Rabbit Mesenchymal Stem Cells Research Articles

BMP-7 accelerates the differentiation of rabbit mesenchymal stem cells into cartilage through the Wnt/β-catenin pathway.

Mesenchymal stem cells (MSCs) are able to differentiate into adipocytes, chondroblasts or cartilage under different stimulation conditions. Identifying a mechanism that triggers the differentiation of MSCs into cartilage may help the development of novel therapeutic approaches for heterotopic ossification, the pathological formation of lamellar bone in soft tissue outside the skeleton that may lead to debilitating immobility. Bone morphogenetic proteins (BMPs), including BMP-7, are the most potent growth factors for enhancing bone formation. The current study aimed to understand the potential involvement of the Wnt/β-catenin signaling pathway in the BMP-7-induced growth of rabbit MSCs (rMSCs). Different concentrations of BMP-7 were applied to cultured rMSCs, and proliferation was evaluated by MTT assay. Changes in the phosphorylation state of glycogen synthase kinase (GSK)-3β, in addition to the expression levels of alkaline phosphatase, β-catenin and runt-related transcription factor 2 were observed by western blot analysis. Following treatment with BMP-7, the phosphorylation of GSK-3β was stimulated and the expression of β-catenin, ALP and Runx2 was increased. Furthermore, inhibiting β-catenin signaling with XAV-939 suppressed the BMP-7-mediated changes. The results indicated that the BMP-7-induced differentiation of rMSCs into cartilage was promoted primarily by the Wnt/β-catenin pathway.

Mesenchymal Stem Cells Reshape and Provoke Proliferation of Articular Chondrocytes by Paracrine Secretion

Coculture between mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represents a promising strategy for cartilage regeneration. This study aimed at elaborating how ACs were regulated by MSCs. Rabbit ACs (rACs) and rabbit MSCs (rMSCs) were seeded separately in a Transwell system to initiate non-contact coculture in growth medium without chondrogenic factors. Cell morphology, cell proliferation, production of extracellular matrix (ECM), and gene expression of rACs were characterized. Upon coculture, rACs underwent a morphological transition from a rounded or polygonal shape into a fibroblast-like one and proliferation was provoked simultaneously. Such effects were dependent on the amount of rMSCs. Along with these changes, ECM production and gene expression of rACs were also perturbed. Importantly, when a ROCK inhibitor (Y27632) was supplemented to coculture, the effects except that on cell proliferation were inhibited, suggesting the involvement of RhoA/ROCK signaling. By applying an inhibitor (BIBF1120) of VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β in coculture, or supplementing FGF-1, VEGF-A and PDGFbb in monoculture, it was confirmed that the paracrine factors by rMSCs mediated the compounding effects on rACs. These findings shed light on MSCs-ACs interactions and might confer an insight view on cell-based cartilage regeneration.

Proliferative activity of skeletal myoblast sheet by paracrine effects of mesenchymal stem cells

ObjectivesThe purpose of this study was to examine the proliferative activity enhancement of skeletal myoblasts in the presence and absence of mesenchymal stem cells (MSC). MethodsWe artificially fabricated two types of cell sheets by co-culturing rabbit skeletal myoblast sheets with rabbit MSCs using type IA collagen gel (MC+), and cell sheets without rabbit MSCs (MC−). ResultsThe results of hematoxylin and eosin staining revealed that MC+ was thicker than MC− on day 7. Immunohistochemical staining revealed a low level of desmin expression in both sheets on day 2. Desmin expression increased at days 7 and 12, and desmin localization was consistent with the stratified area. Reverse transcription PCR revealed the presence of MyoD and PAX7 in both sheets on days 2, 7, and 12. The presence of myogenin was confirmed in both sheets on days 7 and 12. Hepatocyte growth factor expression was evident in MC+ on day 2, and in both sheets on day 7. Measurement of cell proliferative activity based on DNA cell cycle analysis indicated that MC+ had significantly higher cell proliferative activity than MC− on day 7. There were no significant differences in cell proliferative activity between MC+ and MC− on day 12. ConclusionsThis study demonstrated that the presence of MSCs could transiently enhance the proliferative activity of myoblasts, but that this enhancement is ultimately diminished due to contact inhibition.

Fluorescence molecular tomography of DiR-labeled mesenchymal stem cell implants for osteochondral defect repair in rabbit knees.

To assess labelling efficiency of rabbit mesenchymal stem cells (MSCs) using the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and detection of labelled MSCs for osteochondral defect repair in a rabbit model using fluorescence molecular tomography-X-ray computed tomography (FMT-XCT). MSCs were isolated from New Zealand White rabbits and labelled with DiR (1.25-20μg/mL). Viability and induction of apoptosis were assessed by XTT- and Caspase-3/-7-testing. Chondrogenic potential was evaluated by measurement of glycosaminoglycans. Labelled cells and unlabeled controls (n = 3) underwent FMT-XCT imaging before and after chondrogenic differentiation. Osteochondral defects were created surgically in rabbit knees (n = 6). Unlabeled and labelled MSCs were implanted in fibrin-clots and imaged by FMT-XCT. Statistical analyses were performed using multiple regression models. DiR-labelling of MSCs resulted in a dose-dependent fluorescence signal on planar images in trans-illumination mode. No significant reduction in viability or induction of apoptosis was detected at concentrations below 10μg DiR/mL (p > .05); the chondrogenic potential of MSCs was not affected (p > .05). FMT-XCT of labelled MSCs in osteochondral defects showed a significant signal of the transplant (p < .05) with additional high-resolution anatomical information about its osteochondral integration. FMT-XCT allows for detection of stem cell implantation within osteochondral regeneration processes. • DiR-labelling of MSCs shows no toxic side effects or impairment of chondrogenesis. • Fluorescence molecular tomography allows for detection of MSCs for osteochondral defect repair. • FMT-XCT helps to improve evaluation of cell implantation and osteochondral regeneration processes.

Extracellular Matrix (ECM) Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-β3 in Articular Cartilage Repair

Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair.

Oxygen Tension-Controlled Matrices with Osteogenic and Vasculogenic Cells for Vascularized Bone Regeneration In Vivo.

Despite recent progress, segmental bone defect repair is still a significant challenge in orthopedic surgery. While bone tissue engineering approaches using biodegradable matrices along with bone/blood vessel forming cells offered improved possibilities, current regenerative strategies lack the ability to achieve vascularized bone regeneration in critical-sized/segmental bone defects. In this study, we introduced and evaluated a two-pronged approach for vascularized bone regeneration in vivo. The goal was to demonstrate vascularized bone formation using oxygen tension-controlled (OTC) matrices seeded with bone and blood vessel forming cells. OTC matrices were coimplanted with rabbit mesenchymal stem cells (MSCs) and peripheral blood-derived endothelial progenitor cells (PB-EPCs) to demonstrate the osteogenic and vasculogenic differentiation of these cells, postseeding on a matrix, especially deep inside the matrix pore structure. Matrices coimplanted with varied rabbit MSC and PB-EPC ratios (1:4, 1:1, and 4:1) were assessed in a nude mouse subcutaneous implantation model to determine a coimplantation ratio with superior osteogenic as well as vasculogenic properties. The implants were analyzed, at week 8, for endothelial (CD31 and Von Willebrand factor [vWF]) and osteogenic marker (RunX2 and Col I) staining qualitatively and collagen deposition and number of vessel formation quantitatively. Results from these experiments established MSC-to-PB-EPC ratio 1:1 as the best coimplantation ratio. OTC matrix with 1:1 coimplantation ratio was assessed for segmental bone defect repair in a rabbit critical-sized bone defect model. The group under investigation was OTC matrix, and the matrix was seeded with MSCs, EPCs, or MSCs:EPCs in a 1:1 ratio. Explants at week 12 were evaluated for bone defect repair via micro-CT and histology. Results from rabbit in vivo experiments show enhanced mineralization and vascularization for the 1:1 coimplantation group. Overall, the study establishes a two-pronged approach involving OTC matrix and effective progenitors for large-area and vascularized bone regeneration.

Light enhanced bone regeneration in an athymic nude mouse implanted with mesenchymal stem cells embedded in PLGA microspheres.

BackgroundBiodegradable microspheres fabricated from poly (Lactic-co-glycolic acid) (PLGA) have attracted considerable attention in the bone tissue regeneration field. In this study, rabbit mesenchymal stem cells (rMSCs) adherent to PLGA microspheres were implanted into athymic nude mice and irradiated with 647 nm red light to promote bone formation. It was found that irradiating rMSCs with high levels of red light (647 nm) from an LED (light-emitting diode) increased levels of bone specific markers in rMSCs embedded on PLGA microspheres.ResultThese increased expressions were observed by RT-PCR, real time-QPCR, immunohistochemistry (IHC), and von Kossa and Alizarin red S staining. Microsphere matrices coated with rMSCs were injected into athymic nude mice and irradiated with red light for 60 seconds showed significantly greater bone-specific phenotypes after 4 weeks in vivo.ConclusionThe devised PLGA microsphere matrix containing rMSCs and irradiation with red light at 647 nm process shows promise as a means of coating implantable biomedical devices to improve their biocompatibilities and in vivo performances.

In vitro Osteoinduction Potential of a novel Silica Coated Hydroxyapatite Bioscaffold Seeded with Rabbit Mesenchymal Stem Cell

The objective of this study was to assess cyto compatibility of silica coated hydroxyapatite (HASi) bioceramic and rabbit bone marrow derived mesenchymal stem cells (rBMSC) in terms of cell attachment, cell morphology, cell proliferation and osteogenic differentiation. The rBMSC were seeded on HASi blocks and maintained in standard culture conditions. Samples on day 3 and day 17 post-seeding were subjected for scanning electron microscopy (SEM). Day 3 post-seeding samples showed stem cell attachment with a wavy surface morphology and day 17 post-seeding sample showed expansion of attached rBMSCs with cell sheet like morphology and deposition of matrix in the form of crystals. Alizarin red staining of these trypsinized crystals showed positive staining, indicative of presence of calcium. Histological sections of decalcified samples on day 17 post-seeding showed areas of early new bone formation. It was concluded that silica coated hydroxyapatite could induce the osteogenic differentiation of rBMSC by day 17 post-seeding in the absence of any osteogenic induction media which was confirmed by the presence of crystal like matrix deposition on scanning electron microscopic analysis, alizarin red staining of trypsinized crystals and early new bone formation in histological sections.

Chondrogenesis of mesenchymal stem cells in a novel hyaluronate-collagen-tricalcium phosphate scaffolds for knee repair

Scaffolds are expected to play a key role in the induction of chondrogenesis of mesenchymal stem cells (MSCs) for cartilage tissue regeneration. Here, we report the development of a novel tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffold that can function as a stem cell carrier to induce chondrogenesis and promote cartilage repair, and the investigation of chondroinductive properties of scaffolds containing varying amounts of TCP, COL and HA. TCP-COL-HA scaffolds, as well as TCP-COL scaffolds at two different TCP/COL ratios (50:50 and 25:75), were evaluated for their ability to induce cartilage regeneration from rabbit mesenchymal stem cells (rMSCs) in vitro and in vivo. Chondrogenic differentiation was evaluated by sulphated glycosaminoglycan quantification, collagen type II immunohistochemistry, and qRT-PCR. Mechanical strength was evaluated by the compression test. The results showed that the TCP-COL-HA scaffolds enhanced rMSC chondrogenesis to a greater degree than did the TCP-COL scaffolds; for the latter, the scaffold with the lower TCP/COL ratio (25:75) was superior in terms of promoting rMSC chondrogenesis. Similar results were obtained in an ectopic implantation model in nude mice. In a critical-size rabbit osteochondral defect-repair model, rMSCs seeded on TCP-COL-HA scaffolds showed greater cartilage regeneration and integration into surrounding tissue than the TCP-COL groups, in which cartilage repair was more efficient at the 25:75 than at the 50:50 ratio. These results indicate that the addition of HA and different TCP/COL ratios can affect the chondroinductive capacity of scaffolds, and suggest that the TCP-COL-HA scaffold can serve as an effective cell carrier for cartilage regeneration.

Repair of rotator cuff injury in rabbit animal model by in situ implantation of an injectable elastin-like recombinamer and allogeneic mesenchymal stem cells

Event Abstract Back to Event Repair of rotator cuff injury in rabbit animal model by in situ implantation of an injectable elastin-like recombinamer and allogeneic mesenchymal stem cells Alessandra Girotti1, Arturo Ibañez1, José María Trigueros Larrea2, José Francisco Lamus Molina3, Matilde Alonso1, Ana Sánchez García4, Ángel Luis Gato3, Francisco J Santos Martín5, Jose Carlos Rodríguez-Cabello1 and Aurelio Vega Castrillo2 1 University of Valladolid, BIOFORGE, CIBER-BBN, Spain 2 Hospital Clínico Universitario de Valladolid, Spain 3 University of Valladolid, Anatomy and Radiology Department, Spain 4 University of Valladolid, IBGM Institute, Spain 5 University of Valladolid, E.T.S.I.I, Spain Introduction: One of the most common musculoskeletal pathologies is the shoulder pain, the rotator cuff injury affects more than 40% of patients over 60 years and causes disabling pain and loss of shoulder functionality[1]. Despite the recent improvements and advances in the surgery, there remains a failure rate of between 20% and 70%. To obtain the tendon repair augmentation the new treatment strategies are directed to reinforce the mechanical properties and the biological healing capacity by implanting matrices, although the currently in use usually cause inflammatory reaction[2]. To solve this problem, in this study we employed a modified version of an advanced matrix that has been demonstrating high efficiency and biocompatibility as bioactive support[3] in regenerative medicine. It belongs to the family of (ELRs) the Elastin-like recombinamers with high mechanical performance designed as injectable thermogelling systems[4]. The matrix was co-injected with rabbit bone marrow mesenchymal stem cells (rMSCs), under physiological conditions to evaluate the effect of cell therapy on the tendon pathology. Experimental Methods: ELRs were produced by DNA recombinant techniques and E. coli biosynthesis as described elsewhere[5]. Rabbit mesenchymal stem cells (rMSCs) were obtained from rabbit bone marrow and BrdU stained. New Zealand rabbits with critical defect in the infraspinatus tendon were randomly allocated into 3 groups (negative control, treated with ELRs matrix, treated with ELRs matrix and rMSCs). At t 0, 1, 4 and 16 weeks the animals were sacrificed and their tendons removed to perform biomechanical tests and immunohistochemical analysis. Results and Discussion: This study describes the use of injectable thermo-gelling scaffold whose applicability is focused specifically on regenerative medicine. The scaffold generated "in situ" under physiological condition was assayed in combination or not with rMSCs for the forming of an artificial matrix that improved tendon repair capacity. The results at 0, 1, 4 and 16 weeks were compared with which obtained adding ELR matrix and with the normal healing process the after injury of the infraspinatus tendon in rabbits. Figure 1. Showed the BRdU stained rMSCs and the ELR matrix after 1 month in the rabbit tendon implantation. In this work we observed a stronger and more physiological infraspinatus tendon repair using allogeneic rabbit mesenchymal stem cells in combination with an innovative ELRs thermogelling scaffold. Conclusion: The thermo-gelling property of the ELRs matrix has greatly facilitated the surgical technique as it is easier to handle (for injection), especially when used arthroscopically. The bioactivity and biocompatibility of the matrix enhanced the adhesion and proliferation of the rMSCs embedded in ELR-matrix in vitro. The in vivo tendon suture repair of the infraspinatus tendon has been studied in New Zealand rabbits. The rMSCs showed a high proliferation rate in the ELRs matrix when implanted in the tendons and a reinforced structural was obtained as was proved through the biomechanical, histological and immunohistochemical assays. MINECO MAT2012-38043; Castilla y Leon “Consejeria de Sanidad”

Preparation and bioactive properties of bone-repair composite scaffolds based on bioactive glass/hydroxylapatite/chitason

The aim of this work was to develop a bioactive glass/hydroxylapatite/chitason composite material for bone repair. Different proportions of bioactive glass/hydroxylapatite/chitason mixture was make into scaffolds by the freeze-drying method. The microstructure and composition were observed and analysed. The third-generation rabbit mesenchymal stem cells were inoculated into the stent. The adhesion of the stent was detected, and the proliferation of cells on the stent was detected. The compatibility between the stent and cells was evaluated. The size and shapes of the prepared bioactive glass/hydroxylapatite/chitason stent were consistent with moulds. The stent has parous structure and no aggregation of bioactive glass was found. The rabbit mesenchymal stem adhered to the stent, and some cells extended and stretched out pseudopodia. Obvious proliferation of bone marrow stromal cells was found on the stent. The prepared bioactive glass/hydroxylapatite/chitason stents have good porosity, high mechanical strength, and good cell compatibility.

Porous biphasic calcium phosphate ceramics coated with nano-hydroxyapatite and seeded with mesenchymal stem cells for reconstruction of radius segmental defects in rabbits.

The osteoconduction of porous biphasic calcium phosphate (BCP) ceramics has been widely reported. In a previous study, we demonstrated that applying a nano-hydroxyapatite (nHA) coating enhances the osteoinductive potential of BCP ceramics, making these scaffolds more suitable for bone tissue engineering applications. The aim of the present study was to determine the effects of reconstructing radius defects in rabbits using nHA-coated BCP ceramics seeded with mesenchymal stem cells (MSCs) and to compare the bone regeneration induced by different scaffolds. Radius defects were created in 20 New Zealand rabbits, which were divided into four groups by treatment: porous BCP ceramics (Group A), nHA-coated porous BCP ceramics (Group B), porous BCP ceramics seeded with rabbit MSCs (Group C), and nHA-coated porous BCP ceramics seeded with rabbit MSCs (Group D). After in vitro incubation, the cell/scaffold complexes were implanted into the defects. Twelve weeks after implantation, the specimens were examined macroscopically and histologically. Both the nHA coating and seeding with MSCs enhanced the formation of new bone tissue in the BCP ceramics, though the osteoinductive potential of the scaffolds with MSCs was greater than that of the nHA-coated scaffolds. Notably, the combination of nHA coating and MSCs significantly improved the bone regeneration capability of the BCP ceramics. Thus, MSCs seeded into porous BCP ceramics coated with nHA may be an effective bone substitute to reconstruct bone defects in the clinic.

Isolation, Culture and Characterization of New Zealand White Rabbit Mesenchymal Stem Cells Derived from Bone Marrow

Isolation, Culture and Characterization of New Zealand White Rabbit Mesenchymal Stem Cells Derived from Bone Marrow

Enhancement of chondrogenic differentiation of rabbit mesenchymal stem cells by oriented nanofiber yarn-collagen type I/hyaluronate hybrid

Cartilage defects cause joint pain and loss of mobility. It is crucial to induce the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by both biological and structural signals in cartilage tissue engineering. Sponge-like scaffolds fabricated using native cartilage extracellular matrix components can induce the BMSC differentiation by biological signals and limited structural signals. In this study, an oriented poly(L-lactic acid)-co-poly(ε-caprolactone) P(LLA-CL)/collagen type I (Col-I) nanofiber yarn mesh, fabricated by dynamic liquid electrospinning served as a skeleton for a freeze-dried Col-I/hyaluronate (HA) chondral phase (SPONGE) containing both structural and biological signals to guide BMSC chondrogenic differentiation. In vitro results show that the Yarn Col-I/HA hybrid scaffold (Yarn-CH) promotes orientation, adhesion and proliferation of BMSCs better than SPONGE. Furthermore, BMSCs seeded on the Yarn-CH scaffold demonstrated a large increase in the glycosaminoglycan content and expression of collagen type II following a 21-day culture.

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression.

BackgroundThe use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation.MethodsMSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT).ResultsTested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group.ConclusionsThe BCP hybrid scaffold for osteogenesis of MSCs is better than the coralline HA hybrid scaffold by upregulating expression of leptin-R. This was consistent with in vivo data, which indicated that BCP hybrid scaffolds induced more bone formation in a spinal fusion model.

Ghrelin accelerates the growth and osteogenic differentiation of rabbit mesenchymal stem cells through the ERK1/2 pathway.

BackgroundMesenchymal stem cells (MSCs) can differentiate into chondroblasts, adipocytes, or osteoblasts under appropriate stimulation. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), stimulates growth hormone (GH) secretion, and has both orexigenic and adipogenic effects. This study sought to understand the potential involvement of members of MAPK serine/threonine kinases in the ghrelin-induced growth of rabbit MSCs ( rBMSC).MethodsWe applied various concentrations of ghrelin to cultured rBMSC and observed the growth rate of the cells by MTT, changes in the phosphorylation state of ERK1/2, JNK and p38, and the expression levels of ALP, Runx2, and Osterix by wetern blot.ResultsWe found that the growth and osteogenic differentiation of ghrelin-treated rBMSC are promoted primarily by phosphorylated ERK1/2, and that this phosphorylation, as well p38 phosphorylation, is mediated by GHSR.ConclusionsOur study suggests that ghrelin promotes the growth and osteogenic differentiation of rBMSC primarily through the ERK1/2 pathway.

Isolation and Biological Characteristics of Rabbit Bone Marrow Plug-derived Mesenchymal Stem Cells

Though the rabbit is one of most widely used experimental animals for medical regenerative research, it remains difficult to culture mesenchymal stem cells (MSC) on a in large scale due to the extremely lower number and hematopoietic cell contamination. This study was aimed to establish a novel protocol to generate rabbit MSC by culturing bone marrow plugs instead of bone marrow cells so as to obtain a large amount of MSC with higher proliferation and self-renewal properties. The primary MSC were generated from collagenase digested bone marrow plugs and bone marrow cells, respectively. The surface antigen profile of MSC was analyzed with flow cytometry and the cells were induced to differentiate into osteoblasts and adipocytes. The proliferation capacity of MSC were assessed by CCK-8 method. To test their self-renewal property, the colony forming unit-fibroblast assay was performed. Moreover, the cell yields of passage 1, 2, 3 and 4 were calculated. The bone marrow plug-derived MSC shared the typical fibroblast-like morphology same as bone marrow cells derived MSC. Moreover, the ratio of CD45 positive hematopoietic cells in bone marrow plug-derived MSCs was significantly lower than that of bone marrow cell-derived MSC. The results of multi-differentiation experiments showed that bone marrow-plug-derived MSC exhibited similar multi-potent property to their bone marrow counterparts. In addition, the results of CCK-8 and CFU-F assay demonstrated that bone marrow plug-derived MSC grew more robustly and more CFU-F were formed in the culture plates, which indicated that the cells possessed higher proliferation and self-renewal capacities. Promisingly, a larger amount of cells were harvested via using the new protocol. The purity and yields of the bone marrow plug-derived MSC are satisfactory compared with previous rabbit MSC isolation methods. The findings may be helpful for the research of regenerative medicine.

Mechano-growth factor accelerates the proliferation and osteogenic differentiation of rabbit mesenchymal stem cells through the PI3K/AKT pathway.

BackgroundMesenchymal stem cells (MSCs) can differentiate into chondroblasts, adipocytes, or osteoblasts under appropriate stimulation. Mechano-growth factor (MGF) reportedly displays a neuroprotective effect in cerebral regions that were exposed to ischemia and is expressed in stromal cells of the eutopic endometrium and in glandular cells of the ectopic endometrium.ResultsThis study sought to understand the potential involvement of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) in MGF-induced growth of rabbit MSCs (rMSCs). We applied various concentrations of MGF to cultured rMSCs and observed the growth rate of the cells, the changes in the phosphorylation state of AKT and mammalian target of rapamycin (mTOR), and the expression levels of alkaline phosphatase and osteocalcin. We found that the growth and osteogenic differentiation of MGF-induced rMSCs were promoted primarily by phosphorylated AKT, and that this phosphorylation, as well mTOR phosphorylation, was mediated by the MGF receptor.ConclusionOur study suggests that MGF promotes the growth and osteogenic differentiation of rMSCs primarily through the PI3K/AKT pathway.

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.