Tissue engineering and regenerative medicine最新文献

Background: The ultimate goal of regenerative medicine is to restore damaged tissues to a healthy state in the body. Direct reprogramming, also referred to as transdifferentiation, holds significant therapeutic potential by converting abundant somatic cells, such as fibroblasts, into functionally distinct cell types for tissue regeneration. Despite its potential applications in regenerative medicine, direct reprogramming faces major challenges, including low efficiency and poor In vivo applicability. In this study, we propose a novel therapeutic strategy for osteoporosis based on In vivo direct reprogramming using a stepwise delivery approach that first enhances cellular stemness and subsequently induces osteogenic transdifferentiation. Enhancing stemness in lineage-committed cells facilitates their conversion into other functional cell types.

Method: To investigate the efficiency of direct reprogramming via stepwise delivery, we utilized valproic acid (VPA) and tauroursodeoxycholic acid (TUDCA) as reprogramming and bone-stimulating factors, respectively. VPA increased the expression of stemness genes, including Oct4, Nanog, and Sox2, and subsequent treatment of TUDCA enhanced the expression of osteogenic genes in the mouse fibroblast. Targeted delivery of these factors to fibroblasts surrounding bone tissue, enabling subsequent direct reprogramming into osteoblasts, was achieved using bisphosphonate (BP)-conjugated lipid nanoparticles as carriers.

Results: Our findings demonstrate that sequential induction of cell reprogramming and tissue regeneration through stepwise administration of VPA and TUDCA significantly enhances therapeutic efficacy in a mouse model of osteoporosis compared to their simultaneous administration.

Conclusion: This stepwise bone-targeted drug delivery system presents a promising strategy for osteoporosis treatment via In vivo direct reprogramming.

Background: Enamel regeneration and remineralization are critical for restoring enamel integrity, as natural enamel lacks the ability to regenerate due to the absence of ameloblasts. The increasing prevalence of dental caries and the irreversible nature of enamel damage highlight the need for advanced repair strategies.

Methods: This review examines the latest advancements in enamel regeneration and remineralization, focusing on biomaterials, nanotechnology-based approaches, and bioengineering strategies. Google Scholar, Scopus (Elsevier), and PubMed databases were used for the selection of literature. The search included key terms such as "enamel regeneration," "biomimetic enamel repair," "stem cell-based enamel regeneration," "nanotechnology in enamel repair," "hydroxyapatite enamel remineralization," and "biomaterials for enamel remineralization."

Results: Various strategies have been explored for enamel remineralization, including self-assembling peptides, dendrimers, hydrogels, and electrospun mats, each demonstrating varying success in laboratory and preclinical studies. While casein-phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP) combined with fluoride remains a widely used clinical remineralization agent, integrating CPP-ACP with nanotechnology is an emerging area requiring further research. Enamel bioengineering approaches utilizing stem/progenitor cells offer potential, though challenges remain in achieving clinical translation.

Conclusion: Despite advancements, replicating the hierarchical structure and mechanical properties of natural enamel remains challenging. Nanotechnology-driven approaches, bioengineered scaffolds, and interdisciplinary collaboration hold promise for optimizing enamel regeneration techniques. Further research is necessary to enhance clinical applicability and develop scalable, effective treatments for enamel restoration.

Background: Wound healing remains a significant challenge in healthcare, particularly for complex and chronic wounds where conventional treatments often fail to provide effective solutions. Recent advances in nanofiber technology have opened new avenues for wound management by offering biomimetic structures that support tissue regeneration. Due to their high surface area-to-volume ratio and porosity, nanofibers closely resemble the extracellular matrix, facilitating an optimal environment for cell adhesion, proliferation, and differentiation.

Methods: This review examines the role of nanofiber-based wound dressings, highlighting their unique advantages in drug delivery, moisture retention, and antimicrobial protection. Additionally, emerging trends such as smart wound dressings responsive to environmental stimuli and multifunctional nanofiber systems are discussed.

Results and conclusion: Nanofiber technology has demonstrated significant potential in enhancing wound healing outcomes by providing an advanced platform for therapeutic delivery and tissue regeneration. Furthermore, the integration of nanofibers with artificial intelligence and biotechnology offers promising directions for future research. As these innovations continue to evolve, nanofiber-based wound dressings may revolutionize wound care by enabling more personalized and effective treatment strategies.

Background: Recently, regenerative medicine based on cell-based therapies has emerged as a therapeutic possibility for the management of osteoarthritis (OA). Stromal vascular fraction (SVF) is a cellular mixture obtained from lipoaspirate processed through either mechanical or enzymatic separation. SVF has been applied in several countries to treat OA patients without robust supporting evidence or comprehensive evaluation.

Methods: This review purposes to summarize clinical evidence regarding SVF as a therapeutic for OA and to introduce the author's perspective. Eleven studies were found suitable for this review; out of these, seven were randomized clinical trials and four were cohort studies.

Results: A review of controlled studies suggests that SVF may offer better symptomatic relief than placebo or hyaluronic acid in the long term, and the effect of SVF is comparable to that of bone marrow aspirate concentrates.

Conclusion: Prospective studies with improved control over the cell isolation method, dosage, and patient selection are necessary to provide convincing evidence of the benefits of SVF in treating OA.

Background: Effective bone-cartilage integration remains a challenge in orthopedic surgery. Conventional methods often fail to reconstruct the native osteochondral interface. This study explores a scaffold-mediated approach utilizing co-cultured osteoblasts and chondrocytes, with platelet-rich plasma (PRP) as a potential promotor for bone-cartilage interface healing.

Methods: We developed a co-culture system integrating both osteoblasts and chondrocytes on PLGA scaffolds, either with or without PRP supplementation. Cell phenotype maintenance was evaluated by RT-PCR, while morphological analysis was performed by scanning electron microscopy and fluorescence microscopy. To assess healing potential, we created a gap-mimic construct comprising bone, scaffold, and cartilage layers, which was implanted subcutaneously in BALB/c-nude mice. Gap healing was evaluated at 4 and 8 weeks through macroscopic examination, quantitative adhesion analysis, and histological assessment of cellular invasion.

Results: Co-cultured osteoblasts and chondrocytes maintained their phenotypes on PLGA scaffolds, with PRP significantly enhancing cell adhesion (215% increase for chondrocytes, 120% for osteoblasts) and proliferation. In vivo, cell-containing scaffolds demonstrated significantly greater attachment at the bone-cartilage interface compared to acellular constructs. PRP-treated scaffolds exhibited higher attachment rates (82.3% vs 76.7%) and cellular invasion (5/6 vs 3/6 constructs) at 8 weeks, with invasion observed as early as 4 weeks in the PRP group, suggesting accelerated remodeling.

Conclusion: This study demonstrates the feasibility of developing transplantable scaffolds containing co-cultured osteoblasts and chondrocytes while preserving their phenotypes. These scaffolds exhibit significant potential in promoting healing at the bone-cartilage interface, with PRP further enhancing proliferation and improving the scaffold's ability to promote bone-cartilage interface healing.

Background: Choroidal neovascularization (CNV) is a major pathological process underlying retinal degenerative diseases such as wet age-related macular degeneration. While anti-VEGF therapies are widely used, limitations in response and vascular instability necessitate new approaches that promote both antiangiogenic effects and barrier restoration.

Methods: A dual-cell therapy strategy was developed using human umbilical vein endothelial cells (HUVECs) genetically modified to overexpress Tie2 and mesenchymal stem cells (MSCs) engineered to secrete Angiopoietin-1 (Ang1). Antiangiogenic efficacy was evaluated using scratch assays, Transwell migration, and tube formation under VEGF stimulation. A retina-mimetic 2.5D co-culture system incorporating iPSC-derived RPE cells and mCherry-labeled ECs was used to assess endothelial invasion and epithelial barrier preservation.

Results: Tie2/Ang1-modified cells significantly suppressed angiogenic behavior. Transwell migration showed OD595 crystal violet absorbance decreased from 3.54 ± 0.27 (control HUVEC) to 1.28 ± 0.08 (Tie2 overexpressed HUVEC in MSC Ang1 conditioned medium) under VEGF stimulation (p < 0.01). Tube formation area cultured in VEGF dropped from 1.25 ± 0.05 in control group to 0.74 ± 0.07 in Tie2 overexpressed group cultured with MSC-Ang1 conditioned medium (p < 0.01). In the retina-mimetic model, EC infiltration to the RPE monolayer across Transwell membrane decreased from 52.2 ± 8.5% in control HUVEC to 5.6 ± 4.3% with HUVEC-Tie2 + Ang1 conditioned medium under VEGF (p < 0.001).

Conclusion: This study demonstrates that co-delivery of Ang1 and Tie2 via engineered ECs and MSCs synergistically inhibits VEGF-induced angiogenesis and choroidal migration while protecting epithelial barrier function. The retina-mimetic co-culture platform further validates the translational relevance of this dual-cell approach as a regenerative and antiangiogenic strategy in retinal vascular disease.

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.