Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.

Chondrogenesis of human amniotic fluid stem cells in Chitosan-Xanthan scaffold for cartilage tissue engineering.

Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.

Ryanodine receptor 1 mediated dexamethasone-induced chondrodysplasia in fetal rats.

BACKGROUND: Osteoarthritis is caused by cartilage dysplasia and has fetal origin. Prenatal dexamethasone exposure (PDE) induced chondrodysplasia in fetal rats by inhibiting transforming growth factor ß (TGFß) signaling. This study aimed to determine the effect of dexamethasone on fetal cartilage development and illustrate the underlying molecular mechanism. METHODS: Dexamethasone (0.2 mg/kg.d) was injected subcutaneously every morning in pregnant rats from gestational day (GD) 9 to GD21. Harvested fetal femurs and tibias at GD21 for immunofluorescence and gene expression analysis. Fetal chondrocytes were treated with dexamethasone (100, 250 and 500 nM), endoplasmic reticulum stress (ERS) inhibitor, and ryanodine receptor 1 (RYR1) antagonist for subsequent analyses. RESULTS: In vivo, prenatal dexamethasone exposure (PDE) decreased the total length of the fetal cartilage, the proportion of the proliferation area and the cell density and matrix content in fetal articular cartilage. Moreover, PDE increased RYR1 expression and intracellular calcium levels and elevated the expression of ERS-related genes, while downregulated the TGFß signaling pathway and extracellular matrix (ECM) synthesis in fetal chondrocytes. In vitro, we verified dexamethasone significantly decreased ECM synthesis through activating RYR 1 mediated-ERS. CONCLUSIONS: PDE inhibited TGFß signaling pathway and matrix synthesis through RYR1 / intracellular calcium mediated ERS, which ultimately led to fetal dysplasia. This study confirmed the molecular mechanism of ERS involved in the developmental toxicity of dexamethasone and suggested that RYR1 may be an early intervention target for fetal-derived adult osteoarthritis.

The interplay between chondrocyte spheroids and mesenchymal stem cells boosts cartilage regeneration within a 3D natural-based hydrogel.

Articular cartilage (AC) lacks the ability to self-repair and cell-based approaches, primarily based on using chondrocytes and mesenchymal stem cells (MSCs), are emerging as effective technology to restore cartilage functionality, because cells synergic functionality may support the maintenance of chondrogenic phenotype and promote extracellular matrix regeneration. This work aims to develop a more physiologically representative co-culture system to investigate the influence of MSCs on the activity of chondrocytes. A thermo-sensitive chitosan-based hydrogel, ionically crosslinked with ß-glycerophosphate, is optimised to obtain sol/gel transition at physiological conditions within 5 minutes, high porosity with pores diameter <30 µm, and in vitro mechanical integrity with compressive and equilibrium Young's moduli of 37 kPa and 17 kPa, respectively. Live/dead staining showed that after 1 and 3 days in culture, the encapsulated MSCs into the hydrogels are viable and characterised by round-like morphology. Furthermore chondrocyte spheroids, seeded on top of gels that contained either MSCs or no cells, show that the encapsulated MSCs stimulate chondrocyte activity within a gel co-culture, both in terms of maintaining the coherence of chondrocyte spheroids, leading to a larger quantity of CD44 (by immunofluorescence) and a higher production of collagen and glycosaminoglycans (by histology) compared with the mono-culture.

Atomic Force Microscopy on Biological Materials Related to Pathological Conditions.

Atomic force microscopy (AFM) is an easy-to-use, powerful, high-resolution microscope that allows the user to image any surface and under any aqueous condition. AFM has been used in the investigation of the structural and mechanical properties of a wide range of biological matters including biomolecules, biomaterials, cells, and tissues. It provides the capacity to acquire high-resolution images of biosamples at the nanoscale and allows at readily carrying out mechanical characterization. The capacity of AFM to image and interact with surfaces, under physiologically relevant conditions, is of great importance for realistic and accurate medical and pharmaceutical applications. The aim of this paper is to review recent trends of the use of AFM on biological materials related to health and sickness. First, we present AFM components and its different imaging modes and we continue with combined imaging and coupled AFM systems. Then, we discuss the use of AFM to nanocharacterize collagen, the major fibrous protein of the human body, which has been correlated with many pathological conditions. In the next section, AFM nanolevel surface characterization as a tool to detect possible pathological conditions such as osteoarthritis and cancer is presented. Finally, we demonstrate the use of AFM for studying other pathological conditions, such as Alzheimer's disease and human immunodeficiency virus (HIV), through the investigation of amyloid fibrils and viruses, respectively. Consequently, AFM stands out as the ideal research instrument for exploring the detection of pathological conditions even at very early stages, making it very attractive in the area of bio- and nanomedicine.

Ciliated cell observation by SEM on the surface of human incudo-malleolar-joint articular cartilage: are they a new chondrocyte phenotype?

BACKGROUND: Scanning electron microscopy (SEM) study of the human incus bone is scanty whilst, to our knowledge, no information regarding human incudo-malleolar joint articular-cartilage morphology has previously been provided. AIMS/OBJECTIVES: Our aim was to shed some light on this morphological issue and to propose some theoretical perspectives on its functional role. MATERIAL AND METHODS: The human incudo-malleolar joint was documented with field emission SEM on samples recovered during ear surgery procedures after patients' informed consent. RESULTS: Normal articular cartilage chondrocytes, flattened cells with prominent nucleus and short microvilli were observed. Interestingly, cells provided with long cilia were identified. Type A cilia are arranged in a pyramidal formation with extra-long cilia stemming from the cluster, projecting upwards in an antenna-like formation ending with a dilated structure that as a whole, resembles the stereocilia with kinocilium. Types B, C and D cilia resemble those of the genital and respiratory tracts. CONCLUSIONS AND SIGNIFICANCE: It is therefore possible to hypothesize that the observed ciliated cells may be a new chondrocyte phenotype with sensory function. Motile cilia confer the ability to distinguish variations in synovial fluid chemical composition and, in addition, they perhaps may also play some role in the mechanism of sound transmission.

Effects of low-level laser therapy on the organization of articular cartilage in an experimental microcrystalline arthritis model.

The aim of this study was to evaluate the effects of low-level laser therapy using the gallium arsenide laser (λ = 830 nm) on the articular cartilage (AC) organization from knee joint in an experimental model of microcrystalline arthritis in adult male Wistar rats. Seventy-two animals were divided into three groups: A (control), B (induced arthritis), and C (induced arthritis + laser therapy). The arthritis was induced in the right knee using 2 mg of Na4P2O7 in 0.5 mL of saline solution. The treatments were daily applied in the patellar region of the right knee after 48 h of induction. On the 7th, 14th, and 21st days of treatment, the animals were euthanized and their right knees were removed and processed for structural and biochemical analysis of the AC. The chondrocytes positively labeled for the TUNEL reaction were lower in C than in B on the 14th and 21st days. The content of glycosaminoglycans and hydroxyproline in A and C was higher than B on the 21st day. The amount of tibial TNF-α in B and C was lower than in A. The amount of tibial BMP-7 in B and C was higher than in A. The femoral MMP-13 was lower in B and C than for A. The tibial TGF-ß for C was higher than the others. The femoral ADAMT-S4 content of A and C presented similar and inferior data to B on the 21st day. The AsGa-830 nm therapy preserved the content of glycosaminoglycans, reduced the cellular changes and the inflammatory process compared to the untreated group.

Induced lesion and inhibited Ihh-PTHrP signalling pathway activity in the articular cartilage of rats caused by T-2 toxin.

This study aims to investigate the role of Ihh-PTHrP signalling pathway in the articular cartilage injury of rats caused by T-2 toxin. Sixty male Wistar rats were randomly divided into four groups: group A, normal diet; group B, normal diet plus the dissolvent (0.9% sodium chloride sterile aqueous solution containing ethanol); group C, normal diet plus low T-2 toxin (0.1 mg/kg BW/day) and group D, normal diet plus high T-2 toxin (0.2 mg/kg BW/day) by intragastric administration daily for 4 weeks. Histological changes in articular cartilage were assessed by HE staining and scanning electron microscopy. The expression of Ihh and PTHrP in cartilage was assessed by immunohistochemistry. There is a significant difference in average weight gain between group A and group D P < 0.01, groups A and D P < 0.001, respectively. The result of scanning electron microscopy and HE staining showed that the damage of articular cartilage was much severe with the increase of T-2 toxin. Immunohistochemical analysis indicated that the expression of Ihh in group A and group B was higher than that of group C and group D (P < 0.05, <0.01, respectively). However, the expression of PTHrP was lower in group A and group B than that of group C and group D (P < 0.001, <0.001, respectively). These results indicated that T-2 toxin can cause the damage to articular cartilage and weight loss in rats. The effect of T-2 toxin on articular cartilage of rat may be related to the Ihh-PTHrP pathway.

Development of a novel hybrid bioactive hydrogel for future clinical applications.

Three-dimensional hydrogels are ideal for tissue engineering applications due to their structural integrity and similarity to native soft tissues; however, they can lack mechanical stability. Our objective was to develop a bioactive and mechanically stable hydrogel for clinical application. Auricular cartilage was decellularised using a combination of hypertonic and hypotonic solutions with and without enzymes to produce acellular tissue. Methacryloyl groups were crosslinked with alginate and PVA main chains via 2-aminoethylmathacrylate and the entire macromonomer further crosslinked with the acellular tissue. The resultant hydrogels were characterised for its physicochemical properties (using NMR), in vitro degradation (via GPC analysis), mechanical stability (compression tests) and in vitro biocompatibility (co-culture with bone marrow-derived mesenchymal stem cells). Following decellularisation, the cartilage tissue showed to be acellular at a significant level (DNA content 25.33 ng/mg vs. 351.46 ng/mg control tissue), with good structural and molecular integrity of the retained extra cellular matrix (s-GAG= 0.19 µg/mg vs. 0.65 µg/mg ±0.001 control tissue). Proteomic analysis showed that collagen subtypes and proteoglycans were retained, and SEM and TEM showed preserved matrix ultra-structure. The hybrid hydrogel was successfully cross-linked with biological and polymer components, and it was stable for 30 days in simulated body fluid (poly dispersal index for alginate with tissue was stable at 1.08 and for PVA with tissue was stable at 1.16). It was also mechanically stable (Young's modulus of 0.46 ± 0.31 KPa) and biocompatible, as it was able to support the development of a multi-cellular feature with active cellular proliferation in vitro. We have shown that it is possible to successfully combine biological tissue with both a synthetic and natural polymer and create a hybrid bioactive hydrogel for clinical application.

Procaine and saline have similar effects on articular cartilage and synovium in rat knee.

BACKGROUND: Intra-articular local anaesthetics are widely used for providing postoperative analgesia and decreasing the need for opioids. Procaine has proven positive effects in carpal tunnel syndrome and chondromalacia patella. However, the effect of procaine on articular cartilage has not yet been studied. The aim of this study was to evaluate the effects of intra-articular procaine injection on the articular cartilage and the synovium. METHODS: Twenty adult Sprague-Dawley rats were enrolled in the study. After providing anaesthesia and aseptic conditions, 0.25 ml of 10% procaine was injected to the right knee joint, and 0.25 ml of normal saline (as control group) was injected to the left knee joint. Knee joint samples were obtained from four rats in each group after appropriate euthanasia on days 1, 2, 7, 14 and 21. The histological sections of the articular and periarticular regions and the synovium were evaluated by two histologists, and inflammatory changes were graded according to a five-point scale in a blinded manner. The apoptosis of chondrocytes was determined by the caspase-3 indirect immunoperoxidase method. RESULTS: There were no significant differences in inflammation between procaine and saline groups at any of the time intervals. Slight inflammatory infiltration due to injection was seen in both groups on the 1st day. Haemorrhage was observed in both groups at days 1 and 2, and the difference between groups was not found to be significant. No significant difference was detected in the percentage of apoptotic chondrocytes between groups at any of the time intervals. CONCLUSIONS: Injection of procaine seems safe to use intra-articularly based on this in vivo study on rat knee cartilage. However, further studies investigating both the analgesic and histopathological effects of procaine on damaged articular cartilage and synovium models are needed.

Comparison of the effects of exercise with chondroitin sulfate on knee osteoarthritis in rabbits.

BACKGROUND: The aim of the study is to compare the effects of exercise therapy with chondroitin sulfate (CS) therapy in an experimental model of osteoarthritis (OA). METHODS: Twenty-one New Zealand rabbits were randomly divided into four groups: normal group (N group, n = 3); OA control group (C group, n = 6); OA plus medication group (CS group, n = 6); and OA plus exercise group (E group, n = 6). Four weeks after modeling, the rabbits were subjected to exercise (artificial, 30 min/time, 4 times/week) or medicated with CS (2% CS, 0.3 ml/time, once/week) for 4 weeks. Histopathological changes in treated joints were examined after staining. X-ray and scanning electron microscopy was used to evaluate the different therapies by examining the surfaces and joint spaces of the articular cartilage. RT-qPCR was used to assess chondrogenic gene expression including Col2, Col10, mmp-13, il-1ß, adamats-5, and acan in the experimental groups. RESULTS: Histology showed both treatment groups resulted in cartilage that was in good condition, with increased numbers of chondrocytes, and the results of X-ray and scanning electron microscopy showed the therapeutic effect of exercise therapy is equivalent to CS therapy, surface articular cartilage was flat, and the of cartilage layer was thinning. All treated groups induced the expression of Col10 and Col2 and decreased expression of mmp-13, il-1ß, and adamats-5 compared with the control groups. The expression of acan was upregulated in the E group and downregulated in the CS group. Furthermore, expression of Col10 was higher and il-1ß was lower in the exercise group compared to that of the CS group. CONCLUSION: These results indicate that exercise has a positive effect on OA compare with CS, and it also supplies reference for the movement mode to improve function.

Tenascin-C Prevents Articular Cartilage Degeneration in Murine Osteoarthritis Models.

Objective The objective of this study was to determine whether intra-articular injections of tenascin-C (TNC) could prevent cartilage damage in murine models of osteoarthritis (OA). Design Fluorescently labeled TNC was injected into knee joints and its distribution was examined at 1 day, 4 days, 1 week, 2 weeks, and 4 weeks postinjection. To investigate the effects of TNC on cartilage degeneration after surgery to knee joints, articular spaces were filled with 100 µg/mL (group I), 10 µg/mL (group II) of TNC solution, or control (group III). TNC solution of 10 µg/mL was additionally injected twice after 3 weeks (group IV) or weekly after 1 week, 2 weeks, and 3 weeks (group V). Joint tissues were histologically assessed using the Mankin score and the modified Chambers system at 2 to 8 weeks after surgery. Results Exogenous TNC was maintained in the cartilage and synovium for 1 week after administration. Histological scores in groups I and II were better than scores in group III at 4 and 6 weeks, but progressive cartilage damage was seen in all groups 8 weeks postoperatively. Sequential TNC injections (groups IV and V) showed significantly better Mankin score than single injection (group II) at 8 weeks. Conclusion TNC administered exogenously remained in the cartilage of knee joints for 1 week, and could decelerate articular cartilage degeneration in murine models of OA. We also showed that sequential administration of TNC was more effective than a single injection. TNC could be an important molecule for prevention of articular cartilage damage.

Biphasic hierarchical extracellular matrix scaffold for osteochondral defect regeneration.

OBJECTIVE: To investigate the effect of decellularized osteochondral extracellular matrix (ECM) scaffold for osteochondral defect regeneration. DESIGN: We compared the histological features and microstructure of degenerated cartilage to normal articular cartilage. We also generated and evaluated osteochondral ECM scaffolds through decellularization technology. Then scaffolds were implanted to osteochondral defect in rabbit model. After 12 weeks surgery, regeneration tissues were analyzed by histology, immunohistochemistry evaluation. And possible mechanisms of angiogenesis and cell migration were explored. RESULTS: We demonstrated decreased cell numbers, formation of fibrous cartilage, lost microstructure and worse permeability in degenerated cartilage compared to normal cartilage. We also generated an osteochondral ECM scaffold with a hierarchical structure that exhibited low immunogenicity, high bioactivity, and well biocompatibility. We found that the ECM scaffold promoted tissue regeneration in osteochondral defects, which was dependent on the scaffold constituents and stratified three-dimensional microstructure as well as on its ability to inhibit angiogenesis and stimulate cell migration. CONCLUSIONS: Our findings demonstrated that the biphasic hierarchical ECM scaffold represents a novel and effective biomaterial that can be used in the treatment of osteochondral defect.

Decellularization and Recellularization of Cartilage.

Decellularization of cartilage enables the use of cartilage allografts or xenografts as natural scaffolds for repair and regeneration of injured cartilage. The preservation of the extracellular matrix ultrastructure of the graft makes this a promising tool for cartilage tissue engineering. We have optimized the decellularization protocol by enzymatically digesting proteoglycans while preserving the native collagen architecture. Here we describe our methods for cartilage decellularization and cell labeling for the tracking of infiltration for recellularization in detail.

Development of a rat model of knee osteoarthritis by a combination of monoiodoacetate and streptozotocin / Desarrollo de un modelo de rata de osteoartritis de rodilla por una combinación de monoiodoacetato y estreptozotocina

SUMMARY: Osteoarthritis (OA) caused by ageing joints or as a secondary complication of diabetes is a common health problem. We sought to develop an animal model of OA induced by a combination of the chondrocyte glycolytic inhibitor mono-iodoacetate (MIA) and streptozotocin (STZ), the agent that induces diabetes mellitus. We then hypothesized that the extent of damages to the knee joint induced by this model can be greater than OA induced by either MIA or STZ. Rats were either injected with MIA (model 1) or STZ (model 2) or both agents (model 3). After 8 weeks, harvested tissues from the knee joint of these groups were examined using scanning and transmission electron microscopy. In addition, blood samples were assayed for tumor necrosis factor alpha (TNF-α) and interleukin -6 (IL-6) that are known to be modulated in OA and diabetes. Compared to control group, substantial damages to the articular cartilage of the knee joint were observed in the three models with the severest in model 3. In addition, rats in model 3 showed significant (P<0.0001) increase in TNF-α and IL-6 compared to model 1 and 2. Thus, we have developed a new model of knee OA in rats that mimics a type of OA that is common among elderly people who have both, "ageing" joints and diabetes.

RESUMEN: La osteoartritis (OA) es un problema generalizado de salud a causa de un envejecimiento de las articulaciones, o bien de una complicación secundaria de la diabetes. El objetivo de este estudio fue desarrollar un modelo animal de OA inducido por una combinación dos drogas, un inhibidor de los condrocitos glucolíticos, el mono-iodoacetato (MIA), y la estreptozotocina (STZ), agente que induce la diabetes mellitus. Se consideró como hipótesis que el alcance de los daños a la articulación de la rodilla inducida por este modelo puede ser mayor que la OA inducida por MIA o STZ. Las ratas fueron inyectadas con MIA (grupo 1) o STZ (grupo 2) o ambos agentes (grupo 3). Se extrajeron muestras de la articulación de la rodilla de estos grupos al término de 8 semanas, y se examinaron mediante microscopía electrónica de barrido y de transmisión. Además, se analizaron muestras de sangre para el factor de necrosis tumoral alfa (TNF-α) e interleucina-6 (IL-6), que están moduladas en OA y en la diabetes. En comparación con el grupo control, se observaron daños sustanciales en el cartílago articular de la articulación de la rodilla en los tres modelos, encontrándose los daños más severos en el grupo 3. Además, las ratas del grupo 3 mostraron un aumento significativo (P <0,0001) de los niveles de TNF-α e IL- 6, en comparación con los grupos 1 y 2. Hemos desarrollado un nuevo modelo de OA de rodilla en ratas que imita un tipo de OA el cual, además de la diabetes, es común entre las personas mayores con un nivel importante de daño en las articulaciones.

Application of autofluorescence robotic histology for quantitative evaluation of the 3-dimensional morphology of murine articular cartilage.

Murine models of osteoarthritis (OA) are increasingly important for understating pathogenesis and for testing new therapeutic approaches. Their translational potential is, however, limited by the reduced size of mouse limbs which requires a much higher resolution to evaluate their articular cartilage compared to clinical imaging tools. In experimental models, this tissue has been predominantly assessed by time-consuming histopathology using standardized semi-quantitative scoring systems. This study aimed to develop a novel imaging method for 3-dimensional (3D) histology of mouse articular cartilage, using a robotic system-termed here "3D histocutter"-which automatically sections tissue samples and serially acquires fluorescence microscopy images of each section. Tibiae dissected from C57Bl/6 mice, either naïve or OA-induced by surgical destabilization of the medial meniscus (DMM), were imaged using the 3D histocutter by exploiting tissue autofluorescence. Accuracy of 3D imaging was validated by ex vivo contrast-enhanced micro-CT and sensitivity to lesion detection compared with conventional histology. Reconstructions of tibiae obtained from 3D histocutter serial sections showed an excellent agreement with contrast-enhanced micro-CT reconstructions. Furthermore, osteoarthritic features, including articular cartilage loss and osteophytes, were also visualized. An in-house developed software allowed to automatically evaluate articular cartilage morphology, eliminating the subjectivity associated to semi-quantitative scoring and considerably increasing analysis throughput. The novelty of this methodology is, not only the increased throughput in imaging and evaluating mouse articular cartilage morphology starting from conventionally embedded samples, but also the ability to add the third dimension to conventional histomorphometry which might be useful to improve disease assessment in the model.

Biomimetic design and fabrication of multilayered osteochondral scaffolds by low-temperature deposition manufacturing and thermal-induced phase-separation techniques.

Integrative osteochondral repair is a useful strategy for cartilage-defect repair. To mimic the microenvironment, it is necessary that scaffolds effectively mimic the extracellular matrix of natural cartilage and subchondral bone. In this study, biomimetic osteochondral scaffolds containing an oriented cartilage layer, a compact layer, and a three-dimensional (3D)-printed core-sheath structured-bone layer were developed. The oriented cartilage layer was designed to mimic the structural and material characteristics of native cartilage tissue and was fabricated with cartilage matrix-chitosan materials, using thermal-induced phase-separation technology. The 3D-printed core-sheath structured-bone layer was fabricated with poly(L-lactide-co-glycolide)/ß-tricalcium phosphate-collagen materials by low-temperature deposition technology, using a specially designed core-sheath nozzle, and was designed to mimic the mechanical characteristics of subchondral bone and improve scaffold hydrophilicity. The compact layer was designed to mimic the calcified-layer structure of natural cartilage to ensure the presence of different suitable microenvironments for the regeneration of bone and cartilage. A dissolving-bonding process was developed to effectively combine the three parts together, after which the bone and cartilage scaffolds exhibited good mechanical properties and hydrophilicity. Additionally, goat autologous bone mesenchymal stem cells (BMSCs) were isolated and then seeded into the bone and cartilage layers, respectively, and following a 1 week culture in vitro, the BMSC-scaffold constructs were implanted into a goat articular-defect model. Our results indicated that the scaffolds exhibited good biocompatibility, and 24 weeks after implantation, the femoral condyle surface was relatively flat and consisted of a large quantity of hyaloid cartilage. Furthermore, histological staining revealed regenerated trabecular bone formed in the subchondral bone-defect area. These results provided a new method to fabricate biomimetic osteochondral scaffolds and demonstrated their effectiveness for future clinical applications in cartilage-defect repair.

Role of autophagy in the progression of osteoarthritis: The autophagy inhibitor, 3-methyladenine, aggravates the severity of experimental osteoarthritis.

Accumulating evidence suggests that autophagy is closely related to the pathogenesis of osteoarthritis (OA). The aim of this study was to determine the changes in autophagy during the progression of OA and to elucidate the specific role of autophagy in OA. For this purpose, a cellular model of OA was generated by stimulating SW1353 cells with interleukin (IL)-1ß and a rabbit model of OA was also established by an intra-articular injection of collagenase, followed by treatment with the autophagy specific inhibitor, 3-methyladenine (3-MA). Cell viability was analyzed by MTS assay, and the mRNA expression levels of matrix metalloproteinases (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by RT-qPCR. Cartilage degeneration was examined under a light microscope, and autophagosome and chondrocyte degeneration was observed by transmission electron microscopy. The protein expression of Beclin-1 and light chain 3 (LC3)B was evaluated by western blot analysis and immunofluorescence staining. We found that the autophagy was enhanced during the early stages and was weakened during the late stages of experimental OA. The inhibition of autophagy by 3-MA significantly aggravated the degeneration of chondrocytes and cartilage in experimental OA. Our results thus determine the changes in autophagy during different stages of OA, as well as the role of impaired autophagy in the development of OA. Our data suggest that the regulation of autophagy may be a potential therapeutic strategy with which to attenuate OA.

Is there crosstalk between subchondral bone, cartilage, and meniscus in the pathogenesis of osteoarthritis?

OBJECTIVES: This study aims to investigate if there is any crosstalk between subchondral bone, cartilage, and meniscus in the pathogenesis of osteoarthritis. PATIENTS AND METHODS: Twelve female patients (mean age 64 years; range 59 to 71 years) with osteoarthritis in medial compartment were included in the study. The samples of subchondral bone, cartilage and meniscus were obtained during total knee arthroplasty. Degenerated tissue samples obtained from medial compartment were used as the experimental group (12 samples of subchondral bone and cartilage, 1x1 cm each; and 12 samples of meniscus, 1x1 cm each). Healthy tissue samples obtained from lateral compartment were used as the control group (12 samples of subchondral bone and cartilage; 1x1 cm each; and 12 samples of meniscus, 1x1 cm each). After decalcification, tissue samples were evaluated with light and transmission electron microscopy. RESULTS: In the experimental group, light microscopic evaluation of subchondral bone samples demonstrated that the cartilage-to-bone transition region had an irregular structure. Degenerated cartilage cells were observed in the transition region and bone cells were significantly corrupted. In the experimental group, light microscopic evaluation of the meniscus samples demonstrated that the intercellular tissue was partly corrupted. Separation and concentration of the collagen fibers were evident. All findings were supported with ultra structural evaluations. CONCLUSION: Our findings indicate that degeneration of subchondral bone, cartilage, and meniscus probably plays a role in the pathogenesis of osteoarthritis with crosstalk.