Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan

The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 m-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47–51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8–15% of total proteoglycans. Analysis of the extract revealed that 24–50% of its hyaluronan was directly detecable with the probe, while 50–76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-m-thick tissue sections.     

The interactions of cartilage proteoglycans with collagens are determined by their structures.

In the present work, the interaction of aggrecan, decorin and biglycan isolated from pig laryngeal cartilage and of the three squid cartilage proteoglycans with collagen type I and II was studied. The interaction was examined under conditions allowing the formation of collagen fibrils. It was found that biglycan interacted strongly with collagen type II and not with type I and the interaction seemed to proceed exclusively through its core proteins. Decorin interacted with collagen type I but not with type II. Aggrecan interacted very poorly with both collagen types. The two squid proteoglycans of large size, D1D1A and D1D2, interacted only with collagen type I through both glycosaminoglycans and core proteins. The third squid proteoglycan of small size, D1D1B, interacted poorly only with collagen type I. The results suggested that the interactions of cartilage proteoglycans with collagen were mainly due to the primary structure of both molecules, and would contribute to the maintenance of the integrity of the tissue. The biochemical significance of these interactions might be more critical in aged vertebrate cartilage, where loss of aggrecan and increase of the small proteoglycans was observed, a large proportion of which is found in the extracellular matrix free of glycosaminoglycan chains.     

Metabolic kinetics of proteoglycans by embryonic chick sternal cartilage in culture.

Explant cultures of embryonic chick sternum have been widely studied, but the kinetics of biosynthesis of proteoglycans by this tissue in culture has not been characterized. Caudal cartilaginous portions of 16-day-old embryonic chick sterna were cultured for 8 days. Histological examination showed that the fresh cartilage contained morphologically homogenous chondrocytes, which were embedded in a uniform extracellular matrix. After culture for 8 days, the histological appearance of the explant remained unchanged but the tissue increased in size with time as indicated by a progressive increase in DNA content and in the content of glycosaminoglycan and collagen. Rates of degradation and release from the tissue of proteoglycans labeled in ovo with 35S were first order during culture, as were the unlabeled proteoglycans. Proteoglycan synthesis was high during the first 2 days of culture, and this then gradually decreased from this high level during the following 2 days. Synthesis was then maintained at a constant level for the remainder of the culture period. After culture for 2 and 7 days, the proteoglycans synthesized by the explants were identical to the preexisting proteoglycans in hydrodynamic size, glycosaminoglycan chain size, and ability to form aggregates. These findings suggest that the embryonic chick sterna maintained a stable cartilage phenotype during the extended culture periods. The initial rapid rate of matrix turnover was probably attributable to an adaptation of the tissue to ex ovo culture conditions and the subsequent maintenance of cellular activities at a lower level indicated the establishment of a steady-state rate of metabolism.     

Investigation of molecular motion of proteoglycans in cartilage by 13C magnetic resonance.

13C nmr spectral parameters were measured for intact bovine nasal cartilage tissue, the purified proteoglycan aggregate, and chondroitin 4-sulfate. A comparison of integrated intensities obtained for four different samples of fresh tissue with an ethylene glycol standard indicated that at least 80% of the total glycosaminoglycan carbons in the tissue contributed to the spectrum. This result was confirmed by intensity measurements obtained at 56 degrees on fresh tissue and at 37 degrees after extensive papain digestion of fresh tissue. Spin lattice relaxation times and nuclear Overhauser enhancements were analyzed in terms of the following models of molecular motion: (a) single correlation time; (b) log X2 distribution of correlation times; and (c) anisotropic motion. The analysis indicates that the segmental motions of glycosaminoglycan chains are characterized by a broad distribution of correlation times centered at about 50 ns. Slow motion contributions to glycosaminoglycan line widths were reduced by dipolar decoupling (gammaH2/2pi = 65 kHz). Collagen intensity was observed in dipolar decoupled spectra, but not in scalar decoupled spectra of intact tissue, showing that the type II collagen in cartilage undergoes anisotropic motion like the type I collagen in tendon. Only glycosaminoglycan resonances were observed in spectra of a solution of proteoglycan aggregate before and after chondroitinase digestion. After subsequent digestion with papain, protein resonances were observed. These results suggest that the protein portions of the proteoglycan aggregate structure, in contrast with the glycosaminoglycan chains, have restricted backbone mobility and consequently a defined backbone structure.     

Establishment of a reliable method for direct proteome characterization of human articular cartilage

Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.     

Extracellular Matrix of Porcine Pericardium: Biochemistry and Collagen Architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin.     

Biomimetic soluble collagen purified from bones

Type I collagen has been extensively exploited as a biomaterial for biomedical applications and drug delivery; however, small molecular alterations occurring during the isolation procedure and its interaction with residual bone extracellular matrix molecules or proteins might affect the overall material biocompatibility and performance. The aim of the current work is to study the potential alterations in collagen properties and organization associated with the absence of proteoglycans, which mimic pathological conditions associated with age‐related diseases. A new approach for evaluating the effect of proteoglycans on the properties of isolated type I collagen from the bone matrix is described. Additional treatment with guanidine hydrochloride was introduced to remove residual proteoglycans from the collagen matrix. The properties of the isolated collagen with/without guanidine hydrochloride treatment were investigated and compared with a commercial rabbit collagen as control. We demonstrate that the absence of proteoglycans in the isolated type I collagen affects its thermal properties, the extraction into its native structure, and its ability to hydrate and self‐assemble into fibers. The fine control and tuning of all these features, linked to the absence of non‐collagenous proteins as proteoglycans, offer the possibility of designing new strategies and biomaterials with advanced biomimetic properties aimed at regenerating bone tissue in the case of fragility and/or defects.     

Technique to accurately quantify collagen content in hyperconfluent cell culture

Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.     

The stabilization of in vivo assembled collagen fibrils by proteoglycans/glycosaminoglycans

The effects of proteoglycans/glycosaminoglycans on the thermal stability of in vivo assembled collagen fibrils have been examined. The shrinkage temperature of tendon collagen was found to be linearly dependent on the concentration of chondroitin sulphate in the surrounding fluid. Enzymic pretreatment of articular cartilage, to reduce its glycosaminoglycan content, resulted in decreased stability of the collagen present. The stability of the collagen in hyaluronidase-treated cartilage was found to be higher when measured in a solution of chondroitin sulphate (30 g/dl) than in buffer alone. The results of this study demonstrate that the proteoglycans stabilize collagen fibrils in tissues such as articular cartilage.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

In situ interaction of cartilage proteoglycans with matrix proteins

The interaction of proteoglycans with other matrix proteins via thiol-disulphide interchange was explored. Chick sternal cartilage was extracted with 4 M guanidine hydrochloride in the presence and absence of N-ethylmaleimide and the proteoglycans from the centrifugation A2 fractions were isolated. Those from extracts without N-ethylmaleimide were linked with reducible bonds with 10-15 proteins-glycoproteins including the link proteins, the 148 kDa and 36 kDa proteins. The same was observed with extracts of pig laryngeal and sheep nasal cartilage. The linked proteoglycans from sheep amounted to 2-3% of the extractable uronic acid and belonged to two populations. The major fraction was included by Sepharose 6B (Mr 110 000) had twice as long chondroitin sulphate chains, higher 4-sulphated residues and a high content of aspartic acid and leucine-rich protein. The larger proteoglycans had a size and composition similar to those of aggregating proteoglycans.     

Short review. Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

Pleiotrophin inhibits chondrocyte proliferation and stimulates proteoglycan synthesis in mature bovine cartilage

Pleiotrophin (PTN) is a secreted heparin-binding, developmentally regulated protein that is found in abundance in fetal, but not mature, cartilage. SDS-page and glycosaminoglycan (GAG) analysis of sulfate-radiolabeled proteoglycans isolated from the medium of mature cultured chondrocytes treated with PTN showed a threefold increase in the levels of proteoglycan synthesis. In contrast, in cultures of fetal chondrocytes, no changes in proteoglycan synthesis were observed. Thymidine incorporation experiments showed a dose-dependent decrease in proliferation of treated cells compared with control cultures, suggesting that pleiotrophin had an inhibitory effect on growth of chondrocytes. Neither FGF or heparin reversed the inhibitory effect of PTN. Capillary electrophoresis of chondroitinase ABC-digested proteoglycans isolated from mature chondrocytes showed 2-4-fold increases in the amounts of the 4S- and 6S-substituted GAG chains for the PTN-treated chondrocytes. Northern analysis showed a twofold upregulation in the mRNA levels of biglycan and collagen type II, but no difference in the message levels for decorin and aggrecan. These results establish that PTN inhibits cell proliferation, while stimulating the synthesis of proteoglycans in mature chondrocytes in vitro, suggesting that PTN may act directly or indirectly to regulate growth and proteoglycan synthesis in the developing matrix of fetal cartilage.     

Polydispersity and heterogeneity of squid cranial cartilage proteoglycans as assessed by immunochemical methods and electron microscopy

The three populations of squid cranial cartilage proteoglycans, D1D1A, D1D1B and D1D2 appeared to have a high degree of polydispersity. Gel electrophoresis and immunoblotting analysis showed that polydispersity was mainly due to the variable size of chondroitin sulphate E chains. This was further ascertained after rotary shadowing electron microscopy of proteoglycan core proteins and glycosaminoglycan side chains and statistical analysis of the sizes measured for both components. Enzymic treatment of the proteoglycan core proteins produced different peptides from each population, suggesting that the observed heterogeneity of the proteoglycans is due to their core proteins. Antibodies were raised in rabbits against all proteoglycans and enzyme-linked immunosorbent analysis of proteoglycan core proteins revealed that the proteoglycans, even heterogeneous, shared many common epitopes. Part of the common proteoglycan epitopes were found to be located in chondroitin sulphate E chains. Heterogeneity of squid proteoglycans was also investigated by studying their interactions with collagen and it was found that only the two populations of high molecular mass, D1D1A and D1D2, were able to interact with only collagen type I, the latter stronger than the former.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

Summary The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using (i) monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and (ii) cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Microporous acellular extracellular matrix combined with adipose-derived stem cell sheets as a promising tissue patch promoting articular cartilage regeneration and interface integration

BackgroundAcute or chronic injury of articular cartilage leads to localized destruction. Difficulties with interface integration between the implant and native cartilage tissue can lead to an undesirable outcome. To improve cartilage repair and interface integration, we explored the therapeutic efficacy of microporous acellular extracellular matrix (ECM) combined with adipose-derived stem cell (ASC) sheets.MethodsMethods for fabricating ASC sheets and microporous acellular ECM were explored before transplanting the constructed ASC sheet/matrix in vivo and in vitro, respectively. After the operation, distal femur samples were collected at 6 and 12 weeks for further analysis.ResultsThe decellularization process removed 90% of the DNA but retained 82.4% of glycosaminoglycans (GAGs) and 82.8% of collagen, which are the primary components of cartilage matrix. The acellular matrix/ASC sheet construct treatment in vivo showed better interface integration, cartilage regeneration, and collagenous fiber arrangement, which resembles the native structure. There was a significant increase in GAG and collagen accumulation at the zone of regeneration and integration compared to other groups. Gene expression analysis showed that the mRNA level associated with cartilage formation significantly increased in the acellular matrix/ASC sheet group (p<0.05), which is consistent with the histological analysis.DiscussionASC sheets promote interface integration between the implant and native tissue. This effect, together with the acellular matrix as a graft, is beneficial for cartilage defect repair, which suggests that acellular matrix/ASC sheet bioengineered cartilage implants may be a better approach for cartilage repair due to their enhanced integration.