M for the formation of these tissues. We hypothesized that the normal anulus fibrosus tissue contains multipotent progenitor cells, which are able to differentiate into cartilage and/or fibrocartilage cells, osteoblasts, neurons, and blood vessel cells. Methods: We isolated anulus fibrosus cells from the nondegenerative intervertebral discs of adolescent (thirteen to sixteen-year-old) patients with idiopathic scoliosis and cultured the cells in vitro in induction media containing different stimuli. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Expression of markers of adipogenesis, osteogenesis, chondrogenesis, neurogenesis, and differentiation into endothelial lineages was determined with use of immunostaining, cytohistological staining, and reverse transcription-polymerase chain reaction. Results: Anulus fibrosus cells expressed several of the cell surface antigens that are sometimes associated with mesenchymal stem cells, including CD29, CD49e, CD51, CD73, CD90, CD105, CD166, CD184, and Stro-1, and two neuronal stem cell markers, nestin and neuron-specific enolase. Furthermore, varying the stimulants added to the induction media determined whether anulus fibrosus cells differentiated into adipocytes, osteoblasts, chondrocytes, neurons, or endothelial cells. Conclusions: Anulus fibrosus cells isolated from nondegenerative intervertebral discs can differentiate into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells in vitro. Clinical Relevance: Our results, by offering new insights into the biology of anulus fibrosus cells, may assist in future strategies to treat intervertebral disc diseases....

The repair of articular cartilage injuries is impeded by the avascular and non-innervated nature of cartilage. Transplantation of autologous chondrocytes has a limited ability to augment the repair process due to the highly differentiated state of chondrocytes and the risks of donor-site morbidity. Mesenchymal stem cells can undergo chondrogenesis in the presence of growth factors for cartilage defect repair. Growth and differentiation factor-5 (GDF5) plays an important role in chondrogenesis. In this study, we examined the effects of GDF5 on chondrogenesis of adipose-derived stem cells (ADSCs) and evaluate the chondrogenic potentials of GDF5 genetically engineered ADSCs using an in vitro pellet culture model. Rat ADSCs were grown as pellet cultures and treated with chondrogenic media (CM). Induction of GDF5 by an adenovirus (Ad-GDF5) was compared with exogenous supplementation of GDF5 (100 ng/ml) and transforming growth factor-β (TGF-β1; 10 ng/ml). The ADSCs underwent chondrogenic differentiation in response to GDF5 exposure as demonstrated by production of proteoglycan, and up-regulation of collagen II and aggrecan at the protein and mRNA level. The chondrogenic potential of a one-time infection with Ad-GDF5 was weaker than exogenous GDF5, but equal to that of TGF-β1. Stimulation with growth factors or CM alone induced transient expression of the mRNA for collagen X, indicating a need for optimization of the CM. Our findings indicate that GDF5 is a potent inducer of chondrogenesis in ADSCs, and that ADSCs genetically engineered to express prochondrogenic growth factors, such as GDF5, may be a promising therapeutic cell source for cartilage tissue engineering.

Human subcutaneous fat-derived mesenchymal cells recently have been shown to have the potential to differentiate in vitro into a variety of cell types, including adipocytes, osteoblasts, chondrocytes, and myoblasts. This effect suggests that fat tissue may serve as an abundant and easily acquired source of multipotent cells for tissue engineering. The multipotential characteristics of fat-derived mesenchymal cells from the inguinial fat pad of rabbit have not been clearly defined. In this study we have isolated a population of mesenchymal cells from inguinal fat from adult New Zealand white rabbits. The cells that were maintained under various differentiation conditions were shown to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes; this differentiation was demonstrated using gene expression for tissue-specific proteins. We also co-cultured the cells with intervertebral disk tissue from the nucleus pulpous or from the annulus fibrosus. The fat-derived cells co-cultured with nucleus pulposus showed an increase in expression of type II collagen and aggrecan genes, compared with cells in alginate alone and cells co-cultured with annulus fibrosus. The data suggest that the fat-derived mesenchymal cells responded to soluble mediators from the disk. Future studies on intervertebral disk reconstruction could be based on our findings with fat-derived multipotential cells from the inguinal region of the rabbit that were co-cultured with disk tissue and may prove useful in tissue engineering strategies.