Mutations in fibronectin dysregulate chondrogenesis in skeletal dysplasia.

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol, and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFß1) expression, crucial for mesenchymal condensation. Exogenous FN or TGFß1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis.

The collagen-augmented chondrogenesis technique demonstrates superior cartilage repair compared to microfracture for cartilage defects of the knee joint, regardless of age.

PURPOSE: This study investigated whether age affects clinical outcomes and cartilage repair quality in patients who underwent collagen-augmented chondrogenesis. METHODS: The study included patients who underwent either the collagen-augmented chondrogenesis technique or microfracture for cartilage defects of the knee joint of International Cartilage Repair Society grade 3 or 4. Patients were categorised according to an age threshold of 50 years and the treatment method, whether collagen-augmented chondrogenesis technique or microfracture. Group 1 comprised 31 patients aged 50 years or older who received the collagen-augmented chondrogenesis technique, Group 2 consisted of 32 patients under the age of 50 years who received the collagen-augmented chondrogenesis technique and Group 3 included 243 patients aged 50 years or older who received microfracture. Clinical outcomes were assessed using the walking visual analogue scale (VAS) for pain and the Western Ontario McMaster University Osteoarthritis Index scale score (WOMAC) two years after surgery. For patients with magnetic resonance imaging results 1 year postoperatively (Group 1: 30 patients; Group 2: 31 patients; and Group 3: 31 patients), Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) assessment was used to evaluate repaired cartilage lesions. RESULTS: There were no significant differences in the VAS and WOMAC scores between the three groups 2 years after surgery (all n.s.). The MOCART score in patients who underwent MRI at 1 year postoperatively showed significant differences in the degree of defect repair, integration with the border zone, surface of the repaired tissue, adhesion and total score among the three groups (all p < 0.05). Post hoc analysis revealed no difference in the total MOCART scores between Groups 1 and 2. However, Groups 1 and 2 had significantly higher MOCART scores than Group 3 1 year after surgery (all p < 0.05). CONCLUSION: The collagen-augmented chondrogenesis technique group showed improved quality of cartilage repair compared to the microfracture group, regardless of patient age. Compared with simple microfracture treatment, there were no differences in clinical outcomes between the patient groups, related to age. LEVEL OF EVIDENCE: Level Ⅲ.

IL-4 promotes chondrogenesis of bone marrow mesenchymal stem cells and blockade of IL-4Rα retards the endochondral ossification during rat embryonic bone development.

Interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signalling pathways play important roles in the complex process of bone formation and bone remodelling. However, whether IL-4/IL-4Rα participates in skeletogenesis during embryonic development is not completely understood. We used the anti-IL-4Rα monoclonal antibody (anti-IL-4Rα mAb) as a powerful investigational tool to evaluate the potential roles of IL-4/IL-4Rα in the chondrogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Simultaneously, we explored the effect of IL-4/IL-4Rα on bone ossification during rat embryo-fetal development. In this study, we found that, compared to the control group, IL-4 can significantly promote the chondrogenic differentiation of BMSCs. Furthermore, following exposure to anti-IL-4Rα mAb in pregnant rats, unexpected phenomena were observed in fetal bone development, including non-ossification of the fetal sternum, an incomplete ossification centre in long bones and a reduced number of ossification points in digit (toe) bones. To further investigate the underlying mechanism of the phenotype, we studied the rat sternum as the target organ, starting from different time points of sternum development in the embryonic stage. The results indicated that the retardation mainly occurred in the middle and late stages of embryonic development. This retardation was characterized by the inhibition of the differentiation process of mesenchymal stem cells into chondrocytes, resulting in reduced angiogenesis near the ossification centre, failure of osteoblasts to invade the centre of the cartilage body with the blood vessels and delayed formation of the primary ossification centre (POC). Overall, our study demonstrated the significant function of IL-4/IL-4Rα in chondrogenic differentiation of BMSCs and bone ossification during embryo-fetal development.

Cell-free bilayer functionalized scaffold for osteochondral tissue engineering.

Osteochondral tissue engineering using layered scaffolds is a promising approach for treating osteochondral defects as an alternative to microfracture procedure, autologous chondrocyte implantation, and cartilage-bone grafting. The team previously investigated the chondrogenesis of mesenchymal stem cells (MSCs) on a polycaprolactone (PCL)/acetylated hyaluronic acid scaffold. The present study first focused on fabricating a novel osteoconductive scaffold utilizing bismuth-nanohydroxyapatite/reduced graphene oxide (Bi-nHAp/rGO) nanocomposite and electrospun PCL. The osteoconductive ability of the scaffold was investigated by evaluating the alkaline phosphatase (ALP) activity and the osteogenic genes expression in the adipose-derived MSCs. The expression of Runx2, collagen I, ALP, and osteocalcin as well as the result of ALP activity indicated the osteoconductive potential of the Bi-nHA-rGO/PCL scaffold. In the next step, a bilayer scaffold containing Bi-nHAp/rGO/PCL as an osteogenic layer and acetylated hyaluronic acid/PCL as a chondrogenic layer was prepared by the electrospinning technique and transplanted into osteochondral defects of rats. The chondrogenic and osteogenic markers corresponding to the surrounding tissues of the transplanted scaffold were surveyed 60 days later by real-time polymerase chain reaction (PCR) and immunohistochemistry methods. The results showed increased chondrogenic (Sox9 and collagen II) and osteogenic (osteocalcin and ALP) gene expression and augmented secretion of collagens II and X after transplantation. The results strongly support the efficacy of this constructed cell-free bilayer scaffold to induce osteochondral defect regeneration.

Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

The Integral Role of Fibronectin in Skeletal Morphogenesis and Pathogenesis.

Fibronectin (FN) serves as a critical organizer of extracellular matrix networks in two principal isoforms, the plasma FN and the cellular FN. While FN's pivotal role in various organ systems, including the blood vasculature, is well-established, its contribution to the development of the skeletal system is much less explored. Furthermore, the pathomechanisms of spondyloepiphyseal dysplasia caused by FN mutations remain elusive. In this minireview, we discuss findings from our recent two studies using i) an iPSC-based cell culture model to explore how FN mutations in spondyloepiphyseal dysplasia impact mesenchymal cell differentiation into chondrocytes and ii) conditional FN knockout mouse models to determine the physiological roles of FN isoforms during postnatal skeletal development. The data revealed that FN mutations cause severe intracellular and matrix defects in mesenchymal cells and impair their ability to differentiate into chondrocytes. The findings further demonstrate the important roles of both FN isoforms in orchestrating regulated chondrogenesis during skeletal development. We critically discuss the findings in the context of the existing literature.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis.

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

Harnessing Peptide-Based Hydrogels for Enhanced Cartilage Tissue Engineering.

Cartilage tissue engineering remains a formidable challenge due to its complex, avascular structure and limited regenerative capacity. Traditional approaches, such as microfracture, autografts, and stem cell delivery, often fail to restore functional tissue adequately. Recently, there has been a surge in the exploration of new materials that mimic the extracellular microenvironment necessary to guide tissue regeneration. This review investigates the potential of peptide-based hydrogels as an innovative solution for cartilage regeneration. These hydrogels, formed via supramolecular self-assembly, exhibit excellent properties, including biocompatibility, ECM mimicry, and controlled biodegradation, making them highly suitable for cartilage tissue engineering. This review explains the structure of cartilage and the principles of supramolecular and peptide hydrogels. It also delves into their specific properties relevant to cartilage regeneration. Additionally, this review presents recent examples and a comparative analysis of various peptide-based hydrogels used for cartilage regeneration. The review also addresses the translational challenges of these materials, highlighting regulatory hurdles and the complexities of clinical application. This comprehensive investigation provides valuable insights for biomedical researchers, tissue engineers, and clinical professionals aiming to enhance cartilage repair methodologies.

Fibronectin isoforms promote postnatal skeletal development.

Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-ß1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth.

Injectable and self-healing sulfated hyaluronic acid/gelatin hydrogel as dual drug delivery system for circumferential tracheal repair.

Stem cell-based therapies show promise for clinically addressing circumferential tracheal defects (CTD) through tissue engineering. However, creating a tissue-engineered tracheal tube possesses a healthy cartilage matrix and intact tube structure remains a challenge. A solution lies in the use of an injectable hydrogel with shape adaptability and chondrogenic capacity, serving as a practical and dependable platform for tubular tracheal cartilage regeneration. In this study, we developed an injectable hydrogel using modified natural polymers-hydrazide-grafted gelatin (Gelatin-ADH) and aldehyde-modified hyaluronic acid with sulfated groups (HA-CHO-SO3) via Schiff Base interaction. Additionally, aldehyde-modified ß-cyclodextrin (ß-CD-CHO) was introduced into the network during hydrogel formation. The negative sulfated groups and hydrophobic cavities of ß-cyclodextrin facilitated the efficient encapsulation and sustained release of transforming growth factor-ß1 (TGF-ß1) and kartogenin (KGN) within our hydrogel. This synergistically promoted the chondrogenesis of loaded bone marrow stem cells (BMSCs). Subsequently, we employed this TGF-ß1, KGN, and BMSCs loaded hydrogel to form a cartilage ring. This ring was then assembled into an engineered tracheal cartilage tube using our previously reported ring-to-tube strategy. Our results demonstrated that the engineered tracheal cartilage tube effectively repaired CTD in a rabbit model. Hence, this study introduces a novel hydrogel with significant clinical application potential for tracheal tissue engineering.

EVs from cells at the early stages of chondrogenesis delivered by injectable SIS dECM promote cartilage regeneration.

Articular cartilage defect therapy is still dissatisfactory in clinic. Direct cell implantation faces challenges, such as tumorigenicity, immunogenicity, and uncontrollability. Extracellular vesicles (EVs) based cell-free therapy becomes a promising alternative approach for cartilage regeneration. Even though, EVs from different cells exhibit heterogeneous characteristics and effects. The aim of the study was to discover the functions of EVs from the cells during chondrogenesis timeline on cartilage regeneration. Here, bone marrow mesenchymal stem cells (BMSCs)-EVs, juvenile chondrocytes-EVs, and adult chondrocytes-EVs were used to represent the EVs at different differentiation stages, and fibroblast-EVs as surrounding signals were also joined to compare. Fibroblasts-EVs showed the worst effect on chondrogenesis. While juvenile chondrocyte-EVs and adult chondrocyte-EVs showed comparable effect on chondrogenic differentiation as BMSCs-EVs, BMSCs-EVs showed the best effect on cell proliferation and migration. Moreover, the amount of EVs secreted from BMSCs were much more than that from chondrocytes. An injectable decellularized extracellular matrix (dECM) hydrogel from small intestinal submucosa (SIS) was fabricated as the EVs delivery platform with natural matrix microenvironment. In a rat model, BMSCs-EVs loaded SIS hydrogel was injected into the articular cartilage defects and significantly enhanced cartilage regeneration in vivo. Furthermore, protein proteomics revealed BMSCs-EVs specifically upregulated multiple metabolic and biosynthetic processes, which might be the potential mechanism. Thus, injectable SIS hydrogel loaded with BMSCs-EVs might be a promising therapeutic way for articular cartilage defect.

Drop in survivorship 13 years after AMIC procedures in aligned knees: A long-term follow-up.

PURPOSE: Autologous matrix-induced chondrogenesis (AMIC) showed promising short-term results comparable to microfracture. This study aims to assess the 19-year outcomes of AMIC, addressing the lack of long-term data. METHODS: Retrospective cohort of 34 knees treated with AMIC underwent a 19-year follow-up. The primary outcome was AMIC survival, considering total knee arthroplasty as a failure event. Survival analysis for factors that were associated with longer survival of the AMIC was also performed. Clinical and radiological outcome scores were analysed for the AMIC group. RESULTS: Twenty-three knees were available for follow-up analysis. Of these, 14 (61%) underwent revision surgery for total knee arthroplasty (TKA). The mean time was 13.3 ± 2.5 years (range: 9-17 years). Secondary outcomes showed that increased age at surgery (hazard ratio [HR]: 1.05; p = 0.021) and larger defect size (HR: 1.95; p = 0.018) were risk factors for failure. Concomitant proximal tibial osteotomy (HR: 0.22; p = 0.019) was associated with longer survival. The remaining nine knees (39%) were analysed as a single group. The mean clinical score at follow-up of 18.6 ± 0.9 SD years was 79.5 ± 19.7 SD for the Lysholm score, 1.8 ± 1.5 SD for the visual analog scale score, 74.2 ± 22.4 SD for the KOOS score and a median of 3 (range: 3-4) for the Tegner activity scale. CONCLUSIONS: The mean survival time of 13.3 years indicates the durability of AMIC in properly aligned knees. Nonetheless, despite a 61% conversion to TKA, the knees that persisted until the 19-year follow-up remained stable, underscoring the procedure's longevity and consistent clinical outcomes. LEVEL OF EVIDENCE: Level IV.

Alkali-extracted proteins from the tooth dentin matrix as a mixture of bioactive molecules for cartilage repair and regeneration.

Introduction: Dentin matrix extracted protein (DMEP) is a mixture of proteins extracted from the organic matrix of a natural demineralized dentin matrix that is rich in a variety of growth factors. However, the effect of DMEP on cartilage regeneration is unclear. The aim of this study was to investigate the efficacy of DMEP extracted via a novel alkali conditioning method in promoting cartilage regeneration. Methods: Alkali-extracted DMEP (a-DMEP) was obtained from human dentin fragments using pH 10 bicarbonate buffer. The concentration of chondrogenesis-related growth factors in a-DMEP was measured via enzyme-linked immunosorbent assay (ELISA). Human bone marrow mesenchymal stem cells (hBMMSCs) in pellet form were induced with a-DMEP. Alcian blue and Safranin O staining were performed to detect cartilage matrix formation, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess chondrogenic-related gene expression in the pellets. Rabbit articular osteochondral defects were implanted with collagen and a-DMEP. Cartilage regeneration was assessed with histological staining 4 weeks after surgery. Results: Compared with traditional neutral-extracted DMEP, a-DMEP significantly increased the levels of transforming growth factor beta 1(TGF-ß1), insulin-like growth factor-1(IGF-1) and basic fibroblast growth factor (bFGF). After coculture with hBMMSC pellets, a-DMEP significantly promoted the expression of the collagen type II alpha 1(COL2A1) and aggrecan (ACAN) genes and the formation of cartilage extracellular matrix in cell pellets. Moreover, compared with equivalent amounts of exogenous human recombinant TGF-ß1, a-DMEP had a stronger chondrogenic ability. In vivo, a-DMEP induced osteochondral regeneration with hyaline cartilage-like structures. Conclusions: Our results showed that a-DMEP, a compound of various proteins derived from natural tissues, is a promising material for cartilage repair and regeneration.

Autologous matrix-induced chondrogenesis provides better outcomes in comparison to autologous minced cartilage implantation in the repair of knee chondral defects.

PURPOSE: In symptomatic mid-sized focal chondral defects, autologous matrix-induced chondrogenesis (AMIC) and minced cartilage implantation (MCI) offer two versatile treatment options. This study aimed to conduct a matched-patient analysis of patient-reported outcome measures to compare these two surgical treatment methods for focal chondral defects. METHODS: At the first centre, patients underwent a single-stage procedure in which autologous cartilage was hand-minced, implanted into the defect and fixed with fibrin glue. At the second centre, patients underwent AMIC, which was fixed in place with fibrin glue. All patients were seen 2-4 years postoperatively. Postoperative outcomes were assessed using the visual analogue scale for pain (VAS), the Lysholm score and the five domains of the knee osteoarthritis outcome score (KOOS). Patients from each surgical centre were matched by age, sex, defect size and defect localisation. RESULTS: In total, 48 patients from two surgical centres (24 from each site) were matched for sex, age (MCI 30.3 ± 14.9 years vs. AMIC 30.8 ± 13.7 years) and defect size (MCI 2.49 ± 1.5 cm2 vs. AMIC 2.65 ± 1.1 cm2). Significantly better scores in the AMIC cohort were noted for VAS (p = 0.004), Lysholm (p = 0.043) and the KOOS subscales for pain (p = 0.016) and quality of life (p = 0.036). There was a significantly greater proportion of positive responders for Lysholm in the AMIC group (92%) compared with the MCI group (64%). CONCLUSIONS: The AMIC procedure delivers superior patient outcomes compared with hand-minced autologous cartilage implantation. These are mid-term outcomes, with follow-up between 2 and 4 years. LEVEL OF EVIDENCE: Level III.

Temporal modulation of inflammation and chondrogenesis through dendritic nanoparticle-mediated therapy with diclofenac surface modification and strontium ion encapsulation.

Cartilage tissue engineering holds great promise for efficient cartilage regeneration. However, early inflammatory reactions to seed cells and/or scaffolds impede this process. Consequently, managing inflammation is of paramount importance. Moreover, due to the body's restricted chondrogenic capacity, inducing cartilage regeneration becomes imperative. Thus, a controlled platform is essential to establish an anti-inflammatory microenvironment before initiating the cartilage regeneration process. In this study, we utilized fifth-generation polyamidoamine dendrimers (G5) as a vehicle for drugs to create composite nanoparticles known as G5-Dic/Sr. These nanoparticles were generated by surface modification with diclofenac (Dic), known for its potent anti-inflammatory effects, and encapsulating strontium (Sr), which effectively induces chondrogenesis, within the core. Our findings indicated that the G5-Dic/Sr nanoparticle exhibited selective Dic release during the initial 9 days and gradual Sr release from days 3 to 15. Subsequently, these nanoparticles were incorporated into a gelatin methacryloyl (GelMA) hydrogel, resulting in GelMA@G5-Dic/Sr. In vitro assessments demonstrated GelMA@G5-Dic/Sr's biocompatibility with bone marrow stem cells (BMSCs). The enclosed nanoparticles effectively mitigated inflammation in lipopolysaccharide-induced RAW264.7 macrophages and significantly augmented chondrogenesis in BMSCs cocultures. Implanting BMSCs-loaded GelMA@G5-Dic/Sr hydrogels in immunocompetent rabbits for 2 and 6 weeks revealed diminished inflammation and enhanced cartilage formation compared to GelMA, GelMA@G5, GelMA@G5-Dic, and GelMA@G5/Sr hydrogels. Collectively, this study introduces an innovative strategy to advance cartilage regeneration by temporally modulating inflammation and chondrogenesis in immunocompetent animals. Through the development of a platform addressing the temporal modulation of inflammation and the limited chondrogenic capacity, we offer valuable insights to the field of cartilage tissue engineering.

Effectiveness of gellan gum scaffolds loaded with Boswellia serrata extract for in-situ modulation of pro-inflammatory pathways affecting cartilage healing.

In this study, we developed a composite hydrogel based on Gellan gum containing Boswellia serrata extract (BSE). BSE was either incorporated directly or loaded into an MgAl-layered double hydroxide (LDH) clay to create a multifunctional cartilage substitute. This composite was designed to provide anti-inflammatory properties while enhancing chondrogenesis. Additionally, LDH was exploited to facilitate the loading of hydrophobic BSE components and to improve the hydrogel's mechanical properties. A calcination process was also adopted on LDH to increase BSE loading. Physicochemical and mechanical characterizations were performed by spectroscopic (XPS and FTIR), thermogravimetric, rheological, compression test, weight loss and morphological (SEM) investigations. RPLC-ESI-FTMS was employed to investigate the boswellic acids release in simulated synovial fluid. The composites were cytocompatible and capable of supporting the mesenchymal stem cells (hMSC) growth in a 3D-conformation. Loading BSE resulted in the modulation of the pro-inflammatory cascade by down-regulating COX2, PGE2 and IL1ß. Chondrogenesis studies demonstrated an enhanced differentiation, leading to the up-regulation of COL 2 and ACAN. This effect was attributed to the efficacy of BSE in reducing the inflammation through PGE2 down-regulation and IL10 up-regulation. Proteomics studies confirmed gene expression findings by revealing an anti-inflammatory protein signature during chondrogenesis of the cells cultivated onto loaded specimens. Concluding, BSE-loaded composites hold promise as a tool for the in-situ modulation of the inflammatory cascade while preserving cartilage healing.

Collagen-based hydrogels induce stem cell chondrogenesis and hyaline cartilage regeneration: an in vivo study.

Injectable, self-crosslinking collagen-based hydrogels are beneficial for chondrocytes to secrete matrix, positioning them as promising candidates for cartilage tissue engineering. However, previous studies lacked insight into the ability of cell-free collagen-based hydrogels to regenerate hyaline cartilage defect. Therefore, this study aimed to evaluate the potential of collagen-based hydrogels (Col and ColHA) to induce chondrogenic differentiation of stem cells and in situ hyaline cartilage regeneration. Both Col and ColHA hydrogels self-crosslinked in situ and exhibited similar physical properties. In vitro experiments showed they supported the survival, adhesion, spreading, and proliferation of bone marrow stem cells (BMSCs). Moreover, both hydrogels induced ectopic differentiation of BMSCs into chondrocytes when implanted subcutaneously into the back of nude mice. ColHA hydrogel notably enhanced type II collagen secretion. The results of repairing cartilage defects in situ revealed both hydrogels facilitated hyaline cartilage regeneration and maintained cartilage phenotype without exogenous BMSCs. Hydrogels encapsulating BMSCs expedited cartilage repair, and ColHA/BMSC constructs showed better mechanical properties, suggesting their potential for cartilage repair applications. This study implies that collagen-based hydrogels are good candidates for hyaline cartilage regeneration.

The efficacy of autologous matrix-induced chondrogenesis (AMIC) for osteochondral lesions of the talus in the mid-long term: a systematic review and meta-analysis.

PURPOSE: The objective of this study was to provide a comprehensive review of the existing literature regarding the treatment of osteochondral lesions of the talus (OLT) using autologous matrix-induced chondrogenesis (AMIC), while also discussing the mid-long term functional outcomes, complications, and surgical failure rate. METHODS: We searched Embase, PubMed, and Web of Science for studies on OLT treated with AMIC with an average follow-up of at least 2 years. Publication information, patient data, functional scores, surgical failure rate, and complications were extracted. RESULTS: A total of 15 studies were screened and included, with 12 case series selected for meta-analysis and 3 non-randomized controlled studies chosen for descriptive analysis. The improvements in the Visual Analog Scale (VAS), the American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot, and Tegner scores at the last follow-up were (SMD = - 2.825, 95% CI - 3.343 to - 2.306, P < 0.001), (SMD = 2.73, 95% CI 1.60 to 3.86, P < 0.001), (SMD = 0.85, 95% CI 0.5 to 1.2, P < 0.001) respectively compared to preoperative values. The surgery failure rate was 11% (95% CI 8-15%), with a total of 12 patients experiencing complications. CONCLUSION: The use of AMIC demonstrates a positive impact on pain management, functional improvement, and mobility enhancement in patients with OLT. It is worth noting that the choice of stent for AMIC, patient age, and OLT size can influence the ultimate clinical outcomes. This study provides evidences supporting the safety and efficacy of AMIC as a viable treatment option in real-world medical practice.

Amplifications of EVX2 and HOXD9-HOXD13 on 2q31 in mature cystic teratomas of the ovary identified by array comparative genomic hybridization may explain teratoma characteristics in chondrogenesis and osteogenesis.

BACKGROUND: Teratomas are a common type of germ cell tumor. However, only a few reports on their genomic constitution have been published. The study of teratomas may provide a better understanding of their stepwise differentiation processes and molecular bases, which could prove useful for the development of tissue-engineering technologies. METHODS: In the present study, we analyzed the copy number aberrations of nine ovarian mature cystic teratomas using array comparative genomic hybridization in an attempt to reveal their genomic aberrations. RESULTS: The many chromosomal aberrations observed on array comparative genomic hybridization analysis reveal the complex genetics of this tumor. Amplifications and deletions of large DNA fragments were observed in some samples, while amplifications of EVX2 and HOXD9-HOXD13 on 2q31.1, NDUFV1 on 11q13.2, and RPL10, SNORA70, DNASE1L1, TAZ, ATP6AP1, and GDI1 on Xq28 were found in all nine mature cystic teratomas. CONCLUSIONS: Our results indicated that amplifications of these genes may play an important etiological role in teratoma formation. Moreover, amplifications of EVX2 and HOXD9-HOXD13 on 2q31.1, found on array comparative genomic hybridization, may help to explain the characteristics of teratomas in chondrogenesis and osteogenesis.

Long noncoding RNA UCA1 promotes the chondrogenic differentiation of human bone marrow mesenchymal stem cells via regulating PARP1 ubiquitination.

Bone marrow mesenchymal stem cells (BMSCs) possess the potential to differentiate into cartilage cells. Long noncoding RNA (lncRNAs) urothelial carcinoma associated 1 (UCA1) has been confirmed to improve the chondrogenic differentiation of marrow mesenchymal stem cells (MSCs). Herein, we further investigated the effects and underlying mechanisms of these processes. The expression of UCA1 was positively associated with chondrogenic differentiation and the knockdown of UCA1 has been shown to attenuate the expression of chondrogenic markers. RNA pull-down assay and RNA immunoprecipitation showed that UCA1 could directly bind to PARP1 protein. UCA1 could improve PARP1 protein via facilitating USP9X-mediated PARP1 deubiquitination. Then these processes stimulated the NF-κB signaling pathway. In addition, PARP1 was declined in UCA1 knockdown cells, and silencing of PARP1 could diminish the increasing effects of UCA1 on the chondrogenic differentiation from MSCs and signaling pathway activation. Collectively, these outcomes suggest that UCA1 could act as a mediator of PARP1 protein ubiquitination and develop the chondrogenic differentiation of MSCs.