Enhancing bone-vessel coupling to form high-quality vascular-rich peri-implant bone is crucial for improving implant prognosis in elder patients. Notably, hypoxia-inducible factor 1α (HIF1α) is known to promote osteogenesis-angiogenesis coupling; however, this effect remains to be investigated in aged bone owing to the dual effect of HIF1α in different aged organs. In this study, HIF1α inhibitor or activator was applied to aged mice and their bone mesenchymal stem cells (BMSCs) to investigate the effects and inner mechanism of HIF1α on the peri-implant osteogenesis and angiogenesis in senescent status. Cell senescence, along with osteogenic and angiogenic abilities of aged BMSCs, was detected, respectively. Meanwhile, a femur implant implantation model was constructed on aged mice, and the bone-vessel coupling of peri-implant bone was observed. Mandibular bone morphology was also detected to further provide evidence for clinical oral implantation. Furthermore, p53 expression was examined and following HIF1α intervention. A reactive oxygen species (ROS) scavenger was also adopted to further investigate the roles of ROS in the HIF1α-p53 axis. Results showed that the suppression of HIF1α alleviated senescence and osteogenesis-angiogenesis coupling of aged BMSCs, while its activation aggravated these effects. The mandible phenotype and bone-vessel coupling in aged peri-implant bone also changed accordingly upon regulation of HIF1α. Mechanistically, p53 changed in the same direction as HIF1α and . Moreover, the ROS scavenger reversed the HIF1α-p53 relationship and weakened the effect of HIF1α inhibitor on peri-implant bone improvement. In conclusion, in aged mice, highly expressed HIF1α impaired peri-implant bone-vessel coupling and implant osseointegration through p53, and accumulated ROS was a prerequisite for HIF1α to positively regulate p53. These findings provide new insights into the role of HIF1α and the ROS-HIF1α/p53 signaling axis, offering potential therapeutic targets to improve implant outcomes in elderly patients.

N6-methyladenosine (mA) modification is critical in the regulation of osteoporosis (OP). Although ZC3H13 is an important mA methyltransferase, its specific regulatory effects and mechanisms in osteoporosis are not yet fully understood. Therefore, we investigated the impact of ZC3H13 on the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs) in osteoporosis and attempted to elucidate its underlying mechanism. Western blotting, quantitative reverse transcription polymerase chain reaction, and immunohistochemical staining were used to identify changes in ZC3H13 and osteogenic factor (RUNX2 and OPN) expression in osteoporosis. Gain- and loss-of-function experiments were conducted to study the impact of ZC3H13 on the osteogenic differentiation of osteoporotic BMSCs (OP-BMSCs). Transcriptomic sequencing, transmission electron microscopy, and intraperitoneal injection of the ferroptosis inhibitor ferrostatin-1 (Fer-1) were used to elucidate the downstream mechanisms regulated by ZC3H13 in osteoporosis. In addition, rescue assays were performed to elucidate the underlying molecular mechanisms involved. Here, we revealed that ZC3H13 was downregulated in OP-BMSCs and osteoporotic rat femurs, which correlated with the reduced osteogenic differentiation of OP-BMSCs. Functionally, ZC3H13 knockdown resulted in decreased osteogenic differentiation of the BMSCs, whereas ZC3H13 overexpression promoted the osteogenic differentiation of the OP-BMSCs. Furthermore, ZC3H13 knockdown was closely related to metal ion binding, reduced cell proliferation, and altered mitochondrial morphology. Treatment with the ferroptosis inhibitor Fer-1 partially reversed osteoporotic phenotypes . Mechanistically, ZC3H13 was shown to promote osteogenic differentiation in OP-BMSCs by inhibiting ferroptosis. Our study revealed that ZC3H13 promoted the osteogenic differentiation of BMSCs by inhibiting ferroptosis in osteoporosis. This research offers a reliable theoretical foundation for predicting and treating osteoporosis.

Scaffolds made from cartilage extracellular matrix are promising materials for articular cartilage repair, attributed to their intrinsic bioactivity that may promote chondrogenesis. While several cartilage matrix-based scaffolds have supported chondrogenesis and/or , it remains a challenge to balance the biological response (e.g., chondroinductivity) with structural (e.g., robust mechanical performance, >1 MPa in compressive stiffness) and translational (e.g., ease of surgical implantation) considerations. Few studies have evaluated encapsulated cell viability within high-stiffness (>1 MPa) hydrogels. We previously fabricated one formulation of a high-stiffness (>3 MPa) pentenoate-functionalized, solubilized, devitalized cartilage (PSDVC) hydrogel that possessed an injectable, paste-like precursor for easy surgical application. In the current study, the characterization of the PSDVC material was expanded by varying the degree of functionalization (i.e., 0.45-1.09 mmol/g) and amount of crosslinker, dithiothreitol (DTT), to improve the reproducibility of the high compressive moduli and evaluate the viability of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs) in high-stiffness cartilage matrix hydrogels. Prior to crosslinking, specific formulations functionalized with 0.80 mmol/g or less of pentenoate groups retained a paste-like precursor rheology. After crosslinking, these formulations produced hydrogels with greater than 1 MPa compressive stiffness. However, hBMSCs encapsulated in PSDVC hydrogels with lower functionalization (i.e., 0.57 mmol/g, no crosslinker) had a higher stiffness (i.e., 1.4 MPa) but the lowest viability of encapsulated hBMSCs (i.e., 5%). The middle PSDVC functionalization (i.e., 0.70 mmol/g) with DTT (i.e., 0.50 mmol thiols/g) demonstrated high cell viability (77%), high mechanical performance (1.65 MPa, 31% failure strain), and translational features (i.e., paste-like precursor, 1.5 min crosslinking time). For future evaluations of PSDVC hydrogels in cartilage repair, a middle functionalization (i.e., 0.70-0.80 mmol/g) with the addition of a crosslinker (i.e., 0.50 mmol thiols/g) had a desirable balance of high mechanical performance (i.e., >1 MPa compressive stiffness), high viability, and paste-like precursor for surgical translation.

Cartilage injuries are extremely common in the general population, and conventional interventions have failed to produce optimal results. Tissue engineering (TE) technology has been developed to produce neocartilage for use in a variety of cartilage-related conditions. However, progress in the field of cartilage TE has historically been difficult due to the high functional demand and avascular nature of the tissue. Recent advancements in cell sourcing, biostimulation, and scaffold technology have revolutionized the field and made the clinical application of this technology a reality. Cartilage engineering technology will continue to expand its horizons to fully integrate three-dimensional printing, gene editing, and optimal cell sourcing in the future. This review focuses on the recent advancements in the field of cartilage TE and the landscape of clinical treatments for a variety of cartilage-related conditions.

Bioscaffolds composed of extracellular matrix (ECM) have been shown to promote a profound transition in macrophages and T-cells from a proinflammatory to a prohealing phenotype with associated site-appropriate and constructive tissue remodeling rather than scar tissue formation. Matrix-bound nanovesicles (MBV) are a distinct class of extracellular vesicles that can be isolated from the ECM and can recapitulate these immunomodulatory effects on myeloid cells and , as shown in multiple preclinical models of inflammatory-driven diseases. However, the effect of this MBV-mediated immunomodulation upon the ability to mount an adaptive immune response following pathogenic challenge is unknown. The present study assessed the humoral immune response with and without repeated MBV administration in a mouse model of vaccination and infection. Mice were immunized on day 0, followed by an intraperitoneal MBV or methotrexate (MTRX) injection the next day and weekly thereafter for 5 weeks. Antipneumococcal polysaccharide immuglobulin G and immuglobulin M titers were no different between the vaccine + MBV and the vaccine-only groups, in contrast to the decreased titers in the MTRX-treatment group. Fifty percent of animals treated with MBV were protected from lethal septic infection with , and MBV treatment altered the population of immune cells within the lung following sublethal intranasal infection. Macrophages derived from bone marrow mononuclear cells harvested from MBV-treated mice showed persistent immunomodulatory effects following challenge with bacterial antigens. The results of this study show that MBV treatment does not compromise the ability to mount an adaptive immune response and suggest that MBV induce sustained immunomodulation in cells of the myeloid lineage.

The activation of chondrogenic progenitor cells (CPCs) in articular cartilage during a traumatic injury is vital for cartilage regeneration. Although our understanding of the mechanisms underlying CPC chondrogenic activation remains incomplete, there is evidence that exosomal microRNAs (miRNAs or miRs) are involved in tissue healing due to their regulating role of posttranscriptional gene expressions. In this study, we profiled enriched and differential expression of miRNAs in exosomes derived from bovine joint cells (CPCs, chondrocytes, and synoviocytes) via Next Generation Sequencing analysis and validated the potential therapeutic effects of candidate exosomal miRNAs for cartilage regeneration. For CPC-based cartilage regeneration, we tested the impact of administering miR-107, miR-140, and miR-148a on CPCs because we found that these miRNAs were highly and differentially expressed in chondrocytes-derived exosomes (CC-Exo). We found that: (1) miR-140 induced chondrogenic gene expression including SRY-box transcription factor 9, collagen type 2A1, and aggrecan, and (2) miR-107 suppressed catabolic gene expression including matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin motifs 5, and nitric oxide synthase 2. Our findings indicate that transfection of CPCs with specific chondrogenic miRNAs present in CC-Exo have the potential to promote CPC-based cartilage regeneration and could be an important component of posttraumatic osteoarthritis prevention.

Model systems play a crucial role in biological and biomedical research, especially in the search for new treatments for challenging diseases such as glioblastoma multiforme (GBM). Organoids are 3D multicellular "middle-ground" model systems that recapitulate highly organized and heterogeneous organ-like systems, often through stem cell differentiation. Incorporating Matrigel™ or other exogenous extracellular matrices (ECMs) that do not naturally occur in the human body is common practice for organoid generation, ignoring the role of dynamic reciprocity between the cells and the ECM in tissue development. In this study, we describe a method to develop GBM organoids (GBOs) from cells without the need for exogenous ECM encapsulation and without cell culture media changes to produce stable tissue-like organoids that reach a 4 mm diameter in as little as 6 weeks. We observed a transition from homogenous cell populations to tissue-like structures when GBOs were larger than 1 mm in diameter. Transcriptomic analysis revealed that the greatest gene expression changes occurred when GBOs were 2 mm in diameter, with collagen VI as the most upregulated ECM-related gene. Quantitative and histochemical assessments further supported native ECM synthesis with significantly higher levels of glycosaminoglycans and collagen in GBOs compared with spheroids. To our knowledge, this study presents the first reproducibly large GBOs with natively produced ECMs. Organoids with natively synthesized ECMs promise to eliminate artifacts and variability from aged, homogeneic, or xenogeneic scaffolds and to provide insights for ECM-targeted drug development.

The synovium is a loose connective tissue that separates the intra-articular (IA) joint compartments of all diarthrodial joints from the systemic circulation. It can be divided into two layers: the intima, a thin and cell-dense layer atop a more heterogeneous subintima, composed of collagen and various cell types. The subintima contains penetrating capillaries and lymphatic vessels that rapidly clear injected drugs from the joint space which may vary not only with drug size and charge but also with the microstructure and composition of the intima and subintima of the synovium. Prior work has measured the mechanical properties and solute diffusivities in the synovium of porcine, bovine, and human joints. Here, we measured the Young's moduli of synovium from smaller joints of the rat knee, as well as pig and human, using atomic force microscopy (AFM). The format for AFM enabled testing of intima and subintimal regions of synovium in all three species. The Young's moduli of the subintimal regions were similar across all three species (1-1.5 kPa). Furthermore, there was little evidence of differences in Young's moduli between synovium from the intima and subintima in each species. A general similarity of data from AFM testing with moduli measured with bulk testing of pig and human synovium suggests that AFM can be useful to measure the mechanical properties of smaller joint synovium and spatial variations in stiffness with depth. Enzymatic digestion of synovium tissue from the pig was also performed with findings of lower moduli values following treatment with chondroitinase ABC but not collagenase. Although the molecular composition of the synovium is not yet fully characterized and may vary across species, these findings suggest that noncollagenous species contribute to AFM-measured properties in synovium. These are some of the first data to measure mechanical properties in small joint synovium and will be useful in models studying IA drug clearances in joints with pathology and following treatment.

Osteoarthritis, a degenerative disease of articular cartilage and the leading cause of disability, is preceded by acute cartilage injury in a significant proportion of cases. Current auto- and allograft interventions are limited by supply and variability in therapeutic efficacy, prompting interest in tissue engineering solutions. Cell sheet tissue engineering, a scaffold-free regenerative technique, has shown promise in preclinical and clinical trials across various cell types and diseases. Polydactyly-derived juvenile cartilage-derived chondrocyte (JCC) sheets from juvenile patients are a potent cell source for developing allogeneic therapies. JCC sheets have proven safe and effective in animal models and as an add-on therapy in a recent clinical cartilage repair study. However, JCC expansion leads to de-differentiation, contributing to long healing times. This study hypothesized that differentiation of JCC sheets into hyaline-like cartilage constructs could accelerate cartilage regeneration without compromising implant integration. To this end, sheet integration, maturation, and healing of conventionally prepared vs. differentiated JCC sheets were compared in an established nude rat focal chondral defect model. Differentiated JCC sheets exhibit mature cartilage phenotypes prior to transplant. Both conventional and differentiated JCC sheets are reliably transplanted without additional fixation. Histological evaluation reveals that both transplant groups produced equivalent neocartilage regeneration, filling defects with mature hyaline cartilage at 2- and 4-weeks post-transplant. Notably, differentiated JCC sheets respond to signals, undergoing matrix remodeling and integration with adjacent and subchondral tissue. Given equivalent healing outcomes, the future utility of JCC sheet predifferentiation from other JCC donors with different healing capacities should be balanced against their increased culture costs over conventional sheets.

Tissue engineering provides a path forward for emerging personalized medicine therapies as well as the ability to bring about cures for diseases or chronic injuries. Traumatic spinal cord injuries (SCIs) are an example of a chronic injury in which no cure or complete functional recovery treatment has been developed. In part, this has been due to the complex and interconnected nature of the central nervous system (CNS), the cellular makeup, its extracellular matrix (ECM), and the injury site pathophysiology. One way to combat the complex nature of an SCI has been to create functional tissue-engineered scaffolds that replace or replenish the aspects of the CNS and tissue/ECM that are damaged following the immediate injury and subsequent immune response. This can be achieved by employing the tissue-engineering triad consisting of cells, biomaterial(s), and environmental factors. Stem cells, with their innate ability to proliferate and differentiate, are a common choice for cellular therapies. Natural or synthetic biomaterials that have tunable characteristics are normally used as the scaffold base. Environmental factors can range from drugs to growth factors (GFs) or proteins, depending on if the idea would be to stimulate exogeneous or endogenous cell populations or just simply retain cells on the scaffold for effective transplantation. For functional regeneration and integration for SCI, the scaffold must promote neuroprotection and neuroplasticity. Tissue-engineering strategies have shown benefits including neuronal differentiation, axonal regeneration, axonal outgrowth, integration into the native spinal cord, and partial functional recovery. Overall, this review focuses on the background that causes SCI to be so difficult to treat, the individual components of the tissue-engineering triad, and how combinatorial scaffolds can be beneficial toward the prospects of future SCI recovery.

In this 12-month long, preclinical large animal study using a canine model, we report that engineered osteochondral grafts (comprised of allogeneic chondrocyte-seeded hydrogels with the capacity for sustained release of the corticosteroid dexamethasone [DEX], cultured to functional mechanical properties, and incorporated over porous titanium bases), can successfully repair damaged cartilage. DEX release from within engineered cartilage was hypothesized to improve initial cartilage repair by modulating the local inflammatory environment, which was also associated with suppressed degenerative changes exhibited by menisci and synovium. We note that not all histological and clinical outcomes at an intermediary time point of three months paralleled 12-month outcomes, which emphasizes the importance of studies in valid preclinical models that incorporate clinically relevant follow-up durations. Together, our study demonstrates that engineered cartilage fabricated under the conditions reported herein can repair full-thickness cartilage defects and promote synovial joint health and function.

Long bone and craniofacial bone fractures amount to an overwhelming expenditure for patients and health care systems each year. Overall, 5-10% of all bone fractures result in some form of delayed or nonunion fractures. Nonunions occur from insufficient mechanical stabilization or a compromised wound environment lacking in vasculature and progenitor cells. The current standard for treating these critical-sized fractures and defects is the use of autologous bone grafts. However, advancements in tissue engineering have cultivated a shift in scientific efforts toward harnessing the body's own regenerative resources. As such, research on fracture healing has shifted as well. Transforming growth factor-beta 1 (TGFβ-1) has been studied in fracture healing for over 25 years, though many of these studies have been or in small animal models. The few studies in large animals have disagreement due to the heterogeneity within the experimental design. Because TGFβ-1 plays such a crucial role in the bone healing process, this systematic review investigates the application of TGFβ-1 in various carrier vehicles for repairing bone injuries in large animal and rabbit models. A systematic search was conducted in PubMed, Embase, and Web of Science (from database construction-October 2024). A total of 244 articles were screened, and 24 studies were included for review. Most large animal long bone studies used coated titanium implants, while most rabbit long bone studies used some form of degradable polymer constructs. TGFβ-1 doses in large animal long bone studies range from 0.005 to 750 µg, doses in large animal calvaria and mandible studies range from 1 to 5000 µg, and doses in rabbit long bone studies range from 0.05 to 120 µg. Nineteen out of 24 articles reviewed indicate successful use of TGFβ-1 for bone regeneration compared with experimental controls. It is clear that dose and controlled release of growth factor play a crucial role in defect closure, but outcome measures and success criteria were inconsistent across studies. More studies with consistent experimental designs are critical for understanding the therapeutic potential of TGFβ-1 in fracture repair, but overall, this review indicates that TGFβ-1 can be used alone or in conjunction with other growth factors to accelerate successful bone repair.

The high failure rate of surgical repair for tendinopathies has spurred interest in adjunct therapies, including exosomes (EVs). Mesenchymal stromal cell (MSC)-derived EVs (MSCdEVs) have been of particular interest as they improve several metrics of tendon healing in animal models. However, research has shown that EVs derived from tissue-native cells, such as tenocytes, are functionally distinct and may better direct tendon healing. To this end, we investigated the differential regulation of human primary macrophage transcriptomic responses and cytokine secretion by tenocyte-derived EVs (TdEVs) compared with MSCdEVs. Compared with MSCdEVs, TdEVs upregulated TNFa-NFkB and TGFB signaling and pathways associated with osteoclast differentiation in macrophages while decreasing secretion of several pro-inflammatory cytokines. Conditioned media of these TdEV educated macrophages drove increased tenocyte migration and decreased MMP3 and MMP13 expression. In contrast, MSCdEV education of macrophages drove increased gene expression pathways related to INFa, INFg and protection against oxidative stress while increasing cytokine expression of MCP1 and IL6. These data demonstrate that EV cell source differentially impacts the function of key effector cells in tendon healing and that TdEVs, compared with MSCdEVs, promote a more favorable tendon healing phenotype within these cells.

Recently, there has been increased attention on the treatment of cartilage repair. Overall, we constructed PHBVHHx-COL, a composite hydrogel of PHBVHHx-co-PEG and collagen, and evaluated its cartilage repair efficacy through and studies using hydrogel loaded with peripheral blood-derived mesenchymal stem cells (PBMSCs). Rheological properties and compressive mechanical properties of the hydrogels were systematically evaluated. The cytocompatibility of the hydrogels was evaluated using the Cell Counting Kit-8 test, live/dead staining, scratch test, and transwell test. The effect of chondrogenic differentiation of PBMSCs on hydrogels was evaluated using immunofluorescence staining and reverse transcription-polymerase chain reaction. Furthermore, the cartilage repair ability of the hydrogels was confirmed following injections in rabbit chondral defect models. Finally, the induced polarization of the hydrogel scaffold on macrophages was explored by the expression of CD86 and CD206. experimental results confirmed that PHBVHHx-COL-gel led to better cell migration, proliferation, and chondrogenic differentiation than PHBVHHx-PEG and COL hydrogels. Hematoxylin and eosin staining indicated that the tissue of the repaired area in the PHBVHHx-COL group was nearly in fusion with the surrounding normal tissue and the reconstruction of subchondral bone was good. Safranin-O staining and COL-2 immunohistochemistry indicated that the tissue of the repaired area in the PHBVHHx-COL group had more cartilage-specific matrix secretion. The PHBVHHx-COL group exhibited more M2 macrophage infiltration and less M1 macrophage presentation than the other groups. This study demonstrated that PHBVHHx-COL scaffolds loaded with PBMSCs significantly promoted the repair of cartilage injury through immune regulation by M2 polarization and could be potential candidates for cartilage tissue engineering.

Adipose tissue engineering requires effective strategies for regenerating adipose tissue, with adipose-derived stem cells (ASCs) being favored due to their robust self-renewal capacity and multipotent differentiation potential. In this study, the efficacy of poly-L-lactic acid (PLLA) mesh containing collagen sponge (CS), seeded with ASCs to promote adipose tissue formation, was investigated. PLLA-CS implants seeded with GFP-positive ASCs were inserted at high concentration (1 × 10 cells/implant, H-ASC) and low concentration (1 × 10 cells/implant, L-ASC), as were unseeded controls. Adipogenesis was evaluated at 3, 6, and 12 months using a rat inguinal model. At 3 months, the weight and volume of newly formed tissues in the H-ASC group were significantly higher than those in the control group. Histological assessment revealed that the area of all newly formed tissue, including the adipose tissue inside the implants in the H-ASC group, was larger at 6 and 12 months compared with that of the control and L-ASC groups, with the adipose percentage at 12 months being higher in the H-ASC group than in the control group. GFP-positive ASCs in both the L-ASC and H-ASC groups adhered to the CS scaffolds and survived for up to 12 months postimplantation, with spontaneous differentiation into adipocytes observed exclusively in the H-ASC group. Double immunofluorescence confirmed the presence of GFP-positive adipocytes. In summary, this study demonstrated that ASCs coimplanted with PLLA-CS implants could enhance adipose tissue formation within the implants. Uninduced ASCs were capable of spontaneously differentiating into adipocytes within the PLLA-CS implants, with differentiation correlating with the number of implanted cells.

This perspective article draws on lessons learned at the 7th TERMIS World Congress held in Seattle, Washington in June 2024. This gathering of prominent researchers and translational scientists in tissue engineering and regenerative medicine (TERM) from around the world provided a forum to consider the impact of tissue engineering and its future directions. New frontiers are considered in the context of global challenges, including clinical translation and recent advances in pediatric tissue engineering, supercritical fluid technology for scaffold fabrication and sterilization, and learning from successful failures in tissue engineering and regenerative medicine. Bench-to-bedside translational strategies, inclusive research strategies, regulatory hurdles, and ethics linked to navigating responsibilities and innovations, are identified as important drivers in the field.

Allogenic demineralized bone matrix (DBM) is widely used for bone repair and regeneration due to its osteoinductivity and osteoconductivity. The present study utilized acellular dermis microfibers to improve the DBM's clinical handling properties and to enhance bone regeneration. Donated human cadaver skin was de-epidermized and decellularized to be acellular dermal matrix (ADM), which was further processed into microfibers. Donated human bone was micronized and partially demineralized (∼30% calcium removal) for optimal bone regeneration. A flexible ADM/DBM composite foam was fabricated with ADM microfibers and DBM particles. Structural analysis found that the ADM/DBM composite foam had proper porosity with interconnected micropores and rapid wettability, and good stability upon cyclic compressions, whereas cytotoxicity test, collagenase degradation, and rat subcutaneous implantation showed good biocompatibility and biodegradability. The composite foam, used for coculture, significantly increased the alkaline phosphatase activity of C2C12 cells and upregulated the expression of osteogenesis-related genes of human umbilical cord mesenchymal stem cells. Using the rat Φ8 mm calvarium defect repair model, the ADM/DBM composite foam demonstrated superior osteogenicity by rapidly inducing new bone formation and achieving complete closure of the bone defects, as compared with the commercially available bone graft for skull repair (SkuHeal). Therefore, the ADM/DBM composite foam holds promise as a superior DBM-based product for repairing critical bone defects.

In the present study, acellular cartilage matrix (ACM) was modified with poly-l-lysine/hyaluronic acid (PLL/HA) multilayers via detergent-enzyme chemical digestion and layer-by-layer self-assembly technology. This modified ACM was then loaded with Transforming Growth Factor Beta 3 (TGF-β3) and incorporated into a thermosensitive hydrogel (TH) to create a HA/PLL-ACM/TH composite scaffold with sustained-release function. This study aimed to evaluate the efficacy of this novel composite scaffold in promoting chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and facilitating osteochondral defect repair. , isolated, and cultured rat BMSCs were inoculated in equal amounts into TH, ACM/TH, and HA/PLL-ACM/TH groups, with or without TGF-β3 supplementation, for 21 days. Western blot (WB) analysis and immunofluorescence staining were employed to assess the expression levels of collagen II, aggrecan, and SOX-9. , osteochondral defect was created in the Sprague-Dawley rat trochlea using microdrilling. TH, ACM/TH, and HA/PLL-ACM/TH scaffolds, with or without TGF-β3, were implanted into the defect. After 6 weeks, the repairs were evaluated macroscopically, using Micro computed tomography (micro-CT), histological analysis, and immunohistochemistry. The results demonstrated that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 significantly upregulated the expression of collagen II, aggrecan, and SOX-9 compared with the control and other experimental groups. Furthermore, at 6 weeks postsurgery, the HA/PLL-ACM/TH group loaded with TGF-β3 exhibited superior tissue formation on the joint surface, as confirmed by micro-CT and histological evidence, indicating improved osteochondral repair. These findings suggest that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 holds promise as a therapeutic strategy for osteochondral defect and offers a novel approach for utilizing acellular cartilage microfilaments.

The objective of this study is to investigate the influence of exogenous mitochondria (Mt) internalization on the Mt membrane potential of cells. Cationized gelatin nanospheres (cGNS) were prepared to mix Mt at different ratios to prepare Mt associated with cGNS (Mt-cGNS). The Mt internalization depended on the Mt/cGNS mixing ratio to achieve the maximum at the ratio of 3/1. Rho 0 cells of a Mt function-deficient line were prepared to evaluate the enhancement of Mt membrane potential of rho 0 cells after the internalization of Mt-cGNS. When evaluated by using tetramethylrhodamine methyl ester reagent, the mitochondrial membrane potential of rho 0 cells after incubation with Mt-cGNS enhanced compared with that incubated with Mt only and maintained at a significantly higher level even for 6 days. The Mt-cGNS were internalized into rho 0 cells by an actin-dependent pathway, followed by fused with endogenous Mt. It is concluded that association with the cGNS enabled Mt to enhance the cellular internalization, followed by the fusion with endogenous Mt to maintain an enhanced Mt membrane potential.

Senescence and osteogenic differentiation potential loss limited bone nonunion treatment effects of bone marrow-derived mesenchymal stem cells (BMSCs). MiR-100-5p/Lysine(K)-specific demethylase 6B (KDM6B) can inhibit osteogenesis, but their effects on bone union remain unclear. This study aims to investigate the effects of miR-100-5p/KDM6B on osteogenic differentiation and bone defects. Wild-type or microRNA 100 (miR-100) knockdown mice underwent critical-size defect (CSD) cranial surgery and collagen I/poly-γ-glutamic acid scaffold treatment. The crania was observed using microcomputed tomography, hematoxylin and eosin staining, Masson staining, alkaline phosphatase (ALP) staining, immunohistochemistry, and immunofluorescence. Primary-cultured BMSCs transfected with miR-100-5p mimic/inhibitor and KDM6B cDNA were evaluated for osteogenic differentiation using Alizarin Red staining, ALP activity detection, and Western blot analysis. Genetic transcription levels were detected using quantitative reverse transcription polymerase chain reaction. This study found that miR-100 depletion promotes defect healing in mouse calvaria, increases the proportion of new bone and osteoblasts in calvaria, and activates the expression of KDM6B and osteocalcin (OCN) proteins, promoting the transcription of bone morphogenetic protein-2, Runt-related transcription factor 2 (Runx2), OCN, and KDM6B, while methylation of lysine 27 on histone H3 (H3K27me3) decreased. Furthermore, miR-100-5p mimics suppressed osteogenic differentiation by inhibiting KDM6B with increased H3K27me3, ALP, Runx2, OCN, and osteopontin protein expression, while miR-100-5p inhibitors have opposite effects. Moreover, KDM6B can reverse miR-100-5p mimic effects. Notably, scaffolds carrying miR-100-5p mimics/inhibitors transfected BMSCs were placed in CSD mice and found that miR-100-5p inhibitors have a better effect on CSD healing and increase new bone without inflammatory cell infiltration. This study proved that miR-100-5p depletion promotes bone union and osteogenic differentiation of BMSCs via KDM6B/H3K27me3.