The Synergistic Effect of Electrical Stimulation and Dermal Fibroblast Cells-Laden 3D Conductive Hydrogel for Full-Thickness Wound Healing.

Clinically, most patients with poor wound healing suffer from generalized skin damage, usually accompanied by other complications, so developing therapeutic strategies for difficult wound healing has remained extremely challenging until now. Current studies have indicated that electrical stimulation (ES) to cutaneous lesions enhances skin regeneration by activating intracellular signaling cascades and secreting skin regeneration-related cytokine. In this study, we designed different concentrations of graphene in gelatin-methacrylate (GelMa) to form the conductive composite commonly used in wound healing because of its efficiency compared to other conductive thermo-elastic materials. The results demonstrated the successful addition of graphene to GelMa while retaining the original physicochemical properties of the GelMa bioink. In addition, the incorporation of graphene increased the interactions between these two biomaterials, leading to an increase in mechanical properties, improvement in the swelling ratio, and the regulation of degradation characteristics of the biocomposite scaffolds. Moreover, the scaffolds exhibited excellent electrical conductivity, increasing proliferation and wound healing-related growth factor secretion from human dermal fibroblasts. Overall, the HDF-laden 3D electroconductive GelMa/graphene-based hydrogels developed in this study are ideal biomaterials for skin regeneration applications in the future.

Additive manufacturing of Schwann cell-laden collagen/alginate nerve guidance conduits by freeform reversible embedding regulate neurogenesis via exosomes secretion towards peripheral nerve regeneration.

Peripheral nerve injury is a common clinical problem that could be debilitating to one's quality of life. The complex nerve guidance conduits (NGCs) with cells in order to improve nerve regeneration. Therefore, we used freeform reversible embedding of suspended hydrogels to fabricate Schwann cells (SCs)-laden collagen/alginate (Col/Alg) NGCs. First, we evaluated Col influence on the characteristics of NGCs. After which, Wharton's jelly mesenchymal stem cells (WJMSC) are seeded onto the inner channel of NGCs and evaluated neural regeneration behaviors. Results indicated the SCs-laden NGCs with 2.5 % Col found the highest proliferation and secretion of neurotrophic protein. Furthermore, co-culture of SCs promoted differentiation of WJMSC as seen from the increased neurogenic-related protein in NGCs. To determine the molecular mechanism between SCs and WJMSC, we demonstrated the neurotrophic factors secreted by SCs act on tropomyosin receptor kinase A (TrkA) receptors of WJMSC to promote nerve regeneration. In addition, our study demonstrated SCs-derived exosomes had a critical role in regulating neural differentiation of WJMSC. Taken together, this study demonstrates the fabrication of SCs-laden Col/Alg NGCs for nerve regeneration and understanding regarding the synergistic regenerative mechanisms of different cells could bring us a step closer for clinical treatment of large nerve defects.

The Synergistic Effect of Cyclic Tensile Force and Periodontal Ligament Cell-Laden Calcium Silicate/Gelatin Methacrylate Auxetic Hydrogel Scaffolds for Bone Regeneration.

The development of 3D printing technologies has allowed us to fabricate complex novel scaffolds for bone regeneration. In this study, we reported the incorporation of different concentrations of calcium silicate (CS) powder into fish gelatin methacrylate (FGelMa) for the fabrication of CS/FGelMa auxetic bio-scaffolds using 3D printing technology. Our results showed that CS could be successfully incorporated into FGelMa without influencing the original structural components of FGelMa. Furthermore, it conveyed that CS modifications both the mechanical properties and degradation rates of the scaffolds were improved in accordance with the concentrations of CS upon modifications of CS. In addition, the presence of CS enhanced the adhesion and proliferation of human periodontal ligament cells (hPDLs) cultured in the scaffold. Further osteogenic evaluation also confirmed that CS was able to enhance the osteogenic capabilities via activation of downstream intracellular factors such as pFAK/FAK and pERK/ERK. More interestingly, it was noted that the application of extrinsic biomechanical stimulation to the auxetic scaffolds further enhanced the proliferation and differentiation of hPDLs cells and secretion of osteogenic-related markers when compared to CS/FGelMa hydrogels without tensile stimulation. This prompted us to explore the related mechanism behind this interesting phenomenon. Subsequent studies showed that biomechanical stimulation works via YAP, which is a biomechanical cue. Taken together, our results showed that novel auxetic scaffolds could be fabricated by combining different aspects of science and technology, in order to improve the future chances of clinical applications for bone regeneration.

The 3D printed conductive grooved topography hydrogel combined with electrical stimulation for synergistically enhancing wound healing of dermal fibroblast cells.

Patients with extensive cutaneous damage resulting from poor wound healing often have other comorbidities such as diabetes that may lead to impaired skin functions and scar formation. Many recent studies have shown that the application of electrical stimulation (ES) to cutaneous lesions significantly improves skin regeneration via activation of AKT intracellular signaling cascades and secretion of regeneration-related growth factors. In this study, we fabricated varying concentrations of gelatin-methacrylate (GelMa) hydrogels with poly(3,4-ethylenedioxythiophene) (PEDOT): polystyrene sulfonate (PSS), which is a conductive material commonly used in tissue engineering due to its efficiency among conductive thermo-elastic materials. The results showed successful modification of PEDOT:PSS with GelMa while retaining the original structural characteristics of the GelMa hydrogels. In addition, the incorporation of PEDOT:PSS increased the interactions between both the materials, thus leading to enhanced mechanical strength, improved swelling ratio, and decreased hydrophilicity of the scaffolds. Our GelMa/PEDOT:PSS scaffolds were designed to have micro-grooves on the surfaces of the scaffolds for the purpose of directional guiding. In addition, our scaffolds were shown to have excellent electrical conductivity, thus leading to enhanced cellular proliferation and directional migration and orientation of human dermal fibroblasts. In vivo studies revealed that the GelMa/PEDOT:PSS scaffolds with electrical stimulation were able to induce full skin thickness regeneration, as seen from the various stainings. These results indicate the potential of GelMa/PEDOT:PSS as an electro-conductive biomaterial for future skin regeneration applications.

Synergies of Human Umbilical Vein Endothelial Cell-Laden Calcium Silicate-Activated Gelatin Methacrylate for Accelerating 3D Human Dental Pulp Stem Cell Differentiation for Endodontic Regeneration.

According to the Centers for Disease Control and Prevention, tooth caries is a common problem affecting 9 out of every 10 adults worldwide. Dentin regeneration has since become one of the pressing issues in dentistry with tissue engineering emerging as a potential solution for enhancing dentin regeneration. In this study, we fabricated cell blocks with human dental pulp stem cells (hDPSCs)-laden alginate/fish gelatin hydrogels (Alg/FGel) at the center of the cell block and human umbilical vascular endothelial cells (HUVEC)-laden Si ion-infused fish gelatin methacrylate (FGelMa) at the periphery of the cell block. 1H NMR and FTIR results showed the successful fabrication of Alg/FGel and FGelMa. In addition, Si ions in the FGelMa were noted to be bonded via covalent bonds and the increased number of covalent bonds led to an increase in mechanical properties and improved degradation of FGelMa. The Si-containing FGelMa was able to release Si ions, which subsequently significantly not only enhanced the expressions of angiogenic-related protein, but also secreted some cytokines to regulate odontogenesis. Further immunofluorescence results indicated that the cell blocks allowed interactions between the HUVEC and hDPSCs, and taken together, were able to enhance odontogenic-related markers' expression, such as alkaline phosphatase (ALP), dentin matrix phosphoprotein-1 (DMP-1), and osteocalcin (OC). Subsequent Alizarin Red S stain confirmed the benefits of our cell block and demonstrated that such a novel combination and modification of biomaterials can serve as a platform for future clinical applications and use in dentin regeneration.

Effects of Gelatin Methacrylate Bio-ink Concentration on Mechano-Physical Properties and Human Dermal Fibroblast Behavior.

Gelatin-methacryloyl (GelMa) is a very versatile biomaterial widely used in various biomedical applications. The addition of methacryloyl makes it possible to have hydrogels with varying mechanical properties due to its photocuring characteristics. In addition, gelatin is obtained and derived from natural material; thus, it retains various cell-friendly motifs, such as arginine-glycine-aspartic acid, which then provides implanted cells with a friendly environment for proliferation and differentiation. In this study, we fabricated human dermal fibroblast cell (hDF)-laden photocurable GelMa hydrogels with varying physical properties (5%, 10%, and 15%) and assessed them for cellular responses and behavior, including cell spreading, proliferation, and the degree of extracellular matrix remodeling. Under similar photocuring conditions, lower concentrations of GelMa hydrogels had lower mechanical properties than higher concentrations. Furthermore, other properties, such as swelling and degradation, were compared in this study. In addition, our findings revealed that there were increased remodeling and proliferation markers in the 5% GelMa group, which had lower mechanical properties. However, it was important to note that cellular viabilities were not affected by the stiffness of the hydrogels. With this result in mind, we attempted to fabricate 5-15% GelMa scaffolds (20 × 20 × 3 mm3) to assess their feasibility for use in skin regeneration applications. The results showed that both 10% and 15% GelMa scaffolds could be fabricated easily at room temperature by adjusting several parameters, such as printing speed and extrusion pressure. However, since the sol-gel temperature of 5% GelMa was noted to be lower than its counterparts, 5% GelMa scaffolds had to be printed at low temperatures. In conclusion, GelMa once again was shown to be an ideal biomaterial for various tissue engineering applications due to its versatile mechanical and biological properties. This study showed the feasibility of GelMa in skin tissue engineering and its potential as an alternative for skin transplants.

Additive Manufacturing of Nerve Decellularized Extracellular Matrix-Contained Polyurethane Conduits for Peripheral Nerve Regeneration.

The nervous system is the part of our body that plays critical roles in the coordination of actions and sensory information as well as communication between different body parts through electrical signal transmissions. Current studies have shown that patients are likely to experience a functional loss if they have to go through a nerve repair for >15 mm lesion. The ideal treatment methodology is autologous nerve transplant, but numerous problems lie in this treatment method, such as lack of harvesting sites. Therefore, researchers are attempting to fabricate alternatives for nerve regeneration, and nerve conduit is one of the potential alternatives for nerve regeneration. In this study, we fabricated polyurethane/polydopamine/extracellular matrix (PU/PDA/ECM) nerve conduits using digital light processing (DLP) technology and assessed for its physical properties, biodegradability, cytocompatibility, neural related growth factor, and proteins secretion and expression and its potential in allowing cellular adhesion and proliferation. It was reported that PU/PDA/ECM nerve conduits were more hydrophilic and allowed enhanced cellular adhesion, proliferation, expression, and secretion of neural-related proteins (collagen I and laminin) and also enhanced expression of neurogenic proteins, such as nestin and microtubule-associated protein 2 (MAP2). In addition, PU/PDA/ECM nerve conduits were reported to be non-cytotoxic, had sustained biodegradability, and had similar physical characteristics as PU conduits. Therefore, we believed that PU/PDA/ECM nerve conduits could be a potential candidate for future nerve-related research or clinical applications.

Bioprinting of artificial blood vessels.

Vascular networks have an important role to play in transporting nutrients, oxygen, metabolic wastes and maintenance of homeostasis. Bioprinting is a promising technology as it is able to fabricate complex, specific multi-cellular constructs with precision. In addition, current technology allows precise depositions of individual cells, growth factors and biochemical signals to enhance vascular growth. Fabrication of vascularized constructs has remained as a main challenge till date but it is deemed as an important stepping stone to bring organ engineering to a higher level. However, with the ever advancing bioprinting technology and knowledge of biomaterials, it is expected that bioprinting can be a viable solution for this problem. This article presents an overview of the biofabrication of vascular and vascularized constructs, the different techniques used in vascular engineering such as extrusion-based, droplet-based and laser-based bioprinting techniques, and the future prospects of bioprinting of artificial blood vessels.

Mineral Trioxide Aggregate Mixed with 5-Aminolevulinic Acid for the Photodynamic Antimicrobial Strategy in Hard Tissue Regeneration.

Aminolevulinic acid (ALA) based photodynamic antimicrobial strategy can provide good antimicrobial effects and be used for medical applications. The aim of this study was to apply this strategy to Mineral Trioxide Aggregate (MTA), which is commonly used as a filling material for root endings and by doing so, to increase the bactericidal capability of MTA, as well as to investigate its characterization, cytocompatibility, and odontogenic differentiation potential. MTA is known to be a derivative of calcium silicate (CS). In this study, MTA specimens with or without ALA and light treatment were prepared. Diametral tensile strength values (DTS), setting durations, X-ray diffraction (XRD) spectra, apatite-mineralization, and antimicrobial abilities of the MTA, were also analyzed. Human dental pulp cells (hDPCs) can proliferate into the newly formed matrix and differentiate into odontoblasts to reinforce and strengthen the root. Levels of hDPCs proliferation and its odontogenic capabilities when cultured on MTA with ALA and light treatment, and the percentages of cells existing in the various cell cycle stages, were further evaluated in this study. The results indicated that MTA added ALA with light treatment had greater antibacterial ability and cytocompatibility, compared to MTA alone. A higher percentage S phase of the cells cultured on MTA added ALA with light treatment was observed. Furthermore, hDPCs cultured on MTA added ALA with light treatment had the highest expression levels of the odontoblastic differentiation markers. ALA has great antimicrobial efficiency and is a potential material for future medical applications. ALA-based photodynamic antibacterial strategy applied in the MTA has great antibacterial ability, cytocompatibility, and odontoblastic differentiation potential, and can facilitate the development of root canal treatment.

Surface Modification of Calcium Silicate via Mussel-Inspired Polydopamine and Effective Adsorption of Extracellular Matrix to Promote Osteogenesis Differentiation for Bone Tissue Engineering.

Calcium silicate-based cement has garnered huge interest in recent years, due to its versatility and potential in mass fabrication of a variety of bioceramics. For this study, the main objective was to fabricate functionalized calcium silicate (CS) powder integrated with a simple bio-inspired surface modification using polydopamine (PDA), to regulate cellular behaviors such as cellular adhesion, and subsequently cell differentiation and proliferation. For this study, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) techniques were used to analyze the chemical compositions and observe the surface characteristics of our PDA coated CS cements. Such modifications were found to enhance Wharton Jelly's mesenchymal stem cells (WJMSC) in various ways. Firstly, PDA-coated CS cements were found to significantly enhance cell adhesion with higher expressions of cell adhesion markers, such as focal adhesion kinase and integrins. This was further supported by morphology analysis of the cells. This enhanced cell adhesion, in turn, led to significantly higher secretion of extracellular matrix (ECM) proteins, such as collagen I and fibronectin, which directly promoted cell attachments and proliferation. In our osteogenesis assays, it was found that secretion and expression of osteogenesis related genes and proteins were significantly higher and were dependent on the PDA content. Therefore, these results demonstrated that such simple bio-inspired modification techniques of synthetic degradable CS cements can be applied as a future modification, to modify and convert inert surfaces of synthetic bone grafts to enhance and modulate the cell behaviors of WJMSCs. This in turn can be used as a potential alternative for further bioengineering research.

The Physicochemical Properties of Decellularized Extracellular Matrix-Coated 3D Printed Poly(ε-caprolactone) Nerve Conduits for Promoting Schwann Cells Proliferation and Differentiation.

Although autologous nerve grafting remains the gold standard treatment for peripheral nerve injuries, alternative methods such as development of nerve guidance conduits have since emerged and evolved to counter the many disadvantages of nerve grafting. However, the efficacy and viability of current nerve conduits remain unclear in clinical trials. Here, we focused on a novel decellularized extracellular matrix (dECM) and polydopamine (PDA)-coated 3D-printed poly(ε-caprolactone) (PCL)-based conduits, whereby the PDA surface modification acts as an attachment platform for further dECM attachment. We demonstrated that dECM/PDA-coated PCL conduits possessed higher mechanical properties when compared to human or animal nerves. Such modifications were proved to affect cell behaviors. Cellular behaviors and neuronal differentiation of Schwann cells were assessed to determine for the efficacies of the conduits. There were some cell-specific neuronal markers, such as Nestin, neuron-specific class III beta-tubulin (TUJ-1), and microtubule-associated protein 2 (MAP2) analyzed by enzyme-linked immunosorbent assay, and Nestin expressions were found to be 0.65-fold up-regulated, while TUJ1 expressions were 2.3-fold up-regulated and MAP2 expressions were 2.5-fold up-regulated when compared to Ctl. The methodology of PDA coating employed in this study can be used as a simple model to immobilize dECM onto PCL conduits, and the results showed that dECM/PDA-coated PCL conduits can as a practical and clinically viable tool for promoting regenerative outcomes in larger peripheral nerve defects.