Tissue Engineering Part A最新文献

The study aims to enhance the design process of tissue-engineered implants by evaluating the effects of scaffold reinforcement and cultivation conditions on extracellular matrix (ECM) development. The research investigates the hypothesis that mechanical stress drives ECM production and alignment. Furthermore, we have explored the potential of an in silico growth model to complement in vitro findings for accelerated development processes. The study employed fiber-reinforced and nonreinforced scaffolds fabricated using warp-knitted textiles and fibrin gel. Myofibroblasts embedded in the scaffolds were cultivated under static and dynamic conditions. ECM development was evaluated through mechanical testing, hydroxyproline assays, and microscopy, while an in silico growth model was used to predict ECM behavior. Static cultivation resulted in significant ECM development in both reinforced and nonreinforced samples, with nonreinforced scaffolds showing higher collagen content and alignment along the load direction. In contrast, dynamic cultivation inhibited ECM formation, potentially due to cross-contraction and washout effects. Fiber-reinforced scaffolds exhibited higher elasticity and sustained stress across cycles without structural damage. The in silico model provided valuable insights but overestimated mechanical properties due to limited validation data. Reinforced scaffolds maintained geometry and elasticity, suggesting suitability for load-bearing applications. Nonreinforced scaffolds facilitated higher ECM production but were prone to structural damage. Dynamic cultivation requires optimization, such as prestatic cultivation, to support ECM development. The combined in vitro and in silico approach offers a promising framework for scaffold design, reducing the reliance on iterative experimental processes.

The current clinical treatment of large bone defects in humans primarily relies on autologous bone grafts. However, the use of autologous bone grafts can be limited by tissue availability, variable bone quality, and donor site morbidity. In response to these challenges, endochondral bone regeneration has emerged as a promising approach. This method mimics endochondral ossification by chondrogenically differentiating or stimulating cells of various cell sources into 'callus mimics' (CMs). We previously demonstrated the feasibility of endochondral bone regeneration in restoring bone defects using 'mesenchymal stromal cell' (MSC)-derived devitalized CMs in small and large animals. To scale up the size of treated defects using these CMs, we propose the introduction of a vascular supply. In this study, an arteriovenous (AV) loop was introduced as a vascular supply to devitalized 'MSCs'-derived CMs in a centimeter-scale porous chamber in rats. The extent of vascularization and remodeling was evaluated for chambers filled with CMs in the presence or absence of an AV loop at 4 and 8 weeks. While the AV loop's role in vascularization is established, our study uniquely shows that in a challenging in vivo setting with devitalized callus mimics, the AV loop was critical for initiating bone formation. Mineralization was observed in all groups via microCT, but bone tissue formed only in the AV loop group (50% of samples at 8 weeks), underscoring its influential role in supporting both vascular invasion and bone formation.

Notochordal cells (NCs), abundantly found in the developing nucleus pulposus (NP), show potential for intervertebral disc regeneration because of their unique instructive and healthy matrix-producing capacity. However, NCs are lost early in life, and attempts at in vitro expansion have failed because they lose their specific phenotype. Therefore, much effort is focused on the generation of cells resembling the properties of healthy matrix-producing NP-like cells from human induced pluripotent stem cells (hiPSCs). They are considered a promising alternative for employing native NCs. Given the ongoing challenges in the field to fine-tune the differentiation protocol and obtain a high yield of mature matrix-producing cells, this study aims to build on the epigenetic memory and instructive capacity of healthy NP tissue. For this, we employed the epigenetic memory of tissue-specific hiPSCs derived from TIE2⁺ NP progenitor cells (NPPCs) and microenvironmental cues of decellularized porcine NC-derived matrix (dNCM), consisting of matrix components and bioactive factors to differentiate hiPSC into mature, healthy matrix-producing cells for NP repair. As a comparison, donor-matched minimally invasive peripheral blood mononuclear cell-derived hiPSCs were used. The results show that employing NPPC-derived hiPSCs instructed by natural cues provided by dNCM resulted in an increased expression of healthy phenotypic and matrisome-related NP markers. Furthermore, within this in vitro environment, differentiation of blood-derived hiPSC lines led to augmented differentiation into the hematopoietic and neural cell lineage. In conclusion, we demonstrate that hiPSCs derived from NPPCs achieve enhanced differentiation outcomes in the presence of dNCM, highlighting the potential impact of the epigenetic memory.

Tissue engineering offers an alternative for augmentation urethroplasty; however, no ideal material has yet been developed. Recently, materials derived from amniotic tissues appear to exhibit promising properties. Herein, the aim of this study was to provide a proof of concept for the integration of the human umbilical cord vein for urethral reconstructions in rabbits. Rabbits were included in two groups; the control group underwent urethral reconstruction using autograft urethral tissue, and the test group received xenograft tissue (umbilical cord vein) after creating a 1 × 1 cm defect in the proximal urethra. At 3 weeks, endoscopy and biopsy were performed. At 6 weeks, the animals were euthanized, and their urethra and corpus cavernosum were sent for histopathological analysis. The six rabbits exhibited favorable clinical and endoscopic progress with no fistula or stenosis. Biopsy analysis found no lesion of the urothelium and chorion. Final histological analysis found similar results in both groups: normal histology with moderate urothelium vacuolation and a weak inflammatory cellular infiltrate. The present study provides a proof of concept of human umbilical cord vein as a scaffold for urethral regeneration. This could be an alternative to existing urethral tissue grafting procedures that can have difficulties with integration or immunological tolerance; however, further research is required.

While β3GalT2 has been implicated in osteogenic regulation, its synergistic application with bioactive scaffolds remains unexplored. This study pioneers a dual-functional bone regeneration strategy by integrating β3GalT2-engineered bone marrow mesenchymal stem cells (BMSCs-β3GalT2) with nano-hydroxyapatite/polyamide 66 (n-HA/PA66) composites. First, we studied the effect of β3GalT2 on rat BMSCs (rBMSCs) by overexpression the β3GalT2 gene. Following this, we extracted exosomes and verified that β3GalT2 influences osteogenesis of rBMSCs through exosomes. Subsequently, we inoculated these rBMSCs on n-HA/PA66 and demonstrated the effects of β3GalT2 and n-HA/PA66 on osteogenic differentiation of rBMSCs. On this basis, we also explored the molecular mechanism of β3GalT2 regulating M1 polarization through exosomes. Finally, we verified our study by using animal models of skull defect and femur defect. Our results suggest that β3GalT2 promotes osteogenic differentiation of rBMSCs through exosomes. At the same time, rBMSCs-β3GalT2 combined with n-HA/PA66 showed good osteogenic effect in vivo and in vitro. In addition, we also found that β3GalT2 can regulate M1 polarization through exosomes. Our findings establish β3GalT2 as a master regulator of osteogenesis through cellular-exosomal-circuitry mechanisms. The biohybrid system synergistically combines gene-enhanced stem cells with tunable biomaterials, representing a paradigm shift in bone tissue engineering.

Mesenchymal stem cells (MSCs) are widely used for tissue regeneration due to their multilineage differentiation potential and ability to secrete paracrine factors with immunomodulatory and angiogenic functions. Standard MSC differentiation protocols typically rely on two-dimensional (2D) or pellet culture models that are simple to use but not well-suited for translational or clinical applications. To promote better cell survival, tissue deposition, and differentiation of MSCs, a wide variety of three-dimensional (3D) biomaterial scaffolds and platforms have been developed that provide structural support and present a carefully defined set of biochemical and biophysical cues to cells. While biomaterials can guide cell behavior and promote desirable tissue regeneration outcomes, one remaining challenge in the field is inherent donor-to-donor variability in MSC behavior, phenotype, and differentiation capacity. Although MSCs are promising tools for regeneration, the influence of donor variability on MSC differentiation across culture models remains poorly understood. Previous studies typically use cells from a single donor or rely solely on standard culture models. To address these gaps, we compared MSCs from six human donors and assessed differentiation across chondrogenic, osteogenic, and adipogenic lineages using both standard (pellet or 2D) and 3D biomaterial-based culture models. Alginate hydrogels were used to assess chondrogenesis, while gelatin microribbon (µRB) hydrogels were used to evaluate osteogenesis and adipogenesis in 3D. Significant donor-to-donor variability was observed in differentiation outcomes across all three lineages and within both 2D and 3D culture models. By directly comparing donor variability in 2D and 3D, we provide evidence that standard 2D models cannot predict MSC differentiation capacity in 3D biomaterials. Therefore, to improve therapeutic efficacy and advance biomaterial-based strategies for tissue regeneration, it is critical to understand how donor variability affects MSC differentiation patterns across 3D biomaterial-based culture models.

Decellularized extracellular matrix (dECM) plays an important role in tissue engineering by preserving native biochemical and structural cues while removing immunogenic cellular components. Addressing donor shortages, this study develops a standardized, reproducible protocol for producing cell-derived dECM for bone and cartilage applications, focusing on effective deoxyribonucleic acid (DNA) removal to prevent immune responses in 3D-bioprinted hydrogels. We also evaluate dECM's impact on cell viability and differentiation potential. Human dermal fibroblasts were decellularized using nonidet P-40, a nonionic detergent (nonyl phenoxypolyethoxylethanol) (NP-40) lysis buffers (1% or 10%) for 1 or 3 h. Decellularization efficacy was assessed via double-stranded DNA (dsDNA) Qubit assay, gel electrophoresis, immunofluorescence, and bicinchoninic acid protein assay. Hydrogels (5 wt% alginate, 3 wt% gelatin) with/without 1% dECM were extrusion bioprinted. Structural and mechanical properties were analyzed using Raman spectroscopy and rheology. Fibroblast viability within bioprinted constructs was monitored over 21 days. Hoechst staining and Qubit assay confirmed residual DNA after 1-h incubations, but complete removal (<50 ng dsDNA) occurred after 3 h with both NP-40 concentrations. The 10% NP-40/3-h protocol yielded the highest protein content. dECM incorporation did not compromise scaffold properties. Significantly enhanced cell viability and glycosaminoglycan (GAG) content (up to day 6) were observed in dECM hydrogels versus controls. Mechanical testing showed a 33% increase in Young's modulus in dECM-containing hydrogels. Raman spectroscopy confirmed successful dECM integration via a characteristic GAG peak (895 cm^-1). We established an optimized decellularization protocol (10% NP-40, 3 h) that effectively eliminates cellular/nuclear material (DNA <50 ng, RNA undetectable) below immunogenic thresholds while preserving essential extracellular matrix components. Fibroblast-derived dECM significantly enhanced alginate-gelatin hydrogel performance, improving cell viability, GAG synthesis, and early osteogenic markers without compromising structural integrity. This protocol provides a robust and standardized source of bioactive dECM, offering a viable alternative to tissue-derived matrices for advanced bone and cartilage tissue engineering bioinks. While the method demonstrates potential for scale-up, further validation following internationally recognized International Organization for Standardization (ISO) standards would be necessary before production-level implementation.

Osteochondral (OC) defects, involving simultaneous damage to articular cartilage and subchondral bone, remain clinically challenging due to the distinct biological, mechanical, and structural characteristics of each layer. Traditional repair techniques are limited by poor integration and inadequate tissue regeneration. 3D bioprinting has emerged as a promising strategy to fabricate biomimetic OC constructs with precise spatial control over scaffold architecture, cell distribution, and bioactive cues. This review summarizes recent advancements in additive manufacturing techniques and their applications in OC tissue engineering. Scaffold design strategies are discussed, along with the selection of biofunctional materials. Special focus is given to recent progress in bioink development, including the precise incorporation of growth factors, zonal patterning of stem cells to guide region-specific differentiation, and the integration of bioceramics to enhance osteogenic potential while supporting chondrogenic matrix formation.

Nanocellulose has emerged as a promising biomaterial for development of scaffolds for tissue engineering. Incorporation of nanocellulose into a polymer scaffold can increase its stiffness, allowing it to better mimic the mechanical properties of native extracellular matrix. Plant-derived nanocellulose is classified as either cellulose nanofibrils (CNFs) or cellulose nanocrystals (CNCs) depending on particle characteristics and extraction methods. Although both materials have been used in hydrogel composites, the impact of nanocellulose source and morphology on scaffold properties remains unclear. Here, we isolated high aspect ratio CNFs from two macroalgae species and compared them with conventional wood pulp-derived CNFs and CNCs in the preparation of composite gelatin hydrogels. All nanocellulose types increased hydrogel stiffness in a concentration-dependent manner; however, the greatest increase was achieved using brown algae CNF, where the addition of 1.25 wt.% nanocellulose resulted in a 5.2-fold increase in compression modulus relative to neat gelatin. Bioassays showed that nanocellulose improved keratinocyte adhesion and spreading on gelatin scaffolds, with a positive correlation between nanocellulose concentration and surface coverage and inverse with cell circularity. These findings demonstrate the influence of nanocellulose source and morphology on the mechanical and biological properties of composite scaffolds and highlight the potential of novel nanocellulose sources for scaffold development.

Developmentally inspired tissue engineering strategies are increasingly being employed to generate biomimetic articular cartilage (AC) grafts. One such approach leverages the capacity of stem or progenitor cells to self-organize and generate microtissues or organoids, which can then be used as biological building blocks to fabricate larger grafts of clinically relevant size. While human mesenchymal stem/stromal cells (hMSCs) can be used to generate cartilage-like microtissues, they are often fibrocartilaginous in nature and/or have an inherent tendency to become hypertrophic and progress along an endochondral pathway. In this study, a gene silencing approach was explored to engineer hyaline cartilage microtissues by delivering the prochondrogenic factor, antimicro ribonucleic acid 221 (anti-miR-221), using a polymeric nonviral vector. Effective silencing of micro ribonucleic acid 221 (miR-221) was observed for a range of doses, while selected anti-miR-221 concentrations supported type II collagen deposition while simultaneously suppressing the production of type X collagen within the cartilage microtissues. In addition, large numbers of such "silenced" chondrogenic microtissues could be fused into larger grafts, with the resulting constructs again showing no signs of early hypertrophy. To conclude, miR-221-silenced hMSCs support the development of hyaline cartilage microtissues rich in type II collagen, which could be used as in vitro models of AC or as biological building blocks in the engineering of scaled-up regenerative grafts.