OBJECTIVES: This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro. METHODS: ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively. RESULTS: Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression. CONCLUSIONS This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.
Animals ; Mice ; Cartilage/metabolism* ; Cell Differentiation ; Cells, Cultured ; Chondrocytes/metabolism* ; Chondrogenesis/physiology* ; Glycosaminoglycans/pharmacology*

To study the cartilage differentiation of mouse mesenchymal stem cells (MSCs) induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 (0, 10, 20, 50 and 100 ng/mL). After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen II mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen II mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesenchymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.
Bone Marrow Cells/*cytology ; Bone Morphogenetic Proteins/*pharmacology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chondrocytes/*cytology ; Chondrogenesis/drug effects ; Chondrogenesis/physiology ; Mesenchymal Stem Cells/*cytology ; Recombinant Proteins/pharmacology

BACKGROUNDCartilage injury has a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for the cartilage repair using differentiated bone marrow mesenchymal stem cells (BMSCs) is, however, a promising approach to the chondral repair. This study was aimed to explore the chondrogenic potential of the goat BMSCs in the Transwell co-culture system and the poly-laetide-co-glycolide (PLGA) scaffolds.

METHODSThe BMSCs were isolated from the goat iliac crest while the chondrocytes were obtained from the goat's last costal cartilage. In the Transwell co-culture system, the BMSCs co-cultured with chondrocytes were designed as group A, whereas the goat's BMSCs induced with the chondrogenic medium were group B. Both groups A and B were the experimental groups, while group C that only contained BMSCs was the control group. In the PLGA scaffolds co-culture system, BMSCs were seeded into the PLGA scaffolds, which were suspended in the 24-well plate, and the control group was established by presence or absence of chondrocytes at the bottom of the 24-well plate. Toluidine blue staining, Alcian blue staining, collagen II immunofluoresence, collagen II immunochemical staining, collagen I, collagen II, COL2a Q-PCR and osteopontin Q-PCR were used to examine the chondrogenic conditions as well as the expressions of chondrogenic and osteogenic genes.

RESULTSCells isolated from the aspirates of the goat bone marrow proliferated rapidly and gained characteristics of stem cells in Passage 4. However, the differentiations of chondrocytes were not apparent in Passage 3. The results from Toluidine blue staining, collagen II immunofluoresence and PCR showed the transformation of BMSCs to chondrocytes in the Transwell co-culture system and PLGA scaffolds. Although the cartilage gene expressions were upgraded in both chondrogenesis group and co-culture system, the osteopontin gene expression, which represents osteogenic level, was also up-regulated.

CONCLUSIONSThe Transwell co-culture system and the PLGA scaffolds co-culture system can promote the chondrogenic differentiation of the goat's BMSCs, while up-regulated osteopontin gene expression in the Transwell co-culture system implies the osteogenic potential of BMSCs.

Animals ; Bone Marrow Cells ; physiology ; Cell Culture Techniques ; methods ; Chondrocytes ; physiology ; Chondrogenesis ; physiology ; Coculture Techniques ; Goats ; Mesenchymal Stromal Cells ; physiology ; Tissue Engineering ; methods ; Tissue Scaffolds

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III beta tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.
Animals ; *Cell Differentiation ; Chondrogenesis ; Dogs/blood/*physiology ; Fetal Blood/*cytology ; Mesenchymal Stem Cells/*cytology ; Neurons/cytology ; Osteogenesis

OBJECTIVETo explore a method of isolation, culture and chondrogenic phenotype differentiation of mesenchymal stem cell (MSCs) from the bone marrow of rats in vitro and to offer experimental reference for the resources of seeding cells in cartilage tissue engineering.

METHODSMSCs were isolated from bone marrow and purified by density gradient centrifuge and cultured in vitro. The MSC adherence formed and those in passage 3 were chosen to induce into chondrogenic differentiation. After 7, 14, 21 days, immunohistochemical techique was applied to detect the expression of collagen type II. The differentiated cells were implanted on the CPP/PLLA composites. After the cell-scaffold complex was cultured in vitro for one week, the ultrastructure of the scaffold was observed with scanning electron microscopy.

RESULTSThe differentiated cells changed from a spindle-like fibroblastic appearance to a polygonal shape, the capability of proliferation was down markedly. Immunohistochemical staining of collagen II were positive for the pass age, especially in the 21st days. Induced MSCs were well adherent to the scaffold composites and the cells were embedded by the cell-matrix.

CONCLUSIONUnder the induced medium, MSCs can differentiate into chondrogenic phenotype and secrete specificity matrix of cartilage in vitro. MSCs can likely be served as optimal cell source for cartilage tissue engineering.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Cell Differentiation ; Cell Separation ; Chondrocytes ; cytology ; physiology ; Chondrogenesis ; Female ; Male ; Mesenchymal Stromal Cells ; cytology ; physiology ; Rabbits ; Tissue Engineering ; Tissue Scaffolds

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco's modified Eagle's medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.
Adipogenesis ; Amnion/*cytology/physiology ; Animals ; *Cell Differentiation ; *Cell Lineage ; Cell Proliferation ; Chondrogenesis ; Female ; Flow Cytometry/veterinary ; Horses ; Immunophenotyping/veterinary ; Mesenchymal Stromal Cells/*cytology/physiology ; Osteogenesis

Mesenchymal stem cells (MSCs) are progenitors of connective tissues, which have emerged as important tools for tissue engineering due to their differentiation potential along various cell types. In recent years, accumulating evidence has suggested that the regulation of mitochondria dynamics and function is essential for successful differentiation of MSCs. In this paper, we review and provide an integrated view on the role of mitochondria in MSC differentiation. The mitochondria are maintained at a relatively low activity level in MSCs, and upon induction, mtDNA copy number, protein levels of respiratory enzymes, the oxygen consumption rate, mRNA levels of mitochondrial biogenesis-associated genes, and intracellular ATP content are increased. The regulated level of mitochondrial ROS is found not only to influence differentiation but also to contribute to the direction determination of differentiation. Understanding the roles of mitochondrial dynamics during MSC differentiation will facilitate the optimization of differentiation protocols by adjusting biochemical properties, such as energy production or the redox status of stem cells, and ultimately, benefit the development of new pharmacologic strategies in regenerative medicine.
Adipogenesis ; physiology ; Animals ; Cell Differentiation ; physiology ; Chondrogenesis ; physiology ; Humans ; Mesenchymal Stem Cells ; cytology ; metabolism ; Mitochondria ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Osteogenesis ; physiology ; RNA ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Mitochondrial ; Reactive Oxygen Species ; metabolism

This paper is aimed to investigate the feasibility of applying the small intestine submucosa (SIS) as the scaffold in constructing tissue engineering cartilage in vitro. We obtained SIS from the small intestine of specific pathogen-free pigs. Then we isolated tunica submucosa layer from the mucosal, muscular, and serosal layers by gentle mechanic abrasion. The SIS was made acellular by combination of detergent and enzyme digestion. The chondrocytes were seeded onto the SIS and were cultured for 3 weeks. The cell growth, attachment and distribution were detected by histochemical stain, immunohistochemical stain and scan electron microscope. The chondrocytes could adhere and grow well on the matrix surface, and synthesize a large of the GAG and type U collagen. However, the chondrocytes grew only on the surface andsuperficial layer of the scaffold, they did not move into the inner part of the scaffold. It could be concluded that SIS has good cellular compatibility without cytotoxicity and provides temporary substrate to which these anchorage-dependent cells can adhere, and stimulate the chondrocytes anchored on the scaffold to proliferate and keep differentiated phenotype. Further study will be needed to promote the ability of chondrocyte chemotaxis in order to distribute the chondrocytes into the whole scaffold uniformly.
Animals ; Cell Adhesion ; Cell Culture Techniques ; Cell Proliferation ; Chondrocytes ; cytology ; Chondrogenesis ; physiology ; Intestinal Mucosa ; cytology ; Intestine, Small ; cytology ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

AIMThe aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also observed during differentiation.

METHODOLOGYDPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR.

RESULTSThe endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation.

CONCLUSIONAs the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.

Adipocytes ; cytology ; Adipogenesis ; physiology ; Animals ; Anthraquinones ; Azo Compounds ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; physiology ; Chondrocytes ; cytology ; Chondrogenesis ; physiology ; Coloring Agents ; Culture Media ; Dental Pulp ; cytology ; Genetic Markers ; genetics ; Green Fluorescent Proteins ; analysis ; genetics ; Mice ; Mice, Nude ; Mice, Transgenic ; Microscopy, Fluorescence ; Osteoblasts ; cytology ; Osteogenesis ; physiology ; RNA ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; physiology ; Tissue Culture Techniques ; Tolonium Chloride

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, the methods focused on biological repair have aroused interest and several tissue engineering approaches using different cell types have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering, attention has been given to the use of mesenchymal stem cells (MSCs). In this connection, we have made a study on the characteristics of MSCs derived from adult bone marrow and on the feasibility of constructing IVD tissue-engineering cell under a Three-Dimensional Pellet Culture System. The human bone marrow MSCs were isolated and purified with density gradient solution and attachment-independent culture system. MSCs isolated using this method are a homogeneous population as indicated by morphology and other criteria. They have the capacity for self-renewal and proliferation, and the multilineage potential to differentiate.
Adolescent ; Adult ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Humans ; Intervertebral Disc ; Intervertebral Disc Degeneration ; therapy ; Mesenchymal Stromal Cells ; cytology ; Tissue Engineering ; methods ; Young Adult