Superficial Layer Of Articular Cartilage Research Articles

Inhibition of fibroblast activation protein ameliorates cartilage matrix degradation and osteoarthritis progression

Fibroblast activation protein (Fap) is a serine protease that degrades denatured type I collagen, α2-antiplasmin and FGF21. Fap is highly expressed in bone marrow stromal cells and functions as an osteogenic suppressor and can be inhibited by the bone growth factor Osteolectin (Oln). Fap is also expressed in synovial fibroblasts and positively correlated with the severity of rheumatoid arthritis (RA). However, whether Fap plays a critical role in osteoarthritis (OA) remains poorly understood. Here, we found that Fap is significantly elevated in osteoarthritic synovium, while the genetic deletion or pharmacological inhibition of Fap significantly ameliorated posttraumatic OA in mice. Mechanistically, we found that Fap degrades denatured type II collagen (Col II) and Mmp13-cleaved native Col II. Intra-articular injection of rFap significantly accelerated Col II degradation and OA progression. In contrast, Oln is expressed in the superficial layer of articular cartilage and is significantly downregulated in OA. Genetic deletion of Oln significantly exacerbated OA progression, which was partially rescued by Fap deletion or inhibition. Intra-articular injection of rOln significantly ameliorated OA progression. Taken together, these findings identify Fap as a critical pathogenic factor in OA that could be targeted by both synthetic and endogenous inhibitors to ameliorate articular cartilage degradation.

The effect of sucrose supplementation on chondrocyte viability in porcine articular cartilage following vitrification

Vitrification can extend the banking life of articular cartilage (AC) and improve osteochondral transplantation success. Current vitrification protocols require optimization to enable them to be implemented in clinical practice. Sucrose as a non-permeating cryoprotective agent (CPA) and clinical grade chondroitin sulfate (CS) and ascorbic acid (AA) as antioxidants were investigated for their ability to improve a current vitrification protocol for AC. The aim of this study was to assess the impact of sucrose and CS/AA supplementation on post-warming chondrocyte viability in vitrified AC. Porcine osteochondral dowels were randomly vitrified and warmed with one established protocol (Protocol 1) and seven modified protocols (Protocols 2–8) followed by chondrocyte viability assessment. Sucrose supplementation in both vitrification and warming media (Protocol 4) resulted in significantly higher (p = 0.018) post-warming chondrocyte viability compared to the protocol without sucrose (Protocol 1). There was no significant difference (p = 0.298) in terms of post-warming chondrocyte viability between sucrose-supplemented DMEM + CS solution (Protocol 4) and Unisol-CV (UCV) + CS (Protocol 6) solution. Clinical grade CS and AA contributed to similar post-warming chondrocyte viability to previous studies using research grade CS and AA, indicating their suitability for clinical use. The addition of an initial step (step 0) to reduce the initial concentration of CPAs to minimize osmotic effects did not enhance chondrocyte viability in the superficial layer of AC. In conclusion, sucrose-supplemented DMEM + clinical grade CS (Protocol 4) could be an ideal protocol to be investigated for future use in clinical applications involving vitrified AC.

Targeting cartilage epidermal growth factor receptor signaling pathway for osteoarthritis treatment

Purpose: Osteoarthritis (OA) is a widespread chronic joint disease characterized by cartilage degeneration. We previously discovered that EGFR signaling is critical for maintaining the superficial layer of articular cartilage and found that mice with cartilage-specific (Col2-Cre) EGFR deficiency develop spontaneous OA. Here, we designed a two-pronged approach to investigate the effects of positively targeting the EGFR pathway on articular cartilage. First, we genetically enhanced EGFR activity by adopting a Rosa-DTR model.

Divergent effects of distinct perivascular cell subsets for intra-articular cell therapy in posttraumatic osteoarthritis.

Intra-articular injection of mesenchymal stem cells has shown benefit for the treatment of osteoarthritis (OA). However, mesenchymal stem/stromal cells at the origin of these clinical results are heterogenous cell populations with limited cellular characterization. Here, two transgenic reporter mice were used to examine the differential effects of two precisely defined perivascular cell populations (Pdgfrα+ and Pdgfrβ+ cells) from white adipose tissue for alleviation of OA. Perivascular mesenchymal cells were isolated from transgenic Pdgfrα-and Pdgfrβ-CreERT2 reporter animals and delivered as a one-time intra-articular dose to C57BL/6J mice after destabilization of the medial meniscus (DMM). Both Pdgfrα+ and Pdgfrβ+ cell preparations improved metrics of cartilage degradation and reduced markers of chondrocyte hypertrophy. While some similarities in cell distribution were identified within the synovial and perivascular spaces, injected Pdgfrα+ cells remained in the superficial layers of articular cartilage, while Pdgfrβ+ cells were more widely dispersed. Pdgfrβ+ cell therapy prevented subchondral sclerosis induced by DMM, while Pdgfrα+ cell therapy had no effect. In summary, while both cell therapies showed beneficial effects in the DMM model, important differences in cell incorporation, persistence, and subchondral sclerosis were identified.

Application of confocal, SHG and atomic force microscopy for characterizing the structure of the most superficial layer of articular cartilage.

The surface of articular cartilage plays a crucial role in attenuating and transmitting mechanical loads in synovial joints to facilitate painless locomotion. Disruption to the surface of articular cartilage causes changes to its frictional properties instigating the deterioration of the tissue. In this study, we physically peeled the most superficial layer, a transparent membrane of 20.0 ± 4.7 µm thick, from the central loading region of femoral condyles of sheep. The ultrastructure of this layer without interference from the underlying cartilage was independently investigated using confocal, second harmonic generation and atomic force microscopy. We found that the most superficial layer contains chondrocytes, densely packed collagen, coarse elastic fibres and a fine elastic network. The elastic fibres are most prevalent at the surface of the layer, where collagen and chondrocyte densities are lowest. At the interface of this most superficial layer with the underlying bulk cartilage, a dense fibrillar network exists, formed mainly by collagen fibrils and elastin microfibrils. By contrast, the interface of the underlying cartilage with the most superficial layer contains collagen fibrils, fine microfibrils and microfibrils distinctively laced on one side. The findings of this study will play an important role in understanding the mechanical function and wear resistance of articular cartilage, and in developing more promising tissue engineering techniques to treat cartilage defects and osteoarthritis. LAY DESCRIPTION: The chronic pain and dysfuction in synovial joints caused by osteoarthritis can have a debilitating impact on daily activities for sufferers. Osteoarthritis is characterised by the deterioration of the articular cartilage. Despite intensive research, the wear mechanism of articular cartilage and the progression of osteoarthritis remain unclear in the literature. Articular cartilage is a resilient tissue that provides a low friction surface to facilitate painless locomotion. The surface of articular cartilage plays a crucial role in attenuating and transmitting mechanical loads. Disruption at the surface of articular cartilage causes changes to its frictional properties, instigating the deterioration of the tissue. Despite this, the definition of the most superficial layer of articular cartilage, as well as its composition and microstructure, have endured a long history of debate, clouding our understanding of the early progression of osteoarthritis. In order to investigate the surface of articular cartilage independently from the underlying cartilage, we physically peeled a transparent membrane of 20.0± 4.7µm thickness, the most superficial layer, from the central loading region of the femoral condylesofsheep.Using confocal, second harmonic generation and atomic force microscopy, we found that the most superficial layer contains cartilage cells (chondrocytes), densely packed collagen, coarse elastic fibres and a fine elastic network. The coarse elastic fibres are most prevalentatthe surface of the layer where collagen and chondrocyte densities are lowest. Furthermore, we investigated the surfaces at the interface of the most superficial layer with the underlying articular cartilage. At the interface of this most superficial layerwith the underlying bulk cartilage, a dense fibrillar network exists, formed mainly by collagen fibrils and elastin microfibrils. In contrast, the interface of the underlying cartilage with the most superficial layer contains collagen fibrils, finemicrofibrilsand microfibrils distinctively laced on one side. The findings of this study have confirmed that there is a most superficial layer that is able to be removed using a tangential force. Through the application of advanced imaging technologies, we have shown that this most superficial layer is cellular and have detailed its composition and ultrastructure. Due to the close association between the form and function of tissues, the findings of this study will play an important role in understanding the mechanical function and wear mechanism of articular cartilage. This may lead to the development of more promising tissue engineering techniques to treat cartilage defects and osteoarthritis.

Effect of the anisotropic permeability in the frequency dependent properties of the superficial layer of articular cartilage

Articular cartilage is a tissue of fundamental importance for the mechanics of joints, since it provides a smooth and lubricated surface for the proper transfer of loads. From a mechanical point of view, this tissue is an anisotropic poroviscoelastic material: its characteristics at the macroscopic level depend on the complex microscopic architecture. With the ability to probe the local microscopic features, dynamic nanoindentation test is a powerful tool to investigate cartilage mechanics. In this work we focus on a length scale where the time dependent behaviour is regulated by poroelasticity more than viscoelasticity and we aim to understand the effect of the anisotropic permeability on the mechanics of the superficial layer of the articular cartilage. In a previous work, a finite element model for the dynamic nanoindentation test has been presented. In this work, we improve the model by considering the presence of an anisotropic permeability tensor that depends on the collagen fibers distribution. Our sensitivity analysis highlights that the permeability decreases with increasing indentation, thus making the tissue stiffer than the case of isotropic permeability, when solicited at the same frequency. With this improved model, a revised identification of the mechanical and physical parameters for articular cartilage is provided. To this purpose the model was used to simulate experimental data from tests performed on bovine tissue, giving a better estimation of the anisotropy in the elastic properties. A relation between the identified macroscopic anisotropic permeability properties and the microscopic rearrangement of the fiber/matrix structure during indentation is also provided.

Joint Degradation in a Monkey Model of Collagen-Induced Arthritis: Role of Cathepsin K Based on Biochemical Markers and Histological Evaluation.

The role of cathepsin K in joint degradation in a model of collagen-induced arthritis (CIA) in cynomolgus monkey was examined using biochemical markers and histology. Joint swelling, urinary C-telopeptide of type II collagen (CTX-II), deoxypyridinoline (DPD), and N- and C-telopeptides of type I collagen (NTX and CTX-I, resp.) were analyzed. Immunohistochemistry of type II collagen, cathepsin K, and CTX-II were performed using joints. Joint swelling reached peak on day 42 and continued at this level. The CTX-II level peaked on day 28 and declined thereafter, while CTX-I, NTX, and DPD reached plateau on day 43. Joint swelling was positively correlated with CTX-II increases on days 20 and 42/43, with increases in CTX-I and NTX/Cr on days 42/43 and 84, and with DPD increases throughout the study period. Intense cathepsin K staining was observed in osteoclasts and in articular cartilage and synovial tissue in arthritic joints. CTX-II was present in the superficial layer of articular cartilage in CIA monkeys. Evidence from biochemical markers suggests that matrix degradation in the CIA model starts with degradation of cartilage, rather than bone resorption. Cathepsin K expressed in osteoclasts, articular cartilage, and synovial tissue may contribute to degradation of cartilage.

Characterisation of lubricin in synovial fluid from horses with osteoarthritis.

The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. In vitro study. Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral fragments, were analysed for the lubricin glycosylation profiles. Articular cartilage explants were stimulated with or without interleukin-1β for 25 days. Media samples collected at 3-day intervals were analysed by quantitative proteomics, western blot and enzyme-linked immunosorbent assay. O-glycosylation profiles in synovial fluid revealed both Core 1 and 2 O-glycans, with Core 1 O-glycans predominating. Synovial fluid from normal joints (49.5 ± 1.9%) contained significantly lower amounts of monosialylated Core 1 O-glycans compared with joints with osteoarthritis (53.8 ± 7.8%, P = 0.03) or joints with osteochondral fragments (57.3 ± 8.8%, P = 0.001). Additionally, synovial fluid from normal joints (26.7 ± 6.7%) showed higher amounts of disialylated Core 1 O-glycan than from joints with osteochondral fragments (21.2 ± 4.9%, P = 0.03). A C-terminal proteolytic cleavage site in lubricin was found in synovial fluid from normal and osteochondral fragment joints and in media from interleukin-1β stimulated and unstimulated articular cartilage explants. This is the first demonstration of a change in the glycosylation profile of lubricin in synovial fluid from diseased equine joints compared with that from normal joints. We demonstrate an identical proteolytic cleavage site of lubricin both in vitro and in vivo. The reduced sialation of lubricin in synovial fluid from diseased joints may affect the boundary lubricating ability of the superficial layer of articular cartilage and could be one of the early events in the progression of osteoarthritis.

Measuring microscale strain fields in articular cartilage during rapid impact reveals thresholds for chondrocyte death and a protective role for the superficial layer

Articular cartilage is a heterogeneous soft tissue that dissipates and distributes loads in mammalian joints. Though robust, cartilage is susceptible to damage from loading at high rates or magnitudes. Such injurious loads have been implicated in degenerative changes, including chronic osteoarthritis (OA), which remains a leading cause of disability in developed nations. Despite decades of research, mechanisms of OA initiation after trauma remain poorly understood. Indeed, although bulk cartilage mechanics are measurable during impact, current techniques cannot access microscale mechanics at those rapid time scales. We aimed to address this knowledge gap by imaging the microscale mechanics and corresponding acute biological changes of cartilage in response to rapid loading. In this study, we utilized fast-camera and confocal microscopy to achieve roughly 85µm spatial resolution of both the cartilage deformation during a rapid (~3ms), localized impact and the chondrocyte death following impact. Our results showed that, at these high rates, strain and chondrocyte death were highly correlated (p<0.001) with a threshold of 8% microscale strain norm before any cell death occurred. Additionally, chondrocyte death had developed by two hours after impact, suggesting a time frame for clinical therapeutics. Moreover, when the superficial layer was removed, strain – and subsequently chondrocyte death – penetrated deeper into the samples (p<0.001), suggesting a protective role for the superficial layer of articular cartilage. Combined, these results provide insight regarding the detailed biomechanics that drive early chondrocyte damage after trauma and emphasize the importance of understanding cartilage and its mechanics on the microscale.

Human Developmental Chondrogenesis as a Basis for Engineering Chondrocytes from Pluripotent Stem Cells

SummaryJoint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5–6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166low/negBMPR1B+ putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering.

Rotated Hough filtering for automatically distinguishing the collagen bundles in the most superficial layer of articular cartilage.

The structure of the collagen matrix in the most superficial layer of articular cartilage (AC) is particularly critical to the wear and tensile resistance of AC. Disruption of the collagen network leads to rapid wear of the articular surface, which is a major contributory factor of osteoarthritis. Interwoven fiber bundles have been observed in the most superficial layer of healthy AC under confocal microscopy, but gradually disappear with age and pathological change of AC. The image analysis techniques developed in this paper aimed to provide a quantitative description of the relationship between the presence of the fiber bundles in the lamina splendens and health status of AC. The result of this quantitative study confirmed the existence of fiber bundles in healthy AC, and the accuracy of the identified fiber bundles was up to 90%. With the development of confocal arthroscopy for imaging microstructure of AC without biopsy, the image analysis technique can aid to efficiently assess the physiological status of AC for orthopedic clinics.

Degenerative changes in knee joints as determined by OARSI rat scoring system and osteoarthritis-related joint pain in surgically and chemically induced rat models of osteoarthritis

OARSI rat scoring system demonstrated degenerative changes in all rat OA models. Rat MIA model exhibited the loss of chondrocytes, proteoglycans (PG) and collagen matrix in superficial layer of articular cartilage at 2 weeks and in intermediate layer at 4 weeks. Rat ACLT model demonstrated the chondrocyte, PG and collagen matrix loss in superficial layer at 5 weeks. This exacerbated down to tidemark at 10 weeks and was associated with osteophytes and minimal synovial inflammation. Rat MMT + MCLT model exhibited the chondrocyte, PG and collagen matrix loss from superficial layer down to tidemark at 3 and 6 weeks in association with large osteophytes and mild synovial inflammation. Rat ACLT + pMMx model demonstrated the loss of chondrocytes, PG and collagen matrix down to tidemark at 4 and 8 weeks together with large osteophytes and mild synovial inflammation. Static weight bearing decreased in operated knee joints during the first week. Paw withdrawal threshold decreased in operated and MIA-injected knee joints during the first week and at the end of the study.

Trabecular bone structure and spatial differences in articular cartilage MR relaxation times in individuals with posterior horn medial meniscal tears

To analyze knee trabecular bone structure and spatial cartilage T(1ρ) and T(2) relaxation times using 3-T magnetic resonance imaging (MRI) in subjects with and without tears of posterior horn of the medial meniscus (PHMM). 3-T MRI from 59 subjects (>18 years), were used to evaluate PHMM tears based on modified Whole-Organ Magnetic Resonance Imaging Score (WORMS) scoring; and to calculate apparent trabecular bone-volume over total bone volume fraction (app. BV/TV), apparent trabecular number (app. Tb.N), apparent trabecular separation (app. Tb.Sp) and apparent trabecular thickness (app. Tb.Th) for overall femur/tibia and medial/lateral femur/tibia; and relaxation times for deep and superficial layers of articular cartilage. A repeated measures analysis using Generalized Estimating Equation (GEE) was performed to compare trabecular bone and cartilage relaxation time parameters between people with (n = 35) and without (n = 24) PHMM tears, while adjusting for age and knee OA presence. Subjects with PHMM tears had lower app. BV/TV and app. Tb.N, and greater app. Tb.Th, and app. Tb.Sp. They also had higher T(1ρ) times in the deep cartilage layer for lateral tibia and medial femur and higher T(2) relaxation times for the deep cartilage layer across all compartments. PHMM tears are associated with differences in underlying trabecular bone and deep layer of cartilage. Over-load of subchondral bone can lead to its sclerosis and stress shielding of trabecular bone leading to the resorptive changes observed in this study. The results underline the importance of interactions of trabecular bone and cartilage in the pathogenesis of knee OA in people with PHMM tears.

Bupivacaine and Triamcinolone May Be Toxic To Human Chondrocytes: A Pilot Study

Intraarticular injections of corticosteroids combined with local anesthetics are commonly used for management of chronic pain symptoms associated with degenerative joint diseases and after arthroscopic procedures. Several studies suggest chondrotoxicity of local anesthetics whereas others report chondroprotective and cytotoxic effects of corticosteroids on cartilage. Given the frequency of use of these agents, it is important to know whether they are in fact toxic. We asked whether (1) bupivacaine and triamcinolone acetonide, alone and combined, were chondrotoxic to chondrocytes in culture; (2) buffering of the reagents diminished toxicity of the bupivacaine and triamcinolone; and (3) the presence of the superficial layer of articular cartilage protects against toxicity. We obtained cartilage from three patients undergoing arthroplasty. To address triamcinolone acetonide, bupivacaine, and combinatorial toxicity to human chondrocytes, we set up monolayer chondrocyte cultures (n = 8 wells per condition). The question of buffering was addressed by performing the same assays as above, but the reagents were buffered. An MTT assay was used to assess chondrocyte survival in the monolayer. We harvested 21 articular plugs from each of three patients (total 63 plugs) and exposed them to the same reagents as above, including the buffered reagents. A Live/Dead assay was used to determine chondrocyte survival. Triamcinolone acetonide, bupivacaine, and their combination were toxic to human chondrocytes in the monolayer comparisons. The addition of buffering did not mitigate chondrocyte death. With the intact superficial layer in the plug group, bupivacaine was not toxic as compared with for the control group; all the other reagents (triamcinolone, combination bupivacaine/triamcinolone, buffered bupivacaine, buffered triamcinolone, and buffered combination) produced chondrotoxicity. Triamcinolone induced chondrotoxicity in the articular plug and monolayer culture, whereas bupivacaine induced chondrotoxicity only in monolayer culture. The combined used of triamcinolone and bupivacaine did not show additive chondrocyte death in any arm. Buffering of bupivacaine increased its chondrotoxicity. Although not necessarily reflecting in vivo conditions, our data suggest physicians should be cognizant of the potential in vitro chondrotoxicity of bupivacaine and triamcinolone when contemplating intraarticular administration.

Expression of collagen type I, II, X and Ki-67 in osteochondroma compared to human growth plate cartilage.

In order to characterize the consequences for the process of endochondral ossification we performed an immunohistochemical study and compared the expression of collagen type I, II and X as markers of cartilage differentiation and Ki-67 as a marker of cell proliferation in solitary (7-26 years, n=9) and multiple (11-42 years, n=6) osteochondromas with their expression in human fetal and postnatal growth plates. In fetal and young postnatal controls, we found a thin superficial layer of articular cartilage that stained positive for collagen type I while collagen II was expressed in the rest of the cartilage and collagen type X was restricted to the hypertrophic zone. Osteochondromas from children showed lobular collagen type II-positive areas surrounded by collagen type I. In adults, the separation of collagen type I- and type II-positive areas was more blurred, or the cartilaginous cap was missing. Collagen type X was detected in a pericellular distribution pattern within hypertrophic zones but also deeper between bone trabecula. The proliferative activity of osteochondromas from children younger than 14 years of age was comparable to postnatal growth plates, whereas in cartilage from individuals older than 14 years of age, we could not detect significant proliferative activity.

Regulation of the friction coefficient of articular cartilage by TGF‐β1 and IL‐1β

Articular cartilage functions to provide a low-friction surface for joint movement for many decades of life. Superficial zone protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary lubrication. In both cell and explant cultures, TGF-beta1 and IL-1beta have been demonstrated to, respectively, upregulate and downregulate SZP protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF-beta1 or IL-1beta) and a smooth glass surface due to sliding in the boundary lubrication regime was measured with a pin-on-disk tribometer. SZP was quantified using an enzyme-linked immunosorbant assay and localized by immunohistochemistry. Both TGF-beta1 and IL-1beta treatments resulted in the decrease of the friction coefficient of articular cartilage in a location- and time-dependent manner. Changes in the friction coefficient due to the TGF-beta1 treatment corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL-1beta treatment were independent of SZP depth of staining. However, the changes induced by the IL-1beta treatment corresponded to changes in surface roughness, determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of articular cartilage can be modified by TGF-beta1 and IL-1beta treatment and that the friction coefficient depends on multiple factors, including SZP localization and surface roughness.

Binding and localization of recombinant lubricin to articular cartilage surfaces

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.

Poly(vinyl alcohol) Hydrogel as a Biocompatible Viscoelastic Mimetic for Articular Cartilage

The prevalence of suboptimal outcome for surgical interventions in the treatment of full-thickness articular cartilage damage suggests that there is scope for a materials-based strategy to deliver a more durable repair. Given that the superficial layer of articular cartilage creates and sustains the tribological function of synovial joints, it is logical that candidate materials should have surface viscoelastic properties that mimic native articular cartilage. The present paper describes force spectroscopy analysis by nano-indentation to measure the elastic modulus of the surface of a novel poly(vinyl alcohol) hydrogel with therapeutic potential as a joint implant. More than 1 order of magnitude decrease in the elastic modulus was detected after adsorption of a hyaluronic acid layer onto the hydrogel, bringing it very close to previously reported values for articular cartilage. Covalent derivatization of the hydrogel surface with fibronectin facilitated the adhesion and growth of cultured rat tibial condyle chondrocytes as evidenced morphologically and by the observance of metachromatic staining with toluidine blue dye. The present results indicate that hydrogel materials with potential therapeutic benefit for injured and diseased joints can be engineered with surfaces with biomechanical properties similar to those of native tissue and are accepted as such by their constituent cell type.

Del1: A New Protein in the Superficial Layer of Articular Cartilage

Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.