Tissue Engineering Part A最新文献

The liver is a multifunctional organ essential for detoxification, protein synthesis, glucose regulation, bile secretion, and drug metabolism. However, persistent damage leads to chronic inflammation, excessive extracellular matrix deposition, and progressive fibrosis culminating in cirrhosis, for which liver transplantation remains the only curative option. Yet, the scarcity of donor organs and risks of immune rejection underscore the urgent need for physiologically relevant in vitro liver models to investigate pathogenesis and facilitate therapeutic discovery. Current two-dimensional cultures and animal models fail to recapitulate the multicellular interactions that govern liver homeostasis and disease progression. Hepatocytes (Heps) constitute the primary parenchymal population, while hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) coordinate fibrogenic, angiogenic, and regenerative responses. Dysregulation of this crosstalk drives fibrosis and architectural collapse, highlighting the necessity for multicellular systems that mimic native liver complexity. In this study, we established a three-dimensional (3D) microtissue platform that recapitulates both the structural and functional characteristics of the human liver. Human-induced pluripotent stem cells (hiPSCs) were differentiated into Heps, HSCs, and LSECs, which were subsequently cocultured within a self-organizing 3D microenvironment. We successfully reconstructed a miniaturized liver model that maintains hepatic functionality and exhibits steatogenic responses to alcohol exposure. This hiPSC-derived microtissue enables the modeling of chronic liver diseases, intercellular signaling, and fibrogenic pathways, thereby providing a translationally relevant system for mechanistic studies, drug toxicity testing, and personalized therapeutic development.

Alternative therapies are needed for heart failure following myocardial infarction (MI), as ischemic cardiomyopathy remains a major global health concern despite advances in acute MI management. Platelet-rich plasma (PRP), which is enriched with cytokines and growth factors, holds therapeutic potential in ischemic cardiovascular diseases. However, its clinical application remains unrealized due to the absence of a reliable delivery approach. An epicardial patch provides a spatially stable delivery system on the heart surface. This approach becomes particularly attractive when combined with biodegradable controlled-release hydrogels that prolong and localize factor release. Therefore, combining these two modalities into an epicardial hydrogel patch offers a novel and efficient strategy for targeted PRP delivery. This study evaluated the feasibility and therapeutic efficacy of a biodegradable gelatin hydrogel patch, incorporating PRP and designed for epicardial use, in a rat MI model. PRP was prepared via double-spin centrifugation and activated with calcium chloride. In vitro, cytokine and growth factor levels (transforming growth factor-beta 1 [TGF-β1], platelet-derived growth factor-BB [PDGF-BB], insulin-like growth factor-1 [IGF-1], vascular endothelial growth factor [VEGF]) were quantified using enzyme-linked immunosorbent assay. PRP contained TGF-β1, PDGF-BB, and IGF-1; VEGF was undetectable. Release kinetics were measured under nonenzymatic and collagenase conditions. The hydrogel provided controlled release, especially of TGF-β1, for 5 days in vitro. In vivo, MI was induced by ligating the left anterior descending artery in rats. The epicardial hydrogel patch was placed at the infarct center, covering a fibrin-collagen sealant patch. The patch remained in place for 10 days and degraded by day 20. Cardiac function was evaluated via echocardiography through day 28, after which hearts were harvested for histological infarct analysis. Serial echocardiographic evaluations revealed that the PRP group demonstrated less decline in systolic function (fractional area change) from the hyperacute phase (day 1) to the chronic phase (day 28). Morphological assessments demonstrated that the PRP group had smaller left ventricular end-diastolic dimensions from day 7 onward. Histological evaluation confirmed greater infarcted wall thickness and myocardial area within the infarcted region compared with controls. Therefore, the epicardial delivery of PRP via a controlled-release hydrogel patch attenuated cardiac dysfunction from the hyperacute to the chronic phases and mitigated adverse ventricular remodeling.

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.

To provide an optimal wound bed for epidermal regeneration, a viable dermis is needed. As the dermis is destroyed in full-thickness burns, dermal templates (DTs) are used to create a healthy dermis for grafting and other procedures. Neonatal foreskin has exclusively been used as the source of dermal fibroblasts in commercial DTs, as the tissue is readily available and because these cells are assumed to be more proliferative and capable of superior wound healing compared with adult fibroblasts. The goal of this study was to assess the function of adult fibroblasts compared with neonatal fibroblasts for DT construction and epidermal regeneration. Primary fibroblasts were isolated from neonatal or adult surgical discard tissue (n = 4 each). Expression of collagen type 1 A1 (COL1A1) and matrix metalloprotease 1 (MMP1), MMP3, and MMP9 was assessed for each cell strain, and proliferation was quantified in two-dimensional (2D) cultures and 3D DTs. Subsequently, DTs were constructed from each cell strain by inoculating fibroblasts onto electrospun collagen scaffolds. DT contraction, extracellular matrix remodeling, and cell viability were assessed over 7 days in culture, and the ability of the DTs to promote epidermal regeneration was assessed using primary adult keratinocytes. No differences in gene expression were observed in neonatal versus adult fibroblasts in 2D culture. Neonatal fibroblasts were significantly more proliferative at day 7 when cultured in 2D; however, fibroblast proliferation was independent of donor age in 3D culture. Neonatal DTs contracted significantly more than adult DTs (68.8% ± 6.2% vs. 91.7% ± 4.2% original wound area, respectively). Upon seeding with keratinocytes, a robust, stratified epidermis formed in all DT groups, with no statistically significant differences in dermal or epidermal thickness, basal keratinocyte proliferation, epidermal barrier function, or basement membrane deposition. Analysis of gene expression revealed modest differences in the expression of MMP1, COL1A1, and ACTA2 in neonatal versus adult engineered skin in vitro, which were not associated with any discernable histological differences. These results indicate that the fabrication of DTs with adult fibroblasts can promote epidermal regeneration equivalent to that of neonatal fibroblasts but with less in vitro contraction, which may enable the treatment of larger wound areas.

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.

Volumetric muscle loss (VML) is characterized as the loss of muscle tissue that exceeds the muscle's self-repair mechanism, resulting in incomplete restoration of muscle mass and function. Existing treatment modalities, including muscle grafts or autologous muscle transfers, are limited by constraints such as tissue availability and donor site morbidity. Moreover, the inadequate recovery of muscle may lead to fibrosis within the VML site, impeding the process of muscle regeneration and resulting in permanent deficits. Emerging therapeutics, such as hydrogels, show promise in addressing the limitations of current therapeutics and have the potential to significantly reduce fibrosis and facilitate the restoration of muscle form and function following VML injury and repair. This study evaluated the therapeutic potential of repairing a 30% VML injury in the rat tibialis anterior muscle with engineered skeletal muscle units (SMUs), alone, and in combination with a hyaluronic acid-based hydrogel (HyA-HG). Following 1- or 3-months post-implantation, muscle structure and function were assessed. The results indicated that the incorporation of HyA-HG in combination with our SMUs resulted in improvements in force production for VML injuries repaired for 1 month. However, over extended recovery periods (3 months), sustained superior improvements in muscle function with the combination therapy were not observed compared with the repair with just an SMU. Moreover, histological analyses revealed that muscle treated with SMUs and HyA-HG exhibited a greater cross-sectional area and force production in the early stages of recovery (1-month post-surgery) compared with untreated VML sites or those treated with HyA-HG only. However, after 3 months, muscle mass and force production in all experimental groups reached comparable levels, suggesting a transient benefit of the combination therapy. Our findings highlight the potential of HyA-HG and SMU combination therapy to enhance early functional recovery following VML.

For individuals undergoing mastectomy, reconstruction of the nipple-areola complex (NAC) is a critical step in emotional and psychological recovery. However, current clinical approaches-including flap suturing, tattooing, or grafting-are limited by loss of projection, poor mechanical stability, and absence of sensation. Additive manufacturing and tissue engineering offer promising alternatives by enabling the development of hybrid scaffolds that maintain long-term projection and support the potential return of sensation. This review summarizes the state-of-the-art in NAC reconstruction and highlights how advances in additive manufacturing can address existing limitations. Emerging scaffold design strategies allow precise fabrication of biomimetic architectures that replicate the anatomical form and function of the NAC, while supporting tissue integration and mechanical durability. The use of biocompatible polymers such as poly-ε-caprolactone, combined with bioactive coatings and plasma surface modification, enhances cell attachment and vascularization. Additionally, the incorporation of stem cells, multicellular constructs, and conducting polymers is explored to enable multifunctional tissue regeneration and restore sensation through electrical stimulation. By integrating innovations in biomaterials science, regenerative medicine, and advanced fabrication technologies, the field is moving toward nipple reconstructions that are not only more life-like in appearance but also biologically responsive and sensate.

Ischemic cardiac injury, arising due to myocardial infarction (MI), ischemia-reperfusion injury (IRI), and other ischemia-associated forms of cardiac damage, remains a major clinical challenge. The irreversible loss of cardiomyocytes from within the myocardium, together with oxidative stress and inflammation, creates a complex post-MI milieu that is not readily addressed by existing therapeutic strategies. Cardiac tissue engineering solutions that combine advanced biomaterials with either stem cell-derived cardiovascular cells, their derivatives (such as extracellular vesicles and exosomes), or other bioactive compounds (including chemokines and cytokines) are being developed to repair and regenerate the infarcted human heart. This review highlights the state-of-the-art strategies that utilize cutting-edge technologies to develop tissue-inducing biomaterial solutions for cardiac regeneration and repair, with particular emphasis on (i) integrating biomaterials with cells in strategies undergoing clinical investigation, (ii) incorporating cellular derivatives into biomaterial scaffolds, and (iii) designing and evaluating intrinsically functional biomaterials. This review aims to provide both a theoretical foundation and future perspectives for the innovation and optimization of next-generation tissue-inducing biomaterial-based strategies for cardiac tissue regeneration and repair.