The degradation of chondrogenic pellets using cocultures of synovial fibroblasts and U937 cells.

Osteoarthritis (OA) of the knee is often characterized by joint space narrowing on X-ray, knee pain, and a loss of joint function through progressive cartilage degradation and intermittent synovial inflammation. The objective of this work was to develop an in vitro model in a clinically relevant system. Normal human synovial fibroblasts were cultured with U937 cells for 3 days then combined with a chondrogenic stem cell pellet for another 4 days. This culture system mimicked many of the aspects of early stage OA including production of cytokines and degradative enzymes, MMP-1 and MMP-3, resulting in a conditioned medium profile similar to OA synovial fluid. This catabolic environment resulted in the release of glycosaminoglycan (GAG) from the pellet. In a similar manner to early stage OA, the pellet had increased aggrecan and collagen II expression. All of these effects are hallmarks of early stage OA. This relatively simple tissue model containing a 3D cartilage component interacting with synoviocytes and macrophages could be useful to understand early causes and progression of OA. It can be scaled easily thus useful for high throughput screening of disease modifying drugs in a clinically relevant system.

The roles of catabolic factors in the development of osteoarthritis.

Osteoarthritis (OA) is the most prevalent disease of articular joints characterized by joint space narrowing on X-ray, joint pain, and a loss of joint function through progressive cartilage degradation and intermittent synovial inflammation. Current in vitro models of OA are often monolayer cultured primary cells exposed to high concentrations of cytokines or chemokines, usually IL-1ß or TNF-α. IL-1ß could play a role in the early progression or even initiation of OA as evidenced by many of the in vitro studies. However, the inconsistent or outright lack of detectable IL-1ß combined with high concentrations of the natural inhibitor IL-1Ra in the OA synovial fluid makes the idea of OA being IL-1ß-driven questionable. Further, other stimulants, including IL-6 and matrix fragments, have been shown in vitro to cause many of the effects seen in OA at relevant concentrations found in the OA synovial fluid. More work with these stimulants and IL-1ß-independent models needs to be done. Concurrently, research should be conducted with patients with OA as early as possible in the progression of their disease to be able to potentially identify, target, and treat the initiation of the disease.

Murine osteoblasts regulate mesenchymal stem cells via WNT and cadherin pathways: mechanism depends on cell-cell contact mode.

Osteoblasts (OSTs) are derived from mesenchymal stem cells (MSCs) and coexist in close proximity with MSCs in bone during development and remodelling. Interactions between these two cell types remain obscure. Through a well-defined co-culture model, the present work demonstrated that OSTs regulate MSCs through the WNT and cadherin pathways. The regulation mechanism depends on the cell-cell contact mode (indirect or direct) between the two cell types. When physically separated (indirect contact), OSTs express WNTs and stimulate the osteogenic differentiation of MSCs through the activation of the WNT pathway and suppression of the cadherin pathway. This mechanism is evidenced by: (a) the elevation of cytoplasmic and nuclear unphosphorylated beta-catenin protein levels; (b) the suppression of beta-catenin degradation; (c) the increase in WNT-related transcription factor TCF1/LEF1; and (d) the loss of major bone-related cadherins (N-CAD and CAD11). Addition of DKK1 antagonizes the WNT pathway and diminishes the stimulatory effect of OSTs on MSCs. When in direct cell-cell contact, OSTs still secrete WNTs, whose binding still stabilizes the beta-catenin in MSCs. However, direct cell-cell contact induces the upregulation of cadherin pathway in MSCs, which suppresses the WNT pathway by containing cytoplasmic beta-catenin protein at a low level; consequently, the stimulatory effect of OSTs is negated. Regulation of cytoplasmic beta-catenin protein levels through concerted action or crosstalk between the WNT and cadherin pathways is the key to the signalling transduction in these cellular communication networks.

Cartilage tissue engineering with silk scaffolds and human articular chondrocytes.

Adult cartilage tissue has poor capability of self-repair, especially in case of severe cartilage damage due to trauma or age-related degeneration. Autologous cell-based tissue engineering using three-dimensional (3-D) porous scaffolds has provided an option for the repair of full thickness defects in adult cartilage tissue. Mesenchymal stem cells (MSCs) and chondrocytes are the two major cell sources for cartilage tissue engineering. Silk fibroin as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and process-ability has demonstrated strong potential for skeletal tissue engineering. The present study combined adult human chondrocytes (hCHs) with aqueous-derived porous silk fibroin scaffolds for in vitro cartilage tissue engineering. The results were compared with a previous study using the same scaffolds but using MSCs to generate the cartilage tissue outcomes. Culture-expanded hCHs attached to, proliferated and re-differentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-beta1, based on cell morphology, levels of cartilage-related gene transcripts, and the presence of a cartilage-specific ECM. Cell density was critical for the redifferentiation of culture-expanded hCHs in the 3-D aqueous-derived silk fibroin scaffolds. The level of cartilage-related transcripts (AGC, Col-II, Sox 9 and Col-II/Col-I ratio) and the deposition of cartilage-specific ECM were significantly upregulated in constructs initiated with higher seeding density. The hCH-based constructs were significantly different than those formed from MSC-based constructs with respect to cell morphology, zonal structure and initial seeding density needed to successfully generate engineered cartilage-like tissue. These results suggest fundamental differences between stem cell-based (MSC) and primary cell-based (hCH) tissue engineering, as well as the importance of suitable scaffold features, in the optimization of cartilage-related outcomes in vitro. The present work diversifies cell sources in combination with silk fibroin-based tissue engineering applications. Together with our previous studies, the present results show great promise for engineered 3-D silk fibroin scaffolds in autologous cell-based skeletal tissue engineering.

Expansion and osteogenic differentiation of bone marrow-derived mesenchymal stem cells on a vitamin C functionalized polymer.

In human body ascorbic acid plays an essential role in the synthesis and function of skeletal tissues and immune system factors. Ascorbic acid is also a major physiological antioxidant, repairing oxidatively damaged biomolecules, preventing the formation of excessive reactive oxygen species or scavenging these species. We recently reported the synthesis of ascorbic acid-functionalized polymers in which the antioxidant features of the pendant ascorbic acid groups was preserved. In the present work we demonstrate that ascorbic acid-functionalized poly(methyl methacrylate) (AA-PMMA) can modulate the proliferation and osteogenic differentiation of early and late-passage bone marrow-derived human mesenchymal stem cells (MSCs). The covalently coupled ascorbic acid impacted MSCs differently than when ascorbic acid was presented to the cells in soluble form. At optimal concentration, the covalently coupled ascorbic acid and soluble ascorbic acid synergistically promoted and retained the ability of MSCs to respond to osteogenic stimulation over extensive cell expansions in vitro. In the presence of soluble ascorbic acid, AA-PMMA films prepared at optimal concentrations (0.1 mg/ml in the present study) showed a significant promotive effect over other concentrations and tissue culture plastic (TCP) with respect to osteogenic differentiation of both EP (young) and LP (old) MSCs. These results suggest that the coupled ascorbic acid is acting mainly at the extracellular level and, at optimal concentrations, the immobilized extracellular ascorbic acid and soluble ascorbic acid synergistically promote osteogenic differentiation of MSCs. Importantly, the covalently coupled ascorbic acid on the films of optimal concentration was able to preserve the capacity of MSCs to undergo osteogenic differentiation in vitro. These results suggest an important role for functionalized biomaterials with antioxidant features in control of cell physiology and cell aging phenomena.

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells.

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.