Injectable interface-bonded fiber-reinforced thiolated chitosan hydrogels for enhanced cellular activities and cartilage regeneration.

Injectable hydrogels with heterogeneous fibrous structures possessing good mechanical and biological characteristics are attracting increasing research interest in cartilage repair. The integration of nanofibers into hydrogel would largely enhance mechanical property, but impedes the gelation process and formation of hydrogel structures. Construction of biocompatible and mechanical supporting hydrogel with low fiber content remains a challenge. In this study, we developed a chemical cross-linked fibrous hydrogel, namely Thiol chitosan-Poly (lactic-co-glycolic acid)-Polydopamine (CSSH-PP), for facilitating cell proliferation and promoting cartilage tissues regeneration. Compared to conventional CSSH hydrogels, the compressive strength of CSSH-PP scaffolds exhibited a significant increase percentage of 100 %. Incorporation of CSSH-PP upgraded the cell migration with a four-fold increase. Besides, the infiltration of host cells and the formation of new blood vessels were observed in rat models when implanted with CSSH-PP, enhancing the native tissue microenvironmental reconstruction and leading a sustained repair in articular cartilage.

Efficiently directing differentiation and homing of mesenchymal stem cells to boost cartilage repair in osteoarthritis via a nanoparticle and peptide dual-engineering strategy.

Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy.

Glucosamine sulfate-loaded nanofiber reinforced carboxymethyl chitosan sponge for articular cartilage restoration.

Cartilage is severely limited in self-repair after damage, and tissue engineering scaffold transplantation is considered the most promising strategy for cartilage regeneration. However, scaffolds without cells and growth factors, which can effectively avoid long cell culture times, high risk of infection, and susceptibility to contamination, remain scarce. Hence, we developed a cell- and growth factor-dual free hierarchically structured nanofibrous sponge to mimic the extracellular matrix, in which the encapsulated core-shell nanofibers served both as mechanical supports and as long-lasting carriers for bioactive biomass molecules (glucosamine sulfate). Under the protection of the nanofibers in this designed sponge, glucosamine sulfate could be released continuously for at least 30 days, which significantly accelerated the repair of cartilage tissue in a rat cartilage defect model. Moreover, the nanofibrous sponge based on carboxymethyl chitosan as the framework could effectively fill irregular cartilage defects, adapt to the dynamic changes during cartilage movement, and maintain almost 100 % elasticity even after multiple compression cycles. This strategy, which combines fiber freeze-shaping technology with a controlled-release method for encapsulating bioactivity, allows for the assembly of porous bionic scaffolds with hierarchical nanofiber structure, providing a novel and safe approach to tissue repair.

A human organoid drug screen identifies α2-adrenergic receptor signaling as a therapeutic target for cartilage regeneration.

Directed differentiation of stem cells toward chondrogenesis in vitro and in situ to regenerate cartilage suffers from off-target differentiation and hypertrophic tendency. Here, we generated a cartilaginous organoid system from human expanded pluripotent stem cells (hEPSCs) carrying a COL2A1mCherry and COL10A1eGFP double reporter, enabling real-time monitoring of chondrogenesis and hypertrophy. After screening 2,040 FDA-approved drugs, we found that α-adrenergic receptor (α-AR) antagonists, especially phentolamine, stimulated chondrogenesis but repressed hypertrophy, while α2-AR agonists reduced chondrogenesis and induced hypertrophy. Phentolamine prevented cartilage degeneration in hEPSC cartilaginous organoid and human cartilage explant models and stimulated microfracture-activated endogenous skeletal stem cells toward hyaline-like cartilage regeneration without fibrotic degeneration in situ. Mechanistically, α2-AR signaling induced hypertrophic degeneration via cyclic guanosine monophosphate (cGMP)-dependent secretory leukocyte protease inhibitor (SLPI) production. SLPI-deleted cartilaginous organoid was degeneration resistant, facilitating large cartilage defect healing. Ultimately, targeting α2-AR/SLPI was a promising and clinically feasible strategy to regenerate cartilage via promoting chondrogenesis and repressing hypertrophy.

Evaluating Synergistic Effects of Hyaluronic Acid, Human Umbilical Cord-Derived Mesenchymal Stem Cells, and Growth Hormones in Knee Osteoarthritis: A Multi-Arm Randomized Trial.

BACKGROUND: Knee osteoarthritis (OA) significantly affects quality of life and imposes economic burdens due to its prevalence and the disability it causes. The efficacy of current treatments is limited to alleviating the symptoms, and they cannot be used for regenerative purposes. This study aims to evaluate the efficacy and safety of combining hyaluronic acid (HA), human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), and synthetic human growth hormone (somatotropin) in the treatment of knee OA, assessing pain relief, functional improvement, and cartilage regeneration. METHODS: A four-arm, double-blind randomized trial was conducted with 51 knees from 28 subjects aged ≥50 with primary knee OA. The treatments involved were HA alone, HA with hUC-MSCs, HA with somatotropin, and a combination of all three. Efficacy was measured through the International Knee Documentation Committee (IKDC) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and visual analog score (VAS), and MRI T2 mapping of cartilage was conducted on pre-implantation at the 6th and 12th month. RESULTS: All treatment arms showed improvements in the VAS and WOMAC scores over 12 months, suggesting some pain relief and functional improvement. However, MRI T2 mapping showed no significant cartilage regeneration across the groups. CONCLUSIONS: While the combined use of HA, hUC-MSCs, and somatotropin improved symptoms of knee OA, it did not enhance cartilage regeneration significantly. This study highlights the potential of these combinations for symptom management but underscores the need for further research to optimize these therapies for regenerative outcomes.

Polysaccharide-based hydrogels for cartilage regeneration.

Cartilage defect is one of the common tissue defect clinical diseases and may finally lead to osteoarthritis (OA) which threat patients' physical and psychological health. Polysaccharide is the main component of extracellular matrix (ECM) in cartilage tissue. In the past decades, polysaccharide-based hydrogels have shown great potential for cartilage regeneration considering unique qualities such as biocompatibility, enhanced cell proliferation, drug delivery, low toxicity, and many others. Structures such as chain length and chain branching make polysaccharides have different physical and chemical properties. In this review, cartilage diseases and current treatment options of polysaccharide-based hydrogels for cartilage defection repair were illustrated. We focus on how components and structures of recently developed materials affect the performance. The challenges and perspectives for polysaccharide-based hydrogels in cartilage repair and regeneration were also discussed in depth.

Comparative Analysis of Serum and Serum-Free Medium Cultured Mesenchymal Stromal Cells for Cartilage Repair.

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.

Experimental Investigation of the Effect of Polydioxanone Plate and Platelet-Rich Plasma on Cartilage Regeneration.

Objectives: This study aimed to investigate the effectiveness of polydioxanone (PDS) plate and platelet-rich plasma (PRP) on the regeneration of cartilage grafts, which are frequently used in nasal and septal surgery. Methods: Fifteen white New Zealand Albino-type female rabbits were used in the study. Our study was carried out on 4 different applications on each animal: cartilage only, cartilage+PRP, cartilage+PDS plate, and cartilage+PRP+PDS plate, and in 3 different periods: the first month, the second month, and the third month. Results: A significant difference was obtained between the groups using cartilage+PRP and cartilage+PRP+PDS only in the first month. When both application types were compared, a statistically significant decrease was found in the histopathological cartilage viability score after PDS use. In examining peripheral chondrocyte proliferation, a statistically significant difference was found only in the third-month comparison. When the group using only cartilage was compared with the group using cartilage+PDS, it was determined that peripheral chondrocyte proliferation was significantly reduced at the end of the third month with PDS. In evaluating fibrosis and foreign body reaction, a statistically significant increase was detected using a PDS plate. In histopathological cartilage viability score statistical analysis, a significant difference was obtained between the groups using cartilage+PRP and cartilage+PRP+PDS only in the first month. Degeneration in the cartilage structure was observed macroscopically in the specimens where the PDS plate was used. Shape change and cartilage deformation were observed in the PDS plate specimens. Conclusions: When the results were examined, this observation coincided with the statistically significant increase in foreign body reaction and fibrosis in the PDS plate groups. However, these results contradicted our hypothesis before the study and the information in the literature. Our results will help provide preliminary information and guidance for future studies and offer a different perspective.

Advances in cartilage tissue regeneration: a review of stem cell therapies, tissue engineering, biomaterials, and clinical trials.

Cartilage tissue, characterized by its limited regenerative capacity, presents significant challenges in clinical therapy. Recent advancements in cartilage regeneration have focused on integrating stem cell therapies, tissue engineering strategies, and advanced modeling techniques to overcome existing limitations. Stem cells, particularly Mesenchymal Stem Cells (MSCs) and induced pluripotent stem cells (iPSCs), hold promise for cartilage repair due to their ability to differentiate into chondrocytes, the key cells responsible for cartilage formation. Tissue engineering approaches, including 3D models, organ-on-a-chip systems, and organoids, offer innovative methods to mimic natural tissue microenvironments and evaluate potential treatments. MSC-based techniques, such as cell sheet tissue engineering, address challenges associated with traditional therapies, including cell availability and culture difficulties. Furthermore, advancements in 3D bioprinting enable the fabrication of complex tissue structures, while organ-on-a-chip systems provide microfluidic platforms for disease modeling and physiological mimicry. Organoids serve as simplified models of organs, capturing some complexity and enabling the monitoring of pathophysiological aspects of cartilage diseases. This comprehensive review underscores the transformative potential of integrating stem cell therapies, tissue engineering strategies, and advanced modeling techniques to improve cartilage regeneration and pave the way for more effective clinical treatments.

Mitochondrial transfer balances cell redox, energy and metabolic homeostasis in the osteoarthritic chondrocyte preserving cartilage integrity.

Osteoarthrosis (OA) is a leading cause of disability and early mortality, with no disease modifying treatment. Mitochondrial (MT) dysfunction and changes in energy metabolism, leading to oxidative stress and apoptosis, are main drivers of disease. In reaction to stress, mesenchymal stromal/stem cells (MSCs) donate their MT to damaged tissues. Methods: To evaluate the capacity of clinically validated MSCs to spontaneously transfer their MT to human OA chondrocytes (OA-Ch), primary cultured Ch isolated from the articular cartilage of OA patients were co-cultured with MT-labeled MSCs. MT transfer (MitoT) was evidenced by flow cytometry and confocal microscopy of MitoTracker-stained and YFP-tagged MT protein. MT persistence and metabolic analysis on target cells were assessed by direct transfer of MSC-derived MT to OA-Chs (Mitoception), through SNP-qPCR analysis, ATP measurements and Seahorse technology. The effects of MitoT on MT dynamics, oxidative stress and cell viability were gauged by western blot of fusion/fission proteins, confocal image analysis, ROS levels, Annexin V/7AAD and TUNEL assays. Intra-articular injection of MSC-derived MT was tested in a collagenase-induced murine model of OA. Results: Dose-dependent cell-to-cell MitoT from MSCs to cultured OA-Chs was detected starting at 4 hours of co-culture, with increasing MT-fluorescence levels at higher MSC:Ch ratios. PCR analysis confirmed the presence of exogenous MSC-MT within MitoT+ OA-Chs up to 9 days post Mitoception. MitoT from MSCs to OA-Ch restores energetic status, with a higher ATP production and metabolic OXPHOS/Glycolisis ratio. Significant changes in the expression of MT network regulators, increased MFN2 and decreased p-DRP1, reveal that MitoT promotes MT fusion restoring the MT dynamics in the OA-Ch. Additionally, MitoT increases SOD2 transcripts, protein, and activity levels, and reduces ROS levels, confering resistance to oxidative stress and enhancing resistance to apoptosis. Intra-articular injection of MSC-derived MT improves histologic scores and bone density of the affected joints in the OA mouse model, demonstrating a protective effect of MT transplantation on cartilage degradation. Conclusion: The Mitochondria transfer of MSC-derived MT induced reversal of the metabolic dysfunction by restoring the energetic status and mitochondrial dynamics in the OA chondrocyte, while conferring resistance to oxidative stress and apoptosis. Intra-articular injection of MT improved the disease in collagenase-induced OA mouse model. The restoration of the cellular homeostasis and the preclinical benefit of the intra-articular MT treatment offer a new approach for the treatment of OA.

A Randomized Controlled Trial Comparing "Early" Versus "Late" Periosteal Patch Attachment to Knee Chondral Defects in Autologous Chondrocyte Implantation.

OBJECTIVE: Traditional autologous chondrocyte implantation (ACI) involves arthroscopically harvesting a cartilage biopsy (stage 1), followed by arthrotomy 3 to 4 weeks later to apply a periosteal patch and implant culture-expanded chondrocytes underneath (stage 2). This study aimed to determine if patch application during stage 1 rather than stage 2 improved clinical outcome. DESIGN: A randomized controlled trial was conducted from 1998 to 2001. Patients were randomized to receive either traditional ACI (control/late) or ACI with "early" patch during stage 1 (intervention/early). Clinical outcome (Lysholm score) was assessed pre-operatively and annually post-operatively. RESULTS: Seventy-seven patients were recruited, with 40 patients randomized to the early and 37 to the late patch group. The overall mean pre-operative Lysholm score was 51.8 (range 11-89) and significantly improved by 11.1 points (95% confidence interval [CI] = 4.8 to 17.4) at mean 12.7 years (range 1.5-23.7) follow-up. Latest mean Lysholm scores for the early and late groups were 68.4 (95% CI = 19 to 100) versus 56.7 (95% CI = 18 to 98). Adjusted for covariate imbalances, no evidence was found for a difference between the groups (mean difference = 8.5, 95% CI = -5.2 to 22.2, P = 0.22). Twenty-year survival until any re-operation or arthroplasty was 59.6%/82.1% for the early and 56.8%/69.5% for the late group, with no evidence for a difference. CONCLUSION: ACI is an effective durable treatment for cartilage defects, with high levels of patient satisfaction and low failure rates. No evidence was found that applying the periosteal patch at the time of chondrocyte harvest improved long-term Lysholm scores or survival until any re-operation or arthroplasty.

The Use of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles in the Treatment of Osteoarthritis: Insights from Preclinical Studies.

Osteoarthritis (OA) is a prominent cause of disability, and has severe social and economic ramifications across the globe. The main driver of OA's pervasiveness is the fact that no current medical interventions exist to reverse or even attenuate the degeneration of cartilage within the articular joint. Crucial for cell-to-cell communication, extracellular vesicles (EVs) contribute to OA progression through the delivery of bioactive molecules in the inflammatory microenvironment. By repurposing this acellular means of signal transmission, therapeutic drugs may be administered to degenerated cartilage tissue in the hopes of encouraging regeneration. Positive outcomes are apparent in in vivo studies on this subject; however, for this therapy to prove itself in the clinical world, efforts towards standardizing the characterization, application, biological contents, and dosage are essential.

Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model.

PURPOSE: This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. METHODS: JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated. RESULTS: (1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining." CONCLUSION: The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

Sodium alginate hydrogels co-encapsulated with cell free fat extract-loaded core-shell nanofibers and menstrual blood stem cells derived exosomes for acceleration of articular cartilage regeneration.

This study presents a novel scaffold system comprising sodium alginate hydrogels (SAh) co-encapsulated with cell-free fat extract (CEFFE)-loaded core-shell nanofibers (NFs) and menstrual blood stem cell-derived exosomes (EXOs). The scaffold integrates the regenerative potential of EXOs and CFFFE, offering a multifaceted strategy for promoting articular cartilage repair. Coaxially electrospun core-shell NFs exhibited successful encapsulation of CEFFE and seamless integration into the SAh matrix. Structural modifications induced by the incorporation of CEFFE-NFs enhanced hydrogel porosity, mechanical strength, and degradation kinetics, facilitating cell adhesion, proliferation, and tissue ingrowth. The release kinetics of growth factors from the composite scaffold demonstrated sustained and controlled release profiles, essential for optimal tissue regeneration. In vitro studies revealed high cell viability, enhanced chondrocyte proliferation, and migration in the presence of EXOs/CEFFE-NFs@SAh composite scaffolds. Additionally, in vivo experiments demonstrated significant cartilage regeneration, with the composite scaffold outperforming controls in promoting hyaline cartilage formation and defect bridging. Overall, this study underscores the potential of EXOs and CEFFE-NFs integrated into SAh matrices for enhancing chondrocyte viability, proliferation, migration, and ultimately, articular cartilage regeneration. Future research directions may focus on elucidating underlying mechanisms and conducting long-term in vivo studies to validate clinical applicability and scalability.

Chemical preconditioning escalates chondrogenic activity in explant cultured human dental pulp stem cell study model for future temporomandibular joint regeneration.

Context: Human dental pulp stem cells (hDPSC) derived from dental pulp in conducive environment activated by chemicals can enhance chondrogenic cells for future animal model temporomandibular joint model. Aim: The study aims at evaluating the chemicals preconditioning (curcumin and rapamycin) efficacy toward chondrogenic proliferation of human dental pulp stem cells. Settings and Design: The in vitro study model with 10 premolar teeth extirpated pulp was processed under sterile chemical conditions. The cells viability was checked with calorimetric assay for adipogenic and chondrogenic, osteogenic lineages. The viability of the cells and the concentration of curcumin (CU) and rapamycin (RP) required for cell differentiation toward chondrogenic lineage were assessed. Material and Methods: The hDPSC was evaluated after explant long-term cultivation with characterization and chemical conditioning with dimethyl sulfoxide (DMSO) as control. MTT assay was used for cytotoxicity evaluation, cell viability, and proliferation. The dose optimization was observed with RP and CU. Chondrogenic proliferation was assessed with standard staining method of 0.1% Safranin O and 0.1% Alcian blue. Statistical Design: The flow cytometry analysis revealed good results for CD 90 compared to others. The intergroup analysis was done by ANOVA, and intragroup analysis was done by Post hoc Tukey's test. The intragroup analysis showed P value < 0.05 for RP in comparison between the various preconditioning agents CU and RP. The dosage of 10 µg/ml RP was considered statistically significant. Results: The flow cytometer analysis revealed good results for CD 90 compared to other surface markers. The dosage of 10 µg/ml RP was having good chondrogenic cell proliferation. The intragroup analysis showed P value < 0.05 for RP in comparison between the various preconditioning agents CU and RP. The calorimetric assay (MTT) quantitative analysis of the chondrogenic cells with Safranin O stain the standard deviation (SD = 0.017 for rapamycin), Alcian blue (SD = 0.49 for RP) in comparison to DMSO (control) and CU. Conclusion: RP activates mTOR pathway and hence stabilizes the stem cell maintenance of human dental pulp stem cell and the dose quantified can be used for future animal temporomandibular joint animal model.

High hydrostatic pressure treatment for advanced tissue grafts in reconstructive head and neck surgery.

The increasing importance of regenerative medicine has resulted in a growing need for advanced tissue replacement materials in head and neck surgery. Allo- and xenogenic graft processing is often time-consuming and can deteriorate the extracellular matrix (ECM). High hydrostatic pressure (HHP)-treatment could allow specific devitalization while retaining the essential properties of the ECM. Porcine connective tissue and cartilage were HHP-treated at 100-400 MPa for 10 min. Structural modifications following HHP-exposure were examined using electron microscopy, while devitalization was assessed through metabolism and cell death analyses. Furthermore, ECM alterations and decellularization were evaluated by histology, biomechanical testing, and DNA content analysis. Additionally, the inflammatory potential of HHP-treated tissue was evaluated in vivo using a dorsal skinfold chamber in a mouse model. The devitalization effects of HHP were dose-dependent, with a threshold identified at 200 MPa for fibroblasts and chondrocytes. At this pressure level, HHP induced structural alterations in cells, with a shift toward late-stage apoptosis. HHP-treatment preserved ECM structure and biomechanical properties, but did not remove cell debris from the tissue. This study observed a pressure-dependent increase of markers suggesting the occurrence of immunogenic cell death. In vivo investigations revealed an absence of inflammatory responses to HHP-treated tissue, indicating a favorable biological response to HHP. In conclusion, application of HHP devitalizes fibroblasts and chondrocytes at 200 MPa while retaining the essential properties of the ECM. Prospectively, HHP may simplify the preparation of allo- and xenogenic tissue replacement materials and increase the availability of grafts in head and neck surgery.

Therapeutic effect of three-dimensional hanging drop cultured human umbilical cord mesenchymal stem cells on osteoarthritis in rabbits.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown a positive effect on Osteoarthritis (OA), but the efficacy is still not significant in clinical. Conventional two-dimensional (2D) monolayer culture method is prone to cause MSCs undergoing replication senescence, which may affect the functions of MSCs. Three-dimensional (3D) culture strategy can sustain cell proliferative capacity and multi-differentiation potential. This study aimed to investigate the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured by 3D hanging drop method on OA. METHODS: hUC-MSCs were isolated from umbilical cord and cultured by 3D hanging drop method for 48 h. Scanning electron microscopy (SEM) was used to observe gross morphology 2D and 3D hUC-MSCs. Transcriptome comparison of gene expression differences between 2D and 3D hUC-MSCs. GO enrichment analysis, KEGG pathway enrichment analysis and GSEA enrichment analysis were used to analyze the impact of 3D hanging drop culture on the biological functions of hUC-MSCs. Female New Zealand rabbits (n = 12) were divided into 4 groups: Normal group, Model group, 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group. After 8 weeks, the gross and histological appearance of the cartilage was evaluated by safranin O-fast green staining and Mankin scoring system. The expression of type I collagen and type II collagen was detected by immunohistochemistry. The levels of IL-6, IL-7, TNFα, TGFß1 and IL-10 in the knee joint fluid were tested by ELISA. RESULTS: 3D hanging drop culture changed cell morphology but did not affect phenotype. The MSCs transcriptome profiles showed that 3D hanging drop culture method enhanced cell-cell contact, improved cell responsiveness to external stimuli and immunomodulatory function. The animal experiment results showed that hUC-MSCs could promote cartilage regeneration compared with Model group. 3D hUC-MSCs treatment group had a higher histological score and significantly increased type II collagen secretion. In addition, 3D hUC-MSCs treatment group increased the expression of anti-inflammatory factors TGFß1 and IL-10. CONCLUSION: The above experimental results illustrated that 3D hanging drop culture method could enhance the therapeutic effect of hUC-MSCs, and showed a good clinical application prospect in the treatment of OA.

3D printing and computer-aided design techniques for drug delivery scaffolds in tissue engineering.

INTRODUCTION: The challenge in tissue engineering lies in replicating the intricate structure of the native extracellular matrix. Recent advancements in AM, notably 3D printing, offer unprecedented capabilities to tailor scaffolds precisely, controlling properties like structure and bioactivity. CAD tools complement this by facilitating design using patient-specific data. AREA'S COVERED: This review introduces additive manufacturing (AM) and computer-aided design (CAD) as pivotal tools in advancing tissue engineering, particularly cartilage regeneration. This article explores various materials utilized in AM, focusing on polymers and hydrogels for their advantageous properties in tissue engineering applications. Integrating bioactive molecules, including growth factors, into scaffolds to promote tissue regeneration is discussed alongside strategies involving different cell sources, such as stem cells, to enhance tissue development within scaffold matrices. EXPERT OPINION: Applications of AM and CAD in addressing specific challenges like osteochondral defects and osteoarthritis in cartilage tissue engineering are highlighted. This review consolidates current research findings, offering expert insights into the evolving landscape of AM and CAD technologies in advancing tissue engineering, particularly in cartilage regeneration.

Photobiomodulation associated with alginate-based engineered tissue on promoting chondrocytes-derived biological responses for cartilage regeneration.

Articular cartilage is a connective tissue with limited self-healing potential, frequently affected by trauma and degenerative changes, leading to osteoarthritis. Photobiomodulation paired with engineered tissue can improve cartilage's poor intrinsic healing and overcome its restricted self-regeneration. In this study, alginate-based scaffolds were fabricated with varying concentrations of CaCl2 to achieve optimal mechanical, biocompatible, and biodegradable properties. The fluence-dependence of near-infrared (NIR) laser irradiation (830 nm) on chondrocyte viability and proliferation was investigated in a 2D environment across fluences (2.5-10 J/cm2). Optimal conditions of 3 % w/v CaCl2 and 5 J/cm2 were identified to construct alginate scaffolds and promote chondrocyte growth in 2D and 3D cultures. Single PBM (830 nm, 5 J/cm2) further exhibited a significant relative intensity of collagen type II immunostaining and stimulation of Col2a1 expression in 2D culture. Multiple PBM sessions (830 nm, 5 J/cm2) significantly enhanced chondrocyte proliferation and glycosaminoglycan production in alginate scaffolds, with a protocol of one session every four days being the most effective. Scanning electron microscopy revealed PBM-induced secretory granule formation, corresponding to a significant increase in extracellular vesicle release. Consequently, integrating PBM and alginate-based scaffolds is a promising technique for accelerating and optimizing cartilage regeneration, with potential application in tissue engineering.

ISG15 is involved in chondrogenic differentiation through activation of IFN-γ signaling.

Interferon-gamma (IFN-γ) was found to increase in the synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). However, few studies have been conducted to elucidate the role of IFN-γ in cartilage metabolism and regeneration. In this study, we investigated whether cartilage regeneration is driven by interferon-stimulated gene 15 (ISG15) under the control of IFN-γ. IFN-γ significantly increased ITS-induced chondrogenic differentiation of ATDC5 cells. Knockdown of IFN-γ receptor (IFN-γR) inhibited IFN-γ-induced chondrogenic differentiation and reduced ACAN and Col II expression. In addition, ISG15 expression was highly elevated in response to IFN-γ, whereas its expression was downregulated by knockdown of IFN-γR, indicating that ISG15 is closely related to IFN-γ signaling. Furthermore, chondrogenic differentiation and expression of ACAN and Col II were significantly reduced following knockdown of ISG15 in ATDC5 cells despite the presence of IFN-γ. ISGylation of cellular proteins found in chondrogenic differentiated cells was related to activation of IFN-γ signaling. In addition, ISG15/ISGylation was significantly observed in the regenerated cartilage tissue 7 days after FTCI of young mice compared with sham control. Our findings showed that upregulation of ISG15 and/or ISGylation of cellular proteins may play a critical role in cartilage regeneration through activation of IFN-γ signaling.