Decellularised extracellular matrix (dECM) biomaterials originating from allogeneic and xenogeneic tissues have been broadly studied in the field of regenerative medicine and have already been used in clinical treatments. Allogeneic dECMs are considered more compatible, but they have the drawback of extremely limited human tissue sources. Their availability is also restricted by the health and age of the donors. To investigate the viability of xenogeneic tissues as a substitute for human tissues, we fabricated both porcine decellularised nerve matrix (pDNM) and human decellularised nerve matrix for a comprehensive comparison. Photomicrographs showed that both dECM scaffolds retained the ECM microstructures of native human nerve tissues. Proteomic analysis demonstrated that the protein compositions of both dECMs were also very similar to each other. Their functional ECM contents effectively promoted the proliferation, migration, and maturation of primary human Schwann cells in vitro. However, pDNM contained a few antigens that induced severe host immune responses in humanised mice. Interestingly, after removing the α–galactosidase antigen, the immune responses were highly alleviated and the pre–treated pDNM maintained a human decellularised nerve matrix–like pro–regenerative phenotype. Therefore, we believe that an α–galactosidase–free pDNM may serve as a viable substitute for human decellularised nerve matrix in future clinical applications.
Biodegradable polymer microspheres that can be used as drug carriers are of great importance in biomedical applications, however, there are still challenges in controllable preparation of microsphere surface morphology and improvement of bioactivity. In this paper, firstly, poly(L-lactic acid) (PLLA) was synthesised by ring-opening polymerisation under anhydrous anaerobic conditions and further combined with the emulsion method, biodegradable PLLA microspheres (PM) with sizes ranging from 60–100 μm and with good sphericity were prepared. In addition, to further improve the surface morphology of PLLA microspheres and enhance their bioactivity, functionalised porous PLLA microspheres loaded with magnesium oxide (MgO)/magnesium carbonate (MgCO3) (PMg) were also prepared by the emulsion method. The results showed that the loading of MgO/MgCO3 resulted in the formation of a porous structure on the surface of the microspheres (PMg) and the dissolved Mg2+ could be released slowly during the degradation of microspheres. In vitro cellular experiments demonstrated the good biocompatibility of PM and PMg, while the released Mg2+ further enhanced the anti-inflammatory effect and osteogenic activity of PMg. Functionalised PMg not only show promise for controlled preparation of drug carriers, but also have translational potential for bone regeneration.
The printability of bioink and post-printing cell viability is crucial for extrusion-based bioprinting. A proper bioink not only provides mechanical support for structural fidelity, but also serves as suitable three-dimensional (3D) microenvironment for cell encapsulation and protection. In this study, a hydrogel-based composite bioink was developed consisting of gelatin methacryloyl (GelMA) as the continuous phase and decellularised extracellular matrix microgels (DMs) as the discrete phase. A flow-focusing microfluidic system was employed for the fabrication of cell-laden DMs in a high-throughput manner. After gentle mixing of the DMs and GelMA, both rheological characterisations and 3D printing tests showed that the resulting DM-GelMA hydrogel preserved the shear-thinning nature, mechanical properties, and good printability from GelMA. The integration of DMs not only provided an extracellular matrix-like microenvironment for cell encapsulation, but also considerable shear-resistance for high post-printing cell viability. The DM sizes and inner diameters of the 3D printer needles were correlated and optimised for nozzle-based extrusion. Furthermore, a proof-of-concept bioink composedg of RSC96 Schwann cells encapsulated DMs and human umbilical vein endothelial cell-laden GelMA was successfully bioprinted into 3D constructs, resulting in a modular co-culture system with distinct cells/materials distribution. Overall, the modular DM-GelMA bioink provides a springboard for future precision biofabrication and will serve in numerous biomedical applications such as tissue engineering and drug screening.
There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue. Three-dimensional (3D) printing offers a method of fabricating complex anatomical features of clinically relevant sizes. However, the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging. This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions. The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional (2D). The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices, which were arrayed. These 2D slices with each layer of a defined pattern were laser cut, and then successfully assembled with varying thicknesses of 100 µm or 200 µm. It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions, where the clinically relevant sizes ranging from a simple cube of 20 mm dimension, to a more complex, 50 mm-tall human ears were created. In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure. The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice, where a range of hole diameters from 200 µm to 500 µm were laser cut in an array where cell confluence values of at least 85% were found at three weeks. Cells were also seeded onto a simpler stacked construct, albeit made with micromachined poly fibre mesh, where cells can be found to migrate through the stack better with collagen as bioadhesives. This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) play a crucial role in stem cell therapy and are extensively used in regenerative medicine research. However, current methods for harvesting BM-MSCs present challenges, including a low yield of primary cells, long time of in vitro expansion, and diminished differentiation capability after passaging. Meanwhile mesenchymal stem cells (MSCs) recovered from cell banks also face issues like toxic effects of cryopreservation media. In this study, we provide a detailed protocol for the isolation and evaluation of MSCs derived from in vivo osteo-organoids, presenting an alternative to autologous MSCs. We used recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds to construct in vivo osteo-organoids, which were stable sources of MSCs with large quantity, high purity, and strong stemness. Compared with protocols using bone marrow, our protocol can obtain large numbers of high-purity MSCs in a shorter time (6 days vs. 12 days for obtaining passage 1 MSCs) while maintaining higher stemness. Notably, we found that the in vivo osteo-organoid-derived MSCs exhibited stronger anti-replicative senescence capacity during passage and amplification, compared to BM-MSCs. The use of osteo-organoid-derived MSCs addresses the conflict between the limitations of autologous cells and the risks associated with allogeneic sources in stem cell transplantation. Consequently, our protocol emerges as a superior alternative for both stem cell research and tissue engineering.
A composite scaffold composed of a porous scaffold and hydrogel filling can facilitate engraftment, survival, and retention in cell transplantation processes. This study presents a composite scaffold made of poly(ε-caprolactone) (PCL) and methacrylated hyaluronic acid (MeHA) hydrogel and describes the corresponding physical properties (surface area, porosity, and mechanical strength) and host response (angiogenesis and fibrosis) after subcutaneous transplantation. Specifically, we synthesise MeHA with different degrees of substitution and fabricate a PCL scaffold with different porosities. Subsequently, we construct a series of PCL/MeHA composite scaffolds by combining these hydrogels and scaffolds. In experiments with mice, the scaffold composed of 3% PCL and 10–100 kDa, degree of substitution 70% MeHA results in the least fibrosis and a higher degree of angiogenesis. This study highlights the potential of PCL/MeHA composite scaffolds for subcutaneous cell transplantation, given their desirable physical properties and host response.
The repair of large load-bearing bone defects requires superior mechanical strength, a feat that a single hydrogel scaffold cannot achieve. The objective is to seamlessly integrate optimal microarchitecture, mechanical robustness, vascularisation, and osteoinductive biological responses to effectively address these critical load-bearing bone defects. To confront this challenge, three-dimensional (3D) printing technology was employed to prepare a polycaprolactone (PCL)-based integrated scaffold. Within the voids of 3D printed PCL scaffold, a methacrylate gelatin (GelMA)/methacrylated silk fibroin (SFMA) composite hydrogel incorporated with parathyroid hormone (PTH) peptide-loaded mesoporous silica nanoparticles (PTH@MSNs) was embedded, evolving into a porous PTH@MSNs/GelMA/SFMA/PCL (PM@GS/PCL) scaffold. The feasibility of fabricating this functional scaffold with a customised hierarchical structure was confirmed through meticulous chemical and physical characterisation. Compression testing unveiled an impressive modulus of 17.81 ± 0.83 MPa for the composite scaffold. Additionally, in vitro angiogenesis potential of PM@GS/PCL scaffold was evaluated through Transwell and tube formation assays using human umbilical vein endothelium, revealing the superior cell migration and tube network formation. The alizarin red and alkaline phosphatase staining assays using bone marrow-derived mesenchymal stem cells clearly illustrated robust osteogenic differentiation properties within this scaffold. Furthermore, the bone repair potential of the scaffold was investigated on a rat femoral defect model using micro-computed tomography and histological examination, demonstrating enhanced osteogenic and angiogenic performance. This study presents a promising strategy for fabricating a microenvironment-matched composite scaffold for bone tissue engineering, providing a potential solution for effective bone defect repair.