BMP-12 Treatment of Adult Mesenchymal Stem Cells In Vitro Augments Tendon-Like Tissue Formation and Defect Repair In Vivo

We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ∼80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering

New insight into the effects of lead modulation on antioxidant defense mechanism and trace element concentration in rat bone

Risks of heavy metals-induced severe bone disorders generate interest to their toxicity. The present study was undertaken to monitor the biochemical and antioxidant status of bone of 30 and 80 days old male Wistar rats exposed to 5 week lead treatment. At the end of study, the rats were sacrificed, their long bone i.e. femur were excised, cleaned of soft tissue, minced and homogenized. Nucleic acid content, alkaline phosphatase, lipid peroxidation, catalase, glutathione S-transferase and superoxide dismutase were determined in bone. In both groups of treated animals lead treatment increased the production of malondialdehyde, while reducing activities of catalase, glutathione S-transferase and superoxide dismutase, indicating that it causes oxidative stress. Parallely with these effects lead significantly reduced the nucleic acid content and the activity of alkaline phosphatase, considered as biomarkers of osteoblast's function, conditions and development of bones. Moreover the concentrations of copper, zinc, iron and sodium were reduced in the excised bones. The present study indicates that the lead induced bone toxicity and its deteriorated development is the consequence of a primary oxidative stress. Our results may be helpful in understanding the modulation of biochemical parameters under lead toxicity

Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast-osteocyte differentiation are not yet understood. In this study we cultured MC3T3-E1 cells on (A) substrates of different chemical composition and stiffness, as well as, (B) substrates of identical chemical composition but different stiffness. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro.The authors would like to acknowledge funding from the European Research Council (ERC), Grant no.: 258992 (BONEMECHBIO), National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institute of Health (NIH), Grant nos.: AR041210 and AR057139, and the NUI Galway College of Engineering and Informatics Research Fellowship

Stimulation of systemic bone formation induced by experimental blood loss

Direct physical injury to bone marrow is associated with a systemic osteogenic response. However, blood loss, a condition that stimulates hemopoietic stem cells, also may activate osteoprogenitor cells in the bone marrow. To determine if bleeding induces a systemic osteogenic response, the mineral appositional rates and osteoblast numbers were determined in the bones of rats that were subjected to controlled cardiac bleeding and compared with those of rats subjected to ablation of their tibial bone marrow. In addition, a study of the kinetics of the osteogenic responses during the first 10 days after operative treatment was performed by quantitating the serum levels of biochemical indices known to be associated with systemic bone formation. The results showed that animals that sustained acute blood loss (1% or 3% body weight) or injury to their tibial bone marrow had statistically significant increases in mineral appositional rate, osteoblast number, and serum levels of osteogenic growth peptide. The kinetics studies showed that osteogenic growth peptide levels peaked on the tenth postoperative day and declined sharply thereafter. An enhancement of serum osteocalcin activity occurred only on the second postoperative day, was increased in all experimental groups when compared with untreated control animals, but immediately declined to baseline levels. Alkaline phosphatase activities increased in the experimental groups, peaking on Day 10 after tibial bone marrow ablation and on Day 12 in the group that underwent bleeding. These findings suggest that bleeding alone, independent of any skeletal trauma, may evoke a systemic osteogenic response. This response is similar in its timing and magnitude to that which has been shown to follow direct physical injury to bone marrow. The observation that systemic bone formation follows bone marrow activation induced by two different stimuli suggests that these responses may be mediated by common regulatory mechanisms. The ability to trigger or control these responses may form the basis for future therapeutic strategies to enhance bone formation