Adult Human Cartilage Research Articles

Cell-based cartilage repair

Adult human joint cartilage lesions do not spontaneously heal. Patients with isolated articular cartilage lesions make up perhaps some 1% of the total number of patients examined by arthroscopy. Symptoms such pain and locking, and the suggestion that the untreated lesions may progress to osteoarthritis form the basis for current attempts to repair articular cartilage by cell transplantation and other methods. Animal model work and preliminary experience in humans suggest that some degree of cartilage repair can be accomplished with these methods. However, the long-term fate of the transplants, and the benefits are still uncertain. No randomized, controlled and blinded trials have been presented, and available data do not support any certain conclusions with regard to the superiority of one method of cartilage repair over the other, or indeed over treatment without surgery. Better understanding of the role of morphogens, growth factors, stem cells, cartilage development and biology, and tissue engineering may allow the development of improved therapy. Treatment with these experimental methods should only lie done within the context of strictly designed randomized, controlled and masked clinical trials with several years of follow-up time. (Less)

Splicing patterns of fibronectin mRNA from normal and osteoarthritic human articular cartilage

Fibronectin, a large extracellular glycoprotein, mediates the interaction of cells with the extracellular matrix. Heterogeneity in the structure of fibronectin is largely due to the alternative splicing of three exons (IIIB, IIIA and V) during processing of the fibronectin primary transcript. Osteoarthritis, a degenerative disease of synovial joints, is characterized by a progressive loss of the articular cartilage eventually resulting in pain and loss of joint function. In contrast to the loss of most cartilage matrix proteins accompanying this process, osteoarthritic cartilage contains more fibronectin than disease-free cartilage. We examined the splicing patterns of fibronectin mRNA from adult human articular cartilage of normal and osteoarthritic joints by RNase protection (exon IIIA and exon IIIB) and reversed transcription-polymerase chain reaction (exon V) assays to determine whether or not the increased fibronectin content in osteoarthritic cartilage is also associated with differences in the splicing patterns of these three alternatively spliced exons. The results revealed no gross differences in splicing of these exons between the fibronectin mRNA isolated from adult human articular normal and osteoarthritic cartilage. Thus alterations in the structure of cartilage fibronectin do not appear to correlate with the increased level of fibronectin protein associated with osteoarthritis.

Increased insulin‐like growth factor 1 production by human osteoarthritic chondrocytes is not dependent on growth hormone action

To investigate insulin-like growth factor 1 (IGF-1) production in normal and osteoarthritis (OA) chondrocytes and to further examine the role of growth hormone (GH) in adult human cartilage and, in particular, in diseased tissue. IGF-1 production was measured with a radioimmunoassay. Binding assay, Northern blot, and reverse transcriptase polymerase chain reaction (RT-PCR) techniques were used for GH receptor (GHR) detection. The biological response to GH was estimated via IGF-1 production. We observed that basal levels of IGF-1 production were significantly higher in OA chondrocytes than in normal cells (P < 0.005). Adult human chondrocytes, however, were unresponsive to GH stimulation with regard to IGF-1 production, as shown in dose-response (0-1,000 ng/ml) and time-course (days 1-8) studies. In addition, no specific 125I-GH binding was detected in either cell type. Northern blot analysis revealed a 5.5-kb GHR messenger RNA (mRNA) species, but semiquantitative RT-PCR revealed no difference in GHR mRNA expression by normal and OA chondrocytes. This study indicates that the elevated synthesis of IGF-1 by adult human OA chondrocytes occurs through a GH/GHR-independent mechanism, suggesting that other factors are capable of controlling local IGF-1 production in these cells.

Ageing increases growth factor-induced inorganic pyrophosphate elaboration by articular cartilage

Advanced age is the most common risk factor for the development of calcium pyrophosphate dihydrate (CPPD) crystal-associated arthritis. However, the link between ageing and CPPD crystal formation in cartilage remains unexplained. In CPPD deposition disease, excess extracellular inorganic pyrophosphate (ePPi), generated by articular chondrocytes, accumulates in affected joints and contributes to CPPD crystallogenesis. Transforming growth factor β1 (TGFβ1) is the first known physiologic stimulant of ePPi elaboration by adult porcine and human cartilage. We hypothesized that sensitivity of articular cartilage to the ePPi-stimulatory effects of TGFβ1 may increase with ageing. Accordingly, we compared the effects of TGFβ1 on cartilage ePPi elaboration from juvenile, young adult, and old adult pigs. Cartilage organ cultures from old animals increased ePPi elaboration in response to TGFβ1 to a greater extent than did cartilage from juvenile and young adult animals. Similar results were seen in chondrocyte monolayers. Concurrent exposure to epidermal growth factor (EGF) augmented, but was not necessary for TGFβ1-induced ePPi elaboration by adult cartilage. In contrast, in juvenile cartilage, concurrent exposure to EGF was required to permit TGFβ1-induced ePPi elaboration. Thus, increased cartilage responsiveness to the ePPi-stimulatory effects of TGFβ1 occurs with ageing, and may explain the link between advanced age and CPPD deposition disease.

Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein.

The actions of human recombinant stromelysins-1 and -2, collagenase, gelatinases A and B and matrilysin on neonatal human proteoglycan aggregates were examined. With the exception of gelatinase B, aggrecan was degraded extensively by most metalloproteinases studied, whereas link protein showed only limited proteolysis. Sequencing studies of modified link protein components revealed that stromelysins-1 and -2, gelatinases A and B and collagenase cleaved specifically between His16 and Ile17, and matrilysin, stromelysin-2 and gelatinase A cleaved between Leu25 and Leu26. Cleavage at the former bond generated a link protein component with the same N-terminus as that isolated from newborn human cartilage. Based on previously determined in situ cleavage sites it is evident that matrix metalloproteinases are not solely responsible for the accumulation of link protein degradation products in adult human cartilage, indicating that additional proteolytic agents are involved in the normal catabolism of human cartilage matrix.

Characterization of proteoglycan-reactive T cell lines and hybridomas from mice with proteoglycan-induced arthritis.

Abstract Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.

A matrix protein of Mr 55 000 that accumulates in human articular cartilage with age

Adult human cartilage contains a protein of M r 55 000 which is deficient in newborn cartilage. In the adult the molecule represents one of the most abundant non-collagenous, non-proteoglycan molecules in 4 M guanidinium chloride extracts of the tissue. The molecular size of the protein on SDS-PAGE remains constant under reducing and non-reducing conditions, suggesting that it does not exist a disulphide-bonded multimer, nor do intramolecular disulphide bonds greatly influence its conformation. The protein has the ability to interact with some immunoglobulin preparations making its detection possible by Western blotting with some non-specific antisera. Labeling with [ 3H]leucine in organ culture indicates that protein of this size is being made by the chondrocytes. However, during purification the newly synthesized molecules do not behave as the resident protein on ion-exchange chromatography, suggesting that the protein may accumulate with age rather than being a major synthetic product of the adult chondrocytes. Amino terminal protein sequence analysis that the N-terminus of the protein is blocked. Sequences derived from peptides generated with cyanogen bromide do not show homology with previously characterized proteins. Molecules of a similar size and composition have been described in bovine cartilage.

Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage

Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allow stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes inrreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate.

Effects of human carbonic anhydrase III (CA III) on synovial and muscle fibroblast glycosaminoglycan metabolism.

We investigated the ability of CA III, isolated from adult human skeletal muscle, to regulate cell growth and glycosaminoglycan (GAG) formation in connective tissue cells derived from various human tissues. Unlike muscle, dermal, and cartilage fibroblasts, synovial connective tissue cells were substantially activated by CA III and showed enhanced hyaluronic acid (HA) synthesis. Cell culture experiments showed that CA III induced a 2- to 11-fold increase in [14C]HA synthesis by human synovial fibroblasts (SF) in a dose-dependent manner (P less than 0.001); erythrocyte CA I and CA II were inactive. Exposure of SF and muscle fibroblasts to CA III also resulted in a 20-45% and 16-70% increased 35S incorporation into proteoglycans, respectively. When adult human skin and cartilage fibroblasts were studied in the presence of CA III, no differences in the level of DNA and GAG formation were noted. These latter cell types were clearly activated by a platelet (CTAP-III) growth factor. The potential physiological implications of these observations are discussed.

Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.

Proteoglycan aggregates in adult human costal cartilage

1. 1. A sequential extraction procedure using isotonic KCl and 4.0 M guanidine hydrochloride was used to solubilise proteoglycans from adult human costal cartilage under conditions minimising autolysis. Up to 28% of the total tissue hexuronate was extracted. 2. 2. Proteoglycan fractions were prepared from the extracts by CsCl equilibrium density gradient centrifugation. 3. 3. Each fraction exhibited distinct electrophoretic heterogeneity. 4. 4. Proteoglycans extracted by isotonic solutions are relatively small and may be in vivo degradation products of whole molecules. The major fraction (A 1) from high ionic strength extracts has a composition similar to that of A 1 proteoglycans from adult human articular cartilage. A 2 fractions differ and are highly enriched in protein. 5. 5. A 1 and A 2 fractions high ionic strength extracts contain proteoglycan can aggregates, but to a much lesser extent than found on other cartilages like bovine nasal or porcine epiglottal cartilage. 6. 6. The aggregates can be dissociated and a ‘subunit’ proteoglycan isolated by CsCl density gradient centrifugation in 4.0 M guanidine hydrochloride. ‘Subunits’ can reaggregate partially, when mixed with fractions of lower density from the gradient.

Heterogeneity of proteoglycans from adult human costal cartilage [proceedings

Changes in the glycosaminoglycan composition of human hyaline cartilages during maturation and aging (Kaplan & Meyer, 1959; Mathews & Glagov, 1966; Mason & Wusteman, 1970) suggest changes in the type of proteoglycan synthesized by chondrocytes at different times. Electrophoresis of adult human costal-cartilage proteoglycans on cellulose acetate membranes (Pedrini, 1969) separated two types of molecule; one, a predominently chondroitin sulphate proteoglycan; the other, a keratan sulphate proteoglycan. However, heterogeneity could have arisen from mechanical degradation of native proteoglycans during homogenization of the tissue (Sajdera & Hascall, 1969), or by the action of cartilage proteinases during extraction (Pearson & Mason, 1977). After precautions to prevent either physical or enzymic degradation, it has been shown that the major proteoglycan fraction of bovine nasal cartilage can be separated into two components by electrophoresis in large-pore composite agarose/polyacrylamidegels (Pearson & Mason, 1977). The same methods have been applied to study the heterogeneity of adult human costal-cartilage proteoglycans. Cartilage was obtained at necropsy within 5 h of death, frozen (-2O”C, 2 h), stripped of perichondrium and shredded with a Surform blade. Grossly calcified tissue was discarded. Tissue slices were extracted with O . ~ ~ M K C I followed by 4.0~-guanidine HCI at 1°C in the presence of proteinase inhibitors (see Pearson & Mason, 1977). The tissue residue was then powdered in liquid N2 before extraction again with 4.0~-guanidine HCI. This report deals with the proteoglycans solubilized by the first and second guanidine HCI extracts, which account for 12% and 6% respectively of the total tissue hexuronic acid. A,-proteoglycan fractions (d> 1.70) were isolated from the crude extracts by centrifugation in a caesiuni chloride density gradient under associative conditions, at 10°C, in the presence of proteinase inhibitors (Roughley & Mason, 1976; Pearson & Mason, 1977). Fractions from the first and second extracts are described as ‘AIGuHCI,~’ and ‘A1 GuHC1,Z’ respectively, in the nomenclature of Heinegard (1972). Each fraction contained about 70% of the total hexuronic acid of the extract. The methods for electrophoresis in agaroselpolyacrylamide gels and spectrophotometric scanning of gels have been described (McDevitt & Muir, 1971 ; Roughley & Mason, 1976). Proteoglycan bands stained with Toluidine Blue were cut from the gel, placed in 2.0~-CaCl,, agitated by vortex-mixing, centrifuged and the supernatants retained. The residue was resuspended in 2.0 M-C~CI,, sonicated (MSE sonicator; low power setting, amplitude 3, 2OoC, 30s) and the supernatant recovered after centrifugation. The pooled supernatants were dialysed against 2.0~-CaCl, (1 : 50, v/v; 20°C, 18 h), then against water (1 : 50, v/v; 4”C, 18 h, repeated two times), freeze-dried and analysed for hexosamine content (Mayes et al., 1973). The A,-proteoglycans from four cartilage samples (female, 47 years old; male, 59; male, 64; female, 76) all contained three electrophoretic components with mobilities (RBpB) 0.8,0.9 and 0.96 relative to broniophenol blue (BPB) (Fig. 1). Fractions from the two oldest subjects also contained a fourth component, RBps 1.04. Each electrophoretic component has a characteristic g1ucosamine:galactosamine ratio (Table 1). For a given component in fractions ‘AlGuHC1,l’ or ‘AlGuHC1,2’, from either cartilage, the ratio is very similar. The ratio indicates the relative proportions of keratan sulphate: chondroitin sulphate in a proteoglycan. Since the rate of migration of a multichain proteoglycan in large-pore gels is probably, predominantly, a function of its hydrodynamic size, the data show that the larger proteoglycan species (low RBpB) contain relatively more chondroitin sulphate to keratan sulphate than the smaller species (high RBpB). The glucosamine: galactosamine ratio of fraction ‘AlGuHC1,l’ differs from that of fraction ‘AIGuHC1,2’(Table 1). This difference appears to be due to a difference in the relative amounts of the electrophoretic components present in A,-proteoglycan frac-

Proteoglycans collagen interactions in human costal cartilage

1. 1. Adult human costal cartilage was repeatedly extracted using both dissociative and disruptive techniques. A considerable amount of proteoglycans could not be solubilized by these sequential extractions. 2. 2. This non-extractable proteoglycan fraction was solubilized by treating the cartilage residue with bacterial collagenase. In an attempt to define the interactions between the insoluble collagen matrix and the non-extractable proteoglycan, the collagenase-solubilized proteoglycans were extensively purified by gel filtration, carboxymethyl-cellulose chromatography, cesium chloride density gradient ultracentrifugation, electrophoresis and chromatography on cellulose columns in the presence of quaternary ammonium salts. 3. 3. Density gradient ultracentrifugation of collagenase-solubilized proteoglycans in 4.0 M guanidine·HCl yielded low-density and high-density fractions, both containing considerable amounts of hexuronate and hydroxyproline. 4. 4. Chromatography of the high-density fraction on a cellulose column equilibrated with cetyltrimethylammonium bromide yielded a purified proteoglycan fraction containing only a trace of hydroxyproline. The same treatment separated the low-density fraction into two fractions, one eluted with acetate buffer pH 4.0 and the other 1.25 M MgCl 2. Both fractions contained polysaccharide material and hydroxyproline. These results are thought to indicate the presence of collagen peptides tightly bound to the proteoglycans. 5. 5. Digestion of the acetate-eluted fraction obtained from low-density proteoglycans with testicular hyaluronidase removed most of the chondroitin sulfate chains from the proteoglycan, but the hydroxyproline remained bound to the high molecular weight fraction which contained the protein core of the proteoglycan and keratan sulfate.

Cartilages and ligaments of the adult human larynx.

The present report is of an investigation of the range of normal distribution in the shape, size, and configuration of the human laryngeal cartilages and in the size, symmetry, and placement of the articulations and ligaments of these cartilages. The study involved dissection of the larynges of ten white men, and ten white women with the aid of a binocular dissection microscope. The study indicated significant patterns in the adult human laryngeal cartilage configurations with respect to sex, symmetry, size, and anatomical interrelationships. Speculations on vocal fold length adjustments were made based on examination of the cricoarytenoid and cricothyroid articulations and their ligaments.

Protein-polysaccharide complexes from adult human tracheal cartilage.

Protein-polysaccharide complexes from adult human tracheal cartilage.

Sulfate metabolism in avion and mammalian cartilage extracts

Sulfate metabolism in extracts of embryonic chick and calf cartilage and a human chondrosarcoma of the sternum of a 75-year-old patient have been compared. The chondrosarcoma was able to activate and transfer sulfate to acid mucopolysaccharide, at a level approaching that of the embryonic extracts, when comparison was made with adult human costal cartilage extracts which had feeble activity. CSA-A, 1 1 Abbreviations used: CSA-A and CSA-C, chondroitin sulfates A and C, respectively; PAPS, adenosine 3′-phosphate 5′-sulfatophosphate; APS, adenosine 5′-sulfatophosphate; ATP, adenosine triphosphate. incompletely sulfated, was the only mucopolysaccharide isolated from the myxomatous areas of the chondrosarcoma, but the presence of CSA-C was suggested by infrared spectral data. Keratosulfate was absent, and thus the mucopolysaccharide composition resembled that in newborn costal cartilage rather than the pattern of adult costal cartilage. Incorporation of sulfate-S 35 into acid mucopolysaccharide in vivo and in vitro gave a fraction isolated by paper chromatography, which was excluded from Sephadex G-50. The suggested presence of a minute S 35 subfraction, retained on Sephadex G-50 and therefore of mol. wt. < 8–10,000, was obtained. Sulfate-S 35 was also incorporated into a number of endogenous acceptors present in the cartilage extracts. Phenol sulfokinase was present in both avian and mammalian cartilage, but the avian cartilage extracts were unique in being able to sulfate phenolic steroids and it appears in this case that both simple phenols and the phenolic steroids are sulfated by the same enzyme.

Growth of embryonic mammalian cartilage after freezing.

SINCE the discovery that glycerol will protect cells and tissues against freezing damage1, considerable progress has been made in developing techniques for the preservation of tissues at very low temperatures. Attempts to establish low-temperature banks of adult human cartilage have unfortunately, so far, proved unsuccessful2. Biggers3, however, was able to show that embryonic chick tibiae will grow and differentiate in vitro, after freezing to −79 ° C., in saline containing 15 per cent glycerol. This communication describes briefly some of the work concerned with freezing embryonic rat tibiae to −79° C. A fuller account is being given elsewhere4. Rat fœtuses of a known age were obtained from the Medical Research Council strain of albino rat, bred in the Department. The occurrence of a vaginal copulation plug, or the presence of sperm in the vaginal smear, was taken as the indication of mating. On the seventeenth day post coitum, the conceptuses were excised, and the embryonic tibiae were isolated under a dissecting microscope. The bones were cleaned of as much extraneous tissue as possible, with care to avoid injuring the perichondrium. The structure of these tibiae is being described elsewhere5.