Tissue Engineering Part A最新文献

Previously, chitosan reduces the senescence-related phenotypes in human foreskin fibroblasts through the transforming growth factor beta (TGF-β) pathway, and enhances the proliferation and migration capabilities of these cells are demonstrated. In this study, we examined whether the senescence-delaying effect of chitosan could be applied to primary knee-related fibroblasts, such as human synovial membrane derived cells (SCs) and anterior cruciate ligament fibroblasts (ACLs). These two types of cells were obtained from donors who needed ACL reconstruction or knee replacement. We found that chitosan treatment effectively reduced aging-associated β-galactosidase (SA-β-gal)-positive cells, downregulated the expression of senescence-related proteins pRB and p53, and enhanced the 5-bromo-2'-deoxyuridine (BrdU) incorporation ability of SCs and ACLs. Moreover, chitosan could make SCs secret more glycosaminoglycans (GAGs) and produce type I collagen. The ability of ACLs to close the wound was also enhanced, and the TGF-β and alpha smooth muscle actin (αSMA) protein expression decreased after chitosan treatment. In summary, chitosan not only delayed the senescence but also enhanced the functions of SCs and ACLs, which is beneficial to the application of chitosan in cell expansion in vitro and cell therapy.

Silk fibroin (SF) has garnered significant attention as a natural polymer for fabricating porous scaffolds in various engineering applications. However, the limited osteoinductive property of SF has hindered its efficacy in bone repair applications. In this study, we constructed an SF-based injectable porous microcarrier that is doped with laponite (LAP), containing magnesium ions (Mg²⁺). The influence of freezing temperatures and concentrations of SF and LAP on the structural parameters of SF-LAP microcarriers was investigated. The SF-LAP microcarrier exhibited a porosity of 76.7 ± 1.2% and a controlled pore size of 24.6 ± 4.0 μm. At the 6 weeks of in vitro degradation test, a mild alkaline level in culture medium containing SF-LAP microcarriers was detected. The release of Mg²⁺ from the SF-LAP microcarrier was maintained at a concentration within the range of 1.2-2.3 mM during the 6 weeks. The seeded human adipose-derived stem cells in the SF-LAP microcarrier demonstrated a significant enhancement in osteogenic differentiation compared with cells seeded in the pure SF microcarrier, as evidenced by quantitative alkaline phosphatase activity and the expression of osteogenic marker genes. These findings underscore the potential of the SF-LAP microcarrier as an ideal cell carrier in the treatment of bone defects.

The arteriovenous loop (AVL) model allows the in vivo engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Millions of people suffer from tracheal defect worldwide each year, while autograft and allograft cannot meet existing treatment needs. Tissue-engineered trachea substitutes represent a promising treatment for tracheal defect, while lack of precisely personalized treatment abilities. Therefore, development of an artificial trachea that can be used for personalized transplantation is highly desired. In this study, we report the design and fabrication of an artificial trachea based on sericin microsphere (SM) by microtissue engineering technology and three-dimensional (3D) printing for personalized repair of tracheal defect. The SM possessed natural cell adhesion and promoting cell proliferation ability. Then, the microtissue was fabricated by coincubation of SM with chondrocytes and tracheal epithelial cells. This microtissue displayed good cytocompatibility and could support seed cell adhesion and proliferation. After that, this microtissue was individually assembled to form an artificial trachea by 3D printing. Notably, artificial trachea had an encouraging complete cartilaginous and epithelial structure after transplantation. Furthermore, the artificial trachea molecularly resembled native trachea as evidenced by similar expression of trachea-critical genes. Altogether, the work demonstrates the effectiveness of microtissue engineering and 3D printing for individual construction of artificial trachea, providing a promising approach for personalized treatment of tracheal defect.

Volumetric muscle loss (VML) is the loss of skeletal muscle that exceeds the muscle's self-repair mechanism and leads to permanent functional deficits. In a previous study, we demonstrated the ability of our scaffold-free, multiphasic, tissue-engineered skeletal muscle units (SMUs) to restore muscle mass and force production. However, it was observed that the full recovery of muscle structure was inhibited due to increased fibrosis in the repair site. As such, novel biomaterials such as hydrogels (HGs) may have significant potential for decreasing the acute inflammation and subsequent fibrosis, as well as enhancing skeletal muscle regeneration following VML injury and repair. The goal of the current study was to assess the biocompatibility of commercially available poly(ethylene glycol), methacrylated gelatin, and hyaluronic acid (HA) HGs in combination with our SMUs to treat VML in a clinically relevant large animal model. An acute 30% VML injury created in the sheep peroneus tertius (PT) muscle was repaired with or without HGs and assessed for acute inflammation (incision swelling) and white blood cell counts in blood for 7 days. At the 7-day time point, HA was selected as the HG to use for the combined HG/SMU repair, as it exhibited a reduced inflammation response compared to the other HGs. Six weeks after implantation, all groups were assessed for gross and histological structural recovery. The results showed that the groups repaired with an SMU (SMU-Only and SMU+HA) restored muscle mass to greater degree than the groups with only HG and that the SMU groups had PT muscle masses that were statistically indistinguishable from its uninjured contralateral PT muscle. Furthermore, the HA HG, SMU-Only, and SMU+HA groups displayed notable efficacy in diminishing pro-inflammatory markers and showed an increased number of regenerating muscle fibers in the repair site. Taken together, the data demonstrates the efficacy of HA HG in decreasing acute inflammation and fibrotic response. The combination of HA and our SMUs also holds promise to decrease acute inflammation and fibrosis and increase muscle regeneration, advancing this combination therapy toward clinically relevant interventions for VML injuries in humans.

Challenges remain in simultaneously regenerating the multiple diverse tissues of the tooth root in a spatially organized manner. Previously, our research group has established that scaffold-free tissue engineering approaches enable dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) to self-assemble into dentin-pulp and PDL-cementum organoids, respectively. In this study, we leveraged the innate self-organizing capacity of DPSCs and PDLSCs to now engineer organoids that resemble the full tooth root. Scaffold-free engineered tissues were generated using a heterogeneous mixture of human DPSCs and PDLSCs. Within 2 days of construct formation, PDLSCs and DPSCs became spatially restricted to the periphery and center of the constructs, respectively, emulating their anatomical positions in the tooth root. Histological and microcomputed tomography analyses showed that organoids exhibited a striated mineral pattern with a central unmineralized core, surrounded by a mineralized tissue structure, enclosed within a second peripheral unmineralized tissue, similar to the natural tooth root. Interestingly, DPSCs gave rise to the central unmineralized tissue and the inner portion of the mineralized tissue, and PDLSCs generated the outer portion of the mineralized tissue and the peripheral soft tissue. Quantitative image analysis of immunofluorescent staining revealed increased dentin sialophosphoprotein expression in the region of mineralized tissue associated with DPSCs and increased cementum protein-1 expression in the portion formed by PDLSCs, demonstrating that tooth root organoids comprise two biochemically distinct mineralized tissues characteristic of dentin-like and cementum-like structures, respectively. In addition, PDL-associated protein-1 expression was localized to the peripheral soft tissue, suggesting the formation of a rudimentary PDL-like structure. This study demonstrates that DPSCs and PDLSCs have an inherent ability to orchestrate the formation of a full tooth root-like structure. These organoids present a biomimetic model system to study cellular dynamics driving dental tissue repair or could be utilized therapeutically as biological dental implants.

Extensively researched tissue engineering strategies involve incorporating cells into suitable biomaterials, offering promising alternatives to boost tissue repair. In this study, a hybrid scaffold, Gel-DCM, which integrates a photoreactive gelatin-hyaluronic acid hydrogel (Gel) with an oriented porous decellularized cartilage matrix (DCM), was designed to facilitate chondrogenic differentiation and cartilage repair. The Gel-DCM exhibited excellent biocompatibility in vitro, promoting favorable survival and growth of human adipose-derived stem cells (hADSCs) and articular chondrocytes (hACs). Gene expression analysis indicated that the hACs expanded within the Gel-DCM exhibited enhanced chondrogenic phenotype. In addition, Gel-DCM promoted chondrogenesis of hADSCs without the supplementation of exogenous growth factors. Following this, in vivo experiments were conducted where empty Gel-DCM or Gel-DCM loaded with hACs/hADSCs were used and implanted to repair osteochondral defects in a rat model. In the control group, no implants were delivered to the injury site. Interestingly, macroscopic, histological, and microcomputed tomography scanning results revealed superior cartilage restoration and subchondral bone reconstruction in the empty Gel-DCM group compared with the control group. Moreover, both hACs-loaded and hADSCs-loaded Gel-DCM implants exhibited superior repair of hyaline cartilage and successful reconstruction of subchondral bone, whereas defects in the control groups were predominantly filled with fibrous tissue. These observations suggest that the Gel-DCM can provide an appropriate three-dimensional chondrogenic microenvironment, and its combination with reparative cell sources, ACs or ADSCs, holds great potential for facilitating cartilage regeneration.