Surface Zone Chondrocytes Research Articles

Optimization of Methods for Articular Cartilage Surface Tissue Engineering: Cell Density and Transforming Growth Factor Beta Are Critical for Self-Assembly and Lubricin Secretion.

Lubricin/superficial zone protein (SZP)/proteoglycan4 (PRG4) plays an important role in boundary lubrication in articular cartilage. Lubricin is secreted by superficial zone chondrocytes and synoviocytes of the synovium. The specific objective of this investigation is to optimize the methods for tissue engineering of articular cartilage surface. The aim of this study is to investigate the effect of cell density on the self-assembly of superficial zone chondrocytes and lubricin secretion as a functional assessment. Superficial zone chondrocytes were cultivated as a monolayer at low, medium, and high densities. Chondrocytes at the three different densities were treated with transforming growth factor beta (TGF-β)1 twice a week or daily, and the accumulated lubricin in the culture medium was analyzed by immunoblots and quantitated by enzyme-linked immunosorbent assay (ELISA). Cell numbers in low and medium densities were increased by TGF-β1; whereas cell numbers in high-density cell cultures were decreased by twice-a-week treatment of TGF-β1. On the other hand, the cell numbers were maintained by daily TGF-β treatment. Immunoblots and quantitation of lubricin by ELISA analysis indicated that TGF-β1 stimulated lubricin secretion by superficial zone chondrocytes at all densities with twice-a-week TGF-β treatment. It is noteworthy that the daily treatment of TGF-β1 increased lubricin much higher compared with twice-a-week treatment. These data demonstrate that daily treatment is optimal for the TGF-β1 response in a higher density of monolayer cultures. These findings have implications for self-assembly of surface zone chondrocytes of articular cartilage for application in tissue engineering of articular cartilage surface.

Modulation of superficial zone protein/lubricin/PRG4 by kartogenin and transforming growth factor-β1 in surface zone chondrocytes in bovine articular cartilage

Modulation of superficial zone protein/lubricin/PRG4 by kartogenin and transforming growth factor-β1 in surface zone chondrocytes in bovine articular cartilage

Modulation of Superficial Zone Protein/Lubricin/PRG4 by Kartogenin and Transforming Growth Factor-β1 in Surface Zone Chondrocytes in Bovine Articular Cartilage.

Superficial zone protein (SZP)/lubricin/PRG4 functions as a boundary lubricant in articular cartilage to decrease friction and wear. As articular cartilage lubrication is critical for normal joint function, the accumulation of SZP at the surface of cartilage is important for joint homeostasis. Recently, a heterocyclic compound called kartogenin (KGN) was found to induce chondrogenic differentiation and enhance mRNA expression of lubricin. The objective of this study was to determine whether KGN can stimulate synthesis of SZP in superficial zone, articular chondrocytes. We investigated the effects of KGN and transforming growth factor-β1 (TGF-β1) on articular cartilage and synovium of the bovine knee joint by evaluating SZP secretion by enzyme-linked immunosorbent assay analysis. Monolayer, micromass, and explant cultures of articular cartilage, and monolayer culture of synoviocytes, were treated with KGN. SZP accumulation in the medium was evaluated and mRNA expression was measured through quantitative polymerase chain reaction. TGF-β1 stimulated SZP secretion by superficial zone chondrocytes in monolayer, explant, and micromass cultures as expected. In addition, SZP secretion was inhibited by IL-1β in explant cultures, and enhanced by TGF-β1 in synoviocyte monolayer cultures. Although KGN elicited a 1.2-fold increase in SZP mRNA expression in combination with TGF-β1, KGN neither stimulated any significant increases in SZP synthesis nor prevented catabolic decreases in SZP production from IL-1β. These data suggest that the chondrogenic effects of KGN depend on cellular phenotype and differentiation status, as KGN did not alter SZP synthesis in differentiated, superficial zone articular chondrocytes.

Surface zone articular chondrocytes modulate the bulk and surface mechanical properties of the tissue-engineered cartilage.

The central hypothesis of functional tissue engineering is that an engineered construct can serve as a viable replacement tissue in vivo by replicating the structure and function of native tissue. In the case of articular cartilage, this requires the reproduction of the bulk mechanical and surface lubrication properties of native hyaline cartilage. Cartilage tissue engineering has primarily focused on achieving the bulk mechanical properties of native cartilage such as the compressive aggregate modulus and tensile strength. A scaffold-free self-assembling process has been developed that produces engineered cartilage with compressive properties approaching native tissue levels. Thus, the next step in this process is to begin addressing the friction coefficient and wear properties of these engineered constructs. The superficial zone protein (SZP), also known as lubricin or PRG4, is a boundary mode lubricant that is synthesized by surface zone (SZ) articular chondrocytes. Under conditions of high loading and low sliding speeds, SZP reduces friction and wear at the articular surface. The objective of this investigation was to determine whether increasing the proportion of SZ chondrocytes in cartilage constructs, in the absence of external stimuli such as growth factors and mechanical loading, would enhance the secretion of SZP and improve their frictional properties. In this study, cartilage constructs were engineered through a self-assembling process with varying ratios of SZ and middle zone (MZ) chondrocytes (SZ:MZ): 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs containing different ratios of SZ and MZ chondrocytes did not significantly differ in the glycosaminoglycan composition or compressive aggregate modulus. In contrast, tensile properties and collagen content were enhanced in nearly all constructs containing greater amounts of SZ chondrocytes. Increasing the proportion of SZ chondrocytes had the hypothesized effect of improving the synthesis and secretion of SZP. However, increasing the SZ chondrocyte fraction did not significantly reduce the friction coefficient. These results demonstrate that additional factors, such as SZP-binding macromolecules, surface roughness, and adhesion, need to be examined to modulate the lubrication properties of engineered cartilage.

Interaction between zonal populations of articular chondrocytes suppresses chondrocyte mineralization and this process is mediated by PTHrP

Articular cartilage is separated from subchondral bone by the tidemark and a calcified cartilage zone. Advancement of the calcified region and tidemark duplication are both hallmarks of osteoarthritis (OA). Currently the mechanisms controlling post-natal articular cartilage mineralization are poorly understood. The objective of this study is to test the hypothesis that cellular communication between different cartilage layers regulates articular chondrocyte mineralization. Co-culture models were established to evaluate the interaction of chondrocytes derived from the surface, middle and deep zones of articular cartilage. The cultures were stimulated with triiodothyronine (T3) to promote chondrocyte hypertrophy. The effects of zonal chondrocyte interactions on chondrocyte mineralization were examined over time. Co-culture of deep zone chondrocytes (DZCs) with surface zone chondrocytes (SZCs) suppressed the T3-induced increase in alkaline phosphatase (ALP) activity and related mineralization. Moreover, SZC-DZC co-culture was associated with a significantly higher parathyroid hormone-related peptide (PTHrP) expression when compared to controls. When PTHrP(1-40) was added to the DZC-only culture, it suppressed DZC ALP activity similar to the inhibition observed in co-culture with SZC. In addition, treatment with PTHrP reversed the effect of T3 stimulation on the expression of hypertrophic markers (Indian hedgehog, ALP, matrix metalloproteinases-13, Type X collagen) in the DZC cultures. Moreover, blocking the action of PTHrP significantly increased ALP activity in SZC+DZC co-culture. Our findings demonstrate the role of zonal chondrocyte interactions in regulating cell mineralization and provide a plausible mechanism for the post-natal regulation of articular cartilage matrix organization. These findings also have significant implications in understanding the pathology of articular cartilage as well as devising strategies for functional cartilage repair.

Enhancing tissue integration in cartilage repair procedures

Arguably, the gold standard of biological repair of articular cartilage lesions is autologous chondrocyte transplantation. Although the clinical outcomes appear to range between good and excellent in most cases, there are, nevertheless, both clinical and biological challenges that remain to improve rehabilitation and clinical outcome. One of the major biological problems relates to tissue integration of the reparative tissue into the host tissue at a predictable level. Often within a single lesion, varying degrees of integration can be observed from total integration through to non-integration as one passes through the defect. Here we briefly review some of the literature relating to this problem and include some of our own data drawn from questions we have posed about the biological nature of cartilage/cartilage integration. The nature and status of the tissue that comprises the wound lesion edge is central to tissue integration, and controlling aspects of trauma and free-radical-induced cell death together with matrix synthesis are identified as two components that require further investigation. Interestingly, there appears to be a limited ability of chondrocytes to be able to infiltrate existing cartilage matrices and even to occupy empty chondrocyte lacunae. Proliferation as a result of blunt and sharp trauma shows differential responses. As expected, blunt trauma induces a greater proliferative burst than sharp trauma and is more widespread from the lesion edge. However, in the case of sharp trauma, the basal cells enter proliferation before surface zone chondrocytes, which is not the case in blunt wounds. Regulation of these and associated processes will be necessary in order to devise strategies that can predict successful integration in biological repair procedures.

Localization of insulin-like growth factor-1 receptor in human normal and osteoarthritic cartilage in relation to proteoglycan synthesis and content.

Insulin-like growth factor-1 (IGF-1) plays a key role in the regulation of chondrocyte proteoglycan (PG) metabolism. We investigated whether chondrocyte PG synthetic activity correlates with the presence of chondrocyte IGF-1 receptor in the surface, middle and deeper zones of normal human articular cartilage and in cartilage known to display a shift in chondrocyte metabolism, i.e. cultured cartilage or osteoarthritic (OA) cartilage. Cartilage specimens were obtained post mortem from human knees within 18 h after death from donors without known clinical OA history. The samples were taken from macroscopically normal looking regions as well as from damaged regions with osteoarthritic appearance, yielding a range of OA grades from mild to moderate and severe OA. We examined chondrocyte PG synthesis by in situ autoradiography of incorporated [35S]sulphate and chondrocyte IGF-1 receptor localization by immunohistochemistry, followed by confocal laser scanning microscopical (CLSM) analysis in the same cartilage samples. In normal cartilage, both the amount of chondrocyte PG synthesis and the level of chondrocyte IGF-1 receptor localization are at low levels in the surface zone chondrocytes, but both are high in middle and deeper zone chondrocytes. Furthermore, after culture, the increase in chondrocyte PG synthesis in the surface layer coincides with increase in IGF-1 receptor expression. However, in mild OA particularly high levels of chondrocyte synthetic activity were found in the upper cartilage layer, whereas IGF-1 receptor expression was low in this layer, suggesting that factors other than IGF-1 are involved. High chondrocyte PG synthetic activity and chondrocyte IGF-1 receptor staining were found in the upper and deeper layers of moderate OA cartilage, whereas both low levels of chondrocyte activity as well as IGF-1 receptors were observed in cases of severe OA. Our data indicate that IGF-1 displays cellular heterogeneity in chondrocyte stimulation in the various cartilage zones in normal cartilage. Clear zonal correlation is lost in OA cartilage, and patterns of chondrocyte IGF-1 receptor expression and PG synthesis vary with the stage of OA.

The development and characterization of an in vitro system to study strain-induced cell deformation in isolated chondrocytes.

A model system has been developed to investigate cell deformation of chondrocytes in vitro. Chondrocytes were isolated from bovine articular cartilage by enzymatic digestion and seeded in agarose (type VII) at a final concentration of 2 x 10(6) cells.ml-1 in 3% agarose. Mechanical evaluation of the system showed no change in the tangent modulus of agarose/chondrocyte cultures over a 6-d culture period. The resulting agarose/chondrocyte cultures were subjected to compressive strains ranging from 5-20%. Cell shape was assessed by measuring the dimensions of the cell both perpendicular (x) and parallel (y) to the axis of compression and deformation indices (I = y/x) calculated. Cell deformation increased with the level of strain applied for freshly isolated chondrocytes. The cultures were maintained in medium that inhibits or stimulates matrix production (DMEM and DMEM + 20% FCS, respectively) in order to assess the effect of cartilaginous matrix on chondrocyte deformation. Matrix elaborated by the cells markedly influenced levels of cell deformation, an increase in matrix leading to a decrease in cell deformation. Freshly isolated deep zone chondrocytes were found to deform significantly more than surface zone chondrocytes, although this effect was lost after 6 d in culture. The elaborated matrix also altered the recovery characteristics of the chondrocytes following constant compressive strain of 15% for 24 h. Cells that had elaborated matrix took several hours to return to unloaded shape, while cells without matrix returned to the unloaded shape instantly.

Chondrocyte IGF-1 receptor expression and responsiveness to IGF-1 stimulation in mouse articular cartilage during various phases of experimentally induced arthritis.

To examine the distribution of insulin like growth factor-1 (IGF-1) receptors and the biological response to IGF-1 stimulation in articular cartilage of normal mouse knee joints and arthritic joints taken at various stages of experimentally induced arthritis. In situ IGF-1 receptor expression and responsiveness to IGF-1 stimulation were examined in murine articular cartilage at different phases in two models of experimentally induced arthritis. IGF-1 receptor expression was visualised in joint sections with the use of anti-IGF-1 receptor antibodies and quantified by confocal laser scanning microscopy. Chondrocyte proteoglycan (PG) synthesis was measured by incorporation of 35S-sulphate. In control cartilage, the majority of IGF-1 receptors were found on chondrocytes localised in the middle and deeper zones of the cartilage, whereas receptor expression in surface zone chondrocytes was very low. During culture of normal articular cartilage, IGF-1 was able to maintain chondrocyte PG synthesis at the in vivo level. Concurrently with the development of arthritis, cartilage lost its capacity to react to IGF-1, but IGF-1 stimulation recovered when the inflammatory response waned. Shortly after induction of arthritis, IGF-1 receptor expression initially declined, but it had returned to normal levels by day 1 and remained increased thereafter. The distribution of IGF-1 receptor expression in the different zones of normal articular cartilage reflects IGF-1 stimulation and metabolic activity of chondrocytes in these layers. This correlation is disturbed in arthritic cartilage, suggesting inadequate or overruled signalling.