Effects of various antimicrobial agents on multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells

Antimicrobial drugs of several classes play an important role in the treatment of bone and joint infections. In addition to fighting pathogenic microorganisms, the effects of drugs on local tissues and cells are also related to the course and prognosis of bone and joint infections. The multi-directional differentiation potential of bone marrow-derived mesenchymal stem cells (MSCs) is essential for tissue repair after local injury, which is directly related to the recovery of bone, cartilage, and medullary adipose tissue. Our previous studies and the literature indicate that certain antimicrobial agents can regulate the differentiation potential of bone marrow-derived MSCs. Here, in order to systematically analyze the effects of various antimicrobial drugs on local tissue regeneration, we comprehensively review the studies on the effects of these drugs on MSC differentiation, and classify them according to the three differentiation directions (osteogenesis, chondrogenesis, and adipogenesis). Our review demonstrates the specific effects of different antimicrobial agents on bone marrow-derived MSCs and the range of concentrations at which they work, and provides a basis for drug selection at different sites of infection.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

Biological characteristics of muscle-derived satellite cells isolated from rats at different postnatal days

This study investigated the in vitro growth characteristics and differential potential of muscle-derived satellite cells (MDSCs) derived from rats at different postnatal (P) stages, in order to expand the range of source material for tissue engineering. Rat MDSCs were isolated from P5, P10, P15, P21 and P42 rat skeletal muscles using double enzyme digestion and differential adherent culture. Neurogenic, osteogenic and myogenic induction media were used to induce directed differentiation. Differentiated nerve cells, osteoblasts and myotubes were identified by their morphology and immunohistochemical staining. Most cells transformed into spindle-shaped mononuclear cells after 48 h and proliferated rapidly. MDSCs were difficult to isolate from P42 rats. After neurogenesis, four groups MDSCs formed neuron-specific enolase positive polygonal-shaped dendritic cells. After osteogenesis, P5, P10, P15 and P21 MDSCs formed Alizarin red- and osteocalcin-positive bone nodules. After myogenesis, myotubes were formed and were fast muscle myosin-positive. MDSCs derived from P5, P10, P15 and P21 rat skeletal muscle are easy to isolate, culture and amplify in vitro, which increases the range of source material available for tissue engineering.     

Beta-tricalcium phosphate exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway

Beta-tricalcium phosphate (β-TCP) has been clinically used as a bone graft substitute for decades because of its excellent osteoconductivity. However, the exact mechanism(s) by which β-TCP exerts osteoconductivity are not fully documented. This study was aimed to investigate the molecular mechanism(s) by which β-TCP modulates the biological response of primary human osteoblasts (HOBs). It was showed that HOBs seeded into the β-TCP scaffolds expressed significantly higher levels of osteogenic genes, compared to those cultured on tissue culture plastic; meanwhile these cells showed 7-fold increase in α2 integrin subunit gene expression and the activation of the mitogen-activated protein kinase (MAPK)/extracellular related kinase (ERK) signaling pathway. In addition, the osteogenic conduction by β-TCP scaffolds was attenuated directly by inhibiting MAPK/ERK or indirectly by blocking the α2β1 integrin signaling pathway. We concluded that β-TCP scaffold exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway, suggesting a feasible approach to consider when designing or fabricating the scaffolds for bone tissue engineering.     

Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.     

Failure to Process Dentin Matrix Protein 1 (DMP1) into Fragments Leads to Its Loss of Function in Osteogenesis

Dentin matrix protein 1 (DMP1), an acidic protein important to the formation of bone and dentin, primarily exists as the processed NH₂-terminal and COOH-terminal fragments in the extracellular matrix of the two tissues. Previous in vitro studies showed that the substitution of residue Asp²¹³ by Ala²¹³ (D213A) at a cleavage site blocked the processing of mouse DMP1 in cells. In this study, we generated transgenic mice expressing mutant D213A-DMP1 (WT/D213A-Tg mice) to test the hypothesis that the proteolytic processing of DMP1 is an activation step essential to osteogenesis. By crossbreeding WT/D213A-Tg mice with Dmp1 knock-out (Dmp1-KO) mice, we obtained mice expressing D213A-DMP1 in a Dmp1-KO background; these mice will be referred to as “Dmp1-KO/D213A-Tg” mice. Biochemical, radiological, and morphological approaches were used to characterize the skeletal phenotypes of Dmp1-KO/D213A-Tg mice compared with wild-type mice, Dmp1-KO mice, and Dmp1-KO mice expressing the normal Dmp1 transgene. Protein chemistry analyses showed that DMP1 was barely cleaved in the bone of the Dmp1-KO/D213A-Tg mice, indicating that D213A substitution effectively blocked the proteolytic processing of DMP1 in vivo. While the expression of the normal Dmp1 transgene completely rescued the phenotypic skeletal changes of the Dmp1-KO mice, the expression of the mutant D213A-Dmp1 transgene failed to do so. These results indicate that the full-length form of DMP1 is an inactive precursor and its proteolytic processing is an activation step essential to the biological functions of this protein in osteogenesis.