Chao Wan

Articular cartilage is characterized by its avascular, aneural, and alymphatic nature, which confers a limited intrinsic capacity for self-repair. Current regenerative strategies primarily focus on alleviating pain, mitigating symptoms, and restoring joint function. However, their long-term efficacy remains uncertain. Cartilage tissue engineering has emerged as a promising alternative to conventional therapies, offering innovative solutions for articular cartilage regeneration. Central to this approach is the development of functional biomaterials capable of supporting chondrogenic cell adhesion, proliferation, and differentiation, thereby facilitating effective cartilage repair. In this study, we introduce a novel protein-based recombinant spider silk (RSS) as a potential biomaterial for modulating chondrocyte behavior and enabling engineered cartilage formation both in vitro and in vivo. RSS was generated through molecular cloning and processed into silk fibers using biomimetic spinning and acidic coagulation techniques. In micromass cultures of murine chondrocytes, RSS significantly promoted cell aggregation, resulting in increased cell density. Alcian blue and Oil Red O staining demonstrated that RSS-treated cultures produced abundant glycosaminoglycans, a hallmark of chondrogenic activity, while exhibiting minimal lipid accumulation. These findings suggest that RSS supports chondrogenic differentiation and suppresses adipogenic lineage commitment. Real-time PCR analysis revealed upregulation of the chondrogenesis-related gene Sox9 and downregulation of the adipogenic marker PPARγ and the hypertrophic marker Runx2 in RSS-treated micromass cultures. RNA sequencing further corroborated these observations, underscoring the role of RSS in modulating extracellular matrix (ECM) remodeling in chondrocytes. In a subcutaneous transplantation model using severe combined immunodeficiency (SCID) mice, chondrocytes encapsulated in three-dimensional hydrogel scaffolds containing RSS exhibited significantly enhanced ECM accumulation compared to RSS-free controls, indicating that RSS supports the maintenance of the chondrocyte phenotype and promotes cartilage formation in vivo, and underscoring its promising potential as a component of hydrogel composite systems. These findings highlight the potential of RSS as a functional biomaterial to preserve chondrocyte functionality and advance engineered cartilage formation, presenting a promising avenue for cartilage tissue engineering and regeneration.

Current therapies for articular cartilage defects or degeneration (osteoarthritis) remain unsatisfactory. There are significant clinical demands for the development of more efficient approaches to enhance the repair or regeneration of articular cartilage lesions. In this study, a newly defined alginate-gelatin (A-G) composite bioink was synthesized using the carbodiimide chemistry crosslinking method. Then, the dual-crosslinking (DC) bioscaffolds containing both chemical and ionic networks were fabricated by 3D-bioprinting technique and Ca2+ treatment. The optimized networks formed in the DC bioscaffolds provided a desirable Young’s modulus and geometric constraints, which were superior to that of the single-crosslinking (SC) control group to mimic the mechanical properties of the pericellular matrix (PCM) of native articular cartilage. Chondrocytes exhibited above 95% viability and higher proliferation rate in the 3D printed A-G-DC bioscaffolds than in the alginate-only group after 7 days in vitro culture. Chondrogenic differentiation in vitro was improved in the A-G-DC bioscaffolds than that of the alginate-only group, indexed by upregulation of chondrogenic marker gene expression including Sox9, Col2a1, Comp and Aggrecan. In the subcutaneous transplantation model and osteochondral defect model in mice with severe combined immunodeficiency (SCID), the A-G-DC bioscaffolds exhibited enhanced chondrogenesis and repair capacity than the alginate-only or none-implant control group, characterized by the increased expression of SOX9 and collagen type II (Col II) and higher ICRS II scores, respectively. These findings demonstrated that the biomechanically inspired A-G composite bioink and the 3D-printed A-G-DC bioscaffolds possess excellent cell biocompatibility, satisfactory biomechanical properties and chondrogenesis promotion capacity, which have the potential to be applied to enhance cartilage regeneration for patients with articular cartilage lesions.

Articular cartilage is an avascular tissue with very limited capacity of self-regeneration. Trauma or injury-related defects, inflammation, or aging in articular cartilage can induce progressive degenerative joint diseases such as osteoarthritis. There are significant clinical demands for the development of effective therapeutic approaches to promote articular cartilage repair or regeneration. The current treatment modalities used for the repair of cartilage lesions mainly include cell-based therapy, small molecules, surgical approaches, and tissue engineering. However, these approaches remain unsatisfactory. With the advent of three-dimensional (3D) bioprinting technology, tissue engineering provides an opportunity to repair articular cartilage defects or degeneration through the construction of organized, living structures composed of biomaterials, chondrogenic cells, and bioactive factors. The bioprinted cartilage-like structures can mimic native articular cartilage, as opposed to traditional approaches, by allowing excellent control of chondrogenic cell distribution and the modulation of biomechanical and biochemical properties with high precision. This review focuses on various hydrogels, including natural and synthetic hydrogels, and their current developments as bioinks in 3D bioprinting for cartilage tissue engineering. In addition, the challenges and prospects of these hydrogels in cartilage tissue engineering applications are also discussed.

Purpose: Currently, the early diagnosis and treatment of osteoarthritis (OA) remain a challenge. In the present study, we attempted to explore potential biomarkers for the diagnosis and treatment of OA.Methods: The differentially expressed genes (DEGs) were identified based on three mRNA datasets of synovial tissues for OA patients and normal controls downloaded from the Gene Expression Omnibus (GEO) database. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used for evaluating gene function related categories. Then, miRNA sequencing was performed for differentially expressed miRNAs’ identification. Finally, weighted gene co-expression network analysis (WGCNA) was performed for genes detected by the three mRNA datasets and a competing endogenous RNA (ceRNA) network with DEGs and differentially expressed microRNAs (miRNAs) was constructed for central genes identification. In addition, the relationship between central gene expression and immune infiltration was analyzed, and the candidate agents for OA were predicted based on the Connectivity Map database. Quantitative RT-PCR (qRT-PCR), Western blotting analysis, and immunofluorescent staining were performed to validate the expression levels of differentially expressed miRNAs and differentially expressed target genes in normal and OA tissues and chondrocytes. MiRNA–mRNA network was also validated in chondrocytes in vitro.Results: A total of 259 DEGs and 26 differentially expressed miRNAs were identified, among which 94 miRNA–mRNA interactions were predicted. The brown module in WGCNA was most closely correlated with the clinical traits of OA. After overlapping the brown module genes with miRNA–mRNA pairs, 27 miRNA–mRNA pairs were obtained. A ceRNA network was constructed with 5505 lncRNA–miRNA–mRNA interactions. B-cell translocation gene 2(BTG2), Abelson-related gene (ABL2), and vascular endothelial growth factor A (VEGFA) were identified to be the central genes with good predictive performance, which were significantly correlated with immune cell infiltration in OA, reflected by declined activated dendritic cells (aDCs), and elevated contents of B cells, macrophages, neutrophils, and T helper cells. Anisomycin, MG-132, thapsigargin, and lycorine were predicted to be the potential candidate agents for OA intervention. In vitro, the expression levels of differentially expressed miRNAs and biomarkers identified in the present study were consistent with the results obtained in normal or OA knee cartilage tissues and chondrocytes. Furthermore, BTG2 was identified to be negatively regulated by miR-125a-5p.Conclusion: BTG2, ABL2, and VEGFA can be regarded as potential predictive and treatment biomarkers for OA, which might guide the clinical therapy of OA.

The involvement of histone modifications in cartilage development, pathology and regeneration is becoming increasingly evident. Understanding the molecular mechanisms and consequences of histone modification enzymes in cartilage development, homeostasis and pathology provides fundamental and precise perspectives to interpret the biological behavior of chondrocytes during skeletal development and the pathogenesis of various cartilage related diseases. Candidate molecules or drugs that target histone modifying proteins have shown promising therapeutic potential in the treatment of cartilage lesions associated with joint degeneration and other chondropathies. In this review, we summarized the advances in the understanding of histone modifications in the regulation of chondrocyte fate, cartilage development and pathology, particularly the molecular writers, erasers and readers involved. In addition, we have highlighted recent studies on the use of small molecules and drugs to manipulate histone signals to regulate chondrocyte functions or treat cartilage lesions, in particular osteoarthritis (OA), and discussed their potential therapeutic benefits and limitations in preventing articular cartilage degeneration or promoting its repair or regeneration.

We report the synthesis of a series of elastin-like polypeptide (ELP)-based molecular networks through the combined use of the covalent bond-forming SpyTag/SpyCatcher chemistry, physically entangled p53dim domains (Xs), and site-directed mutagenesis. The resulting networks shared similar chemical composition but differed significantly in their viscoelasticity. These materials exhibited excellent compatibility toward encapsulated fibroblasts and stem cells. These results point to a versatile strategy for designing viscoelastic materials by tapping into diverse protein–protein interactions.

Utilization of small molecules in modulation of stem cell self-renewal is a promising approach to expand stem cells for regenerative therapy. Here, we identify Icaritin, a phytoestrogen molecule enhances self-renewal of mouse embryonic stem cells (mESCs). Icaritin increases mESCs proliferation while maintains their self-renewal capacity in vitro and pluripotency in vivo. This coincides with upregulation of key pluripotency transcription factors OCT4, NANOG, KLF4 and SOX2. The enhancement of mESCs self-renewal is characterized by increased population in S-phase of cell cycle, elevation of Cylin E and Cyclin-dependent kinase 2 (CDK2) and downregulation of p21, p27 and p57. PCR array screening reveals that caudal-related homeobox 2 (Cdx2) and Rbl2/p130 are remarkably suppressed in mESCs treated with Icaritin. siRNA knockdown of Cdx2 or Rbl2/p130 upregulates the expression of Cyclin E, OCT4 and SOX2, and subsequently increases cell proliferation and colony forming efficiency of mESCs. We then demonstrate that Icaritin co-localizes with estrogen receptor alpha (ERα) and activates its nuclear translocation in mESCs. The promotive effect of Icaritin on cell cycle and pluripotency regulators are eliminated by siRNA knockdown of ERα in mESCs. The results suggest that Icaritin enhances mESCs self-renewal by regulating cell cycle machinery and core pluripotency transcription factors mediated by ERα.

Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin+Lepr +Sca-1+CD146+ and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo.

Poly(N-isopropylacrylamide) (PNIPAM)-based polymers and gels are widely known and studied for their thermoresponsive property. In the biomaterials category, they are regarded as a potential cell culture substrate, not only because of their biocompatibility, but also their special character of allowing controlled detachment of cells via temperature stimulus. Previous research about PNIPAM-based substrates mostly concentrated on their effects in cell adhesion and proliferation. In this study, however, we investigate the influence of the PNIPAM-based substrate on the differentiation capacity of stem cells. Especially, we choose P(NIPAM-AA) microgels as a culture dish coating and mesenchymal stem cells (MSCs) are cultured on top of the microgels. Interestingly, we find that the morphology of MSCs changes remarkably on a microgel-coated surface, from the original spindle form to a more stretched and elongated cell shape. Accompanied by the alternation in morphology, the expression of several osteogenesis-related genes is elevated even without inducing factors. In the presence of full osteogenic medium, MSCs on a microgel substrate show an enhancement in the expression level of osteopontin and alizarin red staining signals, indicating the physical property of substrate has a direct effect on MSCs differentiation.