Proteoglycan Retention Research Articles

Improved Cartilage Protection with Low Molecular Weight Hyaluronic Acid Hydrogel.

Traumatic joint injuries are common, leading to progressive tissue degeneration and the development of osteoarthritis. The post-traumatic joint experiences a pro-inflammatory milieu, initiating a subtle but deteriorative process in cartilage tissue. To prevent or even reverse this process, our group previously developed a tissue-penetrating methacrylated hyaluronic acid (MeHA) hydrogel system, crosslinked within cartilage to restore and/or protect the tissue. In the current study, we further optimized this approach by investigating the impact of biomaterial molecular weight (MW; 20, 75, 100 kDa) on its integration within and reinforcement of cartilage, as well as its ability to protect tissue degradation in a catabolic state. Indeed, the low MW MeHA integrated and reinforced cartilage tissue better than the high MW counterparts. Furthermore, in a 2 week IL-1β explant culture model, the 20 kDa MeHA demonstrated the most protection from biphasic mechanical loss, best retention of proteoglycans (Safranin O staining), and least aggrecan breakdown (NITEGE). Thus, the lower MW MeHA gels integrated better into the tissue and provided the greatest protection of the cartilage matrix. Future work will test this formulation in a preclinical model, with the goal of translating this therapeutic approach for cartilage preservation.

Characterization of the surface and interfacial properties of the lamina splendens

Joint disease affects approximately 52.5 million patients in the United States alone, costing 80.8 billion USD in direct healthcare costs. The development of treatment programs for joint disease and trauma requires accurate assessment of articular cartilage degradation. The articular cartilage is the interfacial tissue between articulating surfaces, such as bones, and acts as low-friction interfaces. Damage to the lamina splendens, which is the articular cartilage’s topmost layer, is an early indicator of joint degradation caused by injury or disease. By gaining comprehensive knowledge on the lamina splendens, particularly its structure and interfacial properties, researchers could enhance the accuracy of human and animal biomechanical models, as well as develop appropriate biomimetic materials for replacing damaged articular cartilage, thereby leading to rational treatment programs for joint disease and injury. Previous studies that utilize light, electron, and force microscopy techniques have found that the lamina splendens is composed of collagen fibers oriented parallel to the cartilage surface and encased in a proteoglycan matrix. Such orientation maximizes wear resistance and proteoglycan retention while promoting the passage of nutrients and synovial fluid. Although the structure of the lamina splendens has been explored in the literature, the low-friction interface of this tissue remains only partially characterized. Various functional models are currently available for the interface, such as pure boundary lubrication, thin films exuded under pressure, and sheets of trapped proteins. Recent studies suggest that each of these lubrication models has certain advantages over one another. Further research is needed to fully model the interface of this tissue. In this review, we summarize the methods for characterizing the lamina splendens and the results of each method. This paper aims to serve as a resource for existing studies to date and a roadmap of the investigations needed to gain further insight into the lamina splendens and the progression of joint disease.

Inner and outer annulus fibrosus cells exhibit differentiated phenotypes and yield changes in extracellular matrix protein composition in vitro on a polycarbonate urethane scaffold.

Since there is currently no optimal treatment for chronic neck or back pain that restores full spine functionality and disc height, recent research has focused on developing a regenerative medicine approach. This requires a better understanding of the phenotype nature of the different cell types present in disc tissues. In particular, there is very little known about the differentiated states of cells that co-exist within the annulus fibrosus (AF), despite the critical importance of this tissue in maintaining disc functionality. Maintenance of the differentiated states of these AF cells is imperative to the production of appropriate extracellular matrix (ECM) macromolecules and to the engineering of functional AF tissues. The objective of this study was to determine whether inner annulus fibrosus (IAF) cells when grown on polycarbonate urethane (PU) scaffolds in vitro will produce ECM molecules characteristic of IAF cells and different from outer annulus fibrosus (OAF) cells. OAF and IAF cells isolated from bovine coccygeal intervertebral disc were grown on nanofibrous PU for approximately 14 days. The effect of culture time on ECM gene expression, DNA content, and the synthesis and retention of proteoglycans and collagens were evaluated for both OAF and IAF cells. The ECM accumulated was also characterized by immunostaining and Western blot. The tensile strengths of the tissue/scaffolds were evaluated at 14 days. Both OAF and IAF cells both attached to PU and had similar DNA contents over time. IAF cells maintained relatively higher levels of COL2A1, ACAN and VCAN gene expression, and relatively lower levels of COL1A1 gene expression when compared with OAF cells, by 10 days of culture. IAF cells synthesized and retained similar amounts of total collagen and proteoglycans when compared with OAF cells. While both OAF and IAF cells accumulated type I collagen, only IAF cells accumulated type II collagen. Both cell types accumulated similar amounts of aggrecan but IAF cells accumulated higher amounts of versican as determined by immunostaining. In conclusion, nanofibrous PU scaffolds enabled the maintenance of most of the characteristic features of the IAF cell phenotype, and were different from those of OAF cells.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

Expansion in the presence of FGF-2 enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs

MSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. Fibroblast growth factor-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential. The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5 ng/ml) supplementation. Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents.MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after 21 days for FGF-2 expanded MSCs, but decreased between day 21 and 42. By day 42, FGF-2 expanded MSCs had still accumulated ∼1.4 fold higher sGAG and ∼1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ∼2.4 fold higher for FGF-2 expanded MSCs, with only ∼25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose cultures resulting in superior mechanical functionality.

The Effects of Exogenous Crosslinking on Hydration and Fluid Flow in the Intervertebral Disc Subjected to Compressive Creep Loading and Unloading

In vitro study of genipin crosslinking effect on disc water content changes under compressive loading and unloading. To investigate the influence of collagen crosslinking on hydration and fluid flow in different regions of intact discs, and to evaluate the nutritional implications. Age-related reductions of nutrient supply and waste product removal are critically important factors in disc pathogenesis. Diffusion and fluid flow are blocked by subchondral bone thickening, cartilaginous endplate calcification, loss of hydrophilic proteoglycans, and clogging of anular pores by degraded matrix molecules. Previous studies demonstrated increased hydraulic permeability and macromolecular transport through crosslinked collagenous matrices. Genipin has also demonstrated the capability to increase retention of proteoglycans. A total of 57 bovine lumbar motion segments were divided randomly into phosphate buffered saline and 0.33% genipin-soaked treatment groups. Water content changes were measured using a mass-loss technique in 3 intervertebral disc regions following successive stages of compressive loading and unloading (post-treatment, after 1 hour 750 N compression, and after a subsequent 24-hour period of nominal loading). Net flow of fluid into or out of a region was determined from the percentage change in mean water content from successive groups. Fluid flow to and from the nucleus doubled with genipin crosslinking. Relative to the buffer-only controls, overall net fluid flow increased 103% in the nucleus pulposus, 36% in the inner anulus, and was 31% less in the outer anulus of genipin treated discs. The effects of genipin crosslinking on matrix permeability and proteoglycan retention can alter hydration levels and fluid flow in the intervertebral disc. Resulting increases in fluid flow, including a doubling of flow to and from the nucleus, could lead to enhanced nutritional inflow and waste product outflow for the disc, and may have implications for emerging cell-based therapies.

Cartilage tissue enhances proteoglycan retention by nucleus pulposus cells in vitro

To investigate the effect of cartilage on nucleus pulposus (NP) tissue in an in vitro model. Cells were isolated from bovine NP or articular cartilage and allowed to form tissue in vitro. The NP tissue was grown either alone or in the presence of cartilage tissue (coculture) for up to 4 weeks and examined for histologic appearance, gene expression, and biochemical composition. For selected experiments, NP tissue was grown in coculture with fragments of cartilage end-plate. Coculture of in vitro-formed NP tissue with cartilage end-plate tissue resulted in a significant increase in proteoglycan content in the NP tissue by 2 weeks, compared with NP tissue grown alone. Substituting in vitro-formed cartilage tissue for cartilage end-plate also had a positive effect on the NP tissue, suggesting that it was an appropriate substitute for cartilage end-plate. Coculture of NP with in vitro-formed cartilage for 2 weeks increased aggrecan and collagen gene expression compared with that in NP tissue grown alone, and also reduced expression of matrix metalloproteinase 3 (MMP-3), MMP-13, and ADAMTS-5. NP cells from older and younger animals responded similarly to in vitro-formed cartilage. Expression of genes for tumor necrosis factor α (TNFα) and TACE in NP cells was higher when grown in the absence of cartilage. This corresponded with increased TNFα protein levels in the absence of cartilage. The data suggest that chondrocytes may secrete a factor(s) that positively enhances tissue growth, perhaps by inhibiting TNFα production. This could be a potential mechanism explaining how loss of the cartilage end-plate may contribute to the development of NP degenerative changes.

Genipin-Based Tissue Modification for Degenerative Disc Disease

Testing completed over the previous decade indicates that chemical tissue modification can address the core, underlying problems related to chronic back pain and disability --nutritional deficiency, loss of hydration, and mechanical insufficiency of the disc --while also addressing the cause of pain directly by reducing joint instability and disc bulge under load. These tests have shown that a genipin based collagen crosslinking reagent can be expected to immediately increase hydraulic permeability and macromolecular transport (nutrition), increase retention of proteoglycans (hydration), increase tissue strength, stiffness, fatigue resistance and tear resistance (mechanical insufficiency), increase joint stability and decrease disc bulge under load (pain).

The influence of exogenous cross-linking and compressive creep loading on intradiscal pressure

This study involves a biomechanical evaluation of a prospective injectable treatment for degenerative discs. The high osmolarity of the non-degenerated nucleus pulposus attracts water contributing to the hydrostatic behavior of the tissue. This intradiscal pressure is known to drop as fluid is exuded from the matrix due to compressive loading. The objective of this study was to compare the changes in intradiscal pressure in control and genipin cross-linked intervertebral discs. Thirty bovine lumbar motion segments were randomly divided into a phosphate-buffered saline control group and a 0.33% genipin group and soaked at room temperature for 2 days. A needle pressure sensor was held in the center of the disc while short-term and static creep compressive loads were applied. The control group demonstrated a 25% higher average intradiscal pressure compared to genipin-treated discs under 750 N compressive load (p=0.029). Depressurization during static compressive creep was 56% higher in the control than in the genipin group (p=0.014). These results suggest cross-linking induced changes in the poroelastic properties of the involved tissues affected the mechanics of compressive load support in the disc with lower levels of nucleus pressure, a corresponding decrease in the elastic expansion of the annulus, and an increased axial compressive loading of the inner and outer annulus tissues. It is possible that concurrent changes in hydraulic permeability and proteoglycan retention known to be associated with genipin cross-linking were also contributors to poroelastic changes. Reduction of peak pressures and moderation of pressure fluctuations could be beneficial relative to discogenic pain.

Development of anin vitromodel to test the efficacy of novel therapies for IVD degeneration

Low back pain (LBP) is a major cause of disability worldwide that has been linked to intervertebral disc (IVD) degeneration. An improved understanding of the pathogenesis of disc degeneration is now developing, which is leading to the development of a number of possible future therapies targeted at the underlying pathology and regeneration strategies. Although results thus far are promising, the investigation of such therapies in an environment that mimics the mechanical environment of the human disc in vivo is problematic. The development of an in vitro model system that can maintain metabolically active IVD tissue within a loading environment pertaining to that of the human spine is crucial for testing the efficacy of future cell-based and tissue-engineering therapies for IVD degeneration. Here, using our novel loading rig, capable of mimicking the loading environment experienced within the human spine, we have cultured nucleus pulposus tissue explants, applied a daily hydrostatic loading regime for up to 2 weeks and investigated proteoglycan retention, metabolic activity and cellular phenotype. IVD tissue cultured under a loading environment pertaining to the in vivo loading environment maintained metabolic cell activity, proteoglycan content and cellular phenotype. Indeed, all parameters were improved in IVD tissue cultured with load compared to unloaded controls. Such a model is invaluable for investigations assessing the feasibility and efficacy of future therapeutic approaches to inhibiting degeneration or stimulating regeneration of the IVD, where the in vivo loading environment may be crucial to their success or failure.

The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix.

The use of a cell-seeded biomatrix for tissue engineering of the intervertebral disc. To evaluate the ability of a biomatrix to support the viability of intervertebral disc cells and to accumulate the extracellular matrix that they produce. Intervertebral disc degeneration is a common occurrence during adult life that has adverse economic consequences on the health care system. Current surgical treatments are aimed at removing or replacing the degenerate tissue, which can alter the biomechanics of the spine and result in degeneration at adjacent disc levels. The ideal treatment of the degenerate disc would involve biologic repair, and tissue-engineering techniques offer a means to achieve this goal. Scaffolds of type I collagen and hyaluronan were seeded with bovine nucleus pulposus or anulus fibrosus cells and maintained in culture for up to 60 days in the presence of fetal calf serum or a variety of growth factors to try to generate a tissue whose properties could mimic those of the nucleus pulposus with respect to proteoglycan content. During the culture period, various proteoglycans (aggrecan, decorin, biglycan, fibromodulin, and lumican) and collagens (types I and II) accumulated in the scaffold. Proteoglycan accumulation in the scaffold was greatest under conditions in which transforming growth factor-beta1 was present, but under all conditions, more proteoglycan was lost into the culture medium than retained in the scaffold. Both the nucleus and anulus cells behaved in a similar manner with respect to their ability to synthesize matrix macromolecules and have them retained in the scaffold. By day 60 of culture, the proteoglycan content of the scaffolds never exceeded 10% of that present in the mature nucleus pulposus, although this figure could have been considerably increased if most of the proteoglycan being synthesized could have been retained. Furthermore, proteoglycan retention was not uniform within the scaffold, but increased near its periphery. This work demonstrates that although it is possible to maintain functional disc cells in a biomatrix, it will be necessary to optimize proteoglycan synthesis and retention if any resulting tissue is to be of value in the biologic repair of the degenerate disc. The ability of the anulus cells to replicate the matrix production of the nucleus cells, at least in the collagen/hyaluronan scaffold, suggests that repair may not be limited to the availability of authentic nucleus cells.

Latrunculin and cytochalasin decrease chondrocyte matrix retention.

The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure.

Antisense Inhibition of Hyaluronan Synthase-2 in Human Articular Chondrocytes Inhibits Proteoglycan Retention and Matrix Assembly

In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes.

Retention of hyaluronic acid in alginate beads: aspects for in vitro cartilage engineering.

Alginate has been used successfully for three-dimensional chondrocyte cultures and may be important for cartilage transplant formation. However, alginate is not a natural component of the cartilage matrix. The aim of this study was (a) to supplement alginate with the extracellular matrix component hyaluronic acid; and (b) to analyze the hyaluronic acid retention in different alginate gels. Hyaluronan is assumed to improve proteoglycan retention and may be important for in vitro matrix formation, tissue turgor, and biomechanical quality. Alginate and hyaluronan were mixed with chondrocytes and polymerized as were alginate, hyaluronan, and fibrinogen. [3H]hyaluronan was used to quantitate the leakage of hyaluronan from the gel beads. After 28 days in culture, 1.2% alginate beads supplemented with 0.26% hyaluronan contained only 9% of the initial amount of hyaluronan whereas 2.4% alginate beads still contained about 55% of the initial 0.22% hyaluronan. Release of hyaluronan from the beads was significantly lower if the beads additionally contained fibrin. Alginate beads supplemented with hyaluronan or fibrin showed increased chondrocyte proliferation compared to controls. Supplemented hyaluronan greatly diffuses out of alginate gels of lower densities. It must be assumed also that most of the hyaluronan newly synthesized by chondrocytes in these cells diffuses into the surrounding culture medium. The in vitro development of a sufficiently hygroscopic cartilage ground substance therefore may be very limited. Sufficient hyaluronic acid retention can be achieved in alginate gels with concentrations above 1.2% or by addition of fibrin.

Matrix accumulation and retention in embryonic cartilage and in vitro chondrogenesis.

Since hyaluronan anchors the proteoglycan-rich pericellular matrix to chondrocytes, hyaluronan-cell interactions may direct cartilage matrix assembly. To test this hypothesis, the competitive binding of hyaluronan hexasaccharides for native hyaluronan during matrix assembly, accumulation and retention in embryonic cartilage was studied. Chondrocytes released from explants with collagenase P retained pericellular matrices, but chondrocytes appeared "matrix-free" when released from hexasaccharide-treated explants. Decreased safranin O staining was also observed in the hexasaccharide-treated explants. This loss of proteoglycan retention was demonstrated quantitatively in the cartilage extracts and recovered in the media. The continual presence of hexasaccharides in micromass cultures resulted in decreased proteoglycan deposition. Increased proteoglycan retention, indicative of matrix repair, occurred following hexasaccharide wash-out. Thus, native hyaluronan-chondrocyte interactions are important for the assembly and maintenance of cartilage matrix.

Characterization of proteoglycan accumulation during formation of cartilagenous tissue in vitro

In order to study proteoglycan retention and accumulation, we optimized a chondrocyte cell culture system in which isolated bovine articular chondrocytes accumulate extracellular matrix to form a continuous layer of cartilagenous tissue. The tissue can attain a thickness of up to 110 microns by 35 days. The cells synthesize large keratan sulfate containing proteoglycans and type II collagen indicating that the chondrocytes maintain their phenotype in these culture conditions. Matrix accumulation is enhanced by increased cell density and the presence of serum and ascorbic acid. The amount of proteoglycans synthesized by the chondrocytes increases up to day 21 and then decreases to the same levels as are synthesized during the first week of culture. The percentage of newly synthesized proteoglycans retained in the matrix increases from 20% on day 6 to a maximum of 85% by day 35. The proteoglycan and collagen content in the tissue increases with time in culture. The changes in the percentage of proteoglycans retained parallels the increase in proteoglycan content. After day 35, there is no further increase in the amount of proteoglycans and collagen nor in the percentage of newly synthesized proteoglycans retained in the extracellular matrix. These studies demonstrate that the cultures are going through two phases: one of matrix accumulation and then one of maintaining the existing matrix. The period of matrix accumulation occurs between days 10-21 whereas matrix maintenance is observed after day 35. Using this culture system to study proteoglycan accumulation and maintenance during these culture periods may prove useful in identifying the mechanisms regulating these processes.

Proteoglycan depletion of intact articular cartilage by retinoic acid is irreversible and involves loss of hyaluronate

Intact sesamoid bones from bovine metacarpophalangeal joints were cultured with retinoic acid for 9 days and allowed to recover in control medium for up to 17 days. Retinoic acid (300 ng/ml) induced 91.8% inhibition of glycosaminoglycan (GAG) synthesis and 50.6% loss of sulfated GAGs from the cartilage. Retinoic acid also induced 38.2% loss of hyaluronate from the matrix. The synthesis and content of the large aggregating proteoglycan (aggrecan) were preferentially decreased compared with that of the small nonaggregating species. The aggrecan synthesized was similar to control aggrecan in size, aggregation capacity, and composition of its GAGs. GAG synthesis was almost completely restored in control medium within the next 6 days. The GAGs synthesized during recovery were slightly shorter than control GAGs and showed a higher ratio of chondroitin-6-sulfate over chondroitin-4-sulfate. Neither the proteoglycan content nor the hyaluronate content recovered within 17 days. The aggregation capacity of newly synthesized aggrecan was normal. However, the retention of proteoglycans synthesized in the recovery period was much lower in treated cartilage than in control cartilage (T1/2 of 17 and 38 days, respectively). In conclusion, the retinoic-acid-induced proteoglycan depletion was irreversible in spite of the restored synthesis of aggrecan with a normal aggregation capacity. The reduced retention of newly synthesized aggrecan during recovery might be caused by a lack of hyaluronate. This model seems suitable to study aspects of cartilage destruction and repair.

Enhanced sulfated‐proteoglycan core protein synthesis by incubation of rabbit chondrocytes with recombinant transforming growth factor‐β1

Rabbit articular chondrocytes were incubated with recombinant transforming-growth-factor-beta 1 (rhTGF-beta 1) and its effect on newly synthesized proteoglycan measured. rhTGF-beta 1 stimulated proteoglycan synthesis at a concentration as low as 5 ng/ml without further increases in radiosulfate incorporation up to 50 ng/ml. The quantitative increase in radiosulfate incorporation in rh-TGF-beta 1-treated chondrocytes was greater in the cell-associated culture compartment than in the medium compartment. rhTGF-beta 1 promoted an increased proteoglycan retention in the cell-associated compartment as evidenced by an increase in the t1/2 of retention from 8 h to 11 h. Specific enhanced synthesis of [35S]-methionine-labeled core proteins was seen in rh-TGF-beta 1-treated chondrocytes. rh-TGF-beta 1 increased the synthesis of the 2 core proteins derived from hydrodynamically large proteoglycans. They possessed apparent molecular weights of greater than 480 kD and 390 kD after 3-5% acrylamide gel electrophoresis. A compartmental analysis revealed that the cell-associated culture compartment contained only the larger of the 2 core proteins derived from large proteoglycans. Two other core proteins with apparent molecular weights 52 kD and 46 kD were also stimulated by rhTGF-beta 1. These results indicated that TGF-beta probably plays a significant role in stimulating proteoglycan core protein synthesis in articular chondrocytes and therefore may be an important growth factor in the restoration of cartilage extracellular matrix after injury.

Chondrons From Articular Cartilage (II): Analysis of the Glycosaminoglycans in the Cellular Microenvironment of Isolated Canine Chondrons

A chondron rich preparation was isolated from mature canine tibial cartilage using low-speed homogenization techniques. Proteoglycans were extracted from this preparation by exhaustive treatment with 4M guanidine-HCl. A significant proportion of the total proteoglycan, measured as uronic acid, was resistant to extraction and represented 27.9% in intact cartilage chips and 18.6% in the chondron fraction. Histochemical examination of chondrons confirmed that extraction resistant proteoglycans remained within the capsule of the chondron after 4M guanidine-HCl treatment. Electrophoretic analysis of the glycosaminoglycans extracted from intact cartilage chips and the chondron fraction showed approximately equivalent amounts of chondroitin sulphate (79.3%), keratan sulphate (16.3%) and hyaluronic acid (4.3%) present. In contrast, the extraction resistant residue in the chondron fraction was significantly enriched for hyaluronic acid (10.5%, p less than 0.05) but was depleted of chondroitin sulphate (70.9%, p less than 0.05). The major chondroitin sulphate isomer in the resistant fraction was chondroitin 6-sulphate while in the soluble fraction, the quantities of the two isomers were approximately equivalent. Comparison with previously published data suggests a role for minor collagens in the retention of proteoglycans in the cellular microenvironment.