Tissue engineering and regenerative medicine最新文献

Background: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a severe congenital disorder characterized by vaginal hypoplasia caused by dysplasia of the Müllerian duct. Patients with MRKH syndrome often require nonsurgical or surgical treatment to achieve satisfactory vaginal length and sexual outcomes. The extracellular matrix has been successfully used for vaginal reconstruction.

Methods: In this study, we developed a new biological material derived from porcine vagina (acellular vaginal matrix, AVM) to reconstruct the vagina in Bama miniature pigs. The histological characteristics and efficacy of acellularization of AVM were evaluated, and AVM was subsequently transplanted into Bama miniature pigs to reconstruct the vaginas.

Results: Macroscopic analysis showed that the neovaginas functioned well in all Bama miniature pigs with AVM implants. Histological analysis and electrophysiological evidence indicated that morphological and functional recovery was restored in normal vaginal tissues. Scanning electron microscopy showed that the neovaginas had mucosal folds characteristics of normal vagina. No significant differences were observed in the expression of CK14, HSP47, and α-actin between the neovaginas and normal vaginal tissues. However, the expression of estrogen receptor (ER) was significantly lower in the neovaginas than in normal vaginal tissues. In addition, AVM promoted the expression of β-catenin, c-Myc, and cyclin D1. These results suggest that AVM might promotes vaginal regeneration by activating the β-catenin/c-Myc/cyclin D1 pathway.

Conclusion: This study reveals that porcine-derived AVM has potential application for vaginal regeneration.

Background: Diabetic foot ulcer (DFU) is a major debilitating complication of diabetes. The lack of effective diabetic wound dressings has been a significant problem in DFU management. In this study, we aim to establish a phlorotannin-incorporated nanofibre system and determine its potential in accelerating hyperglycaemic wound healing.

Methods: The effective dose of Ecklonia cava phlorotannins (ECP) for hyperglycaemic wound healing was determined prior to phlorotannin nanofibre fabrication using polyvinyl-alcohol (PVA), polyvinylpyrrolidone (PVP), and ECP. Vapour glutaraldehyde was used for crosslinking of the PVA/PVP nanofibres. The phlorotannin nanofibres were characterised, and their safety and cytocompatibility were validated. Next, the wound healing effect of phlorotannin nanofibres was determined with 2D wound scratch assay, whereas immunofluorescence staining of Collagen-I (Col-I) and Cytokeratin-14 (CK-14) was performed in human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), respectively.

Results: Our results demonstrated that 0.01 μg/mL ECP significantly improved hyperglycaemic wound healing without compromising cell viability and proliferation. Among all nanofibres, PVA/PVP/0.01 wt% ECP nanofibres exhibited the best hyperglycaemic wound healing effect. They displayed a diameter of 334.7 ± 10.1 nm, a porosity of 40.7 ± 3.3%, and a WVTR of 1718.1 ± 32.3 g/m²/day. Besides, the FTIR spectra and phlorotannin release profile validated the successful vapour glutaraldehyde crosslinking and ECP incorporation. We also demonstrated the potential of phlorotannin nanofibres as a non-cytotoxic wound dressing as they support the viability and proliferation of both HDF and HEK. Furthermore, phlorotannin nanofibres significantly ameliorated the impaired hyperglycaemic wound healing and restored the hyperglycaemic-induced Col-I reduction in HDF.

Conclusion: Taken together, our findings show that phlorotannin nanofibres have the potential to be used as a diabetic wound dressing.

Background: Bioglasses are used in applications related to bone rehabilitation and repair. The mechanical and bioactive properties of polysaccharides like alginate and agarose can be modulated or improved using bioglass nanoparticles. Further essential metal ions used as crosslinker have the potential to supplement cultured cells for better growth and proliferation.

Method: In this study, the alginate bioink is modulated for fabrication of tissue engineering scaffolds by extrusion-based 3D bioprinting using agarose, bioglass nanoparticles and combination of essential trace elements such as iron, zinc, and copper. Homogeneous bioink was obtained by in situ mixing and bioprinting of its components with twin screw extruder (TSE) based 3D bioprinting, and then distribution of metal ions was induced through post-printing diffusion of metal ions in the printed scaffolds. The mechanical and 3d bioprinting properties, microscopic structure, biocompatibility of the crosslinked alginate/agarose hydrogels were analyzed for different concentrations of bioglass. The adipose derived mesenchymal stem cells (ADMSC) and osteoblast cells (MC3T3) were used to evaluate this hydrogel's biological performances.

Results: The porosity of hydrogels significantly improves with the incorporation of the bioglass. More bioglass concentration results in improved mechanical (compressive, dynamic, and cyclic) and 3D bioprinting properties. Cell growth and extracellular matrix are also enhanced with bioglass concentration.

Conclusion: For bioprinting of the bioinks, the advanced TSE head was attached to 3D bioprinter and in situ fabrication of cell encapsulated scaffold was obtained with optimized composition considering minimal effects on cell damage. Fabricated bioinks demonstrate a biocompatible and noncytotoxic scaffold for culturing MC3T3 and ADMSC, while bioglass controls the cellular behaviors such as cell growth and extracellular matrix formation.

Background: Mammalian target of rapamycin (mTOR) is known to regulate self-renewal ability and potency of embryonic stem cells (ESCs) and adult stem cells in opposite manners. However, its effects vary even among adult stem cells and are not reported in fetal stem/progenitor cells. This study investigated the role of mTOR in the function of human fetal cartilage-derived progenitor cells (hFCPCs).

Methods: mTOR activity in hFCPCs was first examined via the level of phosphor-mTOR until passage 19, together with doubling time of cells and senescence-associated b-galactosidase (SA-bGal). Then, the effect of 100 nM rapamycin, the inhibitor of mTOR, was investigated on self-renewal ability, proliferation rate and osteogenic/adipogenic potential of hFCPCs in vitro. Expression of stemness genes (Oct-4, Sox2 and Nanog) and cell cycle regulators (CDK4 and Cyclin D1) was measured at mRNA or protein levels.

Results: mTOR activity was maintained constantly at high levels in hFCPCs until passage 19, while their proliferation rate was decreasing from 48 h at passage 13 to 70 h at passage 9 and senescent cells were observed at passage 18 (8.3 ± 1.2%) and 19 (15.6 ± 1.9%). Inhibition of mTOR in hFCPCs impaired their colony forming frequency (CFU-F) by 4 folds, while showing no change in their doubling time and expression of CDK4 and Cyclin D1. Upon mTOR inhibition, Oct4 expression decreased by 2 folds and 4 folds at the mRNA and protein levels, respectively, while that of Sox2 and Nanog did not change significantly. Finally, mTOR inhibition reduced osteogenic and adipogenic differentiation of hFCPCs in vitro.

Conclusion: This study has shown that mTOR plays an important role in the self-renewal ability of hFCPCS but not in their proliferation, The effect of mTOR appears to be associated with Oct-4 expression and important in the osteogenic and adipogenic differentiation ability of hFCPCs.

Background: This study aimed to explore the effect of a nanomaterial-based miR-320a inhibitor sustained release system in trauma-induced osteonecrosis of the femoral head (TIONFH).

Methods: The miR-320a inhibitor-loaded polyethylene glycol (PEG)- Poly(lactic-co-glycolic acid) (PLGA)- Poly-L-lysine (PLL) nanoparticles were constructed using the double emulsion method. The TIONFH rabbit model was established to observe the effects of miR-320a inhibitor nanoparticles in vivo. Hematoxylin-eosin staining and microcomputed tomography scanning were used for bone morphology analysis. Bone marrow mesenchymal stem cells (BMSCs), derived from TIONFH rabbits, were used for in vitro experiments. Cell viability was determined using the MTT assay.

Results: High expression of miR-320a inhibited the osteogenic differentiation capacity of BMSCs in vitro by inhibiting the expression of the osteoblastic differentiation markers ALP and RUNX2. MiR-320a inhibitor-loaded PEG-PLGA-PLL nanoparticles were constructed with a mean loading efficiency of 1.414 ± 0.160%, and a mean encapsulation efficiency of 93.45 ± 1.24%, which released 50% of the loaded miR-320a inhibitor at day 12 and 80% on day 18. Then, inhibitor release entered the plateau. After treatment with the miR-320a inhibitor nanoparticle, the empty lacunae were decreased in the femoral head tissue of TIONFH rabbits, and the osteoblast surface/bone surface (Ob.S/BS), osteoblast number/bone perimeter (Ob.N/B.Pm), bone volume fraction, and bone mineral density increased. Additionally, the expression of osteogenic markers RUNX2 and ALP was significantly elevated in the TIONFH rabbit model.

Conclusion: The miR-320a inhibitor-loaded PEG-PLGA-PLL nanoparticle sustained drug release system significantly contributed to bone regeneration in the TIONFH rabbit model, which might be a promising strategy for the treatment of TIONFH.

Fabrication of functional organs is the holy grail of tissue engineering and the possibilities of repairing a partial or complete liver to treat chronic liver disorders are discussed in this review. Liver is the largest gland in the human body and plays a responsible role in majority of metabolic function and processes. Chronic liver disease is one of the leading causes of death globally and the current treatment strategy of organ transplantation holds its own demerits. Hence there is a need to develop an in vitro liver model that mimics the native microenvironment. The developed model should be a reliable to understand the pathogenesis, screen drugs and assist to repair and replace the damaged liver. The three-dimensional bioprinting is a promising technology that recreates in vivo alike in vitro model for transplantation, which is the goal of tissue engineers. The technology has great potential due to its precise control and its ability to homogeneously distribute cells on all layers in a complex structure. This review gives an overview of liver tissue engineering with a special focus on 3D bioprinting and bioinks for liver disease modelling and drug screening.

Background: Collagen is a key component of connective tissue and has been frequently used in the fabrication of medical devices for tissue regeneration. Human-originated collagen is particularly appealing due to its low immune response as an allograft biomaterial compared to xenografts and its ability to accelerate the regeneration process. Ethically and economically, adipose tissues available from liposuction clinics are a good resource to obtain human collagen. However, studies are still scarce on the extraction and characterization of human collagen, which originates from adipose tissue. The aim of this study is to establish a novel and simple method to extract collagen from human adipose tissue, characterize the collagen, and compare it with commercial-grade porcine collagen for tissue engineering applications.

Methods: We developed a method to extract the collagen from human adipose tissue under quasi-Good Manufacturing Practice (GMP) conditions, including freezing the tissue, blood removal, and ethanol-based purification. Various techniques, including protein quantification, decellularization assessment, SDS-PAGE, FTIR, and CD spectroscopy analysis, were used for characterization. Amino acid composition was compared with commercial collagen. Biocompatibility and cell proliferation tests were performed, and in vitro tests using collagen sponge scaffolds were conducted with statistical analysis.

Results: Our results showed that this human adipose-derived collagen was equivalent in quality to commercially available porcine collagen. In vitro testing demonstrated high cell attachment and the promotion of cell proliferation.

Conclusion: In conclusion, we developed a simple and novel method to extract and characterize collagen and extracellular matrix from human adipose tissue, offering a potential alternative to animal-derived collagen for xeno-free tissue engineering applications.

Background: Current polymer-based drug-eluting stents (DESs) have fundamental issues about inflammation and delayed re-endothelializaton of the vessel wall. Substance-P (SP), which plays an important role in inflammation and endothelial cells, has not yet been applied to coronary stents. Therefore, this study compares poly lactic-co-glycolic acid (PLGA)-based everolimus-eluting stents (PLGA-EESs) versus 2-methacryloyloxyethyl phosphorylcholine (MPC)-based SP-eluting stents (MPC-SPs) in in-vitro and in-vivo models.

Methods: The morphology of the stent surface and peptide/drug release kinetics from stents were evaluated. The in-vitro proliferative effect of SP released from MPC-SP is evaluated using human umbilical vein endothelial cell. Finally, the safety and efficacy of the stent are evaluated after inserting it into a pig's coronary artery.

Results: Similar to PLGA-EES, MPC-SP had a uniform surface morphology with very thin coating layer thickness (2.074 μm). MPC-SP showed sustained drug release of SP for over 2 weeks. Endothelial cell proliferation was significantly increased in groups treated with SP (n = 3) compared with the control (n = 3) and those with everolimus (n = 3) (SP: 118.9 ± 7.61% vs. everolimus: 64.3 ± 12.37% vs. the control: 100 ± 6.64%, p < 0.05). In the animal study, the percent stenosis was higher in MPC-SP group (n = 7) compared to PLGA-EES group (n = 7) (MPC-SP: 28.6 ± 10.7% vs. PLGA-EES: 16.7 ± 6.3%, p < 0.05). MPC-SP group showed, however, lower inflammation (MPC-SP: 0.3 ± 0.26 vs. PLGA-EES: 1.2 ± 0.48, p < 0.05) and fibrin deposition (MPC-SP: 1.0 ± 0.73 vs. PLGA-EES: 1.5 ± 0.59, p < 0.05) around the stent strut. MPC-SP showed more increased expression of cluster of differentiation 31, suggesting enhanced re-endothelialization.

Conclusion: Compared to PLGA-EES, MPC-SP demonstrated more decreased inflammation of the vascular wall and enhanced re-endothelialization and stent coverage. Hence, MPC-SP has the potential therapeutic benefits for the treatment of coronary artery disease by solving limitations of currently available DESs.

Background: Thermal traumas impose a huge burden on healthcare systems. This merits the need for advanced but cost-effective remedies with clinical prospects. In this context, we prepared a regenerative 3D-construct comprising of Cassia angustifolia extract (SM) primed adipose-derived stem cells (ASCs) laden amniotic membrane for faster burn wound repair.

Methods: ASCs were preconditioned with SM (30 µg/ml for 24 h), and subsequently exposed to in-vitro thermal injury (51 °C,10 min). In-vivo thermal injury was induced by placing pre-heated copper-disc (2 cm diameter) on dorsum of the Wistar rats. ASCs (2.0 × 10⁵) pre-treated with SM (SM-ASCs), cultured on stromal side of amniotic membrane (AM) were transplanted in rat heat-injury model. Non-transplanted heat-injured rats and non-heat-injured rats were kept as controls.

Results: The significantly upregulated expression of IGF1, SDF1A, TGFβ1, VEGF, GSS, GSR, IL4, BCL2 genes and downregulation of BAX, IL6, TNFα, and NFkB1 in SM-ASCs in in-vitro and in-vivo settings confirmed its potential in promoting cell-proliferation, migration, angiogenesis, antioxidant, cell-survival, anti-inflammatory, and wound healing activity. Moreover, SM-ASCs induced early wound closure, better architecture, normal epidermal thickness, orderly-arranged collagen fibers, and well-developed skin appendages in healed rat-skin transplanted with AM+SM-ASCs, additionally confirmed by increased expression of structural genes (Krt1, Krt8, Krt19, Desmin, Vimentin, α-Sma) in comparison to untreated-ASCs laden-AM transplanted in heat injured rats.

Conclusion: SM priming effectively enabled ASCs to counter thermal injury by significantly enhancing cell survival and reducing inflammation upon transplantation. This study provides bases for development of effective combinational therapies (natural scaffold, medicine, and stem cells) with clinical prospects for treating burn wounds.

Background: Cutaneous wound healing represents a common fundamental phenomenon requiring the participation of cells of distinct types and a major concern for the public. Evidence has confirmed that photobiomodulation (PBM) using near-infrared (NIR) can promote wound healing, but the  cells involved and the precise molecular mechanisms remain elusive.

Methods: Full-thickness skin defects with a diameter of 1.0 cm were made on the back of rats and randomly divided into the control group, 10 J, 15 J, and 30 J groups. The wound healing rate at days 4, 8, and 12 postoperatively was measured. HE and Masson staining was conducted to reveal the histological characteristics. Immunofluorescence staining was performed to label the epidermal stem cells (ESCs) and hair follicle stem cells (HFSCs). Western blot was performed to detect the expressions of proteins associated with ESCs and HFSCs. Cutaneous wound tissues were collected for RNA sequencing. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analysis was performed, and the hub genes were identified using CytoHubba and validated by qRT-PCR.

Results: PBM can promote reepithelialization, extracellular matrix deposition, and wound healing, increase the number of KRT14+/PCNA+ ESCs and KRT15+/PCNA+ HFSCs, and upregulate the protein expression of P63, Krt14, and PCNA. Three hundred and sixty-six differentially expressed genes (DEGs) and 7 hub genes including Sox9, Krt5, Epcam, Cdh1, Cdh3, Dsp, and Pkp3 were identified. These DEGs are enriched in skin development, cell junction, and cadherin binding involved in cell-cell adhesion etc., while these hub genes are related to skin derived stem cells and cell adhesion.

Conclusion: PBM accelerates wound healing by enhancing reepithelialization through promoting ESCs and HFSCs proliferation and elevating the expression of genes associated with stem cells and cell adhesion. This may provide a valuable alternative strategy to promote wound healing and reepithelialization by modulating the proliferation of skin derived stem cells and regulating genes related to cell adhesion.