Tissue Engineering Part A最新文献

In this study, we explored whether embryonic stem cell-derived mesenchymal stem cell (ES-MSC) cellular spheroids in combination with a closed chamber system could be used to create scaffold-free cartilage and endochondral graft tissues. ES-MSC cellular spheroids were cultured in chondrogenic medium for 3-4 days and seeded into a customizable Net Mold chamber system (NCS) and cultured in chondrogenic medium for an additional 18 days to fuse and form a single tissue construct. To assess potential for cartilage repair, cellular spheroids were matured in the NCS for only 7 days before implantation into ex vivo human cartilage defects. To engineer osteochondral tissues, cellular spheroids were initially cultured in chondrogenic medium for 14 days, seeded into one well of the NSC, and cultured together in osteogenic medium for 21 days. For the chondrogenic phase, cellular spheroids were initially cultured in chondrogenic medium for 14 days before seeding in an NCS chamber, adjacent to the osteogenic spheroids. The combined osteogenic and chondrogenic constructs were cultured in serum-free medium for an additional 3 weeks. Cellular spheroids cultured in the NCS developed into neocartilage tissues expressing cartilage-associated genes (COL2A1, ACAN, and COMP) and stained positive for cartilage matrix molecules (glycosaminoglycan and collagen type II). The cartilage-like constructs that were implanted into cartilage defects created in ex vivo osteoarthritic (OA) tissue resulted in repair tissue with an elastic modulus of 46 ± 6 kPa that was histologically integrated with the explant tissues. Spheroids cultured in osteogenic medium produced tissues that were positive for von Kossa stain and for osteopontin immunostaining. Pre-differentiation in chondrogenic and osteogenic medium before placing in the NCS resulted in fused cartilage and bone-like constructs with regional production of chondrogenic and mineralized matrix (Alizarin Red S, von Kossa, and osteopontin positive). Spheroids in stacked NCS chambers produced osteochondral neotissues up to 2 mm in thickness. Our results indicate the potential for cellular spheroids, from a clinically relevant ES-MSC source, to generate scaffold-free chondrogenic or osteochondrogenic graft tissues.

Allogenic demineralized bone matrix (DBM) is widely used for bone repair and regeneration due to its osteoinductivity and osteoconductivity. The present study utilized acellular dermis microfibers to improve the DBM's clinical handling properties and to enhance bone regeneration. Donated human cadaver skin was de-epidermized and decellularized to be acellular dermal matrix (ADM), which was further processed into microfibers. Donated human bone was micronized and partially demineralized (∼30% calcium removal) for optimal bone regeneration. A flexible ADM/DBM composite foam was fabricated with ADM microfibers and DBM particles. Structural analysis found that the ADM/DBM composite foam had proper porosity with interconnected micropores and rapid wettability, and good stability upon cyclic compressions, whereas cytotoxicity test, in vitro collagenase degradation, and rat subcutaneous implantation showed good biocompatibility and biodegradability. The composite foam, used for in vitro coculture, significantly increased the alkaline phosphatase activity of C2C12 cells and upregulated the expression of osteogenesis-related genes of human umbilical cord mesenchymal stem cells. Using the rat Φ8 mm calvarium defect repair model, the ADM/DBM composite foam demonstrated superior osteogenicity by rapidly inducing new bone formation and achieving complete closure of the bone defects, as compared with the commercially available bone graft for skull repair (SkuHeal). Therefore, the ADM/DBM composite foam holds promise as a superior DBM-based product for repairing critical bone defects.

The objective of this study is to investigate the influence of exogenous mitochondria (Mt) internalization on the Mt membrane potential of cells. Cationized gelatin nanospheres (cGNS) were prepared to mix Mt at different ratios to prepare Mt associated with cGNS (Mt-cGNS). The Mt internalization depended on the Mt/cGNS mixing ratio to achieve the maximum at the ratio of 3/1. Rho 0 cells of a Mt function-deficient line were prepared to evaluate the enhancement of Mt membrane potential of rho 0 cells after the internalization of Mt-cGNS. When evaluated by using tetramethylrhodamine methyl ester reagent, the mitochondrial membrane potential of rho 0 cells after incubation with Mt-cGNS enhanced compared with that incubated with Mt only and maintained at a significantly higher level even for 6 days. The Mt-cGNS were internalized into rho 0 cells by an actin-dependent pathway, followed by fused with endogenous Mt. It is concluded that association with the cGNS enabled Mt to enhance the cellular internalization, followed by the fusion with endogenous Mt to maintain an enhanced Mt membrane potential.

The high failure rate of surgical repair for tendinopathies has spurred interest in adjunct therapies, including exosomes (EVs). Mesenchymal stromal cell (MSC)-derived EVs (MSCdEVs) have been of particular interest as they improve several metrics of tendon healing in animal models. However, research has shown that EVs derived from tissue-native cells, such as tenocytes, are functionally distinct and may better direct tendon healing. To this end, we investigated the differential regulation of human primary macrophage transcriptomic responses and cytokine secretion by tenocyte-derived EVs (TdEVs) compared with MSCdEVs. Compared with MSCdEVs, TdEVs upregulated TNFa-NFkB and TGFB signaling and pathways associated with osteoclast differentiation in macrophages while decreasing secretion of several pro-inflammatory cytokines. Conditioned media of these TdEV educated macrophages drove increased tenocyte migration and decreased MMP3 and MMP13 expression. In contrast, MSCdEV education of macrophages drove increased gene expression pathways related to INFa, INFg and protection against oxidative stress while increasing cytokine expression of MCP1 and IL6. These data demonstrate that EV cell source differentially impacts the function of key effector cells in tendon healing and that TdEVs, compared with MSCdEVs, promote a more favorable tendon healing phenotype within these cells.

Adipose tissue engineering requires effective strategies for regenerating adipose tissue, with adipose-derived stem cells (ASCs) being favored due to their robust self-renewal capacity and multipotent differentiation potential. In this study, the efficacy of poly-L-lactic acid (PLLA) mesh containing collagen sponge (CS), seeded with ASCs to promote adipose tissue formation, was investigated. PLLA-CS implants seeded with GFP-positive ASCs were inserted at high concentration (1 × 10⁶ cells/implant, H-ASC) and low concentration (1 × 10⁵ cells/implant, L-ASC), as were unseeded controls. Adipogenesis was evaluated at 3, 6, and 12 months using a rat inguinal model. At 3 months, the weight and volume of newly formed tissues in the H-ASC group were significantly higher than those in the control group. Histological assessment revealed that the area of all newly formed tissue, including the adipose tissue inside the implants in the H-ASC group, was larger at 6 and 12 months compared with that of the control and L-ASC groups, with the adipose percentage at 12 months being higher in the H-ASC group than in the control group. GFP-positive ASCs in both the L-ASC and H-ASC groups adhered to the CS scaffolds and survived for up to 12 months postimplantation, with spontaneous differentiation into adipocytes observed exclusively in the H-ASC group. Double immunofluorescence confirmed the presence of GFP-positive adipocytes. In summary, this study demonstrated that ASCs coimplanted with PLLA-CS implants could enhance adipose tissue formation within the implants. Uninduced ASCs were capable of spontaneously differentiating into adipocytes within the PLLA-CS implants, with differentiation correlating with the number of implanted cells.

Senescence and osteogenic differentiation potential loss limited bone nonunion treatment effects of bone marrow-derived mesenchymal stem cells (BMSCs). MiR-100-5p/Lysine(K)-specific demethylase 6B (KDM6B) can inhibit osteogenesis, but their effects on bone union remain unclear. This study aims to investigate the effects of miR-100-5p/KDM6B on osteogenic differentiation and bone defects. Wild-type or microRNA 100 (miR-100) knockdown mice underwent critical-size defect (CSD) cranial surgery and collagen I/poly-γ-glutamic acid scaffold treatment. The crania was observed using microcomputed tomography, hematoxylin and eosin staining, Masson staining, alkaline phosphatase (ALP) staining, immunohistochemistry, and immunofluorescence. Primary-cultured BMSCs transfected with miR-100-5p mimic/inhibitor and KDM6B cDNA were evaluated for osteogenic differentiation using Alizarin Red staining, ALP activity detection, and Western blot analysis. Genetic transcription levels were detected using quantitative reverse transcription polymerase chain reaction. This study found that miR-100 depletion promotes defect healing in mouse calvaria, increases the proportion of new bone and osteoblasts in calvaria, and activates the expression of KDM6B and osteocalcin (OCN) proteins, promoting the transcription of bone morphogenetic protein-2, Runt-related transcription factor 2 (Runx2), OCN, and KDM6B, while methylation of lysine 27 on histone H3 (H3K27me3) decreased. Furthermore, miR-100-5p mimics suppressed osteogenic differentiation by inhibiting KDM6B with increased H3K27me3, ALP, Runx2, OCN, and osteopontin protein expression, while miR-100-5p inhibitors have opposite effects. Moreover, KDM6B can reverse miR-100-5p mimic effects. Notably, scaffolds carrying miR-100-5p mimics/inhibitors transfected BMSCs were placed in CSD mice and found that miR-100-5p inhibitors have a better effect on CSD healing and increase new bone without inflammatory cell infiltration. This study proved that miR-100-5p depletion promotes bone union and osteogenic differentiation of BMSCs via KDM6B/H3K27me3.

Recently, there has been increased attention on the treatment of cartilage repair. Overall, we constructed PHBVHHx-COL, a composite hydrogel of PHBVHHx-co-PEG and collagen, and evaluated its cartilage repair efficacy through in vitro and in vivo studies using hydrogel loaded with peripheral blood-derived mesenchymal stem cells (PBMSCs). Rheological properties and compressive mechanical properties of the hydrogels were systematically evaluated. The cytocompatibility of the hydrogels was evaluated using the Cell Counting Kit-8 test, live/dead staining, scratch test, and transwell test. The effect of chondrogenic differentiation of PBMSCs on hydrogels was evaluated using immunofluorescence staining and reverse transcription-polymerase chain reaction. Furthermore, the in vivo cartilage repair ability of the hydrogels was confirmed following in situ injections in rabbit chondral defect models. Finally, the induced polarization of the hydrogel scaffold on macrophages was explored by the expression of CD86 and CD206. In vitro experimental results confirmed that PHBVHHx-COL-gel led to better cell migration, proliferation, and chondrogenic differentiation than PHBVHHx-PEG and COL hydrogels. Hematoxylin and eosin staining indicated that the tissue of the repaired area in the PHBVHHx-COL group was nearly in fusion with the surrounding normal tissue and the reconstruction of subchondral bone was good. Safranin-O staining and COL-2 immunohistochemistry indicated that the tissue of the repaired area in the PHBVHHx-COL group had more cartilage-specific matrix secretion. The PHBVHHx-COL group exhibited more M2 macrophage infiltration and less M1 macrophage presentation than the other groups. This study demonstrated that PHBVHHx-COL scaffolds loaded with PBMSCs significantly promoted the repair of cartilage injury through immune regulation by M2 polarization and could be potential candidates for cartilage tissue engineering.

Dental pulp tissue is a desirable cell source for tooth regeneration. However, the extirpation of dental pulp tissue from the tooth root canal causes a partial defect in a sound tooth, which is unacceptable, even for the purpose of tooth regeneration. Moreover, bone or dentine formation by mesenchymal stem cells (MSCs) from dental pulp tissue is slow because of the small number and low proliferative capacity of dental pulp cells containing MSCs. To promote the proliferation and differentiation of MSCs in vitro, a novel accelerator needs to be identified in addition to dexamethasone (Dex), β-glycerophosphate (β-GP), and ascorbic acid (Vc). Therefore, the present in vitro study investigated the effects of L(+)-arginine (Arg) and L(+)-lysine (Lys) as bioactive factors that promote mineralized nodule aggregate formation in MSC subcultures. Bone marrow cells obtained from the femur shafts of rats (rBMCs) were used. Mineralized nodule aggregates were formed by rBMCs in culture medium (MEM: Dulbecco's modified Eagle's medium) for subcultures containing Dex and additional Lys or Arg. Aggregates were decalcified in 10% formic acid to measure the level of Ca²⁺ as an indicator of osteo- or odontogenesis. The results obtained suggest that the addition of Arg to the medium for the rBMC subculture enhanced Dex-induced osteogenesis by rMSCs. The level of Ca²⁺ in calcified nodule aggregates obtained from the rBMC subculture was significantly smaller in MEM containing Dex (MEM-Dex (+)) than in that with 1.150 mmol of Arg (p < 0.001). No significant differences were observed in the level of Ca²⁺ in aggregates formed by rBMCs between MEM-Dex (+) containing 68.4 or 136.8 mmol of Lys or 0.575 mmol of Arg and that without these amino acids (p > 0.05). The level of Ca²⁺ measured following the addition of Arg at 1.150 mmol to 2 mL of MEM-Dex (+) was high. These results indicated that Dex in the medium supplemented with Arg as a cofactor actively promoted the osteogenic activity of MSCs.

Peripheral nerve injury (PNI) is a common disabling condition primarily caused by trauma, such as traffic accidents and occupational injuries. Traditional treatments for PNI have significant limitations. Tissue-engineered nerve grafts (TENGs), which integrate biomaterials, neurotrophic factors, and seed cells, offer a novel solution for nerve regeneration. This review summarizes recent advances in TENGs, focusing on material optimization, preclinical studies, and challenges. Although TENGs show significant potential in repairing long-segment nerve defects, issues such as long-term safety, functional integration, and scalable production require further research. Future multidisciplinary innovations and optimized production processes may enable broader applications of TENGs in nerve regeneration medicine, providing more effective treatment options for patients.

Bone tissue engineering has long been a focal point of research, aiming to address critical large segmental bone defects resulting from severe trauma, tumors, and other bone-related diseases. Despite significant advancements in conventional bone tissue engineering, the simulation of the intricate microenvironment characteristic of natural bone tissue remains inadequate. Natural bone is characterized by intricate macroscopic and microscopic architectures, along with a dynamic microenvironment that facilitates processes such as bone formation, remodeling, and repair. Bone organoids-three-dimensional structures that emulate natural bone tissue derived from stem cells-represent a substantial advancement in both bone tissue engineering and precision medicine. These organoids present a promising pathway for enhancing our understanding of bone biology and disease mechanisms. Their unique potential within precision medicine is underscored by their applications in personalized drug testing, disease modeling, and as platforms for regenerative therapies. As this field continues to progress, bone organoids are poised to play an essential role in developing tailored treatment strategies for disorders related to bones. In this review, we summarize the roles of cell types, biomaterials and culture techniques in the construction of bone organoids, and emphasize the key significance of microenvironment in guiding the maturation of bone organoids. In addition, we will discuss the standardization, current limitations, and future directions of bone organoids to provide insights for research and clinical applications.