The use of multipotent human mesenchymal stem cells (hMSCs) for tissue engineering has been a subject of extensive research. The donor variation in growth, differentiation and in vivo bone forming ability of hMSCs is a bottleneck for standardization of therapeutic protocols. In this study, we isolated and characterized hMSCs from 19 independent donors, aged between 27 and 85 years, and investigated the extent of heterogeneity of the cells and the extent to which hMSCs can be expanded without loosing multipotency. Dexamethasone-induced ALP expression varied between 1.2-and 3.7-fold, but no correlation was found with age, gender, or source of isolation. The cells from donors with a higher percentage of ALP-positive cells in control and dexamethasone-induced groups showed more calcium deposition than cells with lower percentage of ALP positive cells. Despite the variability in osteogenic gene expression among the donors tested, ALP, Collagen type 1, osteocalcin, and S100A4 showed similar trends during the course of osteogenic differentiation. In vitro expansion studies showed that hMSCs can be effectively expanded up to four passages (approximately 10-12 population doublings from a P0 culture) while retaining their multipotency. Our in vivo studies suggest a correlation between in vitro ALP expression and in vivo bone formation. In conclusion, irrespective of age, gender, and source of isolation, cells from all donors showed osteogenic potential. The variability in ALP expression appears to be a result of sampling method and cellular heterogeneity among the donor population. ß

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Tissue engineering of large bone defects is approached through implantation of autologous osteogenic cells, generally referred to as multipotent stromal cells or mesenchymal stem cells (MSCs). Animal-derived MSCs successfully bridge large bone defects, but models for ectopic bone formation as well as recent clinical trials demonstrate that bone formation by human MSCs (hMSCs) is inadequate. The expansion phase presents an attractive window to direct hMSCs by pharmacological manipulation, even though no profound effect on bone formation in vivo has been described so far using this approach. We report that activation of protein kinase A elicits an immediate response through induction of genes such as ID2 and FosB, followed by sustained secretion of bone-related cytokines such as BMP-2, IGF-1, and IL-11. As a consequence, PKA activation results in robust in vivo bone formation by hMSCs derived from orthopedic patients.bone tissue engineering ͉ osteogenesis ͉ PKA signaling

Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.

We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications.