Emerging technology has a brilliant future: the CRISPR-Cas system for senescence, inflammation, and cartilage repair in osteoarthritis.

Osteoarthritis (OA), known as one of the most common types of aseptic inflammation of the musculoskeletal system, is characterized by chronic pain and whole-joint lesions. With cellular and molecular changes including senescence, inflammatory alterations, and subsequent cartilage defects, OA eventually leads to a series of adverse outcomes such as pain and disability. CRISPR-Cas-related technology has been proposed and explored as a gene therapy, offering potential gene-editing tools that are in the spotlight. Considering the genetic and multigene regulatory mechanisms of OA, we systematically review current studies on CRISPR-Cas technology for improving OA in terms of senescence, inflammation, and cartilage damage and summarize various strategies for delivering CRISPR products, hoping to provide a new perspective for the treatment of OA by taking advantage of CRISPR technology.

[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.

Glucosamine and Silibinin Alter Cartilage Homeostasis through Glycosylation and Cellular Stresses in Human Chondrocyte Cells.

Osteoarthritis is more prevalent than any other form of arthritis and is characterized by the progressive mechanical deterioration of joints. Glucosamine, an amino monosaccharide, has been used for over fifty years as a dietary supplement to alleviate osteoarthritis-related discomfort. Silibinin, extracted from milk thistle, modifies the degree of glycosylation of target proteins, making it an essential component in the treatment of various diseases. In this study, we aimed to investigate the functional roles of glucosamine and silibinin in cartilage homeostasis using the TC28a2 cell line. Western blots showed that glucosamine suppressed the N-glycosylation of the gp130, EGFR, and N-cadherin proteins. Furthermore, both glucosamine and silibinin differentially decreased and increased target proteins such as gp130, Snail, and KLF4 in TC28a2 cells. We observed that both compounds dose-dependently induced the proliferation of TC28a2 cells. Our MitoSOX and DCFH-DA dye data showed that 1 µM glucosamine suppressed mitochondrial reactive oxygen species (ROS) generation and induced cytosol ROS generation, whereas silibinin induced both mitochondrial and cytosol ROS generation in TC28a2 cells. Our JC-1 data showed that glucosamine increased red aggregates, resulting in an increase in the red/green fluorescence intensity ratio, while all the tested silibinin concentrations increased the green monomers, resulting in decreases in the red/green ratio. We observed increasing subG1 and S populations and decreasing G1 and G2/M populations with increasing amounts of glucosamine, while increasing amounts of silibinin led to increases in subG1, S, and G2/M populations and decreases in G1 populations in TC28a2 cells. MTT data showed that both glucosamine and silibinin induced cytotoxicity in TC28a2 cells in a dose-dependent manner. Regarding endoplasmic reticulum stress, both compounds induced the expression of CHOP and increased the level of p-eIF2α/eIF2α. With respect to O-GlcNAcylation status, glucosamine and silibinin both reduced the levels of O-GlcNAc transferase and hypoxia-inducible factor 1 alpha. Furthermore, we examined proteins and mRNAs related to these processes. In summary, our findings demonstrated that these compounds differentially modulated cellular proliferation, mitochondrial and cytosol ROS generation, the mitochondrial membrane potential, the cell cycle profile, and autophagy. Therefore, we conclude that glucosamine and silibinin not only mediate glycosylation modifications but also regulate cellular processes in human chondrocytes.

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.

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.

Increased water temperature contributes to a chondrogenesis response in the eyes of spotted wolffish.

Adult vertebrate cartilage is usually quiescent. Some vertebrates possess ocular scleral skeletons composed of cartilage or bone. The morphological characteristics of the spotted wolffish (Anarhichas minor) scleral skeleton have not been described. Here we assessed the scleral skeletons of cultured spotted wolffish, a globally threatened marine species. The healthy spotted wolffish we assessed had scleral skeletons with a low percentage of cells staining for the chondrogenesis marker sex-determining region Y-box (Sox) 9, but harboured a population of intraocular cells that co-express immunoglobulin M (IgM) and Sox9. Scleral skeletons of spotted wolffish with grossly observable eye abnormalities displayed a high degree of perochondrial activation as evidenced by cellular morphology and expression of proliferating cell nuclear antigen (PCNA) and phosphotyrosine. Cells staining for cluster of differentiation (CD) 45 and IgM accumulated around sites of active chondrogenesis, which contained cells that strongly expressed Sox9. The level of scleral chondrogenesis and the numbers of scleral cartilage PCNA positive cells increased with the temperature of the water in which spotted wolffish were cultured. Our results provide new knowledge of differing Sox9 spatial tissue expression patterns during chondrogenesis in normal control and ocular insult paradigms. Our work also provides evidence that spotted wolffish possess an inherent scleral chondrogenesis response that may be sensitive to temperature. This work also advances the fundamental knowledge of teleost ocular skeletal systems.

Equine-assisted service's effect on cartilage and skeletal biomarkers for adults and older adults with arthritis: A pilot study.

OBJECTIVE: The object of this study was to determine the effect of EAS (Equine-Assisted Services) on arthritis conditions, as measured by the sTnT (Skeletal troponin) and COMP (cartilage oligomeric matrix proteins) biomarkers, compared to an exercise attention control intervention. DESIGN: This was a secondary analysis of a randomized clinical trial comparing equine-assisted therapy to exercise education attention-control on cartilage and skeletal biomarkers in adults with arthritis. Twenty-one adults (Mage = 64 years) with arthritis who attended rheumatology clinics in the midwestern United States participated. RESULTS: No changes were found in sTnT from baseline to week six within either intervention nor were there differences in changes between the two groups (p = 0.91). COMP increased from baseline to week six for both conditions, suggesting increased deterioration of cartilage and joints. Although the attention-control condition demonstrated larger increases in cartilage oligomeric matrix proteins level, compared to the EAS condition, these differences were not statistically (p = 0.58) or clinically significant (i.e., trivial effect, d = -0.16). When 3 outliers were removed, the differences in changes between EAT and attention-control group could be arguably of clinical significance (d = - 0.33), suggesting that the attention-control group demonstrated larger increases in levels of COMP than those in the EAS condition, though this difference was not statistically significant (p = 0.28). CONCLUSION: Although equine-assisted therapy may reduce pain and improve quality of life for adults with arthritis, findings here are not fully corroborated with biomarkers.

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.

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.

Skeletal interoception in osteoarthritis.

The interoception maintains proper physiological conditions and metabolic homeostasis by releasing regulatory signals after perceving changes in the internal state of the organism. Among its various forms, skeletal interoception specifically regulates the metabolic homeostasis of bones. Osteoarthritis (OA) is a complex joint disorder involving cartilage, subchondral bone, and synovium. The subchondral bone undergoes continuous remodeling to adapt to dynamic joint loads. Recent findings highlight that skeletal interoception mediated by aberrant mechanical loads contributes to pathological remodeling of the subchondral bone, resulting in subchondral bone sclerosis in OA. The skeletal interoception is also a potential mechanism for chronic synovial inflammation in OA. In this review, we offer a general overview of interoception, specifically skeletal interoception, subchondral bone microenviroment and the aberrant subchondral remedeling. We also discuss the role of skeletal interoception in abnormal subchondral bone remodeling and synovial inflammation in OA, as well as the potential prospects and challenges in exploring novel OA therapies that target skeletal interoception.

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.

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.

Reciprocal negative feedback between Prrx1 and miR-140-3p regulates rapid chondrogenesis in the regenerating antler.

During growth phase, antlers exhibit a very rapid rate of chondrogenesis. The antler is formed from its growth center reserve mesenchyme (RM) cells, which have been found to be the derivatives of paired related homeobox 1 (Prrx1)-positive periosteal cells. However, the underlying mechanism that drives rapid chondrogenesis is not known. Herein, the miRNA expression profiles and chromatin states of three tissue layers (RM, precartilage, and cartilage) at different stages of differentiation within the antler growth center were analyzed by RNA-sequencing and ATAC-sequencing. We found that miR-140-3p was the miRNA that exhibited the greatest degree of upregulation in the rapidly growing antler, increasing from the RM to the cartilage layer. We also showed that Prrx1 was a key upstream regulator of miR-140-3p, which firmly confirmed by Prrx1 CUT&Tag sequencing of RM cells. Through multiple approaches (three-dimensional chondrogenic culture and xenogeneic antler model), we demonstrated that Prrx1 and miR-140-3p functioned as reciprocal negative feedback in the antler growth center, and downregulating PRRX1/upregulating miR-140-3p promoted rapid chondrogenesis of RM cells and xenogeneic antler. Thus, we conclude that the reciprocal negative feedback between Prrx1 and miR-140-3p is essential for balancing mesenchymal proliferation and chondrogenic differentiation in the regenerating antler. We further propose that the mechanism underlying chondrogenesis in the regenerating antler would provide a reference for helping understand the regulation of human cartilage regeneration and repair.

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.

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.

Calcified Cartilage-Guided Identification of Osteogenic Molecules and Geometries.

Calcified cartilage digested by chondroclasts provides an excellent scaffold to initiate bone formation. We analyzed bioactive proteins and microarchitecture of calcified cartilage either separately or in combination and evaluated biomimetic osteogenic culture conditions of surface-coated micropatterning. To do so, we prepared a crude extract from porcine femoral growth plates, which enhanced in vitro mineralization when coated on flat-bottom culture dishes, and identified four candidate proteins by fractionation and mass spectrometry. Murine homologues of two candidates, desmoglein 4 (DSG4) and peroxiredoxin 6 (PRDX6), significantly promoted osteogenic activity based on in vitro mineralization and osteoblast differentiation. Moreover, we observed DSG4 and PRDX6 protein expression in mouse femur. In addition, we designed circular, triangular, and honeycomb micropatterns with 30 or 50 µm units, either isolated or connected, to mimic hypertrophic chondrocyte-sized compartments. Isolated, larger honeycomb patterns particularly enhanced osteogenesis in vitro. Mineralization on micropatterns was positively correlated with the reduction of osteoblast migration distance in live cell imaging. Finally, we evaluated possible combinatorial effects of coat proteins and micropatterns and observed an additive effect of DSG4 or PRDX6 coating with micropatterns. These data suggest that combining a bioactive surface coating with osteogenic micropatterns may recapitulate initiation of bone formation during endochondral ossification.

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.

Expression levels and DNA methylation profiles of the growth gene SHOX in cartilage tissues and chondrocytes.

All attempts to identify male-specific growth genes in humans have failed. This study aimed to clarify why men are taller than women. Microarray-based transcriptome analysis of the cartilage tissues of four adults and chondrocytes of 12 children showed that the median expression levels of SHOX, a growth gene in the pseudoautosomal region (PAR), were higher in male samples than in female samples. Male-dominant SHOX expression was confirmed by quantitative RT-PCR for 36 cartilage samples. Reduced representation bisulfite sequencing of four cartilage samples revealed sex-biased DNA methylation in the SHOX-flanking regions, and pyrosequencing of 22 cartilage samples confirmed male-dominant DNA methylation at the CpG sites in the SHOX upstream region and exon 6a. DNA methylation indexes of these regions were positively correlated with SHOX expression levels. These results, together with prior findings that PAR genes often exhibit male-dominant expression, imply that the relatively low SHOX expression in female cartilage tissues reflects the partial spread of X chromosome inactivation into PAR. Altogether, this study provides the first indication that sex differences in height are ascribed, at least in part, to the sex-dependent epigenetic regulation of SHOX. Our findings deserve further validation.

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.

Sirt1 alleviates osteoarthritis via promoting FoxO1 nucleo-cytoplasm shuttling to facilitate autophagy.

This study aims to investigate the role and underlying mechanisms of Sirt1 in the pathophysiological process of OA. Safranine O and HE staining were utilized to identify pathological changes in the cartilage tissue. Immunohistochemistry was employed to evaluate the expression levels of proteins. IL-1ß treatment and TamCartSirt1flox/flox mice were utilized to induce OA model both in vitro and in vivo. Key autophagy-related transcription factors, autophagy-related genes, and chondrocyte extracellular matrix (ECM) breakdown enzyme markers were examined using multi assays. Immunofluorescence staining revealed subcellular localization and gene expression patterns. ChIP assay and Co-immunoprecipitation assay were conducted to investigate the interactions between FoxO1 and the promoter regions of Atg7 and Sirt1. Our results demonstrate that Sirt1 deficiency exhibited inhibitory effects on ECM synthesis and autophagy, as well as exacerbated angiogenesis. Moreover, Atg7, Foxo1, and Sirt1 could form a protein complex. Sirt1 was observed to facilitate nuclear translocation of FoxO1, enhancing its transcriptional activity. Furthermore, FoxO1 was found to bind to the promoter regions of Atg7 and Sirt1, potentially regulating their expression. This study provides valuable insights into the involvement of Sirt1-Atg7-FoxO1 loop in OA, opening new avenues for targeted therapeutic interventions aiming to mitigate cartilage degradation and restore joint function.