Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained osteoinduction in response to BMP-2 stimulation.

Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing. Therefore, MSC were isolated from femoral heads of healthy and osteoporotic patients. Grouping was carried out by bone mineral densitometry in an age-matched manner. MSC were stimulated with BMP-2. Signal transduction was assessed by western blotting of pSMAD1/5/8 and pERK1/2 as well as by quantitative RT-PCR of Runx-2, Dlx5, and Osteocalcin. Osteogenic differentiation was assessed by quantifying Alizarin Red staining. Osteoporotic MSC featured an accurate phosphorylation pattern of SMAD1/5/8 but a significantly reduced activation of ERK1/2 by BMP-2 stimulation. Furthermore, osteoporotic MSC showed significantly reduced basal expression levels of Runx-2 and Dlx5. However, Runx-2, Dlx5, and Osteocalcin expression showed adequate up-regulation due to BMP-2 stimulation. The global osteogenic differentiation in standard osteogenic differentiation media was reduced in osteoporotic MSC. Nevertheless, osteoporotic MSC were shown to feature an adequate induction of osteogenic differentiation due to BMP-2 stimulation. Taken together, we here demonstrate osteoporosis associated alterations in BMP-2 signaling but sustained specific osteogenic differentiation capacity in response to BMP-2. Therefore, BMP-2 may represent a promising therapeutic agent for the treatment of fractures in osteoporotic patients.

Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation.

Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or inhibits osteogenic differentiation in a time dependent manner indicates an age related alteration in signal transduction of hMSC and requires further investigation.