Tissue engineering and regenerative medicine最新文献

Background: Porcine primary hepatocytes are vital for liver therapy due to their procurement ease and robust functions. However, they rapidly dedifferentiate in vitro, challenging large-scale maintenance. This study aims to enhance the long-term survival and self-renewal of primary porcine hepatocytes by generating spheroids using a rocker system and optimizing conditions with HUVECs and Roof plate-specific spondin 1 (RSPO1).

Methods: Primary hepatocytes were co-cultured with HUVECs in a rocker system using serum-free medium to form spheroids, mimicking their native microenvironment. RSPO1 was added to the media to promote hepatocyte signaling and proliferation. Then pheroids were generated with HUVECs overexpressing RSPO1 (R-HUVECs). The effects of these conditions on the viability, hepatic function, and proliferation of hepatocytes were evaluated.

Results: The 3D environment and RSPO1 synergistically enhanced hepatocyte proliferation and maintained essential liver functions long-term. Co-culture with HUVECs and R-HUVECs promoted spheroid formation, with spheroids surviving and functioning for 28 days.

Conclusion: Large-scale cultured hepatocyte + R-HUVEC spheroids address in vitro challenges of scale, yield, and functional sustainability, promising advances in liver therapeutics and drug development.

Background: The increasing prevalence of neurodegenerative diseases and toxic substance exposure highlights the need for neuronal cell models that closely mimic human neurons in vivo. Compared to traditional models, human pluripotent stem cell (hPSC)-derived three-dimensional models mimic human physiological characteristics and complex nervous system interactions. These models enable patient-specific treatments and improve the predictive accuracy of drug toxicity evaluations. However, differentiation efficiency varies based on organoid size, structure, and cell line characteristics, necessitating standardized protocols for consistent outcome.

Methods: The morphological characteristics of hPSC-derived embryonic bodies (EBs) formed by concave microwells were analyzed at the early stage of neuronal differentiation. Criteria were established to identify cells with high differentiation efficiency, enabling the optimization of differentiation methods applicable across various cell lines. Neuronal organoids were generated using a microfluidic-concave chip, and their suitability for drug toxicity testing was assessed.

Results: EBs, formed in 500 µm concave microwells, exhibited the highest efficiency for neuronal cell differentiation. Cavity-like EBs were more suitable for neuronal differentiation and maturation than cystic-like forms. The optimal neuronal lineage differentiation method was established, and the drug toxicity sensitivity of organoids generated from this method was validated.

Conclusions: This study identified EB structures suitable for neuronal lineage differentiation based on morphological classification. Furthermore, this study suggested an optimal method for generating neuronal organoids. This method can be applied to various cell lines, enabling its precise use in patient-specific treatments and drug toxicity tests.

Background: Human organoid models are invaluable for developmental studies, disease modeling, and personalized medicine research. However, long-term maintenance is challenging due to hypoxia and nutrient limitations as organoids grow. Cutting organoids improves viability, but current methods have low throughput and are prone to causing culture contamination. This study introduces an efficient organoid cutting method to enhance long-term culture and enable high-throughput analyses.

Methods: We employed three-dimensional (3D) printing to fabricate four classes of organoid cutting jigs with blade guides that were compared and optimized for consistent sectioning of human pluripotent stem cell (hPSC)-derived organoids. Organoids were cultured in mini-spin bioreactors and cut every three weeks, beginning on day 35. Organoid health and growth were evaluated by size increase and proliferative marker expression. Additionally, we utilized 3D printed molds to create GelMA or Geltrex-embedded organoid arrays and silicone molds for optimal cutting temperature compound (OCT)-embedding of organoid arrays.

Results: All 3D printed jigs enabled rapid and uniform organoid cutting under sterile conditions. We determined that a flat-bottom cutting jig design had superior cutting efficiency. Cutting improved nutrient diffusion, increased cell proliferation, and enhanced organoid growth during long-term culture. The mold-based approaches enabled the creation of densely packed organoid arrays and cryosections with evenly distributed organoids.

Conclusion: This novel organoid cutting and arraying method overcomes limitations in long-term organoid culture and high-throughput processing. The simplicity of the cutter design and handling make it a versatile tool for diverse types of organoids. By enhancing organoid viability and enabling consistent sample preparation, this approach facilitates improved organ development and disease modeling, drug screening, and high-throughput analyses, including single-cell spatial transcriptomics applications.

Background: Parathyroid hormone (PTH) can promote subchondral bone formation and alleviate temporomandibular joint (TMJ) osteoarthritis (OA), but the effects of PTH on fibrocartilage stem cells (FCSCs) in cartilage surfaces have yet to be studied.

Methods: We established the TMJOA model in rats and administered PTH to treat them. Rat condyles were analyzed using micro-computed tomography, histological, and immunohistochemical staining. To study PTH's effects on FCSCs in vitro, we employed quantitative polymerase chain reaction, Western Blot, and immunofluorescence staining. We also constructed the TMJOA model in tdTomato; Cathepsin K (Ctsk)-Cre mice and rescued them with PTH. EdU and immunofluorescence staining were used to measure the proliferation and chondrogenic differentiation of FCSCs in vivo. Furthermore, after discectomy, we injected diphtheria toxin (DT) into the Ctsk-Cre; diphtheria toxin receptor (DTR) mice to ablate FCSCs. Afterwards, PTH was injected, and we evaluated the Collagen Type II Alpha 1 (COL2A1)-positive area using immunofluorescence staining.

Results: We successfully developed a TMJOA model, and after treatment with PTH, the rat condyles' BV/TV and Tb. Th increased, and the expression of chondrogenic-related genes was elevated. Additionally, PTH promoted the chondrogenic differentiation of FCSCs in vitro. In tdTomato; Ctsk-Cre mice, the Ctsk/EdU and Ctsk/COL2A1 double-positive cells were increased after PTH administration. Moreover, after the ablation of FCSCs by DT, the effects of PTH treatment were notably reduced.

Conclusion: PTH promotes the proliferation and chondrogenic differentiation of condylar FCSCs.

Background: Fillers have become a viable treatment option for addressing volume deficits, whether for aesthetic purposes or due to trauma or congenital deformities. While most fillers effectively maintain volume, promoting adipogenesis remains a significant challenge. This study investigated a biomaterial designed to maintain volume both in the short and long term while promoting adipose tissue formation, focusing on the biological properties of a human acellular adipose matrix (AAM) combined with crosslinked hyaluronic acid (HA) and carboxymethyl cellulose (CMC) gels.

Methods: The AAM was prepared through delipidation and decellularization and evaluated for residual fat and cells. To assess its performance, the AAM was compared with conventional collagen scaffolds for the proliferation and adipogenic differentiation of human adipose-derived stem cells(hADSCs) in vitro. An injectable AAM filler was developed by combining AAM with crosslinked HA and CMC gels for the desired rheological properties. Over 12 weeks, the AAM filler, conventional HA filler, and adipose tissue were compared in a nude mice model, assessing volume retention, cell incorporation, and adipogenesis.

Results: The AAM showed effective fat and cell removal and promoted the viability and adipogenic differentiation of hADSCs in vitro. The AAM filler exhibited six times higher viscosity than HA filler. It also outperformed both HA filler and adipose tissue in volume retention and cell incorporation, and new adipose tissue formation.

Conclusions: These results suggest that AAM filler is a promising biomaterial for soft tissue augmentation, particularly in applications requiring volume retention and adipogenesis.

Background: Osteoarthritis (OA), a widespread chronic joint disorder mainly affecting the elderly, currently lacks a definitive cure. This study investigated the efficacy of exosomes (Exos) extracted from human umbilical cord MSCs (hucMSCs) in the treatment of OA, and preliminarily explored the mechanisms.

Methods: A rat osteoarthritis model was constructed by surgical induction. The cartilage morphology was observed after pathological staining; expression of cartilage matrix protein, autophagy-related protein and β-catenin were detected by immunohistochemistry; and inflammatory factors in serum were tested by ELISA. In cellular experiments, human primary chondrocytes were induced with IL-1β to build the OA microenvironment. The levels of relevant proteins in each group were analyzed.

Results: Comparing to the OA model, the Exos treatment showed positive effects in reducing OARSI score and Mankin score, decreasing joint space stenosis, promoting matrix synthesis, increasing autophagy, and decreasing β-catenin. The results of the cellular experiments were consistent with those from the animal experiments. However, the Wnt/β-catenin pathway was greatly activated, the levels of matrix proteins and autophagy were distinctly reduced in the Exos + LiCl group comparing to the exosome-treated group.

Conclusion: hucMSCs-Exos effectively attenuated the pathological damage of OA cartilage and chondrocytes, promoted the synthesis of cartilage matrix, reduced inflammation, suppressed the Wnt/β-catenin pathway, and enhanced autophagy which promoted the repair of OA cartilage.

Background: Cardiomyocytes derived from pluripotent stem cells (PSCs) hold great promise in heart damage repair in vivo and drug screening in vitro. However, PSC-derived cardiomyocytes exhibit immature structural and functional properties, which hinder their widespread application. To address this challenge, we designed bimetallic gold-platinum nanoparticles (Au@Pt NPs) endowed with intrinsic oxidase-like, peroxidase-like, and catalase-like activities and high electrical conductivity for promoting cardiomyocyte maturation.

Methods: Mouse embryonic stem cell (ESC)-derived and neonatal mouse cardiomyocytes were used to evaluate the effects of Au@Pt NPs on cardiomyocyte maturation. The expression and alignment of cardiomyocyte myofibril proteins were analyzed by qRT-PCR, western blot, and immunofluorescence staining. Cellular functionality was analyzed by the multi-electrode array.

Results: By adding Au@Pt NPs at different stages of cardiac differentiation of mouse ESCs, we found that treatment with Au@Pt NPs at the late stage could promote the maturation of differentiated cardiomyocytes, evidenced by increased expression of mature myofibril protein isoforms, more aligned myofibrils, and enhanced sarcomere length. Additionally, Au@Pt NPs can enhance the expression of mature sarcomere components, increase sarcomere length, and significantly boost beating amplitude and conduction velocity in neonatal mouse cardiomyocytes. Furthermore, Au@Pt NPs promoted cell cycle arrest, increased intracellular reactive oxygen species levels, and promoted contractility by inducing the ERK1/2 signaling pathway.

Conclusion: Our results indicate that the bimetallic Au@Pt NPs with intrinsic oxidase-like, peroxidase-like, and catalase-like activities and high electrical conductivity could promote the maturation of ESCs-derived and neonatal mouse cardiomyocytes, providing a promising approach for cardiomyocyte maturation and cell therapy for cardiovascular disease.

Background: Extracellular vesicles (EVs) have attracted expanded attention as vehicles for the diagnosis and therapy of diseases and regenerative medicine due to their biocompatibility, efficient cellular uptake ability, and capacity to transport biologically active molecules. However, the low secretion yield of EVs and the challenges of large-scale production remain the main barriers to their extensive clinical use.

Methods and results: This review explores recent strategies to enhance EV production in cell culture systems, focusing on chemical stimulation, mechanical stimulation, and structural stimulation. First, we review chemical stimulation strategies for modulating culture conditions using chemical stimulation, including nutrient composition, pH, temperature, oxygen levels, intracellular cholesterol, and oxidative stress. Second, we examine mechanical stimulation strategies, including shear stress, irradiation, and ultrasound. Third, we explore structural stimulation strategies, such as three-dimensional (3D) culture systems involving spheroid-based culture, as well as the use of bioreactors and scaffolds. In addition, cell-derived nanovesicles containing cell membrane and cellular component, which can be more easily mass-produced compared to EVs, are proposed as an alternative to EVs.

Conclusion: Future research should focus on developing cost-effective and scalable EV production methods while improving purification techniques to ensure a high yield without compromising functional integrity. Moreover, integrating optimized stimulation strategies-such as refining 3D culture systems, bioreactor designs, and mechanical stimulation methods-could further enhance EV secretion. Addressing these challenges is essential for advancing EV-based applications in both research and clinical practice.

Background: Recently, magnetic composite biomaterials have raised attention in bone tissue engineering as the application of dynamic magnetic fields proved to modulate the proliferation and differentiation of several cell types.

Methods: This study presents a novel method to fabricate biofunctional magnetic scaffolds by the deposition of superparamagnetic iron oxide nanoparticles (SPIONs) through thermal Drop-On-Demand inkjet printing on three-dimensional (3D) printed scaffolds. Firstly, 3D scaffolds based on thermoplastic polymeric composed by poly-L-lactic acid/poly-caprolactone/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) were fabricated by Fused Deposition Modelling. Then, in a second step, SPIONs were incorporated onto the surface of the scaffolds by inkjet printing following a designed 2D pattern.

Results: A complete characterization of the resulting magnetic scaffolds was carried out attending to the surface SPIONs deposits, demonstrating the accuracy and versatility of the production technique, as well as the stability under physiological conditions and the magnetic properties. Biological evaluation with human bone marrow mesenchymal stems cells demonstrated biocompatibility of the scaffolds and increased osteogenic capability under the application of a magnetic field, due to the activation of mechanotransduction processes.

Conclusion: These results show that the developed 3D magnetic biofunctional scaffolds can be a very promising tool for advanced and personalised bone regeneration treatments.

Background: Liver fibrosis is a reversible but complex pathological condition associated with chronic liver diseases, affecting over 1.5 billion people worldwide. It is characterized by excessive extracellular matrix deposition resulting from sustained liver injury, often advancing to cirrhosis and cancer. As its progression involves various cell types and pathogenic factors, understanding the intricate mechanisms is essential for the development of effective therapies. In this context, extensive efforts have been made to establish three-dimensional (3D) in vitro platforms that mimic the progression of liver fibrosis.

Methods: This review outlines the pathophysiology of liver fibrosis and highlights recent advancements in 3D in vitro liver models, including spheroids, organoids, assembloids, bioprinted constructs, and microfluidic systems. It further assesses their biological relevance, with particular focus on their capacity to reproduce fibrosis-related characteristics.

Results: 3D in vitro liver models offer significant advantages over conventional two-dimensional cultures. Although each model exhibits unique strengths, they collectively recapitulate key fibrotic features, such as extracellular matrix remodeling, hepatic stellate cell activation, and collagen deposition, in a physiologically relevant 3D setting. In particular, multilineage liver organoids and assembloids integrate architectural complexity with scalability, enabling deeper mechanistic insights and supporting therapeutic evaluation with improved translational relevance.

Conclusion: 3D in vitro liver models represent a promising strategy to bridge the gap between in vitro studies and in vivo realities by faithfully replicating liver-specific architecture and microenvironments. With enhanced reproducibility through standardized protocols, these models hold great potential for advancing drug discovery and facilitating the development of personalized therapies for liver fibrosis.