Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients

Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1β and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1β mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1β levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment.     

Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation

Introduction  

Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA.     

Glucosamine prevents <Emphasis Type="Italic">in vitro</Emphasis> collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

Notch signaling is involved in human articular chondrocytes de-differentiation during osteoarthritis

Abstract

Context: During osteoarthritis (OA), chondrocytes undergo de-differentiation, resulting in the acquisition of a fibroblast-like morphology, decreased expression of collagen type II (colII) and aggrecan, and increased expression of collagen type I (colI), metalloproteinase 13 (MMP13) and nitric oxide synthase (eNOS). Notch signaling plays a crucial role during embryogenesis. Several studies showed that Notch is expressed in adulthood. Objective: The aim of our study was to confirm the involvement of Notch signaling in human OA at in vitro and ex vivo levels. Materials and methods: Normal human articular chondrocytes were cultured during four passages either treated or not with a Notch inhibitor: DAPT. Human OA cartilage was cultured with DAPT for five days. Chondrocytes secreted markers and some Notch pathway components were analyzed using Western blotting and qPCR. Results: Passaging chondrocytes induced a decrease in the cartilage markers: colII and aggrecan. DAPT-treated chondrocytes and OA cartilage showed a significant increase in healthy cartilage markers. De-differentiation markers, colI, MMP13 and eNOS, were significantly reduced in DAPT-treated chondrocytes and OA cartilage. Notch1 expression was proportional to colI, MMP13 and eNOS expression and inversely proportional to colII and aggrecan expression in nontreated cultured chondrocytes. Notch ligand: Jagged1 increased in chondrocytes culture. DAPT treatment resulted in reduced Jagged1 expression. Notch target gene HES1 increased during chondrocyte culture and was reduced when treated with DAPT. Conclusion: Targeting Notch signaling during OA might lead to the restitution of the typical chondrocyte phenotype and even to chondrocyte redifferentiation during the pathology.     

Characteristics of cartilage engineered from human pediatric auricular cartilage

In the repair of cartilage defects, autologous tissue offers the advantage of lasting biocompatibility. The ability of bovine chondrocytes isolated from hyaline cartilage to generate tissue-engineered cartilage in a predetermined shape, such as a human ear, has been demonstrated; however, the potential of chondrocytes isolated from human elastic cartilage remains unknown. In this study, the authors examined the multiplication characteristics of human auricular chondrocytes and the ability of these cells to generate new elastic cartilage as a function of the length of time they are maintained in vitro. Human auricular cartilage, harvested from patients 5 to 17 years of age, was digested in collagenase, and the chondrocytes were isolated and cultured in vitro for up to 12 weeks. Cells were trypsinized, counted, and passaged every 2 weeks. Chondrocyte-polymer (polyglycolic acid) constructs were created at each passage and then implanted into athymic mice for 8 weeks. The ability of the cells to multiply in vitro and their ability to generate new cartilage as a function of the time they had been maintained in vitro were studied. A total of 31 experimental constructs from 12 patients were implanted and compared with a control group of constructs without chondrocytes. In parallel, a representative sample of cells was evaluated to determine the presence of collagen. The doubling rate of human auricular chondrocytes in vitro remained constant within the population studied. New tissue developed in 22 of 31 experimental implants. This tissue demonstrated the physical characteristics of auricular cartilage on gross inspection. Histologically, specimens exhibited dense cellularity and lacunae-containing cells embedded in a basophilic matrix. The specimens resembled immature cartilage and were partially devoid of the synthetic material of which the construct had been composed. Analyses for collagen, proteoglycans, and elastin were consistent with elastic cartilage. No cartilage was detected in the control implants. Human auricular chondrocytes multiply well in vitro and possess the ability to form new cartilage when seeded onto a three-dimensional scaffold. These growth characteristics might some day enable chondrocytes isolated from a small auricular biopsy to be expanded in vitro to generate a large, custom-shaped, autologous graft for clinical reconstruction of a cartilage defect, such as for congenital microtia.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

An ex vivo model using human osteoarthritic cartilage demonstrates the release of bioactive insulin‐like growth factor‐1 from a collagen–glycosaminoglycan scaffold

Biomimetic scaffolds hold great promise for therapeutic repair of cartilage, but although most scaffolds are tested with cells in vitro, there are very few ex vivo models (EVMs) where adult cartilage and scaffolds are co‐cultured to optimize their interaction prior to in vivo studies. This study describes a simple, non‐compressive method that is applicable to mammalian or human cartilage and provides a reasonable throughput of samples. Rings of full‐depth articular cartilage slices were derived from human donors undergoing knee replacement for osteoarthritis and a 3 mm core of a collagen/glycosaminoglycan biomimetic scaffold (Tigenix, UK) inserted to create the EVM. Adult osteoarthritis chondrocytes were seeded into the scaffold and cultures maintained for up to 30 days. Ex vivo models were stable throughout experiments, and cells remained viable. Chondrocytes seeded into the EVM attached throughout the scaffold and in contact with the cartilage explants. Cell migration and deposition of extracellular matrix proteins in the scaffold was enhanced by growth factors particularly if the scaffold was preloaded with growth factors. This study demonstrates that the EVM represents a suitable model that has potential for testing a range of therapeutic parameters such as numbers/types of cell, growth factors or therapeutic drugs before progressing to costly pre‐clinical trials. © 2015 The Authors. Cell Biochemistry and Function Published by John Wiley & Sons Ltd.     

Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.Key words: in vitro cartilage, 3-D cell culture, fusion culture technique, tissue engineering, cell differentiation, extracellular matrix, immunohistochemistry     

Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix

Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPalpha expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPalpha on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.     

In vitro comparison of six different matrix systems for the cultivation of human chondrocytes

Summary In recent years, a great variety of different matrix systems for the cultivation of chondrocytes have been developed. Although some of these scaffolds show promising experimental results in vitro, the potential clinical value remains unclear. In this comparative study, we propagated human articular chondrocytes precultivated in monolayer culture on six different scaffolds (collagen gels, membranes and sponges) under standardized in vitro conditions. Mechanical properties of the matrix systems were not improved significantly by cultivation of human chondrocytes under the given in vitro conditions. The gel systems (CaReS, Ars Artho, Germany and Atelocollagen, Koken, Japan) showed a homogeneous cell distribution; chondrocytes propagated on Chondro-Gide (Geistlich Biomaterials, Switzerland) and Integra membranes (Integra, USA) were building multilayers. Only few cells penetrated the two Atelocollagen honeycomb sponges (Koken, Japan). During cultivation, chondrocytes propagated on all systems showed a partial morphological redifferentiation, which was best with regard to the gel systems. In general, only small amounts of collagen type-II protein could be detected in the pericellular region and chondrocytes failed to build a territorial matrix. During the first two weeks of cultivation, the two gel systems showed a significantly higher collagen type-II gene expression and a lower collagen type-I gene expression than the other investigated matrix systems. Although collagen gels seem to be superior when dealing with deep cartilage defects, membrane systems might rather be useful in improving conventional autologous chondrocyte transplantation or in combination with gel systems.     

Protein kinase B and extracellular signal‐regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes

Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal‐regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra‐articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment.     

Potential use of the human amniotic membrane as a scaffold in human articular cartilage repair

The human amniotic membrane (HAM) is an abundant and readily obtained tissue that may be an important source of scaffold for transplanted chondrocytes in cartilage regeneration in vivo. To evaluate the potential use of cryopreserved HAMs as a support system for human chondrocytes in human articular cartilage repair. Chondrocytes were isolated from human articular cartilage, cultured and grown on the chorionic basement membrane side of HAMs. HAMs with chondrocytes were then used in 44 in vitro human osteoarthritis cartilage repair trials. Repair was evaluated at 4, 8 and 16 weeks by histological analysis. Chondrocytes cultured on the HAM revealed that cells grew on the chorionic basement membrane layer, but not on the epithelial side. Chondrocytes grown on the chorionic side of the HAM express type II collagen but not type I, indicating that after being in culture for 3–4 weeks they had not de-differentiated into fibroblasts. In vitro repair experiments showed formation on OA cartilage of new tissue expressing type II collagen. Integration of the new tissue with OA cartilage was excellent. The results indicate that cryopreserved HAMs can be used to support chondrocyte proliferation for transplantation therapy to repair OA cartilage.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.     

OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPα), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPα gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPα expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPα on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.