, we compared the capacity of tri-iodothyronine (T3) with thyroxine (T4) to stimulate expression of the hypertrophic phenotype and matrix calcification in three (B, C, and D) maturationally distinct prehypertrophic chondrocyte subpopulations. The B cell subpopulation was the most mature followed by C and D subpopulations in order of decreasing maturity. Comparisons were made to cultures in fetal calf serum (FCS). In Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, and selenium, both hormones (T3/T4) separately induced, in a dose-dependent manner, chondrocyte maturation to the hypertrophic phenotype characterized by increased type X collagen mRNA and induction of protein synthesis of this molecule, together with increased alkaline phosphatase activity, and eventually calcification of the extracellular matrix. Such cellular maturation to the hypertrophic phenotype was not observed in the absence of T3 or T4 with subpopulations C and D. Only in older fetuses (> 210 days) was this observed and then only in the B subpopulation. Furthermore, T3 was at least 50-fold more potent than T4. The effects of T3 were most pronounced with the most immature cells (subpopulations C and D) where, in the case of the subpopulation C, in contrast to 0.5 nM T3 50 nM T4 was unable to induce expression of the hypertrophic phenotype. Alkaline phosphatase activity was also increased in the C cell subpopulation treated with 1 nM T3 (35.5 U/micrograms of DNA) over that supplemented with 50 nM T4 (7.8 U/micrograms of DNA). Furthermore, matrix calcification, measured by the incorporation of 45Ca2+ into the cell layer, always occurred earlier in cells cultured with T3 compared with T4. Cellular maturation to the hypertrophic phenotype was not accompanied by significant changes in DNA content; this ordinarily increases during culture in the presence of serum. Compared with cells cultured in the presence of serum, either thyroid hormone more potently induced cellular maturation. This study demonstrates that the most immature chondrocytes at the prehypertrophic stage are direct targets for T3 and T4 and, to a much a lesser degree, that either hormone is able to induce full chondrocyte hypertrophy from an early maturational stage leading to matrix calcification. But T3 is much more potent than T4. These studies also offer a new serum-free chemically defined medium containing T3 or T4 for the culture of defined prehypertrophic chondrocytes that supports matrix assembly, hypertrophic expression, followed by matrix calcification.