UNASSIGNED: 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.
UNASSIGNED: The study aims at evaluating the chemicals preconditioning (curcumin and rapamycin) efficacy toward chondrogenic proliferation of human dental pulp stem cells.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

Cartilage defects are frequently caused by trauma, illness and degradation of the cartilage. If these defects are not sufficiently treated, the joints will degrade irreversibly, possibly resulting in disability. Articular cartilage lacks blood vessels and nerves and is unable to regenerate itself, so the repair of cartilage defects is extremely challenging in clinical treatment. Tissue engineering technology is an emerging technology in cartilage repair and cartilage regeneration. 3D-printed hydrogels show great potential in cartilage tissue engineering for the fabrication of 3D cell culture scaffolds to mimic extracellular matrix. In this study, we construct a 3D-printed hydrogel loaded with nanoparticles by electrostatic interaction and photo cross-linking for the regeneration of cartilage, which has adaptable and drug-continuous release behavior. A photopolymerizable bioink was prepared using recombinant collagen, chitosan, nanoclay Laponite-XLG and nanoparticles loaded with Kartogenin (KGN). This bioink was added with KGN, a small molecule drug that promotes cartilage differentiation, and as a result, the 3D-printed CF/CM/3%LAP/KGN scaffolds obtained by extrusion printing is expected to be used for cartilage repair. It was shown that the 3D-printed scaffolds had good cytocompatibility for human bone marrow mesenchymal stem cells (hBMSCs) and exhibited excellent antimicrobial properties, the continuous release of KGN in the scaffold induced the hBMSCs differentiation into chondrocytes, which significantly enhanced the expression of collagen II and glycosaminoglycan. In vivo studies have shown that implantation of KGN-loaded scaffolds into cartilage-injured tissues promoted cartilage tissue regeneration. This study demonstrated that 3D-printed CF/CM/3%LAP/KGN scaffolds can be used for cartilage repair, which is expected to lead to new healing opportunities for cartilage injury-based diseases.

The aim of this study was to evaluate the chondrogenic potential of chondrocyte transplants cultured in vitro on polyethersulfone (PES) membranes. Forty-eight rabbits (96 knee joints) were used in the project. The synthetic, macro-porous PES membranes were used as scaffolds. Fragments of articular cartilage were harvested from non-weight-bearing areas of the joints of the animals. Chondrocytes were isolated and then cultivated on PES scaffolds for 3 weeks. The animals were divided into four groups. All the lesions in the articular cartilage were full thickness defects. In Group I, autogenic chondrocytes on PES membranes were transplanted into the defect area; in Group II, allogenic chondrocytes on PES membranes were transplanted into the defect area; in Group III, pure PES membranes were transplanted into the defect area; and in Group IV, lesions were left untreated. Half of the animals from each group were terminated after 8 weeks, and the remaining half were terminated 12 weeks postoperatively. The samples underwent macroscopic evaluation using the Brittberg scale and microscopic evaluation using the O\'Driscoll scale. The best regeneration was observed in Groups II and I. In Group I, the results were achieved with two surgeries, while in Group II, only one operation was needed. This indicates that allogenic chondrocytes do not require two surgeries, highlighting the importance of further in vivo studies to better understand this advantage. The success of the study and the desired properties of PES scaffolds are attributed mainly to the presence of sulfonic groups in the structure of the material. These groups, similar to chondroitin sulfate, which naturally occurs in hyaline cartilage, likely enable mutual affinity between the scaffold and cells and promote scaffold colonization by the cells.

Background/Objectives: Encouraging results have been reported for Platelet-Rich Plasma (PRP) treatment for knee osteoarthritis (KOA). This study reports the efficacy and safety of a high dose of neutrophile and red-blood-cell-depleted PRP to treat patients with KOA. Methods: A total of 212 consecutive patients diagnosed with Kellgren-Lawrence (KL) grading 1-3 KOA chronic knee pain for at least 1 year were treated with three injections at 15-day intervals with a high dose of neutrophil-depleted PRP (4 billion platelets). Clinical outcomes were retrospectively recorded as the percentage of responders at 3-, 6-, and 12-month follow-up, following the OMERACT-OARSI criteria. Pain, through the VAS score and WOMAC score, was also been recorded. Results: A total of 4 mL of PRP containing 4 × 109 platelets was obtained by single-spin centrifugation and injected intra-articularly into each patient with no preactivation. The overall responder rate of patients responding to the OMERACT-OARSI criteria at 3, 6, and 12 months was 68.9%, 72.7%, and 70.6%, respectively. A significant improvement in VAS and WOMAC scores at 3-, 6-, and 12-month follow-up compared to the pretreatment value (p < 0.01) was observed. The lowest VAS score was observed at 6 months overall and in all three KL-graded groups. The KL2 groups showed the best results regarding pain reduction and their WOMAC score at 6 months (p < 0.01). Conclusions: For KL1-3 KOA, a high dosage of neutrophil-depleted PRP is a successful treatment. It has long-lasting effects that last up to one year, relieves symptoms, and may slow the advancement of the disease.

Stem cell-based therapy holds promise for cartilage regeneration in treating knee osteoarthritis (KOA). Injectable hydrogels have been developed to mimic the extracellular matrix (ECM) and facilitate stem cell growth, proliferation, and differentiation. However, these hydrogels face limitations such as poor mechanical strength, inadequate biocompatibility, and suboptimal biodegradability, collectively hindering their effectiveness in cartilage regeneration. This study introduces an injectable, biodegradable, and self-healing hydrogel composed of chitosan-PEG and PEG-dialdehyde for stem cell delivery. This hydrogel can form in situ by blending two polymer solutions through injection at physiological temperature, encapsulating human adipose-derived stem cells (hADSCs) during the gelation process. Featuring a 3D porous structure with large pore size, optimal mechanical properties, biodegradability, easy injectability, and rapid self-healing capability, the hydrogel supports the growth, proliferation, and differentiation of hADSCs. Notably, encapsulated hADSCs form 3D spheroids during proliferation, with their sizes increasing over time alongside hydrogel degradation while maintaining high viability for at least 10 days. Additionally, hADSCs encapsulated in this hydrogel exhibit upregulated expression of chondrogenic differentiation genes and proteins compared to those cultured on 2D surfaces. These characteristics make the chitosan-PEG/PEG-dialdehyde hydrogel-stem cell construct suitable for direct implantation through minimally invasive injection, enhancing stem cell-based therapy for KOA and other cell-based treatments.

Utilizing transplanted human umbilical cord mesenchymal stem cells (HUMSCs) for cartilage defects yielded advanced tissue regeneration, but the underlying mechanism remain elucidated. Early after HUMSCs delivery to the defects, we observed substantial apoptosis. The released apoptotic vesicles (apoVs) of HUMSCs promoted cartilage regeneration by alleviating the chondro-immune microenvironment. ApoVs triggered M2 polarization in macrophages while simultaneously facilitating the chondrogenic differentiation of endogenous MSCs. Mechanistically, in macrophages, miR-100-5p delivered by apoVs activated the MAPK/ERK signaling pathway to promote M2 polarization. In MSCs, let-7i-5p delivered by apoVs promoted chondrogenic differentiation by targeting the eEF2K/p38 MAPK axis. Consequently, a cell-free cartilage regeneration strategy using apoVs combined with a decellularized cartilage extracellular matrix (DCM) scaffold effectively promoted the regeneration of osteochondral defects. Overall, new mechanisms of cartilage regeneration by transplanted MSCs were unconcealed in this study. Moreover, we provided a novel experimental basis for cell-free tissue engineering-based cartilage regeneration utilizing apoVs.

This study investigated the mechanism of the extracellular matrix-mimicking hydrogel-mediated TGFB1/Nrf2 signaling pathway in osteoarthritis using bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exos). A GMOCS-Exos hydrogel was synthesized and evaluated for its impact on chondrocyte viability and neutrophil extracellular traps (NETs) formation. In an OA rat model, GMOCS-Exos promoted cartilage regeneration and inhibited NETs formation. Transcriptome sequencing identified TGFB1 as a key gene, with GMOCS-Exos activating Nrf2 signaling through TGFB1. Depletion of TGFB1 hindered the cartilage-protective effect of GMOCS-Exos. This study sheds light on a promising therapeutic strategy for osteoarthritis through GMOCS-Exos-mediated TGFB1/Nrf2 pathway modulation.

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.

BACKGROUND: Human adipose-derived stem cells (ADSCs) exert a strong anti-inflammatory effect, and synovium-derived stem cells (SDSCs) have high chondrogenic potential. Thus, this study aims to investigate whether a combination of human ADSCs and SDSCs will have a synergistic effect that will increase the chondrogenic potential of osteoarthritis (OA) chondrocytes in vitro and attenuate the cartilage degeneration of early and advanced OA in vitro.
METHODS: ADSCs, SDSCs, and chondrocytes were isolated from OA patients who underwent total knee arthroplasty. The ADSCs-SDSCs mixed cell ratios were 1:0 (ADSCs only), 8:2, 5:5 (5A5S), 2:8, and 0:1 (SDSCs only). The chondrogenic potential of the OA chondrocytes was evaluated in vitro with a transwell assay or pellet culture with various mixed cell groups. The mixed cell group with the highest chondrogenic potential was then selected and injected into the knee joints of nude rats of early and advanced OA stages in vivo. The animals were then evaluated 12 and 20 weeks after surgery through gait analysis, von frey test, microcomputed tomography, MRI, and immunohistochemical and histological analyses. Finally, the mechanisms underlying these findings were investigated through the RNA sequencing of tissue samples in vivo and Western blot of the OA chondrocyte autophagy pathway.
RESULTS: Among the MSCs treatment groups, 5A5S had the greatest synergistic effect that increased the chondrogenic potential of OA chondrocytes in vitro and inhibited early and advanced OA in vivo. The 5A5S group significantly reduced cartilage degeneration, synovial inflammation, pain sensation, and nerve invasion in subchondral nude rat OA, outperforming both single-cell treatments. The underlying mechanism was the activation of chondrocyte autophagy via the FoxO1 signaling pathway.
CONCLUSIONS: A combination of human ADSCs and SDSCs demonstrated higher potential than a single type of stem cell, demonstrating potential as a novel treatment for OA.

Regeneration of hyaline cartilage in human-sized joints remains a clinical challenge, and it is a critical unmet need that would contribute to longer healthspans. Injectable scaffolds for cartilage repair that integrate both bioactivity and sufficiently robust physical properties to withstand joint stresses offer a promising strategy. We report here on a hybrid biomaterial that combines a bioactive peptide amphiphile supramolecular polymer that specifically binds the chondrogenic cytokine transforming growth factor β-1 (TGFβ-1) and crosslinked hyaluronic acid microgels that drive formation of filament bundles, a hierarchical motif common in natural musculoskeletal tissues. The scaffold is an injectable slurry that generates a porous rubbery material when exposed to calcium ions once placed in cartilage defects. The hybrid material was found to support in vitro chondrogenic differentiation of encapsulated stem cells in response to sustained delivery of TGFβ-1. Using a sheep model, we implanted the scaffold in shallow osteochondral defects and found it can remain localized in mechanically active joints. Evaluation of resected joints showed significantly improved repair of hyaline cartilage in osteochondral defects injected with the scaffold relative to defects injected with the growth factor alone, including implantation in the load-bearing femoral condyle. These results demonstrate the potential of the hybrid biomimetic scaffold as a niche to favor cartilage repair in mechanically active joints using a clinically relevant large-animal model.