Tissue engineering and regenerative medicine最新文献

Background: A 3D-printing technology using polycaprolactone (PCL) has shown promise for the development of patient-customized tracheal constructs. However, no studies have compared the mechanical properties of various grades of PCL using animal experiments under the same conditions. In this study, the mechanical properties and tissue reconstruction abilities of research-grade (RG) and medical-grade (MG) PCL scaffolds were compared in rabbit tracheal defect models.

Method: Customized scaffolds for the rabbit's segmental defect was manufactured using an extrusion-based 3D printing system and two types of PCL. Six months after transplantation into trachea defected rabbits, transplanted areas were excised to evaluate its mechanical properties, and the reconstruction of the damaged tissue were analyzed through endoscope and tissue staining. And, the change in molecular weight of PCL before and after transplantation was compared using Gel permeation chromatography (GPC). Molecular weight changes PCL scaffolds before and after gamma radiation were also compared using GPC.

Results: The medical-grade PCL scaffold (MG) group showed superior ultimate stress, strain, and tissue reconstruction compared with the research-grade PCL scaffold (RG) group, demonstrating better strength, ductility, and mucosal tissue regeneration. However, MG PCL scaffold degrades more rapidly in the body, as indicated by a notable decrease in molecular weight and ultimate stress post-transplantation. Gamma sterilization, which is an essential process for implants, did not affect the molecular weight of PCL, demonstrating its effectiveness in sterilization.

Conclusion: Our results highlight the substantial differences between RG and MG PCL scaffolds, emphasizing the need for researchers to thoroughly evaluate PCL properties before conducting animal studies or clinical trials to ensure the accurate prediction of experimental outcomes.

Background: Regenerative therapy using extracellular vesicles (EVs) is a promising approach for the supportive treatment of chronic limb-threatening ischaemia. Herein, we examined the angiogenic potential of EVs derived from genetically modified mesenchymal stromal cells (MSCs), focusing on the angio-micro RNAs (miRNAs) in EVs.

Methods: Bone marrow-derived MSCs (BM-MSCs) were transfected with lentiviral vectors containing specific angio-miRNAs (miRNA-126, -135b, or -210), and miRNA overexpression was confirmed using quantitative polymerase chain reaction (qPCR). EVs were isolated from the BM-MSC culture medium and characterised using fluorometry, nanoparticle tracking analysis, and ExoScreen assays. In vitro, human umbilical vein endothelial cells (HUVECs) were used to evaluate the angiogenic potential of the EVs. In vivo, EVs were injected into the ischaemic hindlimb muscles of mice, and limb ischaemia severity, blood perfusion, and histological analysis of muscle tissue were performed.

Results: qPCR analysis confirmed the overexpression of angio-miRNAs in MSCs transfected with lentiviral vectors. Isolated EVs expressed CD63 and had consistent protein-to-particle ratios. Tube formation was significantly enhanced when HUVECs were cultured with EV126, EV135b, or their combination (EV126 + EV135b) (p < 0.05), compared to BM-MSC co-culture. In vivo, only the double and triple EV groups significantly improved limb perfusion compared to the EVcontrol (p < 0.05); single EVs showed no significant difference. Histological analysis showed increased capillary density in ischaemic muscles following injection of combined EVs.

Conclusion: EVs derived from genetically modified MSCs promoted angiogenesis both in vitro and in vivo, with a combination of modified EVs demonstrating significantly superior therapeutic effects than single or native EVs.

Background: Endothelial cells (ECs) are key regulators of vascular function, adapting to mechanical forces, such as shear stress to maintain vascular homeostasis. Disruption of this adaptation, particularly in the regions of disturbed flow, contributes to endothelial dysfunction and the development of atherosclerosis later on.

Methods: We prepared a custom-designed PDMS-based flow chamber to apply controlled shear stress (2 or 7 dynes/cm²) to human umbilical vein endothelial cells. ECs were cultured on gelatin-coated coverslips and exposed to different shear flows for up to 12 h. Cell alignment was confirmed by angle measurements using ImageJ. Gene expression of SIRT4, PIEZO1, NOTCH1, and LOX-1 was determined via qPCR, and protein levels were assessed by western blot. Specific gene knockdown was also conducted using siRNAs, targeting either PIEZO1 or SIRT4. Oxidized LDL uptake was evaluated using DiI-labeled Ox-LDL and quantified by fluorescence imaging. Immunofluorescence staining of ECs was performed to visualize VE-cadherin, F-actin, and nuclei. All quantitative data were subjected to statistical analysis.

Results: We demonstrated that the mechanosensitive ion channel PIEZO1, regulates SIRT4 expression in response to shear stress. Under atheroprotective shear stress (7 dyne/cm²), PIEZO1-mediated upregulation of SIRT4 was observed, while atheroprone shear stress (2 dyne/cm²) led to reduced expression. Functional assays showed that SIRT4 protects endothelial cells from Ox-LDL uptake, a key factor in atherosclerosis. SIRT4 silencing increased Ox-LDL accumulation even under protective flow. This effect, and its link to LOX-1, was dependent on PIEZO1 signaling.

Conclusion: Current findings suggest that the PIEZO1-SIRT4 axis may modulate endothelial responses to shear stress, offering a protective mechanism against Ox-LDL-induced dysfunction and pathology. Our study underscores the potential of SIRT4 as a therapeutic target to mitigate vascular disorders associated with oxidative stress and disturbed blood flow.

Background: Osteoarthritis (OA) represents a major global health challenge with no ideal treatment options available. Early-stage treatment typically focuses on symptomatic relief of pain and stiffness; while late-stage patients can only opt for surgical interventions such as joint replacement to improve quality of life. Cell-free therapy based on extracellular vesicles (EVs) has offered a novel therapeutic approach for regulating bone metabolism and repairing cartilage, demonstrating emerging potential.

Methods: Publications related to OA and Cell-free therapy based on EVs were retrieved from the Web of Science Core Collection database from 1991 to 2024. Our study employed bibliometric methods to analyze publication trends, leading contributing countries, institutions, authors, journals, and emerging trends and research directions.

Results: The analysis has revealed a rapid growth in publications since 2019. China dominated both in terms of publication output and citation counts. The most productive institution is Shanghai Jiao Tong University. The most prolific publishing outlet journal was the International Journal of Molecular Sciences, while in terms of citation impact, Biomaterials ranked first. De Girolamo, Laura from Aix-Marseille Universite and Ragni, Enrico from IRCCS Istituto Ortopedico Galeazzi shared the top position in publication output, while Noel, Daniele from the Universite de Montpellier was the author receiving the most citations. Research primarily clustered around key themes including: (1) therapeutic mechanisms of cell-free treatment based on EVs in OA, (2) research advances in cell-free treatment based on EVs, (3) exosome engineering, and (4) a novel drug delivery system for EVs.

Conclusion: This is the first bibliometric study on cell-free therapy based on EVs for OA, providing orthopedic and regenerative medicine experts with comprehensive perspectives on the field's current status and future development directions.

Background: This study investigated a circRNA (Circ_Atp8a1) in regulating intestinal epithelial repair in intestinal mucosal barrier damage.

Methods: A mouse model of intestinal mucosal barrier damage caused by burn injury was constructed. Skin and intestinal histopathologic changes in injured and control mice were compared. Glycolytic enzyme protein expression, lactate production, and glucose consumption in intestinal tissues were detected. Microarray analysis was used to screen differentially expressed circRNAs in mucosal tissues, and RT-qPCR, Sanger sequencing, RNAse R test, nucleoplasmic isolation experiments, and fluorescence in situ hybridization (FISH) were used to characterize the circular structure and localization of Circ_Atp8a1. In Caco-2 cells, adenoviral overexpression vector and small interfering RNA (siRNA) were constructed to regulate Circ_Atp8a1 expression. Cell proliferation and migration were detected by combining with the experiments of CCK-8, EdU, wound healing, and Transwell. The interaction between Circ_Atp8a1 and miR-200b-3p was investigated by dual luciferase reporter assay, RNA pull-down assay, and FISH assay. The target gene of miR-200b-3p was predicted and validated. Finally, the effects of intraperitoneal injection of KD-Circ_Atp8a1 and OE-Circ_Atp8a1 on intestinal mucosal damage in burned mice were observed by in vivo experiments.

Results: Mice with burn-induced intestinal mucosal damage had higher CMDI scores, increased expression of glycolytic enzymes in intestinal tissues, and altered glycolytic processes. A total of 308 aberrantly expressed circRNAs were screened, among which Circ_Atp8a1 was significantly down-regulated and mainly distributed in cytoplasm and jejunal crypts. In Caco-2 cells, overexpression of Circ_Atp8a1 inhibited cell proliferation, migration, and glycolysis, and knockdown of Circ_Atp8a1 did the opposite. Circ_Atp8a1 acted as a sponge for miR-200b-3p, which targeted and inhibited IGF2, which affected glycolysis-related metrics. Circ_Atp8a1 regulated IGF2 indirectly through miR-200b-3p, which in turn regulated intestinal mucosal damage. in vivo experiments showed that overexpression of Circ_Atp8a1 could inhibit miR-200b-3p expression, promote IGF2 expression, reduce intestinal mucosal damage and decrease mucosal permeability.

Conclusion: Circ_Atp8a1 plays a key regulatory role in the process of intestinal mucosal damage and affects the process of glycolysis through adsorption of miR-200b-3p to regulate IGF2. It is expected to be a new target for the treatment of intestinal mucosal damage.

Background: The lacunar-canalicular system (LCS) serves as the mechanobiological foundation for bone tissue metabolism, mechanotransduction, and functional adaptation. However, the impact of solutes with varying molecular weights on LCS mass transfer under gravity remains unclear.

Methods: Rhodamine tracers of varying molecular weights were injected into the peritoneal cavity of SD rats and LCS mass transfer experiments were performed under normal and hypergravity conditions. Femurs were extracted from rats and prepared into bone section samples, which were then observed under a laser scanning confocal microscope to analyze tracer distribution. ImageJ was used to analyze the fluorescence intensity at the lacunae, which indicated the concentration of fluorescent tracer.

Results: Concentrations of a fluorescent tracer in the lacunae gradually decrease with increasing distance from the Haversian canal. Additionally, with the increase in solute molecular weight, concentrations of fluorescent tracers within each lacuna decrease accordingly. Hypergravity (5 g) effectively promotes the solute transfers of varying molecular weights across layers to the lacunae. Larger molecular weight solutes exhibit stronger hypergravity-driven mass transfer augmentation in the LCS.

Conclusion: This study uncovered the effects of solute molecular weights on mass transfer within the LCS under gravitational fields. The higher the molecular weight of the solutes within the bone, the more difficult mass transfer becomes and the more susceptible to gravity. Hypergravity significantly promotes the efficiency of solute mass transfer and ensures normal mass transfer in the LCS. These results not only provide a potential adjuvant strategy for improving bone health but also open up a novel therapeutic pathway for the management of osteoporosis.

Background: Induced pluripotent stem cells (iPSCs) represent a promising source for regenerative therapies, yet allogeneic transplantation is limited by immune rejection. While strategies for generating hypoimmune iPSCs have been proposed, their efficacy after differentiation into lineage-specific cell types remains underexplored.

Methods: A human iPSC line (36A) from peripheral blood mononuclear cells using a Sendai virus-based reprogramming protocol. Hypoimmune properties were conferred via CRISPR-Cpf1-mediated B2M knockout, combined with lentiviral overexpression of HLA-E and CD47. Immune evasion was validated using NK cell cytotoxicity assays. Endothelial differentiation was induced using a defined, stepwise protocol, and in vivo functionality was evaluated in humanized NSG mice.

Results: The hypoimmune iPSCs retained pluripotency, exhibited stable karyotype, and demonstrated > 99% expression of HLA-E/CD47. NK cell-mediated lysis was significantly reduced in edited cells, although IFN-γ levels remained elevated. Upon differentiation, the hypoimmune iPSCs yielded > 98% CD31⁺CD144⁺ endothelial cells, which showed enhanced survival in vivo compared to wild-type controls.

Conclusion: Multiplex gene editing successfully conferred durable immune evasion in both undifferentiated and endothelial-differentiated iPSCs. These findings support the clinical potential of hypoimmune iPSC-derived cell therapies for allogeneic transplantation without immunosuppression.

Background: The human spine relies on intervertebral discs (IVDs) for support and mobility, functioning as shock absorbers that enable friction-free movement. However, IVDs are susceptible to degeneration (IVDD) due to age, excessive strain, and genetic factors, resulting in bulging or herniation that causes pain, stiffness, and nerve compression.

Current treatments: Current treatments primarily focus on symptom management through medication, physical therapy, or surgery in severe cases, without addressing tissue repair.

Emerging therapies: Exosome therapy has recently emerged as a promising regenerative approach for IVDD. Exosomes are small, membrane-bound vesicles released by cells, acting as messengers to transport proteins and RNA that influence recipient cell behavior.

Potential and challenges: Researchers are investigating exosomes for IVDD because they may promote disc repair and regeneration by delivering molecules that stimulate tissue recovery and carry anti-inflammatory agents to reduce inflammation and modulate pain. Engineering strategies, such as loading exosomes with therapeutic cargo or targeting molecules, can further enhance their efficacy. While exosome therapy for IVDD is still in early research stages, ongoing studies are promising, though challenges remain in optimizing isolation methods and ensuring clinical safety.

Conclusion: Exosome-based therapies could offer a safe, effective, and minimally invasive solution for individuals affected by IVDD.

Background: Acellular bi-layer silk fibroin (BLSF) scaffolds represent potential alternatives to autologous tissue grafts for substitution urethroplasty (SU) given their ability to repair focal urethral defects in animal models. However, in patients with a severe fibrotic urethral plate, single or staged SU are often required to restore organ continuity. Currently, the feasibility of tubular BLSF grafts for urethral replacement is unknown. Therefore, the objective of this study was to evaluate the efficacy of BLSF biomaterials for SU using single and staged approaches.

Methods: Single (N = 4) and staged (N = 5) SU with BLSF grafts were carried out in adult male rabbits, and animals were maintained for 3 months. Nonsurgical control animals (NSC, N = 3) were evaluated in parallel.

Results: All rabbits survived until harvest and displayed voluntary voiding after initial catheterization with no evidence of severe complications. At 3 months, retrograde urethrograms revealed relative urethral calibers treated with both single and staged approaches were restored to 80 ± 26% and 129 ± 27% of NSC levels. In addition, staged SU led to significantly higher degrees of scaffold degradation as well as urethral patency in respect to the single stage repairs. Histological and immunohistochemical evaluations demonstrated that both surgical techniques supported the formation of innervated, vascularized neotissues resembling NSC. However, neotissues from single stage repairs presented with elevated levels of fibrosis and reduced smooth muscle relative to NSC and the staged cohort.

Conclusions: Single and staged SU with BLSF grafts are feasible for tubular urethral replacement, but staged reconstruction results in improved functional tissue regeneration.