[Early effectiveness of local injection of multimodal drug cocktail during anterior cruciate ligament reconstruction and its influence on cartilage].

Objective: To explore the early effectiveness and influence on cartilage of local injection of multimodal drug cocktail (MDC) during anterior cruciate ligament reconstruction (ACLR). Methods: Between February 2022 and August 2023, patients undergone arthroscopic ACLR using autologous hamstring tendons were selected as the study subjects. Among them, 90 patients met the selection criteria and were randomly divided into 3 groups ( n=30) according to the different injection drugs after ligament reconstruction. There was no significant difference in baseline data such as gender, age, body mass index, surgical side, disease duration, preoperative thigh circumference, and preoperative levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-1, matrix metalloproteinase 3 (MMP-3), MMP-13, and aggrecan (ACAN) in synovial fluid between groups ( P>0.05). After the ligament reconstruction during operation, corresponding MDC (consisting of ropivacaine, tranexamic acid, and betamethasone in group A, and ropivacaine, betamethasone, and saline in group B) or saline (group C) were injected into the joint and tendon site, respectively. The length of hospital stay, postoperative tramadol injection volume, incidence of complications, degree of knee joint swelling and range of motion, visual analogue scale (VAS) score, International Knee Documentation Committee (IKDC) score, Lyshlom score, and Hospital for Special Surgery (HSS) score were recorded and compared between groups. The T2 * values in different cartilage regions were detected by MRI examination and the levels of TNF-α, IL-6, IL-1, MMP-3, MMP-13, and ACAN in synovial fluid were detected by ELISA method. Results: The patients in group A, B, and C were followed up (12.53±3.24), (13.14±2.87), and (12.82±3.32) months, respectively. All incisions healed by first intention. Compared with group C, group A and group B had shorter length of hospital stay, less tramadol injection volume, and lower incidence of complications, showing significant differences ( P<0.05); there was no significant difference between group A and group B ( P>0.05). The degree of knee swelling in group A was significantly less than that in group B and group C ( P<0.05), but there was no significant difference between group B and group C ( P>0.05). At 3, 6, 12, 24, and 48 hours after operation, VAS scores of group A and group B were significantly lower than those of group C ( P<0.05); at 72 hours after operation, there was no significant difference among the three groups ( P>0.05). At 3 days, 14 days, and 1 month after operation, the range of motion of knee joint in group A were significantly better than those in group C ( P<0.05), and there was no significant difference between the other groups ( P>0.05). At 1 month after operation, the IKDC score of group A and group B was significantly higher than that of group C ( P<0.05); there was no significant difference among the three groups at other time points ( P>0.05). There was no significant difference in Lyshlom score and HSS score among the three groups at each time point ( P>0.05). At 14 days after operation, the levels of IL-1 and IL-6 in the synovial fluid in groups A and B were significantly lower than those in group C ( P<0.05). There was no significant difference in the levels of TNF-α, MMP-3, MMP-13, and ACAN between groups A and B ( P>0.05). At 1 month after operation, there was no significant difference in the above indicators among the three groups ( P>0.05). At 3, 6, and 12 months after operation, there was no significant difference in the T2 * values of different cartilage regions among the three groups ( P>0.05). Conclusion: Injecting MDC (ropivacaine, tranexamic acid, betamethasone) into the joint and tendon site during ACLR can achieve good early effectiveness without significant impact on cartilage.

An Isotonic Drink Containing Pacific Cod (Gadus macrocephalus) Processing Waste Collagen Hydrolysate for Bone and Cartilage Health.

Malnutrition is one of the major factors of bone and cartilage disorders. Pacific cod (Gadus macrocephalus) processing waste is a cheap and highly promising source of bioactive substances, including collagen-derived peptides and amino acids, for bone and cartilage structure stabilization. The addition of these substances to a functional drink is one of the ways to achieve their fast intestinal absorption. Collagen hydrolysate was obtained via enzymatic hydrolysis, ultrafiltration, freeze-drying, and grinding to powder. The lyophilized hydrolysate was a light gray powder with high protein content (>90%), including collagen (about 85% of total protein) and a complete set of essential and non-essential amino acids. The hydrolysate had no observed adverse effect on human mesenchymal stem cell morphology, viability, or proliferation. The hydrolysate was applicable as a protein food supply or a structure-forming food component due to the presence of collagen fiber fragments. An isotonic fitness drink (osmolality 298.1 ± 2.1 mOsm/L) containing hydrolysate and vitamin C as a cofactor in collagen biosynthesis was prepared. The addition of the hydrolysate did not adversely affect its organoleptic parameters. The production of such functional foods and drinks is one of the beneficial ways of fish processing waste utilization.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-ß1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-ß1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Chondrocyte-targeted exosome-mediated delivery of Nrf2 alleviates cartilaginous endplate degeneration by modulating mitochondrial fission.

BACKGROUND: Cartilaginous endplate (CEP) degeneration, which is an important contributor to intervertebral disc degeneration (IVDD), is characterized by chondrocyte death. Accumulating evidence has revealed that dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and dysfunction lead to apoptosis during CEP degeneration and IVDD. Exosomes are promising agents for the treatment of many diseases, including osteoporosis, osteosarcoma, osteoarthritis and IVDD. Despite their major success in drug delivery, the full potential of exosomes remains untapped. MATERIALS AND METHODS: In vitro and in vivo models of CEP degeneration were established by using lipopolysaccharide (LPS). We designed genetically engineered exosomes (CAP-Nrf2-Exos) expressing chondrocyte-affinity peptide (CAP) on the surface and carrying the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). The affinity between CAP-Nrf2-Exos and CEP was evaluated by in vitro internalization assays and in vivo imaging assays. qRT‒PCR, Western blotting and immunofluorescence assays were performed to examine the expression level of Nrf2 and the subcellular localization of Nrf2 and Drp1. Mitochondrial function was measured by the JC-1 probe and MitoSOX Red. Mitochondrial morphology was visualized by MitoTracker staining and transmission electron microscopy (TEM). After subendplate injection of the engineered exosomes, the degree of CEP degeneration and IVDD was validated radiologically and histologically. RESULTS: We found that the cargo delivery efficiency of exosomes after cargo packaging was increased by surface modification. CAP-Nrf2-Exos facilitated chondrocyte-targeted delivery of Nrf2 and activated the endogenous antioxidant defence system in CEP cells. The engineered exosomes inhibited Drp1 S616 phosphorylation and mitochondrial translocation, thereby preventing mitochondrial fragmentation and dysfunction. LPS-induced CEP cell apoptosis was alleviated by CAP-Nrf2-Exo treatment. In a rat model of CEP degeneration, the engineered exosomes successfully attenuated CEP degeneration and IVDD and exhibited better repair capacity than natural exosomes. CONCLUSION: Collectively, our findings showed that exosome-mediated chondrocyte-targeted delivery of Nrf2 was an effective strategy for treating CEP degeneration.

Lumbar instability remodels cartilage endplate to induce intervertebral disc degeneration by recruiting osteoclasts via Hippo-CCL3 signaling.

Degenerated endplate appears with cheese-like morphology and sensory innervation, contributing to low back pain and subsequently inducing intervertebral disc degeneration in the aged population.1 However, the origin and development mechanism of the cheese-like morphology remain unclear. Here in this study, we report lumbar instability induced cartilage endplate remodeling is responsible for this pathological change. Transcriptome sequencing of the endplate chondrocytes under abnormal stress revealed that the Hippo signaling was key for this process. Activation of Hippo signaling or knockout of the key gene Yap1 in the cartilage endplate severed the cheese-like morphological change and disc degeneration after lumbar spine instability (LSI) surgery, while blocking the Hippo signaling reversed this process. Meanwhile, transcriptome sequencing data also showed osteoclast differentiation related gene set expression was up regulated in the endplate chondrocytes under abnormal mechanical stress, which was activated after the Hippo signaling. Among the discovered osteoclast differentiation gene set, CCL3 was found to be largely released from the chondrocytes under abnormal stress, which functioned to recruit and promote osteoclasts formation for cartilage endplate remodeling. Over-expression of Yap1 inhibited CCL3 transcription by blocking its promoter, which then reversed the endplate from remodeling to the cheese-like morphology. Finally, LSI-induced cartilage endplate remodeling was successfully rescued by local injection of an AAV5 wrapped Yap1 over-expression plasmid at the site. These findings suggest that the Hippo signaling induced osteoclast gene set activation in the cartilage endplate is a potential new target for the management of instability induced low back pain and lumbar degeneration.

Transforming growth factor-ß1-loaded RADA-16 hydrogel scaffold for effective cartilage regeneration.

Cartilage repair remains a major challenge in clinical trials. These current cartilage repair materials can not effectively promote chondrocyte generation, limiting their practical application in cartilage repair. In this work, we develop an implantable scaffold of RADA-16 peptide hydrogel incorporated with TGF-ß1 to provide a microenvironment for stem cell-directed differentiation and chondrocyte adhesion growth. The longest release of growth factor TGF-ß1 release can reach up to 600 h under physiological conditions. TGF-ß1/RADA-16 hydrogel was demonstrated to be a lamellar porous structure. Based on the cell culture with hBMSCs, TGF-ß1/RADA-16 hydrogel showed excellent ability to promote cell proliferation, directed differentiation into chondrocytes, and functional protein secretion. Within 14 days, 80% of hBMSCs were observed to be directed to differentiate into vigorous chondrocytes in the co-culture of TGF-ß1/RADA-16 hydrogels with hBMSCs. Specifically, these newly generated chondrocytes can secrete and accumulate large amounts of collagen II within 28 days, which can effectively promote the formation of cartilage tissue. Finally, the exploration of RADA-16 hydrogel-based scaffolds incorporated with TGF-ß1 bioactive species would further greatly promote the practical clinical trials of cartilage remediation, which might have excellent potential to promote cartilage regeneration in areas of cartilage damage.

Bioprinted biomimetic hydrogel matrices guiding stem cell aggregates for enhanced chondrogenesis and cartilage regeneration.

Articular cartilage tissue has limited self-repair capabilities, with damage frequently progressing to irreversible degeneration. Engineered tissues constructed through bioprinting and embedded with stem cell aggregates offer promising therapeutic alternatives. Aggregates of bone marrow mesenchymal stromal cells (BMSCs) demonstrate enhanced and more rapid chondrogenic differentiation than isolated cells, thus facilitating cartilage repair. However, it remains a key challenge to precisely control biochemical microenvironments to regulate cellular adhesion and cohesion within bioprinted matrices simultaneously. Herein, this work reports a bioprintable hydrogel matrix with high cellular adhesion and aggregation properties for cartilage repair. The hydrogel comprises an enhanced cell-adhesive gelatin methacrylate and a cell-cohesive chitosan methacrylate (CHMA), both of which are subjected to photo-initiated crosslinking. By precisely adjusting the CHMA content, the mechanical stability and biochemical cues of the hydrogels are finely tuned to promote cellular aggregation, chondrogenic differentiation and cartilage repair implantation. Multi-layer constructs encapsulated with BMSCs, with high cell viability reaching 91.1%, are bioprinted and photo-crosslinked to support chondrogenic differentiation for 21 days. BMSCs rapidly form aggregates and display efficient chondrogenic differentiation both on the hydrogels and within bioprinted constructs, as evidenced by the upregulated expression of Sox9, Aggrecan and Collagen 2a1 genes, along with high protein levels. Transplantation of these BMSC-laden bioprinted hydrogels into cartilaginous defects demonstrates effective hyaline cartilage repair. Overall, this cell-responsive hydrogel scaffold holds immense promise for applications in cartilage tissue engineering.

Role of Galectin-3 in intervertebral disc degeneration: an experimental study.

BACKGROUND: This study investigated the role of Galectin-3 in the degeneration of intervertebral disc cartilage. METHODS: The patients who underwent lumbar spine surgery due to degenerative disc disease were recruited and divided into Modic I, Modic II, and Modic III; groups. HE staining was used to detect the pathological changes in endplates. The changes of Galectin-3, MMP3, Aggrecan, CCL3, and Col II were detected by immunohistochemistry, RT-PCR, and Western blot. MTT and flow cytometry were used to detect cartilage endplate cell proliferation, cell cycle, and apoptosis. RESULTS: With the progression of degeneration (from Modic I to III), the chondrocytes and density of the cartilage endplate of the intervertebral disc decreased, and the collagen arrangement of the cartilage endplate of the intervertebral disc was broken and calcified. Meanwhile, the expressions of Aggrecan, Col II, Galectin-3, Aggrecan, and CCL3 gradually decreased. After treatment with Galectin-3 inhibitor GB1107, the proliferation of rat cartilage end plate cells was significantly reduced (P < 0.05). GB1107 (25 µmol/L) also significantly promoted the apoptosis of cartilage endplate cells (P < 0.05). Moreover, the percentage of cartilage endplate cells in the G1 phase was significantly higher, while that in the G2 and S phases was significantly lower (P < 0.05). Additionally, the mRNA and protein expression levels of MMP3, CCL3, and Aggrecan in rat cartilage end plate cells were lower than those in the control group. CONCLUSIONS: Galectin-3 decreases with the progression of the cartilage endplate degeneration of the intervertebral disc. Galectin-3 may affect intervertebral disc degeneration by regulating the degradation of the extracellular matrix.

Induced pluripotent stem cells in cartilage tissue engineering: a literature review.

Osteoarthritis (OA) is a long-term, persistent joint disorder characterized by bone and cartilage degradation, resulting in tightness, pain, and restricted movement. Current attempts in cartilage regeneration are cell-based therapies using stem cells. Multipotent stem cells, such as mesenchymal stem cells (MSCs), and pluripotent stem cells, such as embryonic stem cells (ESCs), have been used to regenerate cartilage. However, since the discovery of human-induced pluripotent stem cells (hiPSCs) in 2007, it was seen as a potential source for regenerative chondrogenic therapy as it overcomes the ethical issues surrounding the use of ESCs and the immunological and differentiation limitations of MSCs. This literature review focuses on chondrogenic differentiation and 3D bioprinting technologies using hiPSCS, suggesting them as a viable source for successful tissue engineering. METHODS: A literature search was conducted using scientific search engines, PubMed, MEDLINE, and Google Scholar databases with the terms 'Cartilage tissue engineering' and 'stem cells' to retrieve published literature on chondrogenic differentiation and tissue engineering using MSCs, ESCs, and hiPSCs. RESULTS: hiPSCs may provide an effective and autologous treatment for focal chondral lesions, though further research is needed to explore the potential of such technologies. CONCLUSIONS: This review has provided a comprehensive overview of these technologies and the potential applications for hiPSCs in regenerative medicine.

The Role of Copper-Induced M2 Macrophage Polarization in Protecting Cartilage Matrix in Osteoarthritis.

BACKGROUND The pathological mechanism of osteoarthritis is still unclear. The regulation of the immune microenvironment has been of growing interest in the progression and treatment of osteoarthritis. Macrophages with different phenotypes, producing different cytokines, have been linked to the mechanism of cartilage injury in osteoarthritis. Copper ions play a role in the immune response and are involved in the pathological mechanisms of osteoarthritis by affecting the metabolism of the cartilage matrix. Bioactive glass (BG) is an osteogenic material with superior biocompatibility. Here, we report on the regulatory behavior of macrophages using a copper-based composite BG material. MATERIAL AND METHODS Cu-BGC powder was prepared by sol-gel method, and scaffolds were fabricated and characterized using 3D printing. Macrophage cultures grown with Cu-BGC were examined for cell culture and proliferation. The effect of Cu-BGC on the degradation metabolism of chondrocytes, cultured in the environment of inflammatory cytokine IL-1ß, was determined. In addition, the morphology of macrophages, secretion of inflammatory cytokines, and expression of surface markers were examined. RESULTS The results show that Cu-BGC promotes macrophage proliferation at a range of concentrations and increases the secretion of anti-inflammatory cytokines while inhibiting proinflammatory cytokines. At the same time, M2-type cell surface markers are definitely expressed and the morphology of macrophages is altered. In addition, Cu-BGC inhibited the degradation metabolism of chondrocytes in the inflammatory environment induced by IL-1ß. CONCLUSIONS These results suggest that Cu-BGC induced macrophage polarization into an M2 type anti-inflammatory phenotype, and inhibition of immune injury response may play a role in delaying cartilage matrix damage in osteoarthritis.

Surface functionalization of exosomes for chondrocyte-targeted siRNA delivery and cartilage regeneration.

Osteoarthritis (OA) is the most prevalent degenerative cartilage disease, but no effective treatment is currently available to ameliorate the dysregulation of cartilage catabolism. Cartilage degeneration is closely related to the change in the physiology of chondrocytes: for example, chondrocytes of the OA patients overexpress matrix metallopeptidase 13 (MMP13), a.k.a. collagenase 3, which damages the extracellular matrix (ECM) of the cartilage and deteriorate the disease progression. Inhibiting MMP13 has shown to be beneficial for OA treatments, but delivering therapeutics to the chondrocytes embedded in the dense cartilage is a challenge. Here, we engineered the exosome surface with the cartilage affinity peptide (CAP) through lipid insertion to give chondrocyte-targeting exosomes, CAP-Exo, which was then loaded with siRNA against MMP13 (siMMP13) in the interior to give CAP-Exo/siMMP13. Intra-articular administration of CAP-Exo/siMMP13 reduced the MMP13 level and increased collagen COL2A1 and proteoglycan in cartilage in a rat model of anterior cruciate ligament transection (ACLT)-induced OA. Proteomic analysis showed that CAP-Exo/siMMP13 treatment restored the altered protein levels in the IL-1ß-treated chondrocytes. Taken together, a facile exosome engineering method enabled targeted delivery of siRNA to chondrocytes and chondrocyte-specific silencing of MMP13 to attenuate cartilage degeneration.

Pluronic F127 hydrogel-loaded extracellular vesicles from adipose-derived mesenchymal stem cells promote tracheal cartilage regeneration via SCNN1B delivery.

Extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (AMSC-EVs) have been highlighted as a cell-free therapy due to their regenerative capability to enhance tissue and organ regeneration. Herein, we aimed to examine the mechanism of PF127-hydrogel@AMSC-EVs in promoting tracheal cartilage defect repair. Based on bioinformatics methods, SCNN1B was identified as a key gene for the osteogenic differentiation of AMSCs induced by AMSC-EVs. EVs were isolated from rat AMSCs and then loaded onto thermo-sensitive PF-127 hydrogel to develop PF127-hydrogel@AMSC-EVs. It was established that PF127-hydrogel@AMSC-EVs could effectively deliver SCNN1B into AMSCs, where SCNN1B promoted AMSC osteogenic differentiation. The promotive effect was evidenced by enhanced ALP activity, extracellular matrix mineralization, and expression of s-glycosaminoglycan, RUNX2, OCN, collagen II, PERK, and ATF4. Furthermore, the in vivo experiments revealed that PF127-hydrogel@AMSC-SCNN1B-EVs stimulated tracheal cartilage regeneration in rats through PERK/ATF4 signaling axis activation. Therefore, PF127-hydrogel@AMSC-SCNN1B-EVs may be a novel cell-free biomaterial to facilitate tracheal cartilage regeneration and cartilage injury repair.

Comparison of Minced Cartilage Implantation with Autologous Chondrocyte Transplantation in an In Vitro Inflammation Model.

The current gold standard to treat large cartilage defects is autologous chondrocyte transplantation (ACT). As a new surgical method of cartilage regeneration, minced cartilage implantation (MCI) is increasingly coming into focus. The aim of this study is to investigate the influence of chondrogenesis between isolated and cultured chondrocytes compared to cartilage chips in a standardized inflammation model with the proinflammatory cytokine TNFα. Articular chondrocytes from bovine cartilage were cultured according to the ACT method to passage 3 and transferred to spheroid culture. At the same time, cartilage was fragmented (<1 mm3) to produce cartilage chips. TNFα (20 ng/mL) was supplemented to simulate an inflammatory process. TNFα had a stronger influence on the passaged chondrocytes compared to the non-passaged ones, affecting gene expression profiles differently between isolated chondrocytes and cartilage chips. MCI showed less susceptibility to TNFα, with reduced IL-6 release and less impact on inflammation markers. Biochemical and histological analyses supported these findings, showing a greater negative influence of TNFα on the passaged pellet cultures compared to the unpassaged cells and MCI constructs. This study demonstrated the negative influence of TNFα on chondrogenesis in a chondrocyte spheroid culture and cartilage fragment model. Passaged chondrocytes are more sensitive to cytokine influences compared to non-passaged cells and chondrons. This suggests that MCI may have superior regeneration potential in osteoarthritic conditions compared to ACT. Further investigations are necessary for the translation of these findings into clinical practice.

Topological defects in self-assembled patterns of mesenchymal stromal cells in vitro are predictive attributes of condensation and chondrogenesis.

Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration.

TfR1 mediated iron metabolism dysfunction as a potential therapeutic target for osteoarthritis.

OBJECTIVE: Transferrin receptor-1 (TfR1) plays important roles in controlling cellular iron levels, but its role in OA pathology is unknown. Herein we aim to investigate the role of TfR1 in OA progression and its underlying mechanisms. METHODS: TfR1 expression in cartilage during OA development were examined both in vivo and in vitro. Then IL-1ß was used to induce chondrocytes degeneration in vitro and TfR1 siRNA was used for observing the effect of TfR1 in modulating iron homeostasis, mitochondrial function and degrading enzymes expression. Also the inhibitor of TfR1 was exploited to analyze the protective effect of TfR1 inhibition in vivo. RESULTS: TfR1 is elevated in OA cartilage and contributes to OA inflammation condition. Excess iron not only results in oxidative stress damage and sensitizes chondrocytes to ferroptosis, but also triggers c-GAS/STING-mediated inflammation by promoting mitochondrial destruction and the release of mtDNA. Silencing TfR1 using TfR1 siRNA not only reduced iron content in chondrocytes and inhibited oxidative stress, but also facilitated the mitophagy process and suppressed mtDNA/cGAS/STING-mediated inflammation. Importantly, we also found that Ferstatin II, a novel and selective TfR1 inhibitor, could substantially suppress TfR1 activity both in vivo and in vitro and ameliorated cartilage degeneration. CONCLUSION: Our work demonstrates that TfR1 mediated iron influx plays important roles in chondrocytes degeneration and OA pathogenesis, suggesting that maintaining iron homeostasis through the targeting of TfR1 may represent a novel therapeutic strategy for the treatment of OA.

Engineered human osteoarthritic cartilage organoids.

The availability of human cell-based models capturing molecular processes of cartilage degeneration can facilitate development of disease-modifying therapies for osteoarthritis [1], a currently unmet clinical need. Here, by imposing specific inflammatory challenges upon mesenchymal stromal cells at a defined stage of chondrogenesis, we engineered a human organotypic model which recapitulates main OA pathological traits such as chondrocyte hypertrophy, cartilage matrix mineralization, enhanced catabolism and mechanical stiffening. To exemplify the utility of the model, we exposed the engineered OA cartilage organoids to factors known to attenuate pathological features, including IL-1Ra, and carried out mass spectrometry-based proteomics. We identified that IL-1Ra strongly reduced production of the transcription factor CCAAT/enhancer-binding protein beta [2] and demonstrated that inhibition of the C/EBPß-activating kinases could revert the degradative processes. Human OA cartilage organoids thus represent a relevant tool towards the discovery of new molecular drivers of cartilage degeneration and the assessment of therapeutics targeting associated pathways.

Open-label, single-center, clinical study evaluating the safety, tolerability and clinical effects of pentosan polysulfate sodium in subjects with mucopolysaccharidosis I.

Lysosomal enzyme deficiency in mucopolysaccharidosis (MPS) I results in glycosaminoglycan (GAG) accumulation leading to pain and limited physical function. Disease-modifying treatments for MPS I, enzyme replacement, and hematopoietic stem cell therapy (HSCT), do not completely resolve MPS I symptoms, particularly skeletal manifestations. The GAG reduction, anti-inflammatory, analgesic, and tissue remodeling properties of pentosan polysulfate sodium (PPS) may provide disease-modifying treatment for musculoskeletal symptoms and joint inflammation in MPS I following ERT and/or HSCT. The safety and efficacy of PPS were evaluated in four subjects with MPS I aged 14-19 years, previously treated with ERT and/or HSCT. Subjects received doses of 0.75 mg/kg or 1.5 mg/kg PPS via subcutaneous injections weekly for 12 weeks, then every 2 weeks for up to 72 weeks. PPS was well tolerated at both doses with no serious adverse events. MPS I GAG fragment (UA-HNAc [1S]) levels decreased at 73 weeks. Cartilage degradation biomarkers serum C-telopeptide of crosslinked collagen (CTX) type I (CTX-I) and type II (CTX-II) and urine CTX-II decreased in all subjects through 73 weeks. PROMIS scores for pain interference, pain behavior, and fatigue decreased in all subjects through 73 weeks. Physical function, measured by walking distance and dominant hand function, improved at 49 and 73 weeks. Decreased GAG fragments and cartilage degradation biomarkers, and positive PROMIS outcomes support continued study of PPS as a potential disease-modifying treatment for MPS I with improved pain and function outcomes.

Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids.

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

Microbiome dysbiosis by antibiotics protects cartilage degradation in OAOP mice.

The role of this study was to evaluate the impact of gut microbiota depletion on the progression of osteoarthritis (OA) and osteoporosis (OP). We conducted an experimental mouse model of OA and OP over an 8-week period. The model involved destabilization of the medial meniscus and bilateral ovariectomy (OVX). To deplete the gut microbiota, we administered a course of antibiotics for 8 weeks. The severity of OA was assessed through micro-CT scanning, X-rays, and immunohistochemical staining. Microbiome analysis was performed using PCR of 16S DNA on fecal samples, and the levels of serum lipopolysaccharide, interleukin 6, tumor necrosis factor-α (TNF-α), osteocalcin, and estrogen were measured using enzyme-linked immunosorbent assay. We found that in comparison to the OVX+OA group, the OVX+OA+ABT group exhibited increased bone mineral density (P < 0.0001), bone volume fraction (P = 0.0051), and trabecular number (P = 0.0023) in the metaphyseal bone. Additionally, cartilage injury and levels of matrix metalloproteinase 13 were reduced in the OVX+OA+ABT group compared to the OVX+OA group. Moreover, the OVX+OA+ABT group demonstrated decreased relative abundance of Bacteroidetes, serum lipopolysaccharide (P = 0.0005), TNF-α (P < 0.0001), CTX-1 (P = 0.0002), and increased expression of bone formation markers. These findings were further supported by correlation network analyses. Depletion of gut microbiota was shown to protect against bone loss and cartilage degradation by modulating the composition of the gut microbiota in osteoporosis and osteoarthritis.

Anaphylatoxins and their corresponding receptors as potential drivers in cartilage calcification during osteoarthritis progression.

OBJECTIVE: The complement cascade as major fluid phase innate immune system is activated during progression of osteoarthritis (OA). Generated anaphylatoxins and the corresponding receptors C3aR and C5aR1 are associated with the calcification of blood vessels and involved in osteogenic differentiation. This study aims on elucidating whether complement activation products contribute to cartilage calcification of OA cartilage. METHOD: Human articular chondrocytes were osteogenically differentiated in vitro in the presence or absence of C3a, C5a, and bone morphogenetic protein (BMP) 2. Furthermore, macroscopically intact (OARSI grade ≤ 1) and highly degenerated human cartilage (OARSI grade ≥ 3) was used for C3aR and C5aR1 histochemistry. Calcification of the cartilage was assessed by Alizarin Red S and von Kossa staining. RESULTS: C3a and C5a amplified matrix mineralization during in vitro osteogenesis, while inhibition of the corresponding receptors impaired calcium deposition. Moreover, C3aR and C5aR1 expression was upregulated during osteogenic differentiation and also in degenerated cartilage. Additionally, anaphylatoxin receptor expression was positively associated with calcification of native cartilage tissue and calcium deposition during osteogenic differentiation. Finally, the pro-hypertrophic growth factor BMP2 induced the expression of C5aR1. CONCLUSIONS: Our findings indicate that anaphylatoxins and their receptors play a decisive role in cartilage calcification processes during OA progression.