Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype

Introduction\ud Mesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct.\ud \ud Methods\ud Human MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-β3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-β1 and TIMP1(tissue inhibitor of metalloproteinases-1)).\ud \ud Results\ud Significantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFβ groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-β1 and decreased expression of sox-9 and collagen II relative to the TGFβ group was observed.\ud \ud Conclusions\ud Together, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD

Engineered Osteochondral Grafts Using Biphasic Composite Solid Free-Form Fabricated Scaffolds

Tissue engineering has provided an alternative to traditional strategies to repair cartilage damaged by injury or degenerative disease. A successful strategy to engineer osteochondral tissue will mimic the natural contour of the articulating surface, achieve native mechanical properties and functional load-bearing ability, and lead to integration with host cartilage and underlying subchondral bone. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match articular geometry. The objective of this study was to utilize materials and biological factors in an integrated approach to regenerate a multitissue interface. Biphasic composite scaffolds manufactured by IBD and SFF fabrication were used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-L-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein 7 (BMP-7) in the ceramic phase and fully differentiated chondrocytes in the polymeric phase. After subcutaneous implantation into mice, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63145/1/ten.2004.10.1376.pd

Skeletal homeostasis in tissue-engineered bone

Tissue-engineering strategies to stimulate bone regeneration may offer an alternative approach to conventional orthopaedic and maxillofacial surgical therapies. Over the last decade, significant advances have been accomplished in developing biomimetic matrices, growth factors, cell transplantation and gene delivery therapeutics to support new bone growth. However, it is not known if tissue-engineered bone recapitulates the biology of normal skeletal tissue in response to physiologic cues. Here, we report that bone formed by the differentiation of transplanted murine bone marrow stromal cells (BMSCs) responds to a systemically delivered calciotropic hormone. Ectopic ossicles in mice exposed to catabolic doses of parathyroid hormone (PTH) had increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts as compared to control mice. In contrast, treatment with anabolic doses of PTH promoted a marked increase in trabecular bone mass as analyzed by microcomputed tomography and histomorphometry. Our findings demonstrate that bone formed from transplanted BMSCs is responsive to normal physiologic signals, and can be augmented by the addition of a systemic anabolic agent. Because multiple and distinct ossicles can be generated in a single animal, this versatile system may be used to: (a) elucidate cellular/molecular mechanisms in bone regeneration; (b) study cell-to-cell interactions in the bone marrow microenvironment in health and disease; and (c) evaluate the efficacy of osteotropic agents that modulate bone turnover in vivo. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34920/1/1100210516_ftp.pd

Update in cartilage bio-engineering.

peer reviewe

Phenotype, function, and differentiation potential of human monocyte subsets

<div><p>Human monocytes have been grouped into classical (CD14⁺⁺CD16⁻), non-classical (CD14^dimCD16⁺⁺), and intermediate (CD14⁺⁺CD16⁺) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.</p></div