Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation.

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Although bone appears to be an apparently simple tissue, it is in reality a unique and very complex tissue composed of a variety of types of cells, including osteoblasts, osteoclasts, osteocytes, chondrocytes, adipocytes, immune cells, and hematopoietic cells [1]. Mesenchymal cells contribute to this diversity of bone tissue because mesenchymal cells are multipotent to differentiate into osteoblasts, chondrocytes, and adipocytes [2,3]. Differentiation processes of mesenchymal cells are harmoniously and dynamically controlled by specifi c signal transduction and transcription factors. In the past decade, transcription factors that specifi cally control the differentiation program of mesenchymal cells have been identifi ed. Genetic studies clearly demonstrate that Runx2 (Cbfa1/Pepb2aA) and Osterix (Sp7) are indispensable transcription factors for osteoblast development [4–6] (Fig. 1). Chondrocyte differentiation requires Sox family members in the early stage and Runx2 in the late stage [7–9] (Fig. 1). C/EBP family members and PPAR-γ play critical roles in adipocyte differentiation from mesenchymal cells [10,11] (Fig. 1). Moreover, recent studies have further advanced our understanding of the molecular basis by which these transcription factors regulate each differentiation program. In particular, biochemical studies have revealed how the expression and function of these transcription factors are controlled or modulated by coactivators, co-repressors, and other transcriptional regulators that assemble large complexes with the transcription factors. The functional roles of these transcription factors are also strictly regulated by signal transduction that links extracellular changes with the nucleus through the cytoplasm. Several cytokines and hormones such as bone morphogenetic protein (BMP), transforming growth factor-β (TGFβ), Wnt, hedgehog, fi broblast growth factors, estrogen, and androgen are involved in the regulation of mesenchymal cell differentiation by stimulating intracellular signaling pathways [1,9]. Specifi c intracellular signaling molecules are activated through phosphorylation, ubiquitination, protein– protein interaction, and conformational change in response to the ligand stimulation. The activated signaling molecules elicit the specifi c transcription factors by upregulating their transcriptional activity and/or translocation into the nucleus. These signaling pathways also engage in cross-talk, forming a complex network system. In this review article, we describe recent progress in describing molecular mechanisms that conduct the differentiation of mesenchymal cells into osteoblasts, chondrocytes, and adipocytes. First, we illustrate the role of BMP, TGF-β, Wnt, and Indian hedgehog (Ihh) signaling in mesenchymal cell differentiation. Second, we introduce the transcriptional regulation associated with the differentiation program of mesenchymal cells.

Osteoblast and adipocyte are differentiated from mesenchymal stem cells and dysregulation of the differentiation might result in disease, such as osteoporosis and diabetes. To find small compounds that induce osteoblast differentiation, we screened an in-house natural compounds library with mouse preosteoblastic MC3T3-E1 cells using alkaline phosphatase (ALP) expression as an early osteoblast marker. We found that phenazine-1-carboxylic acid (PCA), one of the major phenazine derivatives produced by Pseudomonas, induced osteoblast differentiation in the cells at micromolar concentrations. PCA acted synergistically with an agonist of hedgehog signaling in inducing ALP activity in the cells. We also found that 2-hydroxy-PCA (2H-PCA) induced osteoblast differentiation in the cells but 2-methoxy-PCA and 1-hydroxy-phenazine did not. Unexpectedly, treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with PCA or 2H-PCA induced an obvious morphological change. Oil Red O staining and real-time reverse-transcription PCR analysis revealed that PCA induced not osteoblast differentiation but adipocyte differentiation in C3H10T1/2 cells. These compounds could allow us to investigate the mechanism of osteoblast and adipocyte differentiation in the two model cell systems through a chemical biology approach.

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.

BackgroundTo evaluate the effect of several representative decontamination methods of oral biofilms on different implant surfaces.Material and methodsEleven participants wore a hard resin splint carrying 6 rough (GC Aadva® implant; 3.3-mm diameter, 8-mm length) or machined (not commercially available) surface implants for 4 days to accumulate dental plaque naturally on the titanium surfaces of the implants. Apart from surface roughness, the morphology of all implants was identical. After detaching the implants from the splints, the ability of the following decontamination methods—gauze soaked in saline (G), ultrasonic scaler (US), air abrasive (Air), rotary stainless steel instrument (Rot), and Er:YAG laser (Las)—to cleanse the contaminated implant surface for 1 min extra-orally was tested. The control (Cont) group did not receive any decontamination. Scanning electron microscopic (SEM) investigation of one participant’s samples was employed to examine the post-instrumented implant surface for qualitative analysis, and bacterial culture of the remaining 10 participants’ samples was performed to count the number of colony-forming units (CFU) for quantitative analysis. The experimental sequence was initially performed for the rough surface implants and then similarly repeated for the machined surface implants. Bacterial CFU counts among the six groups were analyzed using the Steel-Dwass test, and differences between rough and machined surface implants were determined using the Mann-Whitney U test.ResultsG and Rot eliminated most biofilms on machined surface implants according to SEM analysis. G, Air, and Rot removed significantly more of the biofilms on rough and machined surface implants compared with US according to CFU counts. Moreover, G significantly reduced more biofilms than Las on machined surface implants. The analysis between rough and machined surface implants showed that Cont, G, and US were better able to cleanse biofilms on machined surface implants compared with rough surface implants.ConclusionsGauze soaked in saline and rotary stainless steel instruments may be advantageous for cleansing contaminated implant surfaces based on the qualitative and quantitative analyses. In contrast, air abrasives were not shown to be preferable in the qualitative analyses. Additionally, apart from the Er:YAG laser, the reduction of biofilms assessed in both qualitative and quantitative analyses demonstrated that all decontamination methods were better at cleansing machined surface implants compared with rough surface implants.

Multiple myeloma (MM) is a glutamine (Gln)-auxotroph and Gln-addicted cancer, with Gln synthetase (GS)-deficient MM cells avidly taking up extracellular Gln to sustain their metabolism. Thus, MM cells create a peculiar metabolic niche in the patients' bone marrow (BM), where low levels of Gln contribute to the osteolytic bone lesions by inhibiting the osteoblastic differentiation of mesenchymal stromal cells (MSCs). The effects of the altered MM metabolic niche on other BM cell populations remain to be clarified. We demonstrate here that MM cells secrete high amounts of glutamate through the exchange transporter SLC7A11/xCT. In turn, BM MSCs, but neither MM cells nor osteoblasts (OBs), actively take up extracellular glutamate through the transporter EAAT3 (SLC1A1), whose expression decreases during osteogenesis. GS-positive MSCs secrete Gln, a process boosted by extracellular glutamate in undifferentiated MSCs, but not in differentiated OBs. Coculture of MSCs with MM cells promotes the expression of the bidirectional transporter SNAT5 (SLC38A5), suggesting its involvement in Gln efflux. Consistently, MSCs, derived from either patients with MM or healthy donors, sustain MM growth in a low-Gln environment, an effect suppressed by the inhibition or silencing of glutamate uptake or Gln synthesis. In conclusion, a metabolic cycle occurs in MM BM microenvironment, where Gln-auxotroph MM cells extrude glutamate that is converted into Gln by MSC, sustaining in turn MM anabolism through Gln secretion. The inhibition of this metabolic trade-off impairs MM cell growth, thus highlighting novel potential, niche-oriented therapeutic targets.

BackgroundMesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4negCD105+CD90bright and MSCA-1+), in the primary cultures, resulted lower than 2%.Methodology/Principal FindingWe demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID50 = 0.5 µM, p<0.01), while endothelial differentiation was unaffected.ConclusionThe present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.

miRNAs are known as the cellular phenomena regulators that exert their effects in post-transcriptional level. Recent studies highlight the role of miRNAs in mesenchymal stem cells differentiation into osteoblasts. The purpose of this study was to recognize the pattern of miRNA-101a-3p and miRNA-200a expression during osteoblastic differentiation of human adipose tissue-derived mesenchymal stem cells. The cells were incubated in osteoblastic differentiation medium for a period of 21 days. Alizarin red S staining was performed to confirm the successful differentiation of adipose-derived mesenchymal stem cells into osteoblast cells. The expression levels of miRNA-101a-3p and miRNA-200a were analyzed by real-time PCR during 0, 7, 14, and 21 days after differentiation induction. Data exhibited the increase of extracellular red color deposition which was evident at the end of the incubation period. The expression of miRNA-101a-3p and miRNA-200a was up regulated during adipose-derived mesenchymal stem cells trans-differentiation into osteoblast-like cells. These miRNAs could be potential novel biomarkers for monitoring successful differentiation of mesenchymal stem cells toward osteoblasts.

Pivotal role of Twist in skeletal biology and pathology

Histomorphometric Analysis of the Bone-Implant Contact Obtained with 4 Different Implant Surface Treatments Placed Side By Side in the Dog Mandible

Effect of La3+ on osteoblastic differentiation of rat bone marrow stromal cells

Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4-induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8+ T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. SIGNIFICANCE: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression.

Downregulation of the neuregulin 1 receptor ErbB3 by Wnt3a contributes to Wnt-induced osteoblast differentiation in mesenchymal cells

Mesenchymal stem cells (MSCs) and osteoblasts respond to the surface electrical charge and topography of biomaterials. This work focuses on the connection between the roughness of calcium phosphate (CP) surfaces and their electrical potential (EP) at the micro- and nanoscales and the possible role of these parameters in jointly affecting human MSC osteogenic differentiation and maturation in vitro. A microarc CP coating was deposited on titanium substrates and characterized at the micro- and nanoscale. Human adult adipose-derived MSCs (hAMSCs) or prenatal stromal cells from the human lung (HLPSCs) were cultured on the CP surface to estimate MSC behavior. The roughness, nonuniform charge polarity, and EP of CP microarc coatings on a titanium substrate were shown to affect the osteogenic differentiation and maturation of hAMSCs and HLPSCs in vitro. The surface EP induced by the negative charge increased with increasing surface roughness at the microscale. The surface relief at the nanoscale had an impact on the sign of the EP. Negative electrical charges were mainly located within the micro- and nanosockets of the coating surface, whereas positive charges were detected predominantly at the nanorelief peaks. HLPSCs located in the sockets of the CP surface expressed the osteoblastic markers osteocalcin and alkaline phosphatase. The CP multilevel topography induced charge polarity and an EP and overall promoted the osteoblast phenotype of HLPSCs. The negative sign of the EP and its magnitude at the micro- and nanosockets might be sensitive factors that can trigger osteoblastic differentiation and maturation of human stromal cells.

TNF-alpha is known to inhibit osteoblast differentiation in vitro and yet it is essential for bone fracture repair. Roles of TNF-alpha in the bony repair of injured growth plate were examined in young rats treated with a TNF-alpha antagonist. The results show that TNF-alpha mediates p38 activation, which influences the recruitment, proliferation, and osteoblast differentiation of mesenchymal cells and negatively regulates bone formation at the injured growth plate. TNF-alpha inhibits expression of osteoblast differentiation factor cbfa1 and osteoblast differentiation in vitro and yet TNF-alpha signaling is essential for bone fracture healing. Roles of TNF-alpha in the bony repair of injured growth plate cartilage are unknown. Roles of TNF-alpha in the activation of p38 mitogen activated protein (MAP) kinase and the subsequent bony repair of the injured growth plate were examined in young rats receiving the TNF-alpha inhibitor ENBREL or saline control. Activation of p38 was determined by Western blot analysis and immunohistochemistry. Inflammatory cell counts on day 1, measurements of repair tissue proportions, and counting of proliferative mesenchymal cells on day 8 at growth plate injury site were carried out (n = 6). Expression of inflammatory cytokines TNF-alpha and IL-1beta, fibrogenic growth factor (FGF)-2, cbfa1, and bone protein osteocalcin at the injured growth plate was assessed by quantitative RT-PCR. Effects of TNF-alpha signaling on proliferation, migration, and apoptosis of rat bone marrow mesenchymal cells (rBMMCs) and the regulatory roles of p38 in these processes were examined using recombinant rat TNF-alpha, ENBREL, and the p38 inhibitor SB239063 in cultured primary rBMMCs. p38 activation was induced in the injured growth plate during the initial inflammatory response, and activated p38 was immunolocalized in inflammatory cells at the injury site and in the adjacent growth plate. In addition, activation of p38 was blocked in rats treated with TNF-alpha antagonist, suggesting a role of TNF-alpha in p38 activation. Whereas TNF-alpha inhibition did not alter inflammatory infiltrate and expression of TNF-alpha and IL-1beta at the injured growth plate on day 1, it reduced mesenchymal infiltrate and cell proliferation and FGF-2 expression on day 8. Consistently, TNF-alpha increased proliferation and migration of rBMMCs in vitro, whereas p38 inhibition reduced rBMMC proliferation and migration. At the injured growth plate on day 8, TNF-alpha inhibition increased expression of cbfa1 and osteocalcin and increased trabecular bone formation at the injury site. There was a significant inverse correlation between TNF-alpha and cbfa1 expression levels, suggesting a negative relationship between TNF-alpha and cbfa1 in this in vivo model. These observations suggest that TNF-alpha activates p38 MAP kinase during the inflammatory response at the injured growth plate, and TNF-alpha-p38 signaling seems to be required for marrow mesenchymal cell proliferation and migration at the growth plate injury site and in cell culture. Furthermore, TNF signaling has an inhibitory effect on bone formation at the injured growth plate by suppressing bone cell differentiation and bone matrix synthesis at the injury site.