Bone Marrow-derived Stromal Stem Cells Research Articles

The antiapoptotic effects of conditioned medium from bone marrow-derived mesenchymal stromal stem cells on cyclophosphamide-induced testicular damage in rat: An experimental study.

Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy. This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles. BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200-250 gr) were randomly divided into following groups: control, CP, CP+ Dulbecco's Modified Eagle Medium (DMEM), CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction. CP had a negative effect on testis histology (p 0.001) and sperm quality (p 0.001). It changed the expression of genes associated with apoptosis (p 0.001). Treatment with CM reduced the expression of Bax (p 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p 0.001), motility (p 0.001), spermatogonial count (p 0.001), and epithelial thickness of testicular tubules (p = 0.02). These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP.

Connexin43 overexpression promotes bone regeneration by osteogenesis and angiogenesis in rat glucocorticoid-induced osteonecrosis of the femoral head

Glucocorticoids induced osteonecrosis of the femoral head (GIONFH) is a devastating orthopedic disease. Previous studies suggested that connexin43 is involved in the process of osteogenesis and angiogenesis. However, the role of Cx43 potentiates in the osteogenesis and angiogenesis of bone marrow-derived stromal stem cells (BMSCs) in GIONFH is still not investigated. In this study, BMSCs were isolated and transfected with green fluorescent protein or the fusion gene encoding GFP and Cx43. The osteogenic differentiation of BMSCs were detected after transfected with Cx43. In addition, the migration abilities and angiogenesis of human umbilical vein endothelial cells (HUVECs) were been detected after induced by transfected BMSCs supernatants in vitro. Finally, we established GC-ONFH rat model, then, a certain amount of transfected or controlled BMSCs were injected into the tibia of the rats. Immunohistological staining and micro-CT scanning results showed that the transplanted experiment group had significantly promoted more bone regeneration and vessel volume when compared with the effects of the negative or control groups. This study demonstrated for the first time that the Cx43 overexpression in BMSCs could promote bone regeneration as seen in the osteogenesis and angiogenesis process, suggesting that Cx43 may serve as a therapeutic gene target for GIONFH treatment.

Pharmacological inhibition of DKK1 promotes spine fusion in an ovariectomized rat model.

Osteoporosis is common in patients undergoing spine surgery, and carries a considerable risk of adverse outcomes. New methods to positively influence bone regeneration and spine fusion under osteoporotic conditions would be impactful. Neutralizing anti-Dickkopf-1 (DKK1) antibodies has been used as a bone anabolic agent, and recently reported by our group to aid in stem cell-mediated appendicular bone regeneration. Here, a small molecule designed as a DKK1 inhibitor, WAY-262611, was used to induce posterolateral spine fusion in an ovariectomized rat model. In vitro, pharmacological inhibition of DKK1 enhanced osteogenesis and Wnt signaling activity among rat bone marrow-derived stem/stromal cells (BMSCs). In vivo, systemic treatment with WAY-262611 promoted both chondrogenesis and osteogenesis within the spinal fusion site, and ultimately led to significant improvements in lumbar fusion as assessed by XR, μCT, histology and manual palpation assessments. No significant effect on osteoclast numbers or fusion site angiogenesis was detected, suggesting a primary direct effect on mesenchymal cells of the implantation site. Finally, evidence from human stem/stromal cells further demonstrated that pharmacologic inhibition of DKK1 promoted osteogenic differentiation in vitro. Taken together, our results suggest that targeting DKK1 promotes local bone formation and suggests potential clinical value for osteoporotic bone repair.

Mesenchymal Stromal Cells Suppress Hepatic Fibrosis Via Modulating the Expression of Fibronectin and Integrin and Inhibiting DNA Fragmentation in Rats

IntroductionLiver fibrosis is currently the 11th most common cause of death worldwide. Because of self-renewal, available sources for isolation, and high differentiation properties, multipotent mesenchymal stromal stem cells are suggested to be potential tool for treatment of liver fibrosis. In this study, we examined the anti-fibrotic and anti-inflammatory activity of bone marrow-derived multipotent mesenchymal stromal stem cells (MSCs) on liver fibrosis induced by carbon tetrachloride on rats relative to silymarin as a standard drug.Material and methodsThis study was performed on 40 male Sprague Dawley rats divided into 4 groups of ten rats each: Group 1 served as controls, Group 2 served as CCl4 (diseased) group, Group 3 served as silymarin treated group and Group 4 served as MSCs treated group. Liver fibrosis was assessed by determination of liver markers and fibrogenesis related genes together with the anti-inflammatory markers in the liver tissue. DNA fragmentation was assessed by Comet assay.ResultsMSCs treatment reduced all liver fibrosis markers as well as the oxidative stress and inflammatory markers. Additionally, MSCs reduced the expression of integrins and fibronectin compared with the control group as well as decreasing DNA fragmentation.ConclusionsTreatment by MSCs significantly ameliorates liver fibrosis in rats. This amelioration was a result of acting on both the anti-inflammatory and anti-fibrotic activity of hepatocytes.

Hyaluronic Acid Supplement as a Chondrogenic Adjuvant in Promoting the Therapeutic Efficacy of Stem Cell Therapy in Cartilage Healing.

The main aim of this study is to investigate the therapeutic efficacy of direct intra-articular injection of bone-marrow-derived stem/stromal cells (BMSCs) and the adjuvant role of hyaluronic acid (HA) in facilitating rabbit articular cartilage repair. First, rabbit BMSCs were treated with a medium containing different concentrations of HA. Later, HA’s influence on BMSCs’ CD44 expression, cell viability, extracellular glycosaminoglycan (GAG) synthesis, and chondrogenic gene expression was evaluated during seven-day cultivation. For the in vivo experiment, 24 rabbits were used for animal experiments and 6 rabbits were randomly allocated to each group. Briefly, chondral defects were created at the medial femoral condyle; group 1 was left untreated, group 2 was injected with HA, group 3 was transplanted with 3 × 106 BMSCs, and group 4 was transplanted with 3 × 106 BMSCs suspended in HA. Twelve weeks post-treatment, the repair outcome in each group was assessed and compared both macroscopically and microscopically. Results showed that HA treatment can promote cellular CD44 expression. However, the proliferation rate of BMSCs was downregulated when treated with 1 mg/mL (3.26 ± 0.03, p = 0.0002) and 2 mg/mL (2.61 ± 0.04, p = 0.0001) of HA compared to the control group (3.49 ± 0.05). In contrast, 2 mg/mL (2.86 ± 0.3) of HA treatment successfully promoted normalized GAG expression compared to the control group (1.88 ± 0.06) (p = 0.0009). The type II collagen gene expression of cultured BMSCs was significantly higher in BMSCs treated with 2 mg/mL of HA (p = 0.0077). In the in vivo experiment, chondral defects treated with combined BMSC and HA injection demonstrated better healing outcomes than BMSC or HA treatment alone in terms of gross grading and histological scores. In conclusion, this study helps delineate the role of HA as a chondrogenic adjuvant in augmenting the effectiveness of stem-cell-based injection therapy for in vivo cartilage repair. From a translational perspective, the combination of HA and BMSCs is a convenient, ready-to-use, and effective formulation that can improve the therapeutic efficacy of stem-cell-based therapies.

In utero xenotransplantation of mice bone marrow-derived stromal/stem cells into fetal rat liver: An experimental study.

BackgroundAnimals can play an important role in preparing tissues for human through the development of xenotransplantation protocols. The most common problem with liver transplantation like any other organ transplantation is organ supply shortage.ObjectiveTo evaluate the in utero xenotransplantation of mouse bone marrow-derived stromal/stem cells (BMSCs) to the liver of rat fetus to produce mouse liver tissue.Materials and MethodsBMSCs were isolated and confirmed from enhanced green fluorescent protein (eGFP)-genetic labeled mice. Using a microinjection protocol, mice BMSCs were injected into the liver of rat fetuses in utero on day 14 of pregnancy. After birth, livers were collected and the presence of mice eGFP-positive cells in rat livers was evaluated through polymerase chain reaction.ResultsThe eGFP mRNA was detected in the liver of injected infant rats. BMSCs of adult mice were capable to remain functional probably as hepatocyte-like cells in liver of infant rats after in utero xenotransplantation.Conclusion BMSCs have the potential for intrauterine xenotransplantation for the treatment of liver dysfunction before birth. This method can also be used for xenoproduction of liver tissue for transplantation.

Recent Approaches to Isolating and Culturing Mouse Bone Marrowderived Mesenchymal Stromal Stem Cells.

The isolation and culture of murine Bone Marrow-derived Mesenchymal stromal Stem Cells (mBMSCs) have attracted great interest in terms of the pre-clinical applications of stem cells in tissue engineering and regenerative medicine. In addition, culturing mBMSCs is important for studying the molecular mechanisms of bone remodeling using relevant transgenic mice. Several factors have created challenges in the isolation and high-yield expansion of homogenous mBMSCs; these factors include low frequencies of Bone Marrow-derived mesenchymal stromal Stem Cells (BMSCs) in bone marrow, variation among inbred mouse strains, contamination with Haematopoietic Progenitor Cells (HPCs), the replicative senescence phenotype and cellular heterogeneity. In this review, we provide an overview of nearly all protocols used for isolating and culturing mBMSCs with the aim of clarifying the most important guidelines for culturing highly purified mBMSC populations retaining in vitro and in vivo differentiation potential.

Local SDF-1α application enhances the therapeutic efficacy of BMSCs transplantation in osteoporotic bone healing

Bone defect healing is markedly impaired in osteoporotic patient due to poor bone regeneration ability. Stromal cell derived factor-1α (SDF-1α) plays a pivotal role in the repair of various injured tissues including bone. Here, we definite that SDF-1α hydrogels potentiates in vivo osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in osteoporosis. The characteristics of rat primary BMSCs including superficial markers by flow cytometry and multi-lineage differentiation by induction were determined. At different time intervals, the release media from the SDF-1α-releasing hydrogels were collected to identificate SDF-1α exhibited a sustained release profile and maintained its bioactivity after release from the hydrogels to stimulate chemotaxis of BMSCs in a time dependent manner. Bilateral alveolar defects were operated in ovariectomized (OVX) rats and repaired with systemic BMSCs transplantation with or without the hydrogels. Local administration of SDF-1α significantly enhanced BMSCs recruitment and promoted more bone regeneration as well as the expression of OCN and Runx2 compared with the effect of BMSCs transplantation alone. Moreover, after BMSCs transplantation with SDF-1α delivery, macrophage polarization was promoted toward the M2 phenotype, that is identified as an important symbol in tissue regeneration process. Taken together, local SDF-1α application enhances the efficacy of BMSCs transplantation therapy in osteoporotic bone healing, suggesting clinical potential of SDF-1α to serve as a therapeutic drug target for osteoporosis treatment.

Combinatory transplantation of mesenchymal stem cells with flavonoid small molecule in acellular nerve graft promotes sciatic nerve regeneration

Previous animal studies have demonstrated that the flavonoid small-molecule TrkB agonist, 7, 8-dihydroxyflavone (DHF), promotes axon regeneration in transected peripheral nerves. In the present study, we investigated the combined effects of 7, 8-DHF treatment and bone marrow-derived stem/stromal cells (BMSCs) engraftment into acellular nerve allografts (ANAs) and explore relevant mechanisms that may be involved. Our results show that TrkB and downstream ERK1/2 phosphorylation are increased upon 7, 8-DHF treatment compared to the negative control group. Also, 7, 8-DHF promotes proliferation, survival, and Schwann-like cell differentiation of BMSCs in vitro. While selective ERK1/2 inhibitor U0126 suppressed the effect of upregulation of ERK1/2 phosphorylation and decreased cell proliferation, survival, and Schwann-like cell differentiation partially induced by 7, 8-DHF. In vivo, 7, 8-DHF promotes survival of transplanted BMSCs and upregulates axonal growth and myelination in regenerating ANAs. 7, 8-DHF+BMSCs also improved motor endplate density of target musculature. These benefits were associated with increased motor functional recovery. 7, 8-DHF+BMSCs significantly upregulated TrkB and ERK1/2 phosphorylation expression in regenerating ANA, and increased TrkB expression in the lumbar spinal cord. The mechanism of 7, 8-DHF action may be related to its ability to upregulate TrkB signaling, and downstream activation of survival signaling molecules ERK1/2 in the regenerating ANAs and spinal cord and improved survival of transplanted BMSCs. This study provides novel foundational data connecting the benefits of 7, 8-DHF treatment in neural injury and repair to BMSCs biology and function and demonstrates a potential combination approach for the treatment of injured peripheral nerve via nerve graft transplant.

KLF7 overexpression in bone marrow stromal stem cells graft transplantation promotes sciatic nerve regeneration

Objective. Our previous study demonstrated that the transcription factor, Krüppel-like Factor 7 (KLF7), stimulates axon regeneration following peripheral nerve injury. In the present study, we used a gene therapy approach to overexpress KLF7 in bone marrow-derived stem/stromal cells (BMSCs) as support cells, combined with acellular nerve allografts (ANAs) and determined the potential therapeutic efficacy of a KLF7-transfected BMSC nerve graft transplantation in a rodent model for sciatic nerve injury and repair. Approach. We efficiently transfected BMSCs with adeno-associated virus (AAV)-KLF7, which were then seeded in ANAs for bridging sciatic nerve defects. Main results. KLF7 overexpression promotes proliferation, survival, and Schwann-like cell differentiation of BMSCs in vitro. In vivo, KLF7 overexpression promotes transplanted BMSCs survival and myelinated fiber regeneration in regenerating ANAs; however, KLF7 did not improve Schwann-like cell differentiation of BMSCs within in the nerve grafts. KLF7-BMSCs significantly upregulated expression and secretion of neurotrophic factors by BMSCs, including nerve growth factor, ciliary neurotrophic factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in regenerating ANA. KLF7-BMSCs also improved motor axon regeneration, and subsequent neuromuscular innervation and prevention of muscle atrophy. These benefits were associated with increased motor functional recovery of regenerating ANAs. Significance. Our findings suggest that KLF7-BMSCs promoted peripheral nerve axon regeneration and myelination, and ultimately, motor functional recovery. The mechanism of KLF7 action may be related to its ability to enhance transplanted BMSCs survival and secrete neurotrophic factors rather than Schwann-like cell differentiation. This study provides novel foundational data connecting the benefits of KLF7 in neural injury and repair to BMSC biology and function, and demonstrates a potential combination approach for the treatment of injured peripheral nerve via nerve graft transplant.

Compliant substratum modulates vinculin expression in focal adhesion plaques in skeletal cells

The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. In the skeleton system, bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process, which involves chondrocytes, osteoblasts, osteoclasts, osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs). In the current study, we generated stiff (~1 014 ± 56) kPa, Young’s modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios, respectively, and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area, cell cytoskeleton and cell adhesion capacity. The results showed that the cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate. F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group. Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate. This study establishes the potential correlation between microenvironmental mechanics and the skeletal system, and the results regarding changes in cell spreading area, cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

Total body irradiation tremendously impair the proliferation, differentiation and chromosomal integrity of bone marrow-derived mesenchymal stromal stem cells.

Total body irradiation (TBI) is frequently used in hematopoietic stem cell transplantation (HSCT) and is associated with many complications due to radiation injury to the normal cells, including normal stem cells. Nevertheless, the effects of TBI on the mesenchymal stromal stem cell (MSC) are not fully understood. Bone marrow-derived MSCs (BM-MSCs) isolated from normal adults were irradiated with 200cGy twice daily for consecutive 3days, a regimen identical to that used in TBI-conditioning HSCT. The characteristics, differentiation potential, cytogenetics, hematopoiesis-supporting function, and carcinogenicity of the irradiated BM-MSCs were then compared to the non-irradiated control. The irradiated and non-irradiated MSCs shared similar morphology, phenotype, and hematopoiesis-supporting function. However, irradiated MSCs showed much lower proliferative and differentiative potential. Irradiation also induced clonal cytogenetic abnormalities of MSCs. Nevertheless, the carcinogenicity of irradiated MSCs is low in vitro and in vivo. In parallel with the ex vivo irradiation experiments, decreased proliferative and differentiative abilities and clonal cytogenetic abnormalities can also be found in MSCs isolated from transplant recipients who had received TBI-based conditioning previously. Thus, TBI used in HSCT drastically injury MSCs and may contribute to the development of some long-term complications associated with clonal cytogenetic abnormality and poor adipogenesis and osteogenesis after TBI.

Stromal-Cell-Derived Factor (SDF) 1-Alpha Overexpression Promotes Bone Regeneration by Osteogenesis and Angiogenesis in Osteonecrosis of the Femoral Head

Background/Aims: Osteonecrosis of the femoral head (ONFH) is a devastating orthopedic disease. Previous studies suggested that stromal-cell-derived factor (SDF)-1 was involved in osteogenesis and angiogenesis. However, whether SDF-1 potentiates the angiogenesis and osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in ONFH is not clear. Methods: BMSCs were transfected with green fluorescent protein (GFP) or the fusion gene encoding GFP and SDF-1α, and transgenic efficacy was monitored by immunofluorescence. The expression of SDF-1α, runt-related transcription factor 2 (Runx2, osteocalcin (OCN), and alkaline phosphatase (ALP) at the mRNA level was measured by real-time polymerase chain reactions (RT-PCR). The expression of SDF-1α, Runx2, OCN, and p-Smad1/5 were measured at the protein level by Western blot. Transwell migration assay and tube formation assay were utilized to detect the angiogenesis in vitro, whereas the in vivo angiogenesis was monitored by angiography. Immunohistological staining and micro-CT scanning were conducted to assess the histological changes in morphology. Results: In vitro, SDF-1α overexpression in BMSCs promoted osteogenic differentiation and upregulated the expression of osteogenic-related proteins, such as ALP, Runx2, OCN, and p-Smadl/5. In the methylprednisolone induced ONFH rat model used in our investigation, the overexpression of SDF-1α in BMSCs promoted significantly more bone regeneration and the expression of OCN and Runx2 as compared with the effect of vehicle overexpression. Moreover, the morphology of ONFH was ameliorated after the transplantation of BMSCs with SDF-1α overexpression. Furthermore, SDF-1α overexpression in BMSCs significantly increased osteoblastic angiogenesis as indicated by the increased tube formation ability, CD31 expression, and vessel volume. Conclusion: SDF-1α overexpression in BMSCs promotes bone generation as indicated by osteogenesis and angiogenesis, suggesting SDF-1α may serve as a therapeutic drug target for ONFH treatment.

Articular Cartilage Repair with Mesenchymal Stem Cells After Chondrogenic Priming: A Pilot Study.

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess a protocol that involved autologous transplantation of BMSCs into full-thickness cartilage defects in sheep following isolation, expansion, and a short period (4 days) of chondrogenic priming. The impact of oxygen tension during preimplantation culture was investigated. It was hypothesized that chondrogenically primed BMSCs would produce superior cartilaginous repair tissue relative to control defects, and that culture under hypoxia would yield improved repair tissue in comparison to normoxia. Ovine BMSCs were isolated, expanded to passage 2, seeded within a hyaluronic acid (HYAFF) scaffold, and primed ex vivo in chondrogenic medium for 4 days under normoxia (21% oxygen) or hypoxia (3% oxygen). Full-thickness, 7-mm-diameter articular cartilage defects were created in the femoral condyles of five sheep. Twenty defects were treated with normoxia-cultured, autologous BMSC-seeded scaffolds (eight); hypoxia-cultured, autologous BMSC-seeded scaffolds (eight); cell-free scaffolds (two); or no implants (two). Preimplantation priming was evaluated through gene expression analysis using reverse transcription quantitative polymerase chain reaction. After 6 months, histological assessment was performed on repair tissues with a modified O'Driscoll scoring system and tissue dimension analysis. Priming of preimplantation BMSC-seeded scaffolds in chondrogenic medium for 4 days resulted in significantly increased gene expression of hyaline cartilage-related collagen II and aggrecan relative to unprimed BMSCs (p < 0.05). Defects implanted with chondrogenically primed BMSC-seeded scaffolds developed cartilaginous repair tissues that contained safranin O-positive proteoglycans, and had significantly larger repair tissue areas, higher percentages of defect fill, and improved histological scores than cell-free controls (p < 0.05). Although hypoxic culture improved the preimplantation gene expression profile, a consistent difference in histological scores was not found between normoxia- and hypoxia-seeded BMSC-seeded scaffolds after 6 months (p = 0.90). This study demonstrates in a sheep model that (1) chondrogenic priming ex vivo improves the gene expression profile of BMSCs; (2) chondrogenically primed BMSCs are associated with the development of superior cartilaginous tissue to cell-free controls within cartilage defects; and (3) oxygen tension during preimplantation ex vivo culture does not consistently modulate cartilaginous repair tissue formation following BMSC transplantation into cartilage defects.

Effects of Carthamus Tinctorius Extract on Adipogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stromal Stem Cells

Effects of Carthamus Tinctorius Extract on Adipogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stromal Stem Cells

Nurse's A-Phase Material Enhance Adhesion, Growth and Differentiation of Human Bone Marrow-Derived Stromal Mesenchymal Stem Cells.

The purpose of this study was to evaluate the bioactivity and cell response of a well-characterized Nurse’s A-phase (7CaO·P2O5·2SiO2) ceramic and its effect compared to a control (tissue culture polystyrene-TCPS) on the adhesion, viability, proliferation, and osteogenic differentiation of ahMSCs in vitro. Cell proliferation (Alamar Blue Assay), Alizarin Red-S (AR-s) staining, alkaline phosphatase (ALP) activity, osteocalcin (OCN), and collagen I (Col I) were evaluated. Also, field emission scanning electron microscopy (FESEM) images were acquired in order to visualise the cells and the topography of the material. The proliferation of cells growing in a direct contact with the material was slower at early stages of the study because of the new environmental conditions. However, the entire surface was colonized after 28 days of culture in growth medium (GM). Osteoblastic differentiation markers were significantly enhanced in cells growing on Nurse’s A phase ceramic and cultured with osteogenic medium (OM), probably due to the role of silica to stimulate the differentiation of ahMSCs. Moreover, calcium nodules were formed under the influence of ceramic material. Therefore, it is predicted that Nurse’s A-phase ceramic would present high biocompatibility and osteoinductive properties and would be a good candidate to be used as a biomaterial for bone tissue engineering.

Editorial review: Journal of Bone and Mineral Research “CRMP 4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling”

Collapsin response-mediated protein 4 (CRMP4) regulates the differentiation of mouse bone marrow skeletal stem cells (mBMSCs) into osteoblastic cells. CRMP4 was shown to control the early commitment of murine bone marrow-derived stromal stem cells into the osteogenic lineage. The inhibitory effect of CRMP4 on bone mass revealed its direct involvement in mediating BMP2-induced osteogenesis signaling and a role in osteoblast adhesion and proliferation via regulation of cytoskeletal dynamics in a RhoA-focal adhesion kinase (FAK)-dependent mechanism (1). In this editorial, we discuss the details of Rho and its downstream targets, especially Rho-kinase and CRMP families.

Xeno-Free Cryopreservation of Bone Marrow-Derived Multipotent Stromal Cells from Callithrix jacchus.

In the previous decade, numerous biobanks were established and have created large markets for the storage of bioactive compounds, cells, and tissues for medical and diagnostic applications. For in vivo clinical and therapeutic purposes, it is critical to use well-defined and xeno-free components during cultivation, preservation, and transplantation of biological material. Safe and efficacious storage of bioactive molecules, cells, and tissues, without the addition of undefined medium components, minimizes risks of zoonotic disease transmission and is thus an essential and desirable prerequisite for biobanks. This gives rise to a need for well-characterized and serum-free freezing media for application in cryopreservation. For this purpose, cryobiological additives such as methylcellulose, poloxamer-188, and α-tocopherol, which have previously been shown to exhibit a cytoprotective activity, have been investigated for cryoprotection on stem cells. With this strategy, the application of fetal bovine serum (FBS) could be avoided and the concentration of toxic cryoprotective agents such as dimethyl sulfoxide (DMSO) could be reduced. Our results suggest that the viability, as well as the adipogenic and osteogenic differentiation capacity of the thawed bone marrow-derived multipotent stromal stem cells, could be maintained using a freezing medium without FBS consisting of methylcellulose, poloxamer, and α-tocopherol with only 2.5% DMSO (% v/v).

Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization.

Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN-deficient hMSC showed that several genes and genetic pathways associated with cell differentiation, including regulation of actin cytoskeleton and focal adhesion pathways, were downregulated. Our data demonstrate that TAGLN has a role in generating committed progenitor cells from undifferentiated hMSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application.