Tissue Engineering Part A最新文献

Objectives: N6-methyladenosine (m⁶A) modification is critical in the regulation of osteoporosis (OP). Although ZC3H13 is an important m⁶A 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. Materials and Methods: 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. Results: 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 in vivo. Mechanistically, ZC3H13 was shown to promote osteogenic differentiation in OP-BMSCs by inhibiting ferroptosis. Conclusions: 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.

Volumetric muscle loss (VML) due to traumatic injury results in the abrupt loss of contractile units, stem cells, and connective tissue, leading to long-term muscle dysfunction and reduced regenerative potential. Muscle connective tissue contains a proregenerative extracellular matrix (ECM), and our lab harnesses the regenerative capacity of decellularized muscle matrix (DMM) to treat VML, a condition with limited treatment options. However, a major limitation is that muscle often comes from aged donors. Previous work from our lab showed that aged donor muscle contains higher levels of advanced glycation end-product (AGE) cross-links compared to muscle from younger donors. This study aimed to determine whether increased AGE cross-links reduce the regenerative capacity of DMM. To test this, we first generated AGEs in DMM with direct D-ribose incubation. We then removed ∼35% of the gastrocnemius muscle in a model and treated it with either AGE-DMM or standard DMM (no AGEs), comparing results to controls. Although muscle force results remained unchanged between AGE-DMM and DMM, AGEs led to reduced muscle mass in histological sections, fewer fibers, and smaller fiber diameters. AGEs also increased collagen levels in histology, but protein assays showed reduced collagen production. We investigated the canonical receptor for AGEs, the receptor for AGEs (RAGE), and found elevated levels in AGE-treated VML compared to DMM alone, along with increased levels of the noncanonical receptor galectin-3. Both RAGE and galectin-3 are associated with inflammation, and proteomics revealed higher inflammatory markers in AGE-treated muscle than in DMM alone. In conclusion, our data suggest that AGEs impair the regenerative potential of DMM, highlighting the importance of considering donor age when sourcing muscle for DMM therapies.

Background and Aims: Cell therapy approaches to treating chronic liver disease provide only transient improvements, mainly due to loss of hepatocytes after infusion. Microencapsulation in alginate has been shown to protect transplanted cells from physical stress and rejection, but the poor biocompatibility of alginate can lead to graft failure. This study aimed to evaluate a biocompatible poly(vinyl alcohol) (PVA)-based microcapsule against standard alginate for improved transplantation outcome of liver spheroids. Materials and Methods: Human hepatocyte spheroids were microencapsulated in alginate or PVA hydrogel microspheres. Viability and function (albumin secretion and CYP activity) of the encapsulated spheroids were assessed in vitro at 3, 10, and 30 days postencapsulation and compared with unencapsulated spheroids. Spheroids were implanted intraperitoneally into immunodeficient mice, and human albumin levels in serum were monitored over 30 days. Cell-free microspheres were implanted in immune-competent mice to assess material biocompatibility. Results: Unencapsulated spheroids aggregated extensively beyond 10 days, precluding day 30 assessment. At day 30, PVA spheroids showed significantly higher CYP1A1 induction, albumin secretion, and metabolic activity compared with alginate. Mice receiving PVA spheroids had significantly higher serum albumin after 30 days compared with alginate and unencapsulated spheroids. Empty PVA microspheres showed less evidence of foreign body response in vivo, whereas thicker regions of inflamed tissue were observed in the alginate group. Conclusions: PVA-encapsulated hepatocyte spheroids maintained better overall viability, metabolic activity, and function compared with alginate-encapsulated cells both in vitro and in vivo. Both encapsulated groups demonstrated substantially improved outcomes compared with unencapsulated cells.

Angiogenesis is critical for effective wound healing and relies on the successful coordination of various cell types, including endothelial cells, macrophages, and fibroblasts. Adipose-derived stem cell extracellular vesicles (ADSC-EVs) have demonstrated proangiogenic properties and have been posited as a novel therapeutic to aid wound healing; however, their functional impact within human-derived multicellular models remains largely uncharacterized. This study explores the development and application of a 3D multicellular in vitro model to assess the effects of ADSC-EVs on vascularization in the context of wound healing. 3D multicellular in vitro models were developed by coculturing human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages, and fibroblasts within Matrigel to recapitulate the in vivo wound healing microenvironment. A five-color confocal microscopy panel was developed to visualize each cell type and EVs within the models. The optimized models were then treated with ADSC-EVs or control to determine their impact on angiogenesis and cell colocalization. We determined that vessel formation was significantly enhanced when HUVECs were cocultured in multicellular models compared with monocultures, with the greatest effect observed in the full three-cell-type model. This effect was even more pronounced with the addition of ADSC-EVs. ADSC-EV treatment also enhanced macrophage colocalization within endothelial structures. This study developed a multicellular model that can be used for future work assessing wound healing in vitro and will be additive to currently used single-cell and in vivo models. We have applied these models to demonstrate that ADSC-EVs significantly enhance tube formation in HUVECs and the development of tissue-like structures in multicell systems, highlighting their potential as a promising therapeutic approach for improving wound healing.

Diabetic nonunion is a major clinical challenge with unclear molecular mechanisms. This study systematically investigated the key genes and molecular mechanisms of bone nonunion after finger replantation induced by high glucose using Gene Expression Omnibus (GEO), bioinformatics, and experimental analyses. In total, 179 differentially expressed mRNAs and one lncRNA (DElncRNA) were identified using the GEO dataset. Functional enrichment analysis showed that these genes were mainly involved in the regulation of autophagy and metabolism. Protein-protein interaction network analysis identified five core genes (Peroxiredoxin 2 [PRDX2], FK506 binding protein 8 [FKBP8], SHANK-associated RH domain interactor [SHARPIN], WD repeat domain 45 [WDR45], and gamma-aminobutyric acid type A receptor-associated protein like 2 [GABARAPL2]), three of which exhibited good binding affinities for potential therapeutic agents. Immune infiltration analysis revealed significant differences in the CD8+ T cell proportions between nonunion and healthy samples. We constructed a competitive endogenous RNA network (long intergenic non-protein coding RNA 687 [LINC00687]-miR-4443-PRDX2) and verified its direct regulatory interaction using a dual-luciferase reporter assay. FKBP8, PRDX2, SHARPIN, WDR45, and GABARAPL2 were overexpressed in tissue samples from patients with type 2 diabetes mellitus fracture nonunion. Animal experiments further confirmed that LINC00687 upregulated PRDX2 expression by sponging miR-4443 in a hyperglycemic environment, thereby inhibiting bone healing. This study not only identified PRDX2 and other genes as potential biomarkers of diabetic nonunion but also clarified the regulatory role of the LINC00687/miR-4443/PRDX2 axis in hyperglycemia-induced nonunion, providing a new molecular target for clinical prevention and treatment. Impact Statement 1. PRDX2, KBP8, SHARPIN, WDR45, and GABARAPL2 were potential biomarkers for this study. 2. LINC00687-miR-4443-PRDX2 participated in high glucose-induced nonunion in this study. 3. Autophagy process and metabolic pathways contribute to the progression in this study.

Successful osseointegration is crucial for dental implant stability, yet it remains challenging due to adverse local microenvironments, particularly infection and inflammation. While carbon monoxide (CO) has been recognized as a promising gaseous signaling molecule with diverse therapeutic properties, its clinical application faces significant limitations due to dose control challenges. To address this issue, we developed a polyetheretherketone (PEEK)-based photo-responsive implant system with surface-immobilized manganese carbonyl nanocrystals, enabling precisely controlled near-infrared light-triggered CO release. The system demonstrated efficient photoresponsiveness, achieving 13.83 ± 1.16 μM CO release within 10 min under optimal illumination conditions. In vitro studies revealed that low-dose CO significantly enhanced bone marrow mesenchymal stem cell osteogenic differentiation with upregulated expression of key markers, including Runx2, ALP, and OCN. In a rat femoral defect model, implants with controlled CO release exhibited significantly improved osseointegration. Comprehensive biosafety assessments confirmed the system's excellent biocompatibility without detectable organ toxicity. This research provides compelling evidence for controlled low-dose CO as an innovative strategy to enhance osseointegration, offering new possibilities for dental and orthopedic implant development, particularly for challenging clinical scenarios with compromised bone healing. Impact Statement This study introduces a novel approach for improving implant osseointegration through controlled carbon monoxide delivery, potentially offering a new strategy for enhancing the success rate of dental implant procedures.

Osteochondral explants can serve as valuable ex vivo models for investigating joint development and testing therapeutic interventions in osteoarthritis (OA). The incorporation of synovial tissue in coculture settings more closely reproduces the inflammatory milieu characteristic of OA joints; however, no report exists regarding the culture media that can support such ex vivo systems. We investigated the reactivity of osteochondral explants using two media types: Dulbecco's modified essential medium (DMEM) and chondrogenic medium (CHONDRO). Additionally, we tested the potential therapeutic effect of serum-free conditioned media (CM) derived from allogeneic adipose-derived stem cells (ADSCs) in the context of OA. Osteochondral fragments with or without homologous synovium were cultured in DMEM and CHONDRO for up to 30 days. A subset of explants received treatment with CM. Explant reactivity was assessed by cytokine release, synovial cellularity, and osteochondral protein content using Western blot and immunohistochemistry. Explants kept in DMEM displayed diminished levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα), together with increased Collagen II (Col II) expression. Notably, consistent suppression of TNFα was observed following CM treatment. Conversely, the CHONDRO-kept samples exhibited an increased prevalence of chondrocyte clusters; heightened Perlecan presence as well as IL-1β levels in response to CM treatment and synovial tissue-dependent fluctuations in Col II levels. Remarkably, significantly increased β-galactosidase levels could be detected in osteochondral tissues treated with CM, regardless of the culture media type. In the experimental conditions created, DMEM provided a neutral milieu and was less prone to confounding experimental outcomes, rendering it suitable for evaluating potential therapies. CHONDRO apparently increased chondrocyte clusters and facilitated extracellular matrix synthesis; however, its usage requires caution due to potential interference with experimental readouts. CM could exert an antisenescence effect, an effect that warrants further investigation.

Over the past 20 years, endothelial keratoplasty procedures have revolutionized the treatment of corneal endothelial disorders. These conditions have now become the leading indication for corneal transplantation in Western countries and account for half of all donor cornea usage. Despite their undeniable success, the global shortage of donor tissues and major disparities between nations justify the development of alternatives to donor grafts. Cell therapy using injections of suspended endothelial cells has proven effective, and tissue-engineered endothelial keratoplasty (TEEK), comprising a membrane coated with cultured endothelial cells, is under development to better mimic the native endothelial graft. Our team utilizes a femtosecond-laser-cut lens capsule disc as a bioengineering scaffold, taking advantage of this novel tissue's biocompatibility, transparency, curvature, and availability. In the present study, we provide proof of concept, in 12 TEEKs, that it is possible to control the final endothelial cell density (ECD) by varying the seeding density per mm². Cell characterization was performed through morphometric analysis of the endothelial mosaic stained with anti-NCAM (a lateral membrane marker used as a differentiation marker), using the CellPose artificial intelligence algorithm specifically trained for in vitro endothelium segmentation. Five criteria related to pleomorphism, polymorphism, and elongation were combined into a single endothelial quality score. The median cell viability at 28 days of culture, assessed by Hoechst 33342 and Calcein-AM staining, reached 98% (range: 83-99%). The median viable ECD (number of live cells per surface unit) in the highest-density group was 3.245 cells/mm² (range: 2.778-3.753), paving the way for the bioengineering of supra-physiological TEEKs, or "super TEEKs".

Spatially and temporally controlled drug delivery is an important field to address the limitations of conventional pharmaceutical administration. While many effective controlled drug delivery systems exist, the repertoire of systems that additionally present a beneficial mechanical environment to cells remains scarce. To address this, a comprehensive release study of fluorescein as a model drug, and the corticosteroid dexamethasone, from poly(N-isopropylacrylamide)/polypyrrole (pNIPAM/PPy) conducting polymer hydrogels is presented within this study. Cyclic voltammetry and scanning electron microscopy (SEM) indicated that having the pNIPAM hydrogel phase present and doping with drugs reduced PPy thickness and shifted/suppressed redox peaks to some degree but not enough to prevent release. Fluorescein release was initiated by constant reduction, with a maximum of 54.5 ± 6.8 µg/cm² from PPy films and 6.3 ± 1.1 µg/cm² from pNIPAM/PPy. The quantity of fluorescein released was shown to be tunable by modulating the charge passed during PPy electropolymerization. Fluorescein-loaded pNIPAM/PPy samples were capable of multiple cycles of depletion and reloading via re-incorporation through re-oxidation in a fluorescein solution. The stability of pNIPAM/PPy regarding drug release was demonstrated, with no difference in release profiles and quantities after soaking samples for 1, 8, and 15 days. Interestingly, constant reduction did not elicit release of dexamethasone, while a biphasic pulsed potential of ±0.8 V at 0.5 Hz was effective. Minimal leaching of dexamethasone without stimulation was shown, alongside a multi-day, multi-triggerable release profile upon short stimulations. pNIPAM/PPy conducting polymer hydrogels are a promising platform for on/off drug delivery, with a nondegrading matrix, minimal passive drug-leaching, and where the drug payload can be reloaded, all while providing a suitable mechanical environment to interface with living cells.

Cell and tissue engineering therapies provide promise for regenerating damaged intervertebral disc (IVD) tissue and resolving the low back pain that often accompanies it. However, these treatments remain experimental and unavailable for patients. Furthermore, the large body of work characterizing and utilizing mesenchymal stromal cells (MSCs) for these applications has, unfortunately, not resulted in any FDA-approved spinal therapies. Herein, we characterized DiscGenics's human cadaver-derived discogenic nucleus pulposus (NP) progenitor cells and, for the first time, their discogenic annulus fibrosus (AF) progenitor cells. We then used these discogenic NP and AF cells to create biomimetic human-sized total tissue-engineered IVD replacements, also known as endplate-modified angle ply structures (eDAPS), and compared these with eDAPS formulated with goat or human MSCs. Prior to eDAPS fabrication, discogenic cells were expanded using either two-dimensional attachment culture or three-dimensional suspension culture. Currently, no data exist as to how these discogenic progenitor cells deposit extracellular matrix in a 3D culture environment, nor do data exist characterizing whether the unique expansion environment influences subsequent discogenic cell behavior. Our data support that NP and AF discogenic cells occupy unique niches and serve distinct functions, both in the IVD and in an in vitro 3D culture environment. As a result, discogenic cells deposited more matrix overall than did MSCs. That matrix was distinct between the NP and AF analogs of the tissue-engineered IVDs while also being more homogeneous within each region. Most importantly, unlike both MSC groups, discogenic cells deposited little to no collagen X, suggesting that discogenic eDAPS possess a more stable regional phenotype that will be less susceptible to hypertrophy and downstream calcification. Overall, DiscGenics's discogenic NP and AF cells made compositionally and mechanically superior eDAPS when compared with both human and goat MSCs, with only minor differences between attachment- and suspension-derived discogenic cell eDAPS, supporting their use as a cell source for the creation of human-scale living whole disc replacements.