Non-cartilaginous Tissues Research Articles

Incompletely Decellularized Tracheal Matrix Scaffold for Tissue Engineering.

Dense cartilaginous extracellular matrix makes decellularization and repopulation of tracheal cartilage difficult. However, the dense matrix isolates cartilaginous antigens from the recipient's immune system. Therefore, allorejection may be avoided by removing antigens from noncartilaginous tissues. In this study, incompletely decellularized tracheal matrix scaffolds were developed for tracheal tissue engineering. Brown Norway rat tracheae were decellularized with 4% sodium deoxycholate treatment. The cell and antigen removal efficacy, histoarchitecture, surface ultrastructure, glycosaminoglycan, collagen contents, mechanical properties, and chondrocyte viability of the scaffold were evaluated in vitro. Brown Norway rat tracheal matrix scaffolds ( n = 6) were implanted subcutaneously into Lewis rats and observed for 4 weeks. Brown Norway rat tracheae ( n = 6) and Lewis rat scaffolds ( n = 6) were implanted as controls. Histologic analysis of macrophage and lymphocyte infiltration was performed. One decellularization cycle removed all cells and antigens from noncartilaginous tissue. Incomplete decellularization preserved the structural integrity of the tracheal matrix and chondrocyte viability. Except for 31% glycosaminoglycan loss, the scaffold had comparable collagen content and tensile and compressive mechanical properties to those of the native trachea. The allogeneic scaffold showed remarkably reduced CD68 + , CD8 + , and CD4 + cell infiltration compared with the allografts and demonstrated similar cell infiltration to the syngeneic scaffold. It also maintained the three-dimensional tracheal structure and cartilage viability in vivo. Incompletely decellularized trachea did not induce immunorejection and maintained the integrity and viability of cartilage in vivo. Tracheal decellularization and repopulation can be simplified for urgent tracheal replacement. The present study describes the development of an incomplete decellularization protocol that creates a decellularized matrix scaffold for tracheal tissue engineering, aiming to provide preliminary data that this method may generate suitable tracheal scaffolds for use in tracheal replacement.

Hemodynamic stress shapes subchondral bone in osteoarthritis: An emerging hypothesis.

Osteoarthritis (OA) is no longer regarded as a simple wear-and-tear problem of articular cartilage. Instead, OA is a whole joint disorder involving both cartilaginous and non-cartilaginous tissues such as subchondral bone and synovium. Among them, subchondral bone undergoes constant remodeling in response to the changes of mechanical environment. Current understanding of subchondral bone disturbance in OA is limited to its link with an altered local mechanical loading as a result of ligament or meniscus injury. Very recently, hypertension, the most common vascular morbidity, has been emerged as an independent risk factor of OA. It might suggest a plausible role of systemic hemodynamic mechanical stress in subchondral bone remodeling and the pathogenesis of OA. However, their relationship remains not fully understood. Based on our preliminary clinical observation on the association of hemodynamic parameters with subchondral bone mass and microstructure in late-stage knee OA patients, we formulate a vascular etiology hypothesis of OA from a mechanobiology perspective. Noteworthily, hemodynamic stress associated with subchondral bone mineral density; yet compressive mechanical loading does not. Furthermore, hemodynamic parameters positively correlated with subchondral plate-like trabecular bone volume but negatively associated with rod-like trabecular bone volume. In contrast, compressive mechanical loading tends to increase both plate-like and rod-like trabecular bone volume. Taken together, it warrants further investigations into the distinct role of hemodynamic or compressive stress in shaping subchondral bone in the pathophysiology of OA.The Translational potential of this articleThis work provides a new insight, from the angle of biomechanics, into the emerging role of vascular pathologies, such as hypertension, in the pathogenesis of OA. It might open up a new avenue for the development of a mechanism-based discovery of novel diagnostics and therapeutics.

Evaluating the structural effects of intra-articular sprifermin on cartilage and non-cartilaginous tissue alterations, based on sqMRI assessment over 2 years

Sprifermin (recombinant human fibroblast growth factor-18), a potential disease-modifying osteoarthritis (OA) drug, demonstrated dose-dependent effects on femorotibial cartilage thickness (by quantitative magnetic resonance imaging [MRI]) in the phase II FORWARD study. This post-hoc analysis evaluated the potential effects of sprifermin on several articular structures in the whole joint over 24 months using semi-quantitative MRI assessment. Patients aged 40-85 years with symptomatic radiographic knee OA, Kellgren-Lawrence grade 2 or 3, and medial minimum joint space width ≥2.5mm in the target knee were randomized (1:1:1:1:1) to receive three double-blinded, once-weekly, intra-articular injections of sprifermin 30μg or 100μg or placebo every 6 (q6mo) or 12 months. 1.5- or 3T MRIs were read using the Whole-Organ Magnetic Resonance Imaging Score (WORMS) system at baseline and 24 months. Change from baseline at 24 months on compartment and/or whole knee level was assessed for cartilage morphology, bone marrow lesions (BMLs), and osteophytes by delta-subregional and delta-sum (DSM) approaches. Menisci, Hoffa-synovitis, and effusion-synovitis were also evaluated for worsening. 549 patients were included. Dose-dependent treatment effects from baseline to 24 months were observed on cartilage morphology (sprifermin 100μg q6mo vs placebo; mean DSM (95% confidence interval [CI])-0.6 (-1.5, 0.2); less cartilage worsening) in the entire knee and BMLs sprifermin 100μg q6mo vs placebo; mean DSM (95% CI)-0.2 (-0.5, 0.1) in the patellofemoral compartment. No effects over 24 months were observed on osteophytes, menisci, Hoffa-synovitis or effusion-synovitis. Positive effects associated with sprifermin were observed for cartilage morphology changes, and BML improvement. There were no meaningful negative or positive effects associated with sprifermin in the other joint tissues examined.

The neoacetabulum in developmental dysplasia of the hip is covered with hyaline cartilage

The lack of adequate mechanical stimulation and appropriate contact between acetabulum and femoral head results with developmental dysplasia of the hip (DDH). In DDH, hip joint forms normally during the organogenesis, but deforms during the fetal development. Acetabulum remains comparable in width with normal acetabulum, but has increased length and decreased depth, resulting in a poor coverage of the femoral head. In cases of severe hip subluxation and luxation due to DDH, the femoral head articulates with the external side of the iliac bone, forming a neoacetabulum in the position that was genetically predetermined to become bony tissue. A neoacetabulum is therefore formed under intermittent mechanical pressure, but never has the depth of a physiological acetabulum due to different forces at this new location. Over time, the depth of the neoacetabulum increases, and a crest is formed that obstructs reposition of the femoral head into the anatomic acetabulum.We hypothesize that the neoacetabulum on the iliac bone in DDH patients is formed of hyaline cartilage, despite the lack of genetic predisposition for hyaline cartilage formation in this area. We assume that as the femoral head migrates during development in such patients, joint capsular tissue interposes between the external side of the iliac bone and the femoral head, and a cartilaginous metaplasia of the capsule follows. This results in elongation of the acetabular cartilage in the same direction as the femoral head migrated. This assumption is based on the finding that in patients with hip luxation such interposed joint capsule showed signs of cartilaginous transformation. Furthermore, in the inner part of such joint capsules, proteoglycan production was notably higher than that of other non-cartilaginous tissue. Also, high expression of cartilaginous genes, which are usually not expressed in this tissue, was observed. Confirmation of this hypothesis would put a new perspective on the pathogenesis of DDH and could lead to better management or even prevention of this condition.

Pathogenetic mechanisms of biological agents in managing of relapsing polychondritis

Relapsing polychondritis (RPC) is an autoimmune disease characterized by the inflammation of cartilaginous tissues and other proteoglycan rich tissues. A concomitant disease, particularly myelodysplasia or systemic autoimmune disease can be detected in one-third of the patients with RPC. Unlike adults in children, RPC is less often associated with other autoimmune diseases. The diagnosis of RPC is established using the criteria of Mc Adam (1976) or Damiani (1979). The basis of the pathogenesis of RPC is an autoimmune reaction, which is initially directed against cartilage and then spreads to non-cartilaginous tissues. One of the elements in the pathogenesis of RPC is the mechanical trauma of cartilage, resulting in the release of pro-inflammatory cytokines (tumor necrosis factor alpha, interferon-γ, interleukin-8, and macrophage inflammatory protein 1) and local inflammation followed by the formation of autoantibodies in a patient with a genetic predisposition. In the treatment of RPC, steroids, non-steroidal anti-inflammatory drugs, colchicine are used and, if they are ineffective, immunosuppressants are prescribed. The most effective anti-cytokine drugs used in the treatment of RPC are tumor necrosis factor-alpha (TNF-α) inhibitors, IL-1 receptor antagonists, an inhibitor of the costimulatory pathway of T-lymphocyte activation, monoclonal antibodies against the IL-6 receptor. Given the fact that management of these patients is very complex, the aim of the study is to review available data on pathogenetic mechanisms of biological agents in managing of relapsing polychondritis.

Activation of mTORC1 in chondrocytes does not affect proliferation or differentiation, but causes the resting zone of the growth plate to become disordered

There are several pitfalls associated with research based on transgenic mice. Here, we describe our interpretation and analysis of mTORC1 activation in growth plate chondrocytes and compare these to a recent publication (Yan et al., Nature Communications 2016, 7:11151). Both laboratories employed TSC1-floxed mice crossed with collagen type 2-driven Cre (Col2-Cre), but drew substantially different conclusions. It was reported that activation of mechanistic target of rapamycin complex 1 (mTORC1) via Tsc1 ablation promotes the hypertrophy of growth plate chondrocytes, whereas we observe only disorganization in the resting zone, with no effect on chondrocyte hypertrophy or proliferation. Here, we present our data and discuss the differences in comparison to the earlier phenotypic characterization of TSC1 ablation in cartilage. Importantly, we detect Col2-Cre activity in non-cartilaginous tissues (including the brain) and discuss it in relation to other studies reporting non-cartilaginous expression of collagen alpha(1) II. Altogether, we conclude that mouse phenotypes following genetic ablation using Col2-Cre should be interpreted with care. We also conclude that activation of mTORC1 by TSC1 ablation in postnatal chondrocytes with inducible Col2-Cre (Col2-CreERt) leads to disorganization of the resting zone but causes no changes in chondrocyte proliferation or differentiation.

Structural effects of sprifermin in knee osteoarthritis: a post-hoc analysis on cartilage and non-cartilaginous tissue alterations in a randomized controlled trial.

BackgroundA recent publication on efficacy of Sprifermin for knee osteoarthritis (OA) using quantitatively MRI-defined central medial tibio-femoral compartment cartilage thickness as the structural primary endpoint reported no statistically significant dose response. However, Sprifermin was associated with statistically significant, dose-dependent reductions in loss of total and lateral tibio-femoral cartilage thickness. Based on these preliminary promising data a post-hoc analysis of secondary assessment and endpoints was performed to evaluate potential effects of Sprifermin on semi-quantitatively evaluated structural MRI parameters. Aim of the present analysis was to determine effects of sprifermin on several knee joint tissues over a 12 month period.Methods1.5 T or 3 T MRIs were acquired at baseline and 12 months follow-up using a standard protocol. MRIs were read according to the Whole-Organ Magnetic Resonance Imaging Score (WORMS) scoring system (in 14 articular subregions) by four muskuloskeletal radiologists independently. Analyses focused on semiquantitative changes in the 100 μg subgroup and matching placebo of multiple MRI-defined structural alterations. Analyses included a delta-subregional and delta-sum approach for the whole knee and the medial and lateral tibio-femoral (MTFJ, LTFJ), and patello-femoral (PFJ) compartments, taking into account number of subregions showing no change, improvement or worsening and changes in the sum of subregional scores. Mann–Whitney − Wilcoxon tests assessed differences between groups.ResultsFifty-seven and 18 patients were included in the treatment and matched placebo subgroups. Less worsening of cartilage damage was observed from baseline to 12 months in the PFJ (0.02, 95 % confidence interval (CI) (−0.04, 0.08) vs. placebo 0.22, 95 % CI (−0.05, 0.49), p = 0.046). For bone marrow lesions (BMLs), more improvement was observed from 6 to 12 months for whole knee analyses (−0.14, 95 % CI (−0.48, 0.19) vs. placebo 0.44, 95 % CI (−0.15, 1.04), p = 0.042) although no significant effects were seen from the baseline visit, or in Hoffa-synovitis, effusion-synovitis, menisci and osteophytes.ConclusionsIn this post-hoc analysis cartilage showed less worsening from baseline to 12 months in the PFJ, and BMLs showed more improvement from 6 to 12 months for the whole knee.Trial registrationClinicalTrials.gov identifier: NCT01033994.

Relapsing polychondritis: A 2016 update on clinical features, diagnostic tools, treatment and biological drug use.

Relapsing polychondritis (RP) is a very rare autoimmune disease characterised by a relapsing inflammation of the cartilaginous tissues (joints, ears, nose, intervertebral discs, larynx, trachea and cartilaginous bronchi), which may progress to long-lasting atrophy and/or deformity of the cartilages. Non-cartilaginous tissuesmay also be affected, such as the eyes, heart, aorta, inner ear and skin. RP has a long and unpredictable course. Because no randomised therapeutic trials are available, the treatment of RP remains mainly empirical. Minor forms of the disease can be treated with non-steroidal anti-inflammatory drugs, whereas more severe forms are treated with systemic corticosteroids. Life-threatening diseasesand corticosteroid-dependent or resistant diseases are an indication for immunosuppressant therapy such as methotrexate, azathioprine, mycophenolate mofetil and cyclophosphamide. Biologics could be given as second-line treatment in patients with an active disease despite the use of steroids and immunosuppressive drugs. Although the biologics represent new potential treatment for RP, very scarce information is available to draw any firm conclusion on their use in RP.

Mitochondrial DNA Deletions are Associated with Upregulated Articular Nitrotyrosine in Human Femoral Head Cartilage

Background: The pathogenesis of osteoarthritis (OA) has been linked with the elaboration of increased amounts of nitrotyrosine in cartilage as a stable marker of tyrosine nitration by nitric oxide. Reactive oxygen species (ROS) are known to damage mitochondrial DNA (mtDNA); this process is associated with aging of non-cartilaginous tissues. This study aimed to link intraarticular nitrotyrosine content, with mtDNA lesions and OA severity in order to gain insights into the contribution of nitrotyrosine and mitochondria to cartilage aging and the development of OA. Methods: Femoral heads were prospectively obtained from individuals undergoing hip arthroplasty or postmortem autopsy (22 male, 26 female, age range 16-93 years). OA severity was graded radiographically, and histologically by means of the Mankin score. Articular chondrocytes were isolated, and the ‘common’ mtDNA deletion quantified with a Polymerase Chain Reaction (PCR) technique. mtDNA copy numbers per chondrocyte were determined by quantitative PCR. Articular nitrotyrosine content in chondrocytes was quantified by ELISA. Multivariate associations between parameters were computed by linear regression analysis or Spearman rank correlations, as appropriate. Results: Articular nitrotyrosine concentrations were independently correlated with both, the age of the subjects (r=0.39, p=0.01) and the presence of the common deletion (r=0.48, p=0.004), but not with the Mankin Score (p=0.84) or wild type mtDNA copy numbers. The severity of hip OA (Mankin score) however was only correlated with patient age, but not with articular nitrotyrosine, mtDNA deletions or wild type mtDNA copy numbers. Conclusions: mtDNA lesions are correlated with articular nitrotyrosine content in hip cartilage, but a link of mitochondrial mutagenesis and NO-mediated ROS formation with the age of the subjects, or with OA severity cannot be demonstrated in this study.

Be it resolved: plain radiography or MRI – which is better in assessing outcome in clinical trials?

Be it resolved: plain radiography or MRI – which is better in assessing outcome in clinical trials?

Characterization of a murine type IIB procollagen-specific antibody

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1+ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1+ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.

Deboning broiler chicken legs and wings by dislocation of articular cartilage followed by stripping periosteum

The yield of deboned meat is an important economic factor affecting the profit of the meat industry. This study was undertaken to determine whether the yield of boneless meat from broiler chicken leg (thigh and drumstick) and wing (drumette and winglet) is improved by introducing a new deboning method consisting of articular cartilage dislocation followed by stripping periosteum. A total of 44 broiler chicken carcasses were used in the deboning experiment. Right and left legs or wings from the first 22 carcasses were assigned to the new and ordinary hand deboning methods, respectively. For the remaining 22 carcasses, right and left legs or wings were assigned to the ordinary and new methods, respectively. The weight of residue, composed of bone and small amounts of cartilage and noncartilaginous tissues obtained after deboning, was then compared between the right and left legs or wings to see the difference between the 2 methods. The removal of tibia, fibula, humerus, radius, or ulna resulted in formation of a hollow in boneless meat obtained. There was no difference (P > 0.05) between the right and left legs or wings in the weight of residue obtained after deboning as expected. The weight of residue was less (P < 0.05) with the new method compared with the ordinary method in all chicken parts examined. The difference of residue weight between the 2 methods accounted for 10, 12, 14, and 21% of the weight of residue obtained by the ordinary method in thigh, drumstick, drumette, and winglet, respectively. The new method may be useful to deboners at home kitchens as well as the poultry meat industry. The present study also showed the development of a secondary ossification center at the proximal end of the carpometacarpus of chickens. This is, to our knowledge, the first report of development of secondary ossification center in chicken wings.

RNAi silencing of type V collagen in Schistosoma japonicum affects parasite morphology, spawning, and hatching

Type V collagen is a component of non-cartilaginous tissues and is important in the determination of fibril structure and matrix organization, although its functions are still poorly understood. In this report, RNA interference (RNAi) approaches were used to investigate the effects of knockdown of the schistosome type V collagen (SjColV) gene. In this study, three different short interfering (si) RNAs targeting different regions of the gene were designed to suppress the expression of SjColV in Schistosoma japonicum using a soaking method. By establishing controls for measuring off-target RNAi effects, we found that different siRNA sequences had different levels of effectiveness. Although all the siRNAs tested reduced SjColV transcript levels, the S1 siRNA consistently reduced SjColV expression to >99 % of the control. In the following experiments, S1 siRNA was adapted to inhibit SjColV expression, and the silencing effects were detected by real-time PCR and Western blot. The spawning and egg hatching of parasites were calculated, while the worms' morphology was taken by scanning electron microscopy. The results show that silencing the expression of SjColV significantly affects the spawning and egg hatching of S. japonicum, and it also affects the worms' morphology.

Collagen Type V Polymorphism in Spontaneous Quadriceps Tendon Ruptures

Spontaneous simultaneous bilateral quadriceps tendon rupture is associated with multiple medical conditions and pharmacological treatments; however, identifying prior risk factors is impossible in most cases. Achilles tendon and anterior cruciate ligament ruptures are associated with collagen, type V, alpha 1 (COL5A1) polymorphism. This genetic variant may be implicated quadriceps tendon rupture. The COL5A1 encodes the protein for pro-α1 chains of the low-abundance heterotrimeric type V collagen. In most noncartilaginous tissues, type V collagen is a quantitatively minor component of type I collagen that has been implicated in the regulation of the size and configuration of type I collagen fibrils. The functional significance of COL5A1 polymorphism in relation to type V collagen expression or activity has not been determined.This article describes a patient with COL5A1 polymorphism and spontaneous simultaneous quadriceps tendon rupture. However, genetic and histologic studies performed on blood and tendon tissues and 3 consecutive sex- and age-matched controls showed a statistically significant reduction in collagen type V expression and an alteration in collagen structure in the tendon. These findings might explain the pathomechanisms of spontaneous tendon ruptures associated with COL5A1 polymorphism.

Relapsing polychondritis: a description of a case and review article

Relapsing polychondritis (RPC) is a rare autoimmune disease in which the cartilaginous tissues are the target for inflammation and destruction, the associated immune reaction causes inflammation in non-cartilaginous tissues like kidney and blood vessels. This article provides a description of a case of RPC and a review article about the disease.

CCAAT/Enhancer-binding Protein β and NF-κB Mediate High Level Expression of Chemokine Genes CCL3 and CCL4 by Human Chondrocytes in Response to IL-1β*

A large set of chemokines is highly up-regulated in human chondrocytes in response to IL-1β (Sandell, L. J., Xing, X., Franz, C., Davies, S., Chang, L. W., and Patra, D. (2008) Osteoarthr. Cartil. 16, 1560-1571). To investigate the mechanism of transcriptional regulation, deletion constructs of selected chemokine gene promoters, the human CCL3 (MIP-1α) and CCL4 (MIP-1β), were transfected into human chondrocytes with or without IL-1β. The results show that an IL-1β-responsive element is located between bp -300 and -140 of the CCL3 promoter and between bp -222 and -100 of the CCL4 promoter. Because both of these elements contain CCAAT/enhancer-binding protein β (C/EBPβ) motifs, the function of C/EBPβ was examined. IL-1β stimulated the expression of C/EBPβ, and the direct binding of C/EBPβ to the C/EBPβ motif was confirmed by EMSA and ChIP analyses. The -300 bp CCL3 promoter and -222 bp CCL4 promoter were strongly up-regulated by co-transfection with the C/EBPβ expression vector. Mutation of the C/EBPβ motif and reduction of C/EBPβ expression by siRNA decreased the up-regulation. Additionally, another cytokine-related transcription factor, NF-κB, was also shown to be involved in the up-regulation of chemokines in response to IL-1β, and the binding site was identified. The regulation of C/EBPβ and NF-κB was confirmed by the inhibition by C/EBPβ and NF-κB and by transfection with C/EBPβ and NF-κB expression vectors in the presence or absence of IL-1β. Taken together, our results suggest that C/EBPβ and NF-κB are both involved in the IL-1β-responsive up-regulation of chemokine genes in human chondrocytes. Time course experiments indicated that C/EBPβ gradually and steadily induces chemokine up-regulation, whereas NF-κB activity was highest at the early stage of chemokine up-regulation.

Use of Autologous Bronchial Cartilage to Repair an Intraoperative Bronchial Airway Defect After Pneumonectomy for Lung Mesothelioma

Tracheobronchial defects may occur intraoperatively because of surgical trauma after resection of thoracic tumors. The repair of large, circumferential tracheobronchial defects with tissue loss may pose a major challenge. In an effort to solve this problem, different techniques have been used with little success. Attempts with foreign materials, nonviable tissues, autogenous noncartilaginous tissues, tissue engineering, and allotransplantation have all been associated with disappointing or suboptimal results. In each category, biologic problems are noted. Hence, the reconstruction and substitution of large, circumferential tracheobronchial defects has so far remained an unsolved surgical dilemma. We report a unique case in which an airway defect in the bronchus, made inadvertently intraoperatively, was repaired using autologous bronchial cartilage obtained from the resected specimen. In the literature, autologous bronchial cartilage has never been used before in reconstructing a large tracheobronchial defect.

Expression pattern of two collagen type 2 α1 genes in the Japanese inshore hagfish (Eptatretus burgeri) with special reference to the evolution of cartilaginous tissue

Collagen type 2 alpha1 (Col2A1) protein is a major component of the cartilaginous extracellular matrix (ECM) in vertebrates. Over the past two decades, the evolutionary origin of Col2A1 has been studied at the biochemical and molecular levels in extant jawless vertebrates (hagfishes and lampreys). Although these studies have contributed to our understanding of ECM protein evolution, the expression profile of the Col2A1 gene in hagfishes has not been fully described. We have performed molecular cloning and analyzed the gene expression pattern of the Col2A1 gene in the Japanese inshore hagfish (Eptatretus burgeri). We succeeded in isolating two Col2A1 genes, EbCol2A1A and EbCol2A1B, in which EbCol2A1A was expressed in the noncartilaginous connective tissues whereas EbCol2A1B was detected in some cartilaginous elements. Based on these results, we discuss the evolutionary history of Col2A1 genes in early vertebrates.

Extraction, Isolation and Analysis of Chondroitin Sulfate Glycosaminoglycans

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS) and chondroitin sulfate/dermatan sulfate (CS/DS) copolymers are anionic straight chain polysaccharides. They are galactosamine containing GAGs (galactosaminoglycans) having wide range of applications in pharmaceutical, cosmetic and food industries. This article reviews techniques to isolate and characterize these galactosaminoglycans from animal and poultry tissues. Patent based information is also discussed. Cartilaginous tissues are the major source of CS consisting entirely of D-glucuronosyl-N-acetylgalactosamine repeating disaccharide units, in which the galactosamine is sulfated at C4 or C6. In contrast, most galactosaminoglycans in non-cartilaginous connective tissues (e.g. skin and tendon) are CS/DS copolymers comprised of varying proportions of D-glucuronosyl-N-acetylgalactosamine and L-iduronosyl-N-acetylgalactosamine. Tissues are digested with proteinase (e.g. papain) to liberate GAGs, which are fractionated to isolate and purify galactosaminoglycans. Common techniques used for fractionation of GAGs include: precipitation with different concentrations of ethanol; solubilization of GAG precipitated as GAG-quarternary ammonium compound complexes with different concentrations of NaCl; anion exchange chromatography and gel filtration chromatography. Purified galactosaminoglycans are examined by various methods including chondroitinase digestion, high performance liquid chromatography and electrophoresis. Histological methods are used to localize galactosaminoglycans in tissues. The patent information on the CS hydrolase and ultraviolet irradiation may be useful for the preparation of CS oligosaccharide.

On the Horizon From the ORS

On the Horizon From the ORS