Tissue Engineering Part A最新文献

Bone-related pathologies due to injuries, trauma, and disease are a burden on the current health system that will only continue to grow as the population's life expectancy increases. The field of biomaterials aims to address these concerns by exploring, investigating, and optimizing bioregenerative grafts. In the context of bone regeneration, many biomaterials aim to achieve autograft-level regenerative properties, such as osteoconduction, osteoinduction, and low immunogenicity but also aim to address the disadvantages, such as the need for a secondary operation, donor site burden, and limited donor availability. Chitosan (CS) is a natural polymer well-studied in the field of biomaterials; it is known for its ease of fabrication, biocompatibility, antibacterial nature, and being a nonproteinaceous polysaccharide, which offers the advantage of low immunogenicity. However, CS lacks any osteogenic potential and is often combined with a bioceramic, creating a biocomposite scaffold. Bioceramics are ceramics specifically designed to aid bone regeneration due to their potential osteogenic properties. Although CS-bioceramic composites have been extensively studied, most research emphasizes their physicochemical properties, with limited attention to biological performance and in vivo outcomes. This review presents current findings on the regenerative potential of various CS-bioceramic composites, with a particular focus on in vitro and in vivo studies.

The aim of this study was to grow axially vascularized soft tissue flaps in sheep using the arteriovenous loop (AVL) technique to be transplanted for defect reconstruction. This technique may be a promising alternative to conventional free flaps to further reduce flap donor site morbidity. In this pilot study, AVLs (n = 12) were created in the groins of six sheep, placed into an isolation chamber, and embedded in Matriderm®. Tissue volume, vascularization, and cell proliferation were assessed on postoperative day (POD) 28 using immunohistochemical staining and microcomputed tomography (µCT). Four AVL free flaps were microsurgically anastomosed to the neck vessels in a standardized defect sheep model on POD 28. Defect closure and intrinsically vascularized scaffold-based bioengineered flaps (IVSBs) flap perfusion were studied by angiography and histology 10 days after transplantation. One IVSB flap was lost due to chamber infection. At POD 28, the remaining 11 IVSB flaps had filled the isolation chamber. Histological examination and µCT analysis of seven IVSB flaps verified homogeneous microvascular networks within the flaps. The mean number of microvessels, vessel volume, and the percentage of proliferating cells increased significantly over time. In the defect model, all four transplanted flaps showed macroscopically, angiographically, and histologically stable defect closure 10 days after transplantation, with homogeneous vascular integration into the surrounding tissue. This pilot study demonstrates that in a large animal model complex, defects can be reconstructed using free IVSB flaps with a clinically relevant tissue volume. These data provide the preclinical proof prior to human application.

In vitro models aim to recapitulate human physiological processes, improving upon and replacing the need for animal-based models. Modeling bone formation via endochondral ossification in vitro is a very complex process due to the large number of cell types involved. Most current models are limited to mimicking the initial stages of the process (i.e., cartilage template formation and mineralization of the matrix), using a single cell type. Chondroclasts/osteoclasts are key players in cartilage resorption during endochondral ossification, but their introduction into in vitro models has thus far proven challenging. In this study, we aimed toward a new level of model complexity by introducing human monocyte-derived osteoclasts into 3D in vitro-cultured cartilage templates undergoing mineralization. Chondrogenic and mineralized chondrogenic pellets were formed from human pediatric bone marrow stromal cells and cultured in the presence of transforming growth factor-β3 (TGF-β) and TGF-β/β-glycerophosphate, respectively. These pellets have the capacity to form bone if implanted in vivo. To identify suitable in vitro co-culture conditions and investigate cell interactions, pellets were co-cultured with CD14+ monocytes in an indirect (transwell) or direct setting for up to 14 days, and osteoclastogenesis was assessed by means of histological stainings, osteoclast counting, and gene expression analysis. Upon direct co-culture, we achieved effective osteoclast formation in situ in regions of both mineralized and unmineralized cartilages. Notably, in vitro-generated osteoclasts showed the ability to form tunnels in the chondrogenic matrix and infiltrate the mineralized matrix. Addition of osteoclasts in human in vitro models of endochondral ossification increases the physiological relevance of these models. This will allow for the development of robust 3D human in vitro systems for the study of bone formation, disease modeling, and drug discovery, further reducing the need for animal models in the future. Impact Statement In vitro bone formation models of endochondral ossification are currently limited to the recapitulation of the initial stages of the process. In this article, we present a novel in vitro endochondral ossification model where osteoclasts were incorporated into mineralized hypertrophic cartilage templates, adding a new level of complexity toward the modeling of cartilage resorption during endochondral ossification.

Model systems play a crucial role in biological and biomedical research, especially in the search for new treatments for challenging diseases such as glioblastoma multiforme (GBM). Organoids are 3D in vitro multicellular "middle-ground" model systems that recapitulate highly organized and heterogeneous in vivo organ-like systems, often through stem cell differentiation. Incorporating Matrigel™ or other exogenous extracellular matrices (ECMs) that do not naturally occur in the human body is common practice for organoid generation, ignoring the role of dynamic reciprocity between the cells and the ECM in tissue development. In this study, we describe a method to develop GBM organoids (GBOs) from cells without the need for exogenous ECM encapsulation and without cell culture media changes to produce stable tissue-like organoids that reach a 4 mm diameter in as little as 6 weeks. We observed a transition from homogenous cell populations to tissue-like structures when GBOs were larger than 1 mm in diameter. Transcriptomic analysis revealed that the greatest gene expression changes occurred when GBOs were 2 mm in diameter, with collagen VI as the most upregulated ECM-related gene. Quantitative and histochemical assessments further supported native ECM synthesis with significantly higher levels of glycosaminoglycans and collagen in GBOs compared with spheroids. To our knowledge, this study presents the first reproducibly large GBOs with natively produced ECMs. Organoids with natively synthesized ECMs promise to eliminate artifacts and variability from aged, homogeneic, or xenogeneic scaffolds and to provide insights for ECM-targeted drug development. Impact Statement Glioblastoma multiforme (GBM) is the most common and deadly brain tumor due to its complex tissue heterogeneity. Drug development for GBM is difficult because GBM models are not very translatable and are limited, leading to the need of GBM organoids (GBOs). Current GBO development is highly laborious and of questionable relevance because of the reliance on non-native, animal-derived extracellular matrix (ECM). This study describes a scalable and reproducible method of developing GBOs with natively generated ECMs. These GBOs allow for both the study of the early stages of GBM that are currently inaccessible and a quicker and more translatable tool for GBM drug screening and development.

The activation of chondrogenic progenitor cells (CPCs) in articular cartilage during a traumatic injury is vital for cartilage regeneration. Although our understanding of the mechanisms underlying CPC chondrogenic activation remains incomplete, there is evidence that exosomal microRNAs (miRNAs or miRs) are involved in tissue healing due to their regulating role of posttranscriptional gene expressions. In this study, we profiled enriched and differential expression of miRNAs in exosomes derived from bovine joint cells (CPCs, chondrocytes, and synoviocytes) via Next Generation Sequencing analysis and validated the potential therapeutic effects of candidate exosomal miRNAs for cartilage regeneration. For CPC-based cartilage regeneration, we tested the impact of administering miR-107, miR-140, and miR-148a on CPCs because we found that these miRNAs were highly and differentially expressed in chondrocytes-derived exosomes (CC-Exo). We found that: (1) miR-140 induced chondrogenic gene expression including SRY-box transcription factor 9, collagen type 2A1, and aggrecan, and (2) miR-107 suppressed catabolic gene expression including matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin motifs 5, and nitric oxide synthase 2. Our findings indicate that transfection of CPCs with specific chondrogenic miRNAs present in CC-Exo have the potential to promote CPC-based cartilage regeneration and could be an important component of posttraumatic osteoarthritis prevention. Impact Statement Chondrocytes, chondrogenic progenitor cells (CPCs), and synoviocytes secrete exosomal microRNAs (miRNAs) that contribute to joint health and disease. These miRNAs could also have important implications for improving cartilage repair and regeneration. In this study, we identified candidate miRNAs that were enriched in chondrocytes-derived exosomes and found that these miRNAs induced chondrogenic gene expression or suppressed catabolic gene expression in a CPC monolayer culture system. These findings suggest that miRNA-based cartilage repair strategies could be developed to regenerate damaged and diseased cartilage.

Bioscaffolds composed of extracellular matrix (ECM) have been shown to promote a profound transition in macrophages and T-cells from a proinflammatory to a prohealing phenotype with associated site-appropriate and constructive tissue remodeling rather than scar tissue formation. Matrix-bound nanovesicles (MBV) are a distinct class of extracellular vesicles that can be isolated from the ECM and can recapitulate these immunomodulatory effects on myeloid cells in vitro and in vivo, as shown in multiple preclinical models of inflammatory-driven diseases. However, the effect of this MBV-mediated immunomodulation upon the ability to mount an adaptive immune response following pathogenic challenge is unknown. The present study assessed the humoral immune response with and without repeated MBV administration in a mouse model of Streptococcus pneumoniae vaccination and infection. Mice were immunized on day 0, followed by an intraperitoneal MBV or methotrexate (MTRX) injection the next day and weekly thereafter for 5 weeks. Antipneumococcal polysaccharide immuglobulin G and immuglobulin M titers were no different between the vaccine + MBV and the vaccine-only groups, in contrast to the decreased titers in the MTRX-treatment group. Fifty percent of animals treated with MBV were protected from lethal septic infection with S. pneumoniae, and MBV treatment altered the population of immune cells within the lung following sublethal intranasal infection. Macrophages derived from bone marrow mononuclear cells harvested from MBV-treated mice showed persistent immunomodulatory effects following ex vivo challenge with bacterial antigens. The results of this study show that MBV treatment does not compromise the ability to mount an adaptive immune response and suggest that MBV induce sustained immunomodulation in cells of the myeloid lineage. Impact Statement The current study shows the immunomodulatory effect of matrix-bound nanovesicles (MBV) on vaccinated mice, while demonstrating their compatibility with the adaptative immune system. Furthermore, results of this study suggest a sustained MBV-mediated immunomodulation on myeloid lineages, which could be used in the development of future vaccines and immunomodulatory therapies.

Tissue-inducing biomaterials, which promote tissue regeneration without the addition of exogenous cells and/or bioactive factors, have recently attracted increasing interest in the repair of nonosseous tissues. As a key strategy for transforming data into actionable evidence, evidence-based biomaterials research plays a critical role in guiding material development. In this study, evidence mapping method was employed to systematically analyze and visualize animal study designs, material characteristics, outcome indicators, and evaluation methods, aiming to identify current research trends and emerging focal areas. The results revealed a wide diversity of experimental animal species, with a predominance of small animal models. Among the 19 types of nonosseous tissues investigated, skin, abdominal wall, cartilage, and blood vessels were the most frequently studied. Materials were mainly classified into bio-derived materials, polymers, and composites. Outcome indicators span from macroscopic to molecular levels, with tissue-level indicators being the most commonly applied. Histological analysis served as the primary method for validating inductive effects, supported by gross observation, imaging analysis, molecular biology assays, and biomechanical testing. Overall, tissue-inducing biomaterials show promising potential for nonosseous tissue regeneration. However, challenges remain, including limitations of animal models, short follow-up periods, and insufficient evaluation systems. Future studies should strengthen the alignment between functional validation and clinical needs to promote the translation of these materials from experimental research to clinical application.

Removal of major xenoantigens of the Galα1-3Gal (α-Gal) epitope and the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) is essential to eliminate xenoimmunogenicity and optimize recellularization for cardiac xenografts. The aim of this study was to evaluate the safety and efficacy of α-galactosidase for removal of α-Gal xenoantigen and peptide N-glycosidase F (PNGase-F) for removal of non-α-Gal xenoantigen combined with optimal decellularization, and the potential of in vitro recellularization was assessed with coculturing human mesenchymal stem cells and human umbilical vein endothelial cells for major xenoantigen-free cardiac xenografts. We investigated the biomechanical properties and efficacy for xenoantigen removal with expression of carbohydrate-binding lectins in porcine pericardia decellularized and treated with α-galactosidase and PNGase-F. There were no histological changes depending on α-galactosidase and PNGase-F treatment. There was no difference in tensile stress, tensile displacement, tensile strain at break, and permeability test following enzymatic treatments. Both enzyme-treated xenografts were stained with Jacalin, Maackia amurensis lectin I, wheat germ agglutinin, Ricinus communis agglutinin, Griffonia simplicifolia lectin (GSL), erythrina cristagalli lectin, peanut agglutinin, soybean agglutinin, Wisteria floribunda lectin, and Datura stramonium lectin and showed synergistic effects for low fluorescence qualitatively and quantitatively. The enzymatic treatments for decellularization significantly reduced lectin expression, demonstrating the synergistic effect of both enzymes and decellularization. In vitro recellularization for decellularized and both enzymes-treated xenografts was assessed with vimentin, calponin, fibronectin, and CD31 staining. Stronger signals were detected in decellularized xenografts, and decellularized xenografts treated with both enzymes showed significantly faster mesenchymal cell infiltration into the tissue, leading to accelerated recellularization. We have successfully produced major xenoantigen-free scaffolds by demonstrating the safety and the synergistic effect of α-galactosidase and PNGase-F treatments and proved effective recellularization for the xenoantigen-free scaffolds not previously reported in the literature.

Bone regeneration remains a significant challenge in regenerative medicine. In this context, fibrin hydrogels have attracted attention as a promising biomaterial that regulates the inflammatory response and promotes tissue repair by influencing macrophages. In this study, we investigated the immunomodulatory effects of fibrin hydrogels on macrophage polarization and their subsequent impact on bone regeneration. It is widely recognized that M1 macrophages produce tumor necrosis factor alpha (TNF-α), while M2 macrophages produce interleukin-10 (IL-10). When undifferentiated mouse bone marrow-derived macrophages were stimulated with lipopolysaccharides (LPS), a marked increase in the proinflammatory cytokine TNF-α was observed. However, coculture with fibrin hydrogels in the presence of LPS significantly suppressed TNF-α production while enhancing the secretion of the anti-inflammatory cytokine IL-10. Furthermore, in a rat calvarial defect model, tissue analysis 1-week postimplantation of fibrin hydrogels revealed an upregulation of M2 macrophage markers (CD163, CD204, and CD206), indicating a shift toward an anti-inflammatory phenotype. Notably, 11 weeks after implantation, the fibrin hydrogel-treated sites exhibited enhanced bone regeneration. These findings highlight the potential of fibrin hydrogels as an immunomodulatory biomaterial that facilitates bone repair by promoting M2 macrophage polarization and modulating the local inflammatory microenvironment.

Revascularization remains a challenge for regenerative medicine strategies. Extensive research has been done to identify key moments of the dynamic wound healing cascade where targeted therapies can elicit a proregenerative response. However, the influence of oxygenation, temperature, and their temporal variation during healing are often challenging to promote tissue regeneration. This study investigated the effects of temporally varied oxygenation and temperature conditions on angiogenesis using an in vitro model of rat-derived, intact microvascular fragments in a collagen type-I hydrogel. By generating culture conditions that are similar to the accepted wound healing time course, the angiogenic response depended critically on both the timing of stimulus initiation and the magnitude of deviation from model conditions. Dynamic stimuli activated distinct biological pathways, as evidenced by qPCR analysis, revealing mechanistic links between environmental perturbations and the angiogenic response. This work emphasizes the need for regenerative medicine strategies to consider varying environmental stimuli to improve revascularization outcomes. Impact Statement This work demonstrated the impact of time-varying oxygenation and temperature conditions on self-assembling three-dimensional microvascular networks in vitro that mimic the physiological time course of wound healing. These findings suggest an important temporal relationship in angiogenesis where unresolved oxygen and temperature environments inhibit vascular network formation, cellular viability, proliferation, and environment-specific transcriptional factors.