Rabbit Bone Marrow-derived Mesenchymal Stem Research Articles

Injectable Peptide Decorated Functional Nanofibrous Hollow Microspheres to Direct Stem Cell Differentiation and Tissue Regeneration.

Injectable microspheres are attractive stem cell carriers for minimally invasive procedures. For tissue regeneration, the microspheres need to present the critical cues to properly direct stem cell differentiation. In natural extracellular matrix (ECM), growth factors (GFs) and collagen nanofibers provide critical chemical and physical cues. However, there have been no reported technologies that integrate synthetic nanofibers and GFs into injectable microspheres. In this study, we synthesized functional nanofibrous hollow microspheres (FNF-HMS), which can covalently bind GF-mimicking peptides. Two different GF-mimicking peptides, Transforming Growth Factor-β1 mimicking peptide Cytomodulin (CM) and Bone Morphogenetic Protein-2 mimicking peptide P24, were separately conjugated onto the FNF-HMS to induce distinct differentiation pathways of rabbit bone marrow-derived mesenchymal stem cells (BMSCs). While no existing biomaterials were reported to successfully deliver CM to induce chondrogenesis, the developed FNF-HMS were shown to effectively present CM to BMSCs and successfully induced their chondrogenesis for cartilage formation in both in vitro and in vivo studies. In addition, P24 was conjugated onto the newly developed FNF-HMS and was capable of retaining its bioactivity and inducing ectopic bone formation in nude mice. These results demonstrate that the novel FNF-HMS can effectively deliver GF-mimicking peptides to modulate stem cell fate and tissue regeneration.

Poly-ε-caprolactone composite scaffolds for bone repair.

Synthetic biomaterials combined with cells and osteogenic factors represent a promising approach for the treatment of a number of orthopedic diseases, such as bone trauma and congenital malformations. To guarantee optimal biological properties, bone substitutes are prepared with a 3D structure and porosity grade functional to drive cell migration and proliferation, diffusion of factors, vascularization and cell waste expulsion. In this study, synthetic hydroxyapatite (HA) or rat bone extracellular matrix (BP) were examined in an effort to optimize the mechanical properties and osteogenic activity of poly-ε-caprolactone scaffolds prepared with alginate threads (PCL-AT). Using rabbit bone marrow-derived mesenchymal stem cells (rMSCs), the effects of PCL composite substrates on cell adhesion, growth and osteogenic differentiation were evaluated. Micro-CT analysis and scanning electron microscopy evidenced that porous PCL scaffolds containing HA or BP acquire a trabecular bone-like structure with interconnected pores homogenously distributed and are characterized by a pore diameter of approximately 10 µm (PCL-AT-BP) or ranging from 10 to 100 µm. Although the porosity grade of both PCL-AT-HA and PCL-AT-BP promoted optimal conditions for the cell growth of rMSCs at the early phase, the presence of BP was crucial to prolong the cell viability at the late phase. Moreover, a precocious expression of Runx2 (at 7 days) was observed in PCL-AT-BP in combination with osteogenic soluble factors suggesting that BP controls better than HA the osteogenic maturation process in bone substitutes.

Efficient In vitro labeling rabbit bone marrow-derived mesenchymal stem cells with SPIO and differentiating into neural-like cells.

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca2+]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.

Expansion, characterization, and differentiation of rabbit bone marrow-derived mesenchymal stem cells in serum-free medium

Mesenchymal stem cells (MSCs) derived from rabbit bone marrow are routinely investigated in cardiovascular and orthopedic models for regenerative medicine application. However, classical medium used for generating rabbit MSCs (rbMSCs) was supplemented with fetal bovine serum (FBS), which would raise several safety concerns and inconsistencies in the generation of MSCs. Here, we tested a serum-free medium for culture of rbMSCs through the investigation of multiple parameters including cell morphology, expansion, phenotype, and trilineage differentiation. RbMSCs were isolated using the gradient centrifugation method, and expanded in StemPro® MSC SFM in the presence of substrates and basic fibroblast growth factor (bFGF). Cells were then characterized by phase contrast microscopy observation, cell proliferation analysis, immunocytochemistry staining, RT-PCR, and qPCR analysis. Similar to that achieved with FBS-containing culture medium, the viability and proliferation of rbMSCs were improved in serum-free medium supplemented with bFGF and pre-coated with CELLstartTM. Serum-free expanded rbMSCs showed the fibroblastic spindle-shape morphology, and were positive for CD29, CD44, and CD73, and negative for CD34, CD45, and CD166. After the corresponding differentiation, rbMSCs from serum-free condition were demonstrated to differentiate into adipogenic, osteogenic, and chondrogenic lineages. In addition, differences were also observed between rbMSCs cultured with or without serum in terms of their morphology, proliferation, and gene expression levels of early differentiation transcription factors. In conclusion, this study demonstrates that rbMSCs can be expanded in a serum-free condition while maintaining the original characteristics of MSCs, which provides a useful tool to understand the basic biological properties of rbMSCs in culture.

Comparison of cellular responses of mesenchymal stem cells derived from bone marrow and synovium on combined silk scaffolds

As a brand new member in mesenchymal stem cells (MSCs) families, synovium-derived mesenchymal stem cells (SMSCs) have been increasingly regarded as a promising therapeutic cell species for musculoskeletal regeneration. However, there are few reports mentioning ligamentogenesis of SMSCs and especially null for their engineering use towards ligament regeneration. The aim of this study was to investigate and compare the cellular responses of MSCs derived from bone marrow and synovium on combined silk scaffolds that can be used to determine the cell source most appropriate for tissue-engineered ligament. Rabbit SMSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro for two weeks after seeding on the combined silk scaffolds. Samples were studied and compared for their cellular morphology, proliferation, collagen production, gene, and protein expression of ligament-related extracellular matrix (ECM) markers. In addition, the two cell types were transfected with green fluorescent protein to evaluate their fate after implantation in an intraarticular environment of the knee joint. After 14 days of culturing, SMSCs showed a significant increase in proliferation as compared with BMSCs. The transcript and protein expression levels of ligament-related ECM markers in SMSCs were significantly higher than those in BMSCs. Moreover, 6 weeks postoperatively, more viable cells were presented in SMSC-loaded constructs than in BMSC-loaded constructs. Therefore, based on the cellular response in vitro and in vivo, SMSCs may represent a more suitable cell source than BMSCs for further study and development of tissue-engineered ligament.

Enhancement of Fat Graft Survival by Bone Marrow–Derived Mesenchymal Stem Cell Therapy

Adipose-derived stem cells can improve fat graft survival, but there is no literature reporting whether bone marrow-derived mesenchymal stem cells enhance fat graft survival. The authors explored the feasibility of enhancing fat graft survival using bone marrow-derived mesenchymal stem cells. Third-passage expanded rabbit bone marrow-derived mesenchymal stem cells were characterized by adipocyte and osteocyte differentiation and by CD29 and CD31 expression. Three were three groups of nude mice in this experiment: group A, mesenchymal stem cells; group B, expanded mesenchymal stem cells; group C, Dulbecco's medium as a blank control. The transplanted mixture contained 0.3 ml of adipose granule and 0.2 ml of cell components of 5×10⁶ cells. Four months later, grafts were harvested, weighed, and analyzed. Expanded cells were successfully isolated and identified by fibroblast-like adherent shape and osteogenic and adipogenic differentiation. The results were 24.6±3.4 percent for CD29 and 1.8±0.4 percent for CD31 in group A, and 45.0±4.9 percent for CD29 and 1.6±0.3 percent for CD31 in group B. Fat graft survival rates were 0.2052±0.0015 g, 0.1761±0.0014 g, and 0.1350±0.0020 g in groups A, B, and C, respectively (p<0.05). Fat grafts in group A exhibited the best survival and morphologic integrity, uniform lipid droplets, and rich blood vessels; those in group B exhibited modest survival, less integrity, less uniform lipid droplets, and connective tissue septa; and those in group C exhibited some large bubbles, varied sizes of lipid droplets, and significant fibrous septa (p<0.05). The vascular densities for groups A, B, and C were 30.4±1.5, 27.2±1.3, and 23.3±1.9 capillaries/mm2, respectively (p<0.05). Bone marrow-derived mesenchymal stem cells and expanded bone marrow-derived mesenchymal stem cells are capable of improving fat graft survival; bone marrow-derived mesenchymal stem cells are more potent than expanded bone marrow-derived mesenchymal stem cells for doing so.

Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface

The purpose of this study was to investigate whether rabbit bone marrow-derived mesenchymal stem cells (MSCs) effectively decrease alkali-induced oxidative stress in the rabbit cornea. The alkali (0.15 N NaOH) was applied on the corneas of the right eyes and then rinsed with tap water. In the first group of rabbits the injured corneas remained untreated. In the second group MSCs were applied on the injured corneal surface immediately after the injury and eyelids sutured for two days. Then the sutures were removed. In the third group nanofiber scaffolds seeded with MSCs (and in the fourth group nanofibers alone) were transferred onto the corneas immediately after the injury and the eyelids sutured. Two days later the eyelid sutures were removed together with the nanofiber scaffolds. The rabbits were sacrificed on days four, ten or fifteen after the injury, and the corneas were examined immunohistochemically, morphologically, for the central corneal thickness (taken as an index of corneal hydration) using an ultrasonic pachymeter and by real-time PCR. Results show that in untreated injured corneas the expression of malondialdehyde (MDA) and nitrotyrosine (NT) (important markers of lipid peroxidation and oxidative stress) appeared in the epithelium. The antioxidant aldehyde dehydrogenase 3A1 (ALDH3A1) decreased in the corneal epithelium, particularly in superficial parts, where apoptotic cell death (detected by active caspase-3) was high. (In control corneal epithelium MDA and NT are absent and ALDH3A1 highly present in all layers of the epithelium. Cell apoptosis are sporadic). In injured untreated cornea further corneal disturbances developed: The expressions of matrix metalloproteinase 9 (MMP9) and proinflammatory cytokines, were high. At the end of experiment (on day 15) the injured untreated corneas were vascularized and numerous inflammatory cells were present in the corneal stroma. Vascular endothelial growth factor (VEGF) expression and number of macrophages were high. The results obtained in injured corneas covered with nanofiber scaffolds alone (without MSCs) or in injured corneas treated with MSCs only (transferred without scaffolds) did not significantly differ from the results found in untreated injured corneas. In contrast, in the injured corneas treated with MSCs on nanofiber scaffolds, ALDH3A1 expression remained high in the epithelium (as in the control cornea) and positive expression of the other immunohistochemical markers employed was very low (MMP9) or absent (NT, MDA, proinflammatory cytokines), also similarly as in the control cornea. Corneal neovascularization and the infiltration of the corneal stroma with inflammatory cells were significantly suppressed in the injured corneas treated with MSCs compared to the untreated injured ones. The increased central corneal thickness together with corneal opalescency appearing after alkali injury returned to normal levels over the course of ten days only in the injured corneas treated with MSCs on nanofiber scaffolds. The expression of genes for the proinflammatory cytokines corresponded with their immunohistochemical expression. In conclusion, MSCs on nanofiber scaffolds protected the formation of toxic peroxynitrite (detected by NT residues), lowered apoptotic cell death and decreased matrix metalloproteinase and pro-inflammatory cytokine production. This resulted in reduced corneal inflammation as well as neovascularization and significantly accelerated corneal healing.

The effects of exogenous hyaluronic acid with different molecular weight on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells

Objective To study the effects of exogenous hyaluronic acid (HA) with different molecular weight on chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs).Methods The un-induced MSCs in passage 3 were divided into five groups with different suplements:Group A,basic medium + HA 0.1 g/L (average molecular weight:1000 × 103 ) ; Group B,basic medium + HA 0.1 g/L ( average molecular weight 1800 × 103 ) ; group C,basic medium + HA 0.1 g/L ( average molecular weight 2000 × 103 ) ; Group D,basic medium + transforming growth factor-33 ( TGFβ3) 10 μg/L (positive control) ; Group E,basic medium only (negative control).The morphologic features and proliferation of MSCs were observed under a light microscope.The chondrogenic differentiation was identified by toluidine blue staining as well as immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of collagen Ⅱ respectively.Results The speed of cell proliferation was decelerated after HA was added.The cell morphology changed from long spindle to polygon,oval.The extracellular matrix showed metachromasia with toluidine blue and positive type Ⅱ collagen immunohistochemical staining.The mRNA expression of type Ⅱ collagen was detected by RT-PCR.The relative gene expression of type Ⅱ collagen in group A,B and C was 0.64 ± 0.06,0.72 ±0.03,0.75 ±0.01,respectively,and that in groups D and E was 0.96 ±0.15 and 0.09 ±0.03 respectively.There was no statistically significant difference in the expression of type Ⅱ collagen between groups B and group C.There was significant difference between experimental group and control groups.Conclusion The exogenous HA with different molecular weight can induce chondrogenic differentiation of MSCs with different ability.HA with higher average molecular weight enhances the chondrogenesis of MSCs effectively.But all HA shows weaker ability of inducement than TGF-33. Key words: Hyaluronic acid; Molecular weight; Mesenchymal stem cell; Chondrogenesis

Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro

Objective VEGF and BMP play important roles in angiogenesis and osteogenesis. Combining these two factors may be a promising therapeutic strategy for avascular necrosis of the femoral head (ANFH). Methods Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and purified by density gradient centrifugation combined with attachment culture methods. The purity and characteristics of the BMSCs were detected by cell surface antigen identification. The best MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting, and cell viability was determined by MTT assay. Expression of the genes of interest was detected by GFP gene expression, RT-PCR assay, and ELISA assay. The biological activities of VEGF and BMP were detected by angiogenic and osteogenic assays. Results The best MOI of BMSCs transfected with rAAV was 5 × 10 4 v.g./cell. Cell growth curves showed vigorous cell viability. Expressions of the GFP, VEGF165, and BMP 7 genes were detected 1 day post-transfection and peaked 14 days post-transfection. Expression of the genes of interest was sustained over 1 month. VEGF and BMP proteins secreted from BMSCs transfected with rAAV-hVEGF 165-IRES-hBMP 7 enhanced angiogenesis and osteogenesis in vitro. Conclusion Recombinant adeno-associated viral vectors co-expressing the hVEGF 165 and hBMP 7 genes showed efficient gene expression ability. The VEGF 165 and BMP 7 proteins expressed from the vector have efficient biological activity in vitro.

All-trans retinoic acid promotes smooth muscle cell differentiation of rabbit bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells are multipotent stem cells, an attractive resource for regenerative medicine. Accumulating evidence suggests that all-trans retinoic acid plays a key role in the development and differentiation of smooth muscle cells. In the present study, we demonstrate, for the first time, that rabbit bone marrow-derived mesenchymal stem cells differentiate into smooth muscle cells upon the treatment with all-trans retinoic acid. All-trans retinoic acid increased the expression of myocardin, caldesmon, 22-kDa smooth muscle cell-specific protein (SM22alpha), and SM-myosin heavy chains in rabbit bone marrow-derived mesenchymal stem cells, as detected by reverse transcription polymerase chain reaction (PCR). Immunostaining of SM22alpha and SM-myosin heavy chains using monoclonal antibodies also indicated smooth muscle cell differentiation of rabbit bone marrow-derived mesenchymal stem cells following the treatment with all-trans retinoic acid. In addition, more than 47% of bone marrow-derived mesenchymal stem cells demonstrated the contractile phenotype of smooth muscle cells. Western blot results showed that SM-1 and SM-2 were highly expressed in the differentiated cells. These results suggest that all-trans retinoic acid may serve as a potent agent for functional smooth muscle cell differentiation in tissue engineering.

Evaluation of RGD-Grafted Porous PLLA Scaffold for Chondrogenic Differentiation of Stem Cells

Biodegradable porous poly(L-lactic acid) (PLLA) scaffolds were prepared using gas foaming method. The PLLA scaffolds with a hydrophobic surface were subjected to Ar plasma treatment and in situ acrylic acid (AA) grafting to obtain hydrophilic PLLA scaffold (PLLA-PAA). Cell-adhesive RGD peptide was then immobilized onto the AA-grafted PLLA (PLLA-PAA-RGD). Once rabbit bone marrow-derived mesenchymal stem cells (BM-MSC) were isolated, MSCs were seeded into PLLA control, PLLA-PAA, and PLLA-PAA-RGD scaffold and cultured for up to 4 weeks in chondrogenic medium with the addition of 10 ng/ml transforming growth factor (TGF)-β1. Surface analysis of AA-grafted PLLA identified significant alterations of surface characteristics, including reduced contact angle and different atomic compositions. From WST-1 assay at 4 weeks, cells were found more proliferative in PLLA-PAA than the others. Upon the histological analysis of Safranin O staining, chondrogenic differentiation of MSCs appeared to be progressed more actively in PLLA-PAA. The effect of RGD immobilization on MSC differentiation was barely notable.

Novel gene-modified-tissue engineering of cartilage using stable transforming growth factor-β1-transfected mesenchymal stem cells grown on chitosan scaffolds

Rabbit bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the TGF-beta1 gene in monolayer culture using Lipofectamine 2000. After transfection, the expression of cartilage-specific extracellular matrix was upregulated, whereas matrix metalloproteinases 1 and 3 (MMP 1 and 3) protein expressions and enzymatic activities were downregulated. Autologous MSCs modified with the TGF-beta1 gene were seeded into chitosan scaffolds to construct gene-modified cartilage, which was then implanted into the full-thickness articular cartilage defects of rabbits' knees. Twelve weeks after implantation, the defects were filled with regenerated hyaline-like cartilage tissue as confirmed by the positive immunohistochemical staining of collagen type II and intense toluidine blue staining of proteoglycan. Our findings suggest that the repair of cartilage defects can be enhanced by TGF-beta1 gene-modified-tissue engineering of cartilage on the basis of a strategy using MSCs, chitosan, and liposomal transfection.

Bio-Engineered Mesenchymal Stem Cell-Tricalcium Phosphate Ceramics Composite Augmented Bone Regeneration in Posterior Spinal Fusion

Grafting of autologous iliac crest and decortication approach in posterior spinal fusion surgery has been the “gold standard”. However, the limited source of autograft has prompted extensive research into bone substitute and biological enhancement of the fusion mass. In this study, the application of stem cell therapy by tissue engineering method was investigated to enhance posterior spinal fusion with -tricalcium phosphate ceramics in rabbit model. Rabbit bone marrow derived mesenchymal stem cells were aspirated from trochanter region of proximal femur. The mesenchymal stem cells were grown and directed to differentiate into osteogenic cells by osteogenic supplement (ascorbic acid, -glycerophosphate and dexamethasone) in basal medium (10% FBS in DMEM). The osteogenic cells were seeded on tricalcium phosphate ceramics for one day (MSC group, n=6). The cell-ceramics composite was implanted onto autologous L5 and L6 transverse processes with decortication approach in posterior spinal fusion. The cell free ceramics acts as control (Control group, n=6) and iliac crest autograft as positive control (Autograft group). The spinal segments were harvested at week 7 post-operation. Manual palpation was performed with spinal segments to assess any movement of L5-L6 vertebral joint. The stiffness of the joint was considered as solid fusion. The specimens then were fixed by formalin and transferred to 70% ethanol. The BMC and volume of fusion transverse processes of L5 and L6 was measured by peripheral quantitative computed tomography. In manual palpation, 50% solid fusion was found in MSC group, 60% in autograft group but none in control group. Moreover, the BMC of L5 and L6 transverse processes in MSC group was greater than autograft and control group (45%, 40% respectively, p&lt;0.01). The volume of transverse processes in MSC group was greater than autograft by 45% (p&lt;0.01) and control group by 26% (p&lt;0.05). In conclusion, the mesenchymal stem cells derived osteogenic cells augmented spinal fusion and bone mineralization.

Repair of patellar tendon injuries using a cell–collagen composite

Collagen gels were seeded with rabbit bone marrow-derived mesenchymal stem cells (MSCs) and contracted onto sutures at initial cell densities of 1, 4, and 8 million cells/ml. These MSC–collagen composites were then implanted into full thickness, full length, central defects created in the patellar tendons of the animals providing the cells. These autologous repairs were compared to natural repair of identical defects on the contralateral side. Biomechanical, histological, and morphometric analyses were performed on both repair tissue types at 6, 12, and 26 weeks after surgery. Repair tissues containing the MSC–collagen composites showed significantly higher maximum stresses and moduli than natural repair tissues at 12 and 26 weeks postsurgery. However, no significant differences were observed in any dimensional or mechanical properties of the repair tissues across seeding densities at each evaluation time. By 26 weeks, the repairs grafted with MSC–collagen composites were one-fourth of the maximum stress of the normal central portion of the patellar tendon with bone ends. The modulus and maximum stress of the repair tissues grafted with MSC–collagen composites increased at significantly faster rates than did natural repairs over time. Unexpectedly, 28% of the MSC–collagen grafted tendons formed bone in the regenerating repair site. Except for increased repair tissue volume, no significant differences in cellular organization or histological appearance were observed between the natural repairs and MSC–collagen grafted repairs. Overall, these results show that surgically implanting tissue engineered MSC–collagen composites significantly improves the biomechanical properties of tendon repair tissues, although greater MSC concentrations produced no additional significant histological or biomechanical improvement.