Tissue Engineering Part A最新文献

The current clinical treatment of large bone defects in humans primarily relies on autologous bone grafts. However, the use of autologous bone grafts can be limited by tissue availability, variable bone quality, and donor site morbidity. In response to these challenges, endochondral bone regeneration has emerged as a promising approach. This method mimics endochondral ossification by chondrogenically differentiating or stimulating cells of various cell sources into 'callus mimics' (CMs). We previously demonstrated the feasibility of endochondral bone regeneration in restoring bone defects using 'mesenchymal stromal cell' (MSC)-derived devitalized CMs in small and large animals. To scale up the size of treated defects using these CMs, we propose the introduction of a vascular supply. In this study, an arteriovenous (AV) loop was introduced as a vascular supply to devitalized 'MSCs'-derived CMs in a centimeter-scale porous chamber in rats. The extent of vascularization and remodeling was evaluated for chambers filled with CMs in the presence or absence of an AV loop at 4 and 8 weeks. While the AV loop's role in vascularization is established, our study uniquely shows that in a challenging in vivo setting with devitalized callus mimics, the AV loop was critical for initiating bone formation. Mineralization was observed in all groups via microCT, but bone tissue formed only in the AV loop group (50% of samples at 8 weeks), underscoring its influential role in supporting both vascular invasion and bone formation.

While β3GalT2 has been implicated in osteogenic regulation, its synergistic application with bioactive scaffolds remains unexplored. This study pioneers a dual-functional bone regeneration strategy by integrating β3GalT2-engineered bone marrow mesenchymal stem cells (BMSCs-β3GalT2) with nano-hydroxyapatite/polyamide 66 (n-HA/PA66) composites. First, we studied the effect of β3GalT2 on rat BMSCs (rBMSCs) by overexpression the β3GalT2 gene. Following this, we extracted exosomes and verified that β3GalT2 influences osteogenesis of rBMSCs through exosomes. Subsequently, we inoculated these rBMSCs on n-HA/PA66 and demonstrated the effects of β3GalT2 and n-HA/PA66 on osteogenic differentiation of rBMSCs. On this basis, we also explored the molecular mechanism of β3GalT2 regulating M1 polarization through exosomes. Finally, we verified our study by using animal models of skull defect and femur defect. Our results suggest that β3GalT2 promotes osteogenic differentiation of rBMSCs through exosomes. At the same time, rBMSCs-β3GalT2 combined with n-HA/PA66 showed good osteogenic effect in vivo and in vitro. In addition, we also found that β3GalT2 can regulate M1 polarization through exosomes. Our findings establish β3GalT2 as a master regulator of osteogenesis through cellular-exosomal-circuitry mechanisms. The biohybrid system synergistically combines gene-enhanced stem cells with tunable biomaterials, representing a paradigm shift in bone tissue engineering.

Mesenchymal stem cells (MSCs) are widely used for tissue regeneration due to their multilineage differentiation potential and ability to secrete paracrine factors with immunomodulatory and angiogenic functions. Standard MSC differentiation protocols typically rely on two-dimensional (2D) or pellet culture models that are simple to use but not well-suited for translational or clinical applications. To promote better cell survival, tissue deposition, and differentiation of MSCs, a wide variety of three-dimensional (3D) biomaterial scaffolds and platforms have been developed that provide structural support and present a carefully defined set of biochemical and biophysical cues to cells. While biomaterials can guide cell behavior and promote desirable tissue regeneration outcomes, one remaining challenge in the field is inherent donor-to-donor variability in MSC behavior, phenotype, and differentiation capacity. Although MSCs are promising tools for regeneration, the influence of donor variability on MSC differentiation across culture models remains poorly understood. Previous studies typically use cells from a single donor or rely solely on standard culture models. To address these gaps, we compared MSCs from six human donors and assessed differentiation across chondrogenic, osteogenic, and adipogenic lineages using both standard (pellet or 2D) and 3D biomaterial-based culture models. Alginate hydrogels were used to assess chondrogenesis, while gelatin microribbon (µRB) hydrogels were used to evaluate osteogenesis and adipogenesis in 3D. Significant donor-to-donor variability was observed in differentiation outcomes across all three lineages and within both 2D and 3D culture models. By directly comparing donor variability in 2D and 3D, we provide evidence that standard 2D models cannot predict MSC differentiation capacity in 3D biomaterials. Therefore, to improve therapeutic efficacy and advance biomaterial-based strategies for tissue regeneration, it is critical to understand how donor variability affects MSC differentiation patterns across 3D biomaterial-based culture models.

Tissue engineering offers an alternative for augmentation urethroplasty; however, no ideal material has yet been developed. Recently, materials derived from amniotic tissues appear to exhibit promising properties. Herein, the aim of this study was to provide a proof of concept for the integration of the human umbilical cord vein for urethral reconstructions in rabbits. Rabbits were included in two groups; the control group underwent urethral reconstruction using autograft urethral tissue, and the test group received xenograft tissue (umbilical cord vein) after creating a 1 × 1 cm defect in the proximal urethra. At 3 weeks, endoscopy and biopsy were performed. At 6 weeks, the animals were euthanized, and their urethra and corpus cavernosum were sent for histopathological analysis. The six rabbits exhibited favorable clinical and endoscopic progress with no fistula or stenosis. Biopsy analysis found no lesion of the urothelium and chorion. Final histological analysis found similar results in both groups: normal histology with moderate urothelium vacuolation and a weak inflammatory cellular infiltrate. The present study provides a proof of concept of human umbilical cord vein as a scaffold for urethral regeneration. This could be an alternative to existing urethral tissue grafting procedures that can have difficulties with integration or immunological tolerance; however, further research is required.

Polyhydroxyalkanoates are promising biomaterials, but their application in cartilage repair is still limited. In this study, an injectable thermosensitive hydrogel poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate)-Polyethylene Glycol (PEG)/hyaluronic acid/kartogenin was prepared from 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, hyaluronic acid, and kartogenin. The hydrogels are porous, temperature-sensitive, and hydrophilic and have good compressive modulus. Mesenchymal stem cells derived from peripheral blood can proliferate on the hydrogels under two- and three-dimensional cultures. In addition, the hydrogel has the ability to induce chondrogenic differentiation of stem cells and induce M2 differentiation of macrophages. The hydrogel loaded with peripheral blood mesenchymal stem cells can repair cartilage defects in the knee joints of New Zealand rabbits and the newly formed cartilage was identified as type II collagen. Overall, this newly developed system could provide a new treatment option for repairing cartilage defects. Impact Statement In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) was modified with hyaluronic acid and kartogenin to synthesize a thermosensitive injectable hydrogel scaffold. The scaffold has anti-inflammatory and cartilage-promoting effects. This study used the scaffold to carry peripheral blood mesenchymal stem cells to repair cartilage defects in rabbit knee joints, providing a new idea for the treatment of cartilage defects.

Investigating the infection mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the airway epithelium and developing effective defense strategies against infection are important. To achieve this, establishing appropriate infection models is crucial. Therefore, various in vitro models, such as cell lines and primary cultures, and in vivo models involving animals that exhibit SARS-CoV-2 infection and genetically humanized animals have been used as animal models. However, no animal model has been established that allows infection experiments with human cells under the physiological environment of airway epithelia. Therefore, we aimed to establish a novel animal model that enables infection experiments using human cells. Human induced pluripotent stem cell-derived airway epithelial cell-transplanted nude rats (hiPSC-AEC rats) were used, and infection studies were performed by spraying lentiviral pseudoviruses containing SARS-CoV-2 spike protein and the GFP gene on the tracheae. After infection, immunohistochemical analyses revealed the existence of GFP-positive-infected transplanted cells in the epithelial and submucosal layers. In this study, a SARS-CoV-2 infection animal model including human cells was established mimicking infection through respiration, and we demonstrated that the hiPSC-AEC rat could be used as an animal model for basic research and the development of therapeutic methods for human-specific respiratory infectious diseases.

Tracheal cartilage provides structural support to the airways to enable breathing. However, it can become damaged or impaired, sometimes requiring surgical resection and reconstruction. Previously, we clinically applied an artificial trachea composed of a polypropylene mesh and collagen sponge, with a favorable postoperative course. However, the artificial trachea presents a limitation, as the mesh is not biodegradable and cannot be used in pediatric patients. Compared to a polypropylene mesh, regenerated cartilage represents an ideal material for reconstruction of the damaged trachea. The use of mesenchymal stem cells (MSCs) as a source for cartilage regeneration has gained widespread acceptance, but challenges such as the invasiveness of harvesting and limited cell supply persist. Therefore, we focused on the potential of human-induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) for tracheal cartilage regeneration. In this study, we aimed to regenerate tracheal cartilage on an artificial trachea as a preliminary step to replace the polypropylene mesh. iMSCs were induced from hiPSCs through neural crest cells and transplanted with a polypropylene mesh covered with a collagen sponge into the damaged tracheal cartilage in immunodeficient rats. Human nuclear antigen (HNA)-positive cells were observed in all six rats at 4 weeks and in six out of seven rats at 12 weeks after transplantation, indicating that transplanted iMSCs survived within the tracheal cartilage defects of rats. The HNA-positive cells coexpressed SOX9, and type II collagen was detected around HNA-positive cells in four of six rats at 4 weeks and in three of seven rats at 12 weeks after transplantation, reflecting cartilage-like tissue regeneration. These results indicate that the transplanted iMSCs could differentiate into chondrogenic cells and promote tracheal cartilage regeneration. iMSC transplantation thus represents a promising approach for human tracheal reconstruction.

Four human acellular dermal matrices (hADMs) were characterized in a nonhuman primate abdominal wall repair model by evaluating host immune response, vascularization, and incorporation into host tissues. AlloDerm™ (electron beam-sterilized hADM [e-hADM]), AlloMax™ (gamma beam-sterilized hADM, freeze-dried [g-hADM-FD]), DermaMatrix™ (hADM, freeze-dried [hADM-FD]), and FlexHD™ (ethanol-treated hADM [EtOH-hADM]) were each implanted in an abdominal wall-bridging defect in nonhuman primates (n = 3 animals/time point, n = 36 animals). Immunohistochemical and histological assessments were conducted on biopsies from each hADM at 1-, 3-, and 6-months postimplantation to assess vascularization (hematoxylin and eosin [H&E], CD31, alpha smooth muscle actin [αSMA], collagen IV), inflammatory/immune response (H&E, CD3, CD20, CD68), and collagen turnover (H&E, matrix metalloproteinase-9 [MMP-9]). MMP-9 immunolabeling was similar among different hADMs at 1 month; however, hADM-FD and EtOH-hADM showed higher total mean MMP-9-immunopositive areas at approximately 16% compared with <1% for e-hADM and g-hADM at 6 months postimplantation. Cells that stained positively for CD68, CD3, and CD20 were generally higher for hADM-FD and EtOH-hADM compared with other hADMs. The mean CD31-immunopositive area, CD31 vessel density, CD31 vessel diameter, and collagen IV-immunopositive area increased over time. Among all the hADM types, e-hADM had the highest mean (±standard deviation [SD]) CD31-immunopositive area at 1.54% ± 1.01%, vessel density at 7.86 × 10^-5 ± 3.96 × 10^-5 vessels/µm², and collagen IV-immunopositive area at 2.55% ± 0.73% 1-month postimplantation. The pattern of αSMA immunolabeling varied among the hADMs. Histology showed that overall inflammation was mild at 1 month. Overall fibroblast repopulation and collagen remodeling increased over time from 1 to 6 months postimplantation. Fibroblast infiltration was minimal to mild at 1 month, with e-hADM showing the highest mean (±SD) score at 2.00 ± 0.00 compared with other hADMs. Only hADM-FD was not completely replaced by neotissue formation at 6 months postimplantation. All hADMs promoted vascularization, cell infiltration, and incorporation into host tissue, which were associated with acute inflammation and immune responses, within a 6-month period. A trend toward relatively enhanced early vascularization in e-hADM compared with other hADMs was observed. Immunogenic responses among the hADMs in the present study showed a slight distinction toward more quiescent terminally sterilized hADMs (e-hADM, g-hADM-FD) versus aseptically processed hADMs (EtOH-hADM, hADM-FD).

Volumetric muscle loss (VML) injuries are defined by loss of sufficient skeletal muscle to produce persistent deficits in muscle form and function, with devastating lifelong consequences to both soldiers and civilians. There are currently no satisfactory treatments for VML injuries. The work described herein details the implementation of a fully enclosed bioreactor environment (FEBE) system that efficiently interfaces with our existing automated bioprinting and advanced biomanufacturing methods for cell deposition on sheet-based scaffolds for our previously described tissue-engineered muscle repair (TEMR) technology platform. Briefly, the TEMR technology consists of a porcine bladder acellular matrix seeded with skeletal muscle progenitor cells and preconditioned via 10% uniaxial cyclic stretch in a bioreactor. Overall, TEMR implantation in an established rat tibialis anterior (TA) VML injury model can result in 60 to ∼90% functional recovery. However, our original study documented >50% failure rate. That is, more than half of the implanted TEMR constructs produced no functional improvement beyond no treatment/repair. The high failure rate was attributed to the untoward mechanical disruption of TEMR during surgical implantation. In a follow-up study, adjustments were made to the geometry of both the VML injury and the TEMR construct, and the "nonresponder" group was reduced from over half the TEMR-treated animals to just 33%. Nonetheless, additional improvement is needed for clinical applicability. The main objectives of the current study were twofold: (1) explore the use of advanced biomanufacturing methods (i.e., FEBE bioreactor) to further improve TEMR reliability (i.e., increase functional response rate), (2) determine if previously established bioprinting methods, when coupled to the customized FEBE system would further improve the rate, magnitude or amplitude of functional outcomes following TEMR implantation in the same rat TA VML injury model. The current study demonstrates the unequivocal benefits of a customized bioreactor system that reduces manipulation of TEMR during cell seeding and maturation via bioprinting while simultaneously maximizing TEMR stability throughout the biofabrication process. This new biomanufacturing strategy not only accelerated the rate of functional recovery, but also eliminated all TEMR failures. In addition, implementation of bioprinting resulted in more physiomimetic skeletal muscle characteristics of repaired muscle tissue.

To improve bladder compliance in patients with low-compliance bladders, augmentation cystoplasty with the intestinal tract is performed. However, the use of the intestinal tract often leads to serious surgical complications. Tissue engineering technologies have the potential to improve bladder compliance without using the intestinal tract. In this study, we fabricated bi-layered adipose-derived mesenchymal cell (AMC) sheets and then determined whether the bi-layered AMC sheets could improve bladder compliance in rats with spinal cord injury (SCI). The abdominal adipose tissues of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were harvested, and the attached and proliferating cells on type I collagen were used as AMCs. The AMCs were then cultured on temperature-responsive culture dishes. After reaching over-confluence, the AMCs that maintained cell-cell contacts were detached from the dishes and applied to a gelatin hydrogel sheet. Then, another detached AMC monolayer was accumulated on the AMC monolayer-applied gelatin. Prior to 4 weeks of transplantation, the levels of T8-9 in the spinal cords of recipient SD rats were partially transected. After producing the bi-layered AMC sheets and the rats with SCI, the detrusor muscles of the anterior bladder walls of the rats with SCI were incised, and the bi-layered AMC sheet was patch-transplanted onto the exposed bladder epithelium (n = 8). As a control, the sham operation was performed (n = 7). Four weeks after the transplantation, bladder capacity and bladder compliance in AMC sheet-transplanted SCI rats were significantly higher than those in sham-operated control SCI rats. The smooth muscle layers in AMC sheet-transplanted bladders were significantly larger than those in control bladders. In addition, the collagen fibers in the AMC sheet-transplanted bladders were significantly smaller than those in the control bladders. Some GFP-positive transplanted AMCs differentiated into smooth muscle actin- or desmin-positive cells. Furthermore, GFP-positive cells secreted transforming growth factor-β1 or vascular endothelial growth factor. Therefore, this study showed that bi-layered AMC sheets could improve bladder compliance and bladder tissues in SCI rats.