Walter E. Horton

Response of engineered cartilage tissue to biochemical agents as studied by proton magnetic resonance microscopy.

OBJECTIVE To test the hypothesis that magnetic resonance imaging (MRI) results correlate with the biochemical composition of cartilage matrix and can therefore be used to evaluate natural tissue development and the effects of biologic interventions. METHODS Chondrocytes harvested from day-16 chick embryo sterna were inoculated into an MRI-compatible hollow-fiber bioreactor. The tissue that formed over a period of 2-4 weeks was studied biochemically, histologically, and with MRI. Besides natural development, the response of the tissue to administration of retinoic acid, interleukin-1beta (IL-1beta), and daily dosing with ascorbic acid was studied. RESULTS Tissue wet and dry weight, glycosaminoglycan (GAG) content, and collagen content all increased with development time, while tissue hydration decreased. The administration of retinoic acid resulted in a significant reduction in tissue wet weight, proteoglycan content, and cell number and an increase in hydration as compared with controls. Daily dosing with ascorbic acid increased tissue collagen content significantly compared with controls, while the administration of IL-1beta resulted in increased proteoglycan content. The water proton longitudinal and transverse relaxation rates correlated well with GAG and collagen concentrations of the matrix as well as with tissue hydration. In contrast, the magnetization transfer value for the tissue correlated only with total collagen. Finally, the self-diffusion coefficient of water correlated with tissue hydration. CONCLUSION Parameters derived from MR images obtained noninvasively can be used to quantitatively assess the composition of cartilage tissue generated in a bioreactor. We conclude that MRI is a promising modality for the assessment of certain biochemical properties of cartilage in a wide variety of settings.

Bcl‐2 regulates chondrocyte morphology and aggrecan gene expression independent of caspase activation and full apoptosis

Bcl‐2 is widely expressed in a variety of cell types and is known to block apoptosis through a conserved pathway. However, recent reports have demonstrated that Bcl‐2 regulates cell behavior independent of its control of apoptosis. Chondrocytes express a unique set of matrix proteins, including the proteoglycan aggrecan, and have been widely used to study the relationship between trophic factors and apoptosis. In this article, we report that Bcl‐2 affects the morphology and regulates the expression of aggrecan in a rat chondrocyte cell line (IRC). Endogenous Bcl‐2 and aggrecan mRNA were both down‐regulated in response to serum withdrawal in parental IRC cells, while constitutive expression of Bcl‐2 maintained aggrecan levels under conditions of serum withdrawal. In addition, expression of anti‐sense Bcl‐2 resulted in decreased aggrecan mRNA and produced a fibroblastic morphology compared with parental cells. The caspase inhibitor ZVAD‐fmk effectively blocked full apoptosis of IRC cells in response to serum withdrawal or anti‐sense Bcl‐2 but did not prevent the down‐regulation of aggrecan expression from either signal. These results suggest a novel role for Bcl‐2 in regulating the differentiated phenotype of chondrocytes and the expression of a differentiation‐specific gene independent of its control of apoptosis. J. Cell. Biochem. 74:576–586, 1999.

Highly Hydrophobic Electrospun Fiber Mats from Polyisobutylene-Based Thermoplastic Elastomers

This paper is the first report of electrospinning neat polyisobutylene-based thermoplastic elastomers. Two generations of these materials are investigated: a linear poly(styrene-b-isobutylene-b-styrene) (L_SIBS) triblock copolymer and a dendritic poly(isobutylene-b-p-methylstyrene) (D_IB-MS), also a candidate for biomedical applications. Cross-polarized optical microscopy shows birefringence, indicating orientation in the electrospun fibers, which undergo large elongation and shear during electrospinning. In contrast to the circular cross section of L_SIBS fibers, D_IB-MS yields dumbbell-shaped fiber cross sections for the combination of processing conditions, molecular weight, and architecture. Hydrophobic surfaces with a water contact angle as high as 146 ± 3° were obtained with D_IB-MS that had the noncircular fiber cross section and a hierarchical arrangement of nano- to micrometer-sized fibers in the mat. These highly water repellent fiber mats were found to serve as an excellent scaffold for bovine chondrocytes to produce cartilage tissue.

Modification of osteoarthritis in the guinea pig with pulsed low-intensity ultrasound treatment

OBJECTIVE The Hartley guinea pig develops articular cartilage degeneration similar to that seen in idiopathic human osteoarthritis (OA). We investigated whether the application of pulsed low-intensity ultrasound (PLIUS) to the Hartley guinea pig joint would prevent or attenuate the progression of this degenerative process. METHODS Treatment of male Hartley guinea pigs was initiated at the onset of degeneration (8 weeks of age) to assess the ability of PLIUS to prevent OA, or at a later age (12 months) to assess the degree to which PLIUS acted to attenuate the progression of established disease. PLIUS (30 mW/cm(2)) was applied to stifle joints for 20 min/day over periods ranging from 3 to 10 months, with contralateral limbs serving as controls. Joint cartilage histology was graded according to a modified Mankin scale to evaluate treatment effect. Immunohistochemical staining for interleukin-1 receptor antagonist (IL-1ra), matrix metalloproteinase (MMP)-3, MMP-13, and transforming growth factor (TGF)-beta1 was performed on the cartilage to evaluate patterns of expression of these proteins. RESULTS PLIUS did not fully prevent cartilage degeneration in the prevention groups, but diminished the severity of the disease, with the treated joints showing markedly decreased surface irregularities and a much smaller degree of loss of matrix staining as compared to controls. PLIUS also attenuated disease progression in the groups with established disease, although to a somewhat lesser extent as compared to the prevention groups. Immunohistochemical staining demonstrated a markedly decreased degree of TGF-beta1 production in the PLIUS-treated joints. This indicates less active endogenous repair, consistent with the marked reduction in cartilage degradation. CONCLUSIONS PLIUS exhibits the ability to attenuate the progression of cartilage degeneration in an animal model of idiopathic human OA. The effect was greater in the treatment of early, rather than established, degeneration.

Coordinate down-regulation of cartilage matrix gene expression in Bcl-2 deficient chondrocytes is associated with decreased SOX9 expression and decreased mRNA stability.

The anti‐apoptotic protein Bcl‐2 has been shown to function in roles unrelated to apoptosis in a variety of cell types. We have previously reported that loss of Bcl‐2 expression alters chondrocyte morphology and modulates aggrecan expression via an apoptosis‐independent pathway. Here we show that Bcl‐2 is required for chondrocytes to maintain expression of a variety of cartilage‐specific matrix proteins. Using quantitative, real‐time PCR, we demonstrate that Bcl‐2‐deficient chondrocytes coordinately down‐regulate genes coding for hyaline cartilage matrix proteins including collagen II, collagen IX, aggrecan, and link protein. The decrease in steady‐state level of these mRNA transcripts results, in part, from decreased mRNA stability in Bcl‐2‐deficient chondrocytes. Transcriptional regulation is also likely involved because chondrocytes with decreased Bcl‐2 levels show decreased expression of SOX9, a transcription factor necessary for expressing the major cartilage matrix proteins. In contrast, chondrocytes constitutively expressing Bcl‐2 have a stable phenotype when subjected to loss of serum factor signaling. These cells maintain high levels of SOX9, as well as the SOX9 targets collagen II and aggrecan. These results suggest that Bcl‐2 is involved in a pathway important for maintaining a stable chondrocyte phenotype. J. Cell. Biochem. 88: 941–953, 2003.

ECM production of primary human and bovine chondrocytes in hybrid PEG hydrogels containing type i collagen and hyaluronic acid

The development of advanced materials that facilitate hyaline cartilage formation and regeneration in aging populations is imperative. Critical to the success of this endeavor is the optimization of ECM production from clinically relevant cells. However, much of the current literature focuses on the investigation of primary bovine chondrocytes from young calves, which differ significantly than osteoarthritic cells from human sources. This study examines the levels of extracellular matrix (ECM) production using various levels of type I collagen and hyaluronic acid in poly(ethylene glycol) dimethacrylate (PEGDM) hydrogels in total knee arthroplasties, compared with the results from bovine chondrocytes. The addition of type 1 collagen in both the presence and absence of low levels of hyaluronic acid increased ECM production and/or retention in scaffolds containing either bovine or human chondrocytes. These findings are supported consistently with colorimetric quantification, whole mount extracellular matrix staining for both cell types, and histological staining for glycoaminoglycans and collagen of human chondrocyte containing samples. While exhibiting similar trends, the relative ECM productions levels for the primary human chondrocytes are significantly less than the bovine chondrocytes which reinforces the need for additional optimization.

A novel role for Bcl-2 associated-athanogene-1 (Bag-1) in regulation of the endoplasmic reticulum stress response in mammalian chondrocytes.

BAG‐1 (Bcl‐2 associated athanogene‐1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG‐1 is expressed in growth plate and articular cartilage, and the expression of BAG‐1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG‐1 is involved in ER stress‐induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG‐1 expression. In addition, direct suppression of BAG‐1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG‐1 delayed the onset of ER stress‐mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG‐1 deficient chondrocytes. In contrast, overexpression of BAG‐1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG‐1. These results suggest a novel role for BAG‐1 in supporting viability and matrix expression of chondrocytes. J. Cell. Biochem. 102: 786–800, 2007.