Tissue Engineering Part A最新文献

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.

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.

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.

Conventional gene therapy approaches for drug delivery generally rely on constitutive expression of the transgene and thus lack precise control over the timing and magnitude of delivery. Synthetic gene circuits with promoters that are responsive to user-defined stimuli can provide a molecular switch that can be utilized by cells to control drug production. Our laboratory has previously developed a mechanogenetic gene circuit that can deliver biological drugs, such as interleukin-1 receptor antagonist (IL-1Ra), on-demand through the activation of Transient receptor potential family, vanilloid 4 (TRPV4), a mechanosensory ion channel that has been shown to be activated transiently in response to physical stimuli such as physiological mechanical loading or hypo-osmotic stimuli. The goal of this study was to use mutations in TRPV4 to further tune the response of this mechanogenetic gene circuit. Human iPSC-derived chondrocytes harboring targeted gain-of-function mutations of TRPV4 were chondrogenically differentiated. Both mutants-V620I and T89I-showed greater total IL-1Ra production compared with wild type following TRPV4 agonist treatment, as well as mechanical or osmotic loading, but with altered temporal dynamics. Gene circuit output was dependent on the degree of TRPV4 activation secondary to GSK101 concentration or strain magnitude during loading. V620I constructs secreted more IL-1Ra compared with T89I across all experimental conditions, indicating that two mutations that cause similar functional changes to TRPV4 can result in distinct circuit activation profiles that differ from wild-type cells. In summary, we successfully demonstrate proof-of-concept that point mutations in TRPV4 that alter channel function can be used to tune the therapeutic output of mechanogenetic gene circuits.

Cartilage regeneration is hindered due to the low proliferative capacity of chondrocytes and the avascular nature of cartilaginous tissue. Mesenchymal stromal cells (MSCs) are widely studied for cartilage tissue engineering, and the aggregation of MSCs into high-density cell spheroids facilitates chondrogenic differentiation due to increased cell-cell contact. Despite the promise of MSCs, the field would benefit from improved strategies to regulate the chondrogenic potential of MSCs differentiated from induced pluripotent stem cells (iPSCs), which are advantageous for their capacity to yield large numbers of required cells. We previously demonstrated the ability of MSC-secreted extracellular matrix (ECM) to promote MSC chondrogenic differentiation, but the combinatorial effect of iPSC-derived MSC (iMSC) spheroids, iMSC-derived decellularized ECM (idECM), and other stimuli (e.g., oxygen tension and transforming growth factor [TGF]-β) on chondrogenic potential has not been described. Similar to MSCs, iMSCs secreted a collagen-rich ECM. When incorporated into spheroids, idECM increased spheroid diameter and promoted chondrogenic differentiation. The combination of idECM loading, chondrogenic media, and hypoxia enhanced glycosaminoglycan (GAG) content 1.6-fold (40.9 ± 4.6 ng vs. 25.6 ± 3.3 ng, p < 0.05) in iMSC spheroids. Compared with active TGF-β1, the presentation of latent TGF-β1 resulted in greater GAG content (26.6 ± 1.8 ng vs. 41.9 ± 4.3 ng, p < 0.01). Finally, we demonstrated the capacity of individual spheroids to self-assemble into larger constructs and undergo both chondrogenic and hypertrophic differentiation when maintained in lineage-inducing media. These results highlight the potential of idECM to enhance the efficacy of chondrogenic stimuli for improved cartilage regeneration using human MSCs and iMSCs.

Scaffolds made from cartilage extracellular matrix are promising materials for articular cartilage repair, attributed to their intrinsic bioactivity that may promote chondrogenesis. While several cartilage matrix-based scaffolds have supported chondrogenesis in vitro and/or in vivo, it remains a challenge to balance the biological response (e.g., chondroinductivity) with structural (e.g., robust mechanical performance, >1 MPa in compressive stiffness) and translational (e.g., ease of surgical implantation) considerations. Few studies have evaluated encapsulated cell viability within high-stiffness (>1 MPa) hydrogels. We previously fabricated one formulation of a high-stiffness (>3 MPa) pentenoate-functionalized, solubilized, devitalized cartilage (PSDVC) hydrogel that possessed an injectable, paste-like precursor for easy surgical application. In the current study, the characterization of the PSDVC material was expanded by varying the degree of functionalization (i.e., 0.45-1.09 mmol/g) and amount of crosslinker, dithiothreitol (DTT), to improve the reproducibility of the high compressive moduli and evaluate the viability of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs) in high-stiffness cartilage matrix hydrogels. Prior to crosslinking, specific formulations functionalized with 0.80 mmol/g or less of pentenoate groups retained a paste-like precursor rheology. After crosslinking, these formulations produced hydrogels with greater than 1 MPa compressive stiffness. However, hBMSCs encapsulated in PSDVC hydrogels with lower functionalization (i.e., 0.57 mmol/g, no crosslinker) had a higher stiffness (i.e., 1.4 MPa) but the lowest viability of encapsulated hBMSCs (i.e., 5%). The middle PSDVC functionalization (i.e., 0.70 mmol/g) with DTT (i.e., 0.50 mmol thiols/g) demonstrated high cell viability (77%), high mechanical performance (1.65 MPa, 31% failure strain), and translational features (i.e., paste-like precursor, 1.5 min crosslinking time). For future evaluations of PSDVC hydrogels in cartilage repair, a middle functionalization (i.e., 0.70-0.80 mmol/g) with the addition of a crosslinker (i.e., 0.50 mmol thiols/g) had a desirable balance of high mechanical performance (i.e., >1 MPa compressive stiffness), high viability, and paste-like precursor for surgical translation.

The synovium is a loose connective tissue that separates the intra-articular (IA) joint compartments of all diarthrodial joints from the systemic circulation. It can be divided into two layers: the intima, a thin and cell-dense layer atop a more heterogeneous subintima, composed of collagen and various cell types. The subintima contains penetrating capillaries and lymphatic vessels that rapidly clear injected drugs from the joint space which may vary not only with drug size and charge but also with the microstructure and composition of the intima and subintima of the synovium. Prior work has measured the mechanical properties and solute diffusivities in the synovium of porcine, bovine, and human joints. Here, we measured the Young's moduli of synovium from smaller joints of the rat knee, as well as pig and human, using atomic force microscopy (AFM). The format for AFM enabled testing of intima and subintimal regions of synovium in all three species. The Young's moduli of the subintimal regions were similar across all three species (1-1.5 kPa). Furthermore, there was little evidence of differences in Young's moduli between synovium from the intima and subintima in each species. A general similarity of data from AFM testing with moduli measured with bulk testing of pig and human synovium suggests that AFM can be useful to measure the mechanical properties of smaller joint synovium and spatial variations in stiffness with depth. Enzymatic digestion of synovium tissue from the pig was also performed with findings of lower moduli values following treatment with chondroitinase ABC but not collagenase. Although the molecular composition of the synovium is not yet fully characterized and may vary across species, these findings suggest that noncollagenous species contribute to AFM-measured properties in synovium. These are some of the first data to measure mechanical properties in small joint synovium and will be useful in models studying IA drug clearances in joints with pathology and following treatment.

Muscle degeneration after rotator cuff tendon tear is a significant clinical problem. In these experiments, we developed a poly(ethylene glycol)-based injectable granular hydrogel containing two heparin derivatives (fully sulfated [Hep] and fully desulfated [Hep-]) as well as a matrix metalloproteinase-sensitive peptide to promote sustained release of tumor necrosis factor-stimulated gene 6 (TSG-6) over 14+ days in vivo in a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles in vivo, thus facilitating comparisons between delivery from heparin derivatives on the level of tissue repair in two different areas of muscle (near the myotendious junction [MTJ] and in the muscle belly [MB]) that have been shown previously to have differing responses to rotator cuff tendon injury. We hypothesized that sustained delivery of TSG-6 would enhance the anti-inflammatory response following rotator cuff injury through macrophage polarization and that release from Hep would potentiate this effect throughout the muscle. Inflammatory/immune cells, satellite cells, and fibroadipogenic progenitor cells were analyzed by flow cytometry 3 and 7 days after injury and hydrogel injection, while metrics of muscle healing were examined via immunohistochemistry up to day 14. Results showed controlled delivery of TSG-6 from Hep caused heightened macrophage response (day 7 macrophages, 4.00 ± 1.85% single cells, M2a, 3.27 ± 1.95% single cells) and increased markers of early muscle regeneration (embryonic heavy chain staining) by day 7, particularly in the MTJ region of the muscle. This work provides a novel strategy for localized, controlled delivery of TSG-6 to enhance muscle healing after rotator cuff tear.

Osteoarthritis, a degenerative disease of articular cartilage and the leading cause of disability, is preceded by acute cartilage injury in a significant proportion of cases. Current auto- and allograft interventions are limited by supply and variability in therapeutic efficacy, prompting interest in tissue engineering solutions. Cell sheet tissue engineering, a scaffold-free regenerative technique, has shown promise in preclinical and clinical trials across various cell types and diseases. Polydactyly-derived juvenile cartilage-derived chondrocyte (JCC) sheets from juvenile patients are a potent cell source for developing allogeneic therapies. JCC sheets have proven safe and effective in animal models and as an add-on therapy in a recent clinical cartilage repair study. However, JCC ex vivo expansion leads to de-differentiation, contributing to long healing times. This study hypothesized that in vitro differentiation of JCC sheets into hyaline-like cartilage constructs could accelerate cartilage regeneration without compromising implant integration. To this end, sheet integration, maturation, and healing of conventionally prepared vs. differentiated JCC sheets were compared in an established nude rat focal chondral defect model. Differentiated JCC sheets exhibit mature cartilage phenotypes prior to transplant. Both conventional and differentiated JCC sheets are reliably transplanted without additional fixation. Histological evaluation reveals that both transplant groups produced equivalent neocartilage regeneration, filling defects with mature hyaline cartilage at 2- and 4-weeks post-transplant. Notably, differentiated JCC sheets respond to in vivo signals, undergoing matrix remodeling and integration with adjacent and subchondral tissue. Given equivalent healing outcomes, the future utility of in vitro JCC sheet predifferentiation from other JCC donors with different healing capacities should be balanced against their increased culture costs over conventional sheets.