Prasanna Malaviya

Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels

Articular cartilage responds to its mechanical environment through altered cell metabolism and matrix synthesis. In this study, isolated articular chondrocytes were cultured in collagen type I gels and exposed to uniaxial static compression of 0%, 25%, or 50% of original thickness for 0.5, 4, and 24 h, and to oscillatory (25 +/- 4%, 1 Hz) compression for 24 h. The cellular response was assessed through competitive and real-time RT-PCR to quantify expression of genes for collagen type I, collagen type II, and aggrecan core protein, and through radiolabelled proline and sulfate incorporation to quantify protein and proteoglycan synthesis rates. Static compression for 24 h inhibited expression of collagen I and II mRNAs and inhibited 3H-proline and 35S-sulfate incorporation. The mRNA expression exhibited transient fluctuations at intermediate time points. Oscillatory compression had no effect upon mRNA expression, and 24 h after release from static compression, there was no difference in collagen II or aggrecan mRNA, while there was an inhibition of collagen I. We conclude that the chondrocytes maintained some aspects of their ability to sense and respond to static compression, despite a biochemical and mechanical environment which is different from that in tissue. This suggests that mechanical stimuli may be useful in modulating chondrocyte metabolism in tissue engineering systems using fibrillar protein scaffolds.

Long-term Outcome for Large Meniscal Defects Treated With Small Intestinal Submucosa in a Dog Model

Background Large meniscal defects are a common problem for which current treatment options are limited. Hypothesis Treatment of posterior medial meniscal defects in dogs with small intestinal submucosa is superior to partial meniscectomy in terms of clinical limb function, chondroprotection, and amount and type of new tissue in the defect. Study Design Controlled laboratory study. Methods A total of 51 mongrel dogs underwent medial arthrotomy with creation of standardized meniscal defects. The dogs were divided into groups based on defect treatment: small intestinal submucosa meniscal implant (n = 29) or meniscectomy (n = 22). The dogs were assessed for lameness by subjective scoring after surgery and sacrificed at 3, 6, or 12 months and assessed for articular cartilage damage, gross and histologic appearance of the operated meniscus, amount of new tissue in the defect, equilibrium compressive modulus of meniscal tissue, and relative compressive stiffness of articular cartilage. Results Dogs in the meniscectomy groups were significantly (P <. 001) more lame than dogs treated with small intestinal submucosa. Joints treated with small intestinal submucosa had significantly (P <. 001) less articular cartilage damage, based on india ink staining, than did those treated with meniscectomy. Menisci receiving small intestinal submucosa had more tissue filling in the defects than did menisci receiving no implants, and this new tissue was more mature and meniscus-like and better integrated with remaining meniscus. Conclusion Small intestinal submucosa scaffolds placed in large meniscal defects resulted in production of meniscus-like replacement tissue, which was consistently superior to meniscectomy in amount, type, and integration of new tissue; chondroprotection; and limb function in the long term. Clinical Relevance Small intestinal submucosa implants might be useful for treatment of large posterior vascular meniscal defects in humans.

Fluid-Induced Shear Stress Stimulates Chondrocyte Proliferation Partially Mediated via TGF-β1

There is growing evidence that a hydrodynamic environment is beneficial for growing cartilage tissue-engineered constructs; however, the mechanisms by which fluid shear provides for a better construct are not well understood. In this study, we investigated one possible mechanism by which constructs grow faster under fluid shear: fluid shear upregulates chondrocyte proliferation. Further, we investigated if this effect is mediated by TGF-beta1, a known mediator of fluid shear effects in other cell types and a mitogen for chondrocytes. To test the hypotheses, primary bovine articular chondrocytes were cultured in monolayers (approximately 40,000 cells/cm(2)) to 80-85% confluency. After 24 h of growth arrest, cells were exposed to 3.5 Pa fluid shear stress for 96 h. Total DNA was compared between flow and static culture slides. Total TGF-beta1 was quantified in flow-conditioned media (CM) and static culture-CM. Mitogenic capacity of the CM, with or without anti-TGF-beta1 or anti-TbetaRII (TGF beta receptor type II) antibodies, was also assessed. Results show that fluid shear significantly up-regulates chondrocyte proliferation (p < 0.02). Further, total TGF-beta1 in the flow-CM was more than 3.5-fold higher (p < 0.03) and its mitogenicity significantly higher (p < 0.007) as compared to static culture-CM. Adding excess anti-TGF-beta1 or anti-TbetaRII antibodies partially, but significantly depressed mitogenicity (approximately 20% decrease) of the flow-CM. These results show that fluid shear stress upregulates chondrocyte proliferation and that this effect is partially mediated by TGF-beta1.

Fibrochondrogenesis of Free Intraarticular Small Intestinal Submucosa Scaffolds

Naturally occurring biomaterials, such as small intestine submucosa (SIS), are attractive as potential scaffolds for engineering various tissue types. The aim of this study was to determine whether acellular SIS scaffolds can support cell attachment and ingrowth in a diarthroadial joint without significant intraarticular hemorrhage. Disks of porcine SIS were arthoscopically implanted freely within a randomized knee joint of 21 dogs and harvested 1, 2, 3, and 6 weeks postoperatively. Harvested disks were assessed for gross and histologic appearance, cellular infiltration, and immunoreactivity of collagenase and collagen types I and II. Knee synovium and synovial fluid were also evaluated. All disks were thickened and opacified at harvest. Eleven disks (52%) had adhered to intraarticular tissues and cellular infiltration into the disks was positively correlated with tissue adherence. Further, tissue adherence was positively correlated with duration of intraarticular implantation. Five disks (24%) contained focal areas of homogeneous extracellular matrix. A trend toward more collagenase immunoreactivity was noted in the 3-week disks. Collagen type I was present in remaining SIS and extracellular matrix associated with infiltrated cells. Placed freely within a joint, acellular SIS disks underwent cellular and extracellular matrix modification resulting in fibrocartilage-like tissue. Utilization of SIS as a scaffold for intraarticular tissue-engineering applications is supported as cytoconductivity, appropriate residence time, and absence of untoward effects of implantation are desirable criteria for a tissue-engineering biomaterial.