Tissue Engineering Part A最新文献

Advancements in biomaterials design increasingly focus on material-host immune interactions as one of the strategies to promote new bone formation, referred to as osteoimmunomodulation. Recent studies indicate that inflammatory stimuli can synergize with growth factors such as bone morphogenetic protein 2 (BMP-2) to promote bone formation. Pathogen-associated molecular patterns (PAMPs) are motifs expressed by microbes that are recognized by immune cells and induce an immune-stimulatory response. In this study, we combined PAMPs with low-dose BMP-2 on a biphasic calcium phosphate (BCP) scaffold and evaluated its effect on ectopic bone formation in a subcutaneous implantation model. The PAMPs tested include gamma-irradiated whole microbes (γi-Staphylococcus aureus and γi-Candida albicans), a vaccine (Bacillus Calmette-Guérin containing Mycobacterium bovis), bacterial cell wall components (peptidoglycan [PGN], lipopolysaccharide [LPS], lipoteichoic acid, and Pam3CysSerLys4), an exopolysaccharide (Curdlan), and nucleic acid analogues (polyinosinic:polycytidylic acid [Poly(I:C)] and Cytidine-phosphate-guanosine [CpG]-containing oligonucleotides type C). Implants consisting of BCP, PAMPs, and BMP-2 were placed subcutaneously in rabbits and evaluated for ectopic bone formation after 5 weeks. Implants with only BMP-2 served as controls. Of the PAMPs tested, only PGN and BMP-2 showed a positive bone volume compared with the control, with borderline significance (+4.4%, p = 0.08). Decreased bone volume was seen for LPS (-7.4%, p = 0.03) and Poly(I:C) (-6.3%, p = 0.04). Fluorochrome labeling at weeks 2 and 3 assessed mineralization onset, revealing no mineralization in the first 2 weeks and some implants showing onset at week 3. We observed variability in ectopic bone formation across animals, associated with higher osteoclast numbers in those where ectopic bone occurred versus those that did not (p = 0.004). PAMPs can modulate bone formation, but their effects are variable, requiring further refinement to harness their osteoimmunomodulatory properties effectively. Additionally, we highlight osteoclasts' important role in stimulating ectopic bone formation.

Successful bioprinting requires an appropriate combination of bioinks, cells, and a delivery platform. To demonstrate the potential of in situ bioprinting for regeneration of cartilage lesions we combined clinically relevant embryonic-derived mesenchymal stem cells (ES-MSCs) with a fibrin-based bioink that was delivered into chondral defects created in human ex vivo osteoarthritic (OA) tissue using a bioprinting platform. We used an integrated multitool, 6-axis bioprinting system to laser scan and map the surface of chondral defects and bioprint within the cartilage defects in vitro and ex vivo. For cartilage neotissue generation, clinically relevant ES-MSCs were encapsulated at 20 × 10⁶ cells per mL in chondro-inductive bioinks composed of fibrinogen mixed with nanocellulose or fibrinogen mixed with nanocellulose and hyaluronic acid. After bioprinting as free-standing constructs or in situ within chondral defects, gels were cross-linked in thrombin and cultured for up to 8 weeks in chondrogenic medium. Print fidelity was assessed in the free-standing printed constructs after cross-linking and culture. In situ bioprinted constructs were evaluated for cell viability, mechanical properties, histology (Safranin O and collagen type II immunostaining), and gene expression of chondrogenic genes. Adding nanocellulose to fibrinogen significantly improved print fidelity. ES-MSCs in the fibrinogen-based bioink formulations generated cartilage-like neotissues with positive Safranin O and collagen type II staining. Chondrogenic genes (COLA2A1, ACAN, COMP, and SOX9) were significantly upregulated with negligible expression of hypertrophic markers (COL10A1 and RUNX2). The mechanical properties of the printed constructs increased from 30 to 50 kPa after 3 weeks to ∼150 kPa after 8 weeks in culture. We demonstrated the feasibility of combining clinically relevant ES-MSCs with printable fibrin-based hydrogel bioinks and an integrated bioprinting platform for in situ bioprinting that promoted neocartilage tissue generation and repair of ex vivo lesions in human OA tissues.

Development of relevant human induced pluripotent stem cell-derived cardiac organoids is essential to recapitulate myocardium physiology and functionality for the assessment of drug-induced toxicity evaluations. However, the optimal conditions for culturing self-aggregating multicellular cardiac organoids are not well-elucidated, particularly the impact of noncardiomyocytes. In this study, we generated cardiac organoids at varying seeding densities to formulate organoids that meet or exceed the biological diffusion limit. We assessed their morphology, gene expression profiles, beating functionality, viability, and mitochondrial activity over time. Our results show that organoid sizes stabilize by 7 days of culture, regardless of seeding density. However, organoids seeded with 20,000 cells retained a more optimal cardiac signature that promotes cardiac maturity and minimizes fibrotic tendencies, especially when cultured for longer than 7 days. While all organoid populations maintained their beating functionalities, those seeded with 80,000 cells exhibited greater cell shedding and increased apoptosis at long-term culture. In contrast, minimal apoptosis was observed in organoids seeded with 20,000 cells after 7 days. Mitochondrial staining further revealed that organoids seeded with 20,000 cells consistently demonstrated higher metabolic activity. Taken together, organoids seeded with 20,000 cells and cultured for 7 days yielded the healthiest morphology, transcriptional signature, and viability while maintaining robust beating kinetics. Importantly, the organoid model identified in this study demonstrated a selectivity index (SI) that is over an order of magnitude larger than that of two-dimensional cultures, showing improved sensitivity to clinically relevant doxorubicin-induced cardiotoxicity, enabling more accurate dose-response evaluations that better reflect therapeutic conditions.

Full-thickness rotator cuff tears (RCTs) represent a musculoskeletal damage that severely affects shoulder function and quality of life. Current surgical interventions are hindered by limited regenerative capacity of rotator cuff repair implants and high retear rates postoperatively. In this study, we investigated a tendon repair matrix (TRM) product prepared from bovine tendon collagen. The TRM was designed as a regenerative scaffold to improve the healing of damaged rotator cuff. In vitro results showed excellent cytocompatibility of TRM, with significantly enhanced adhesion, proliferation, and spreading of bone marrow stromal cells and tenocyte-like mouse tendon precursor cells,mouse tendon-derived cell line, clone D6 (TT-D6) cells (mouse tendon-derived cell line, clone D6). In a rabbit model of acute full-thickness supraspinatus tendon tear, TRM promoted type I collagen deposition, improved interface tissue formation, and enhanced tendon-to-bone integration. Furthermore, biomechanical test results revealed load-bearing capacity of the TRM group compared with both the empty and native tissue control groups. These findings support the clinical potential of TRM as a regenerative scaffold for the functional reconstruction of RCTs. Impact Statement This study addresses a critical clinical need in sports medicine by evaluating a novel bovine collagen-based tendon repair matrix (TRM) for the repair of acute full-thickness rotator cuff tears (RCTs). The TRM exhibited excellent biocompatibility and significantly enhanced tendon-to-bone healing, as demonstrated by improved fibrocartilaginous tissue formation and biomechanical strength in a rabbit model. These promising results underscore TRM's potential to reduce postoperative retear rates by promoting effective regeneration of the tendon-bone interface. Consequently, this research represents an important advancement toward improving clinical outcomes for RCT patients, offering substantial potential for translation into clinical practice.

Indigenous health and wellness encompasses physical, mental, emotional, and spiritual well-being, with a focus on "the interconnectedness of these aspects and the importance of community and cultural practices." "Regenerative healing," as distinct from "Regenerative medicine," is a similarly wholistic term that has emerged from conversations with selected First Nations and Métis Knowledge Holders from across Canada. Impact Statement Building trust with patients and the broader public who support health and medical research requires continuous engagement with the public. "Regenerative Healing" may be a more welcoming and more humble framework to launch the conversation.

Periodontal ligament (PDL) is a thin connective tissue that connects the tooth to the bony socket and plays a crucial role in the regeneration and maintenance of homeostasis of periodontal tissues by supplying stem/progenitor cells. Induced pluripotent stem cells (iPSCs) are highly anticipated in regenerative medicine because of their differentiation potential into a wide variety of cell types. In this study, we investigated the effects of humoral factors on iPSC differentiation by culturing iPSCs in the presence of PDL cell-derived culture supernatants. Changes in gene expression were analyzed using quantitative real-time PCR, reverse-transcription PCR, and RNA sequencing. The marker protein expression on the cell surface was assessed using flow cytometry. Periodontal regeneration was verified by microcomputed tomography and histomorphological observation in a periodontal defect model using male F344/NJcl-rnu/rnu rats. When iPSCs were cultured in the PDL culture supernatant, some cells formed clumps, and spindle-shaped cells grew out from them. Upon passaging, spindle cells increased further, and by the fifth passage, these cells occupied the entire culture. These cells (iPS-PDLs) expressed genes such as periostin and Asporin/PLAP1, and their comprehensive gene expression patterns resembled those of PDL cells. iPS-PDL cells exhibited a cell surface antigen profile of CD90+, CD73+, CD105+, CD44+, CD29+, CD14-, CD34-, CD45-, and CD19- and differentiation potential into osteoblasts, adipocytes, and chondrocytes. Transplantation of iPS-PDLs into rat periodontal defects increased the height of newly formed bone and enhanced periodontal tissue regeneration after 4 weeks. Our results showed that iPSCs differentiated into cells with properties similar to those of PDL cells in the presence of humoral factors of cultured PDL cells. Additionally, the transplantation of iPS-PDL cells into periodontal defects induces periodontal tissue regeneration. These findings provide valuable insights for developing novel periodontal regenerative therapies using iPSCs.

Background: Renal tubule cells lose differentiated characteristics in artificial culture, limiting their application in medical research and cell therapy. We previously showed that adding inhibitors of transforming growth factor-β (TGF-β) signaling to cell culture media increased specific transport functions characteristic of differentiated tubule cells. Transport in proximal tubule cells is energetically demanding; in vivo, these cells rely primarily on oxidative phosphorylation of fatty acids for adenosine triphosphate (ATP) generation. We examined whether TGF-β inhibition, with or without metformin, altered glycolysis and oxidative phosphorylation compared with standard culture conditions. Approach: Primary renal tubule cells (PRTC) were cultured with or without an inhibitor of TGF-β receptor I and with or without metformin in a 2 × 2 factorial design. First, expression of proteins in fatty acid transport and the electron transport chain was compared between conditions. The relative contributions of glycolysis and oxidative phosphorylation to ATP generation were assessed by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). We also tested substrate-specific contributions using inhibitors of pyruvate, glutamine, and carnitine mitochondrial entry. Finally, OCR and transport were measured after 48 weeks in culture to determine durability of culture phenotype. Results: Metformin and SB431542 increased expression and phosphorylation of proteins in the electron transport chain and involved in fatty acid transport. Metformin and TGF-β inhibition increased oxidative phosphorylation. Metformin decreased glucose dependency, while combination with TGF-β inhibition increased fatty acid dependency. Differences in OCR and transport between treatment conditions persisted at 48 weeks in culture. Discussion: Renal tubule cell transport is energetically demanding, so cellular differentiation requires matching increases in energetic machinery. We found that metformin and inhibition of TGF-β increased oxygen consumption and utilization of fatty acids in cultured primary tubule cells. These data support the hypothesis that TGF-β inhibition in vitro not only increases expression of a broad array of transporters characteristic of the proximal tubule, as we previously showed, but also improves the supply of energy to support active transport.

Composite scaffolds combining polysaccharides and bioceramics represent next-generation scaffolds extensively investigated in tissue engineering (TE) and biomedical applications. Polysaccharides such as chitosan, hyaluronic acid, and pectin mimic the extracellular matrix components with their tunable physicochemical properties, enabling a favorable microenvironment for cell adhesion, proliferation, and cell-matrix interactions. On the other hand, bioceramics, including calcium phosphate, hydroxyapatite, and bioactive glasses, enhance the mechanical properties of the material and offer structural integrity and osteoconductive properties. While they have generally been preferred to be used in bone TE and dental applications, various studies have also demonstrated their potential in cartilage regeneration, wound healing, and broader biomedical applications. Recent advancements in material design and scaffold fabrication techniques, particularly 3D printing and electrospinning, have provided precise engineering of materials and fabrication of scaffolds for desirable mechanical properties and biological performance. These innovations foster the development of patient-specific scaffolds, thereby paving the way for applications in personalized medicine. This review critically summarizes alternative polysaccharides, bioceramics, and composite materials used in TE and biomedical applications. It also highlights advanced fabrication strategies and finally explores the translational potential of these biocomposites. By integrating emerging technologies, this review aims to provide alternative and sustainable materials for the development of next-generation scaffolds that meet clinical needs. Impact Statement This study introduces polysaccharide-bioceramic composites with enhanced mechanical and biological properties for tissue engineering. Beyond bone and dental repair, their applications increasingly extend to wound healing, cartilage, cardiac, and muscle regeneration with drug delivery, angiogenesis, and neurogenesis. By mimicking the native extracellular matrix, these composites support cell growth and tissue regeneration, offering a versatile platform for advanced regenerative therapies.

Enhancing bone-vessel coupling to form high-quality vascular-rich peri-implant bone is crucial for improving implant prognosis in elder patients. Notably, hypoxia-inducible factor 1α (HIF1α) is known to promote osteogenesis-angiogenesis coupling; however, this effect remains to be investigated in aged bone owing to the dual effect of HIF1α in different aged organs. In this study, HIF1α inhibitor or activator was applied to aged mice and their bone mesenchymal stem cells (BMSCs) to investigate the effects and inner mechanism of HIF1α on the peri-implant osteogenesis and angiogenesis in senescent status. Cell senescence, along with osteogenic and angiogenic abilities of aged BMSCs, was detected, respectively. Meanwhile, a femur implant implantation model was constructed on aged mice, and the bone-vessel coupling of peri-implant bone was observed. Mandibular bone morphology was also detected to further provide evidence for clinical oral implantation. Furthermore, p53 expression was examined in vivo and in vitro following HIF1α intervention. A reactive oxygen species (ROS) scavenger was also adopted to further investigate the roles of ROS in the HIF1α-p53 axis. Results showed that the suppression of HIF1α alleviated senescence and osteogenesis-angiogenesis coupling of aged BMSCs, while its activation aggravated these effects. The mandible phenotype and bone-vessel coupling in aged peri-implant bone also changed accordingly upon regulation of HIF1α. Mechanistically, p53 changed in the same direction as HIF1α in vivo and in vitro. Moreover, the ROS scavenger reversed the HIF1α-p53 relationship and weakened the effect of HIF1α inhibitor on peri-implant bone improvement. In conclusion, in aged mice, highly expressed HIF1α impaired peri-implant bone-vessel coupling and implant osseointegration through p53, and accumulated ROS was a prerequisite for HIF1α to positively regulate p53. These findings provide new insights into the role of HIF1α and the ROS-HIF1α/p53 signaling axis, offering potential therapeutic targets to improve implant outcomes in elderly patients.