Tuan-Ti Hsu

In scaffold-regulated bone regeneration, most three-dimensional (3D)-printed scaffolds do not provide physical stimulation to stem cells. In this study, a magnetic scaffold was fabricated using fused deposition modeling with calcium silicate (CS), iron oxide nanoparticles (Fe₃O₄), and poly-ε-caprolactone (PCL) as the matrix for internal magnetic sources. A static magnetic field was used as an external magnetic source. It was observed that 5% Fe₃O₄ provided a favorable combination of compressive strength (9.6 ± 0.9 MPa) and degradation rate (21.6 ± 1.9% for four weeks). Furthermore, the Fe₃O₄-containing scaffold increased in vitro bioactivity and Wharton’s jelly mesenchymal stem cells’ (WJMSCs) adhesion. Moreover, it was shown that the Fe₃O₄-containing scaffold enhanced WJMSCs’ proliferation, alkaline phosphatase activity, and the osteogenic-related proteins of the scaffold. Under the synergistic effect of the static magnetic field, the CS scaffold containing Fe₃O₄ can not only enhance cell activity but also stimulate the simultaneous secretion of collagen I and osteocalcin. Overall, our results demonstrated that Fe₃O₄-containing CS/PCL scaffolds could be fabricated three dimensionally and combined with a static magnetic field to affect cell behaviors, potentially increasing the likelihood of clinical applications for bone tissue engineering.

Tooth loss or damage is a common problem affecting millions of people worldwide, and it results in significant impacts on one’s quality of life. Dental regeneration with the support of stem cell-containing scaffolds has emerged as an alternative treatment strategy for such cases. With this concept in mind, we developed various concentrations of calcium silicate (CS) in a gelatin methacryloyl (GelMa) matrix and fabricated human dental pulp stem cells (hDPSCs)-laden scaffolds via the use of a bioprinting technology in order to determine their feasibility in promoting odontogenesis. The X-ray diffraction and Fourier transform-infrared spectroscopy showed that the incorporation of CS increased the number of covalent bonds in the GelMa hydrogels. In addition, rheological analyses were conducted for the different concentrations of hydrogels to evaluate their sol–gel transition temperature. It was shown that incorporation of CS improved the printability and printing quality of the scaffolds. The printed CS-containing scaffolds were able to release silicate (Si) ions, which subsequently significantly enhanced the activation of signaling-related markers such as ERK and significantly improved the expression of odontogenic-related markers such as alkaline phosphatase (ALP), dentin matrix protein-1 (DMP-1), and osteocalcin (OC). The calcium deposition assays were also significantly enhanced in the CS-containing scaffold. Our results demonstrated that CS/GelMa scaffolds were not only enhanced in terms of their physicochemical behaviors but the odontogenesis of the hDPSCs was also promoted as compared to GelMa scaffolds. These results demonstrated that CS/GelMa scaffolds can serve as cell-laden materials for future clinical applications and use in dentin regeneration.

Tissue engineering and scaffolds play an important role in tissue regeneration by supporting cell adhesion, proliferation, and differentiation. The design of a scaffold is critical in determining its feasibility, and it is critical to note that each tissue is unique in terms of its morphology and composition. However, calcium-silicate-based scaffolds are undegradable, which severely limits their application in bone regeneration. In this study, we developed a biodegradable mesoporous calcium silicate (MS)/calcium sulfate (CS)/poly-ε-caprolactone (PCL) composite and fabricated a composite scaffold with 3D printing technologies. In addition, we were able to load bone morphogenetic protein-2 (BMP-2) into MS powder via a one-step immersion procedure. The results demonstrated that the MS/CS scaffold gradually degraded within 3 months. More importantly, the scaffold exhibited a gradual release of BMP-2 throughout the test period. The adhesion and proliferation of human dental pulp stem cells on the MS/CS/BMP-2 (MS/CS/B) scaffold were significantly greater than that on the MS/CS scaffold. It was also found that cells cultured on the MS/CS/B scaffold had significantly higher levels of alkaline phosphatase activity and angiogenic-related protein expression. The MS/CS/B scaffold promoted the growth of new blood vessels and bone regeneration within 4 weeks of implantation in rabbits with induced critical-sized femoral defects. Therefore, it is hypothesized that the 3D-printed MS/CS/B scaffold can act both as a conventional BMP-2 delivery system and as an ideal osteoinductive biomaterial for bone regeneration.

Recent advances in three-dimensional printing technology enable facile and on-demand fabrication of patient-specific bone scaffolds. However, there is still an urgent need for printable biomaterials with osteoinductivity. In the present study, we propose an approach to synthesize fibroblast growth factor-2 loaded-mesoporous calcium silicate nanoparticles. The growth factor loaded-nanoparticles served as fillers of polycaprolactone and then the composite scaffolds with a controlled pore structure were obtained through a fused deposition modeling technique. To evaluate the feasibility of the composite scaffolds in bone tissue engineering, drug release kinetic, bioactivity, cell proliferation, differentiation, and animal study were conducted. Our findings illustrate that utilization of mesoporous calcium silicate allowed the introduction of fibroblast growth factor-2 into the composite scaffolds through a simple soaking process and then gradually released from the scaffold to facilitate proliferation and osteogenesis differentiation of human Wharton’s jelly mesenchymal stem cells. Additionally, the in vivo femur defect experiments also indicate that the co-existence of calcium silicate and fibrous growth factor-2 synergistically accelerated new bone formation. These results demonstrate that the fibroblast growth factor-2-loaded mesoporous calcium silicate nanoparticles/polycaprolactone composite scaffolds may serve as potential bone grafts for facilitating repair of defected bone tissues.

There had been a paradigm shift in tissue engineering studies over the past decades. Of which, part of the hype in such studies was based on exploring for novel biomaterials to enhance regeneration. Strontium ions have been reported by others to have a unique effect on osteogenesis. Both in vitro and in vivo studies had demonstrated that strontium ions were able to promote osteoblast growth, and yet at the same time, inhibit the formation of osteoclasts. Strontium is thus considered an important biomaterial in the field of bone tissue engineering. In this study, we developed a Strontium-calcium silicate scaffold using 3D printing technology and evaluated for its cellular proliferation capabilities by assessing for protein quantification and mineralization of Wharton’s Jelly mesenchymal stem cells. In addition, verapamil (an L-type of calcium channel blocker, CCB) was used to determine the mechanism of action of strontium ions. The results found that the relative cell proliferation rate on the scaffold was increased between 20% to 60% within 7 days of culture, while the CCB group only had up to approximately 10% proliferation as compared with the control specimen. Besides, the CCB group had downregulation and down expressions of all downstream cell signaling proteins (ERK and P38) and osteogenic-related protein (Col I, OPN, and OC). Furthermore, CCB was found to have 3–4 times lesser calcium deposition and quantification after 7 and 14 days of culture. These results effectively show that the 3D printed strontium-contained scaffold could effectively stimulate stem cells to undergo bone differentiation via activation of L-type calcium channels. Such results showed that strontium-calcium silicate scaffolds have high development potential for bone tissue engineering.

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.

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.

Electrospinning technology allows fabrication of nano- or microfibrous fibers with inorganic and organic matrix and it is widely applied in bone tissue engineering as it allows precise control over the shapes and structures of the fibers. Natural bone has an ordered composition of organic fibers with dispersion of inorganic apatite among them. In this study, poly (lactic acid) (PLA) mats were fabricated with electrospinning and coated with chitosan (CH)/calcium silicate (CS) mixer. The microstructure, chemical component, and contact angle of CS/CH-PLA composites were analyzed by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. In vitro, various CS/CH-coated PLA mats increased the formation of hydroxyapatite on the specimens’ surface when soaked in cell cultured medium. During culture, several biological characteristics of the human mesenchymal stem cells (hMSCs) cultured on CS/CH-PLA groups were promoted as compared to those on pure PLA mat. Increased secretion levels of Collagen I and fibronectin were observed in calcium silicate-powder content. Furthermore, with comparison to PLA mats without CS/CH, CS10 and CS15 mats markedly enhanced the proliferation of hMSCs and their osteogenesis properties, which was characterized by osteogenic-related gene expression. These results clearly demonstrated that the biodegradable and electroactive CS/CH-PLA composite mats are an ideal and suitable candidate for bone tissue engineering.

Electrospinning is the versatile technique to generate large quantities of micro- or nano-fibers from a wide variety of shapes and sizes of polymer. Natural bone is a hierarchically composites with the dispersion of inorganic ceramic along organic polymer. The aim of this study, the electrospun poly (lactic acid) (PLA) mats coated with chitosan (CH) and calcium silicate (CS) powder were fabricated. The morphology, chemical composition, and surface properties of CS/CH-PLA composites were characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. In vitro, the CS/CH-coated PLA mats increased the formation of apatite on the surface when soaking in cell cultured medium. During culture, the adhesion and proliferation of the human mesenchymal stem cells (hMSCs) cultured on CS/CH-PLA were significantly promoted relative to those on PLA. Collagen I and fibronectin levels and promoted cell adhesion were observed upon an increase in CS content. Further, compared to PLA mats without CS/CH, CS10 and CS15 mats markedly enhanced the proliferation of hMSCs as well as their osteogenesis properties, which was characterized by bone-related gene expression. Our results demonstrated that the biodegradable and electroactive CS/CH-PLA mats had potential application as an ideal candidate for bone tissue engineering. Together, findings from this study clearly demonstrated that PLLA-C2S composite scaffold may function as an ideal candidate for bone tissue engineering.

In this study, we manufacture and analyze bioactive magnesium–calcium silicate/poly-ε-caprolactone (Mg–CS/PCL) 3D scaffolds for bone tissue engineering. Mg–CS powder was incorporated into PCL, and we fabricated the 3D scaffolds using laser sintering technology. These scaffolds had high porosity and interconnected-design macropores and structures. As compared to pure PCL scaffolds without an Mg–CS powder, the hydrophilic properties and degradation rate are also improved. For scaffolds with more than 20% Mg–CS content, the specimens become completely covered by a dense bone-like apatite layer after soaking in simulated body fluid for 1 day. In vitro analyses were directed using human mesenchymal stem cells (hMSCs) on all scaffolds that were shown to be biocompatible and supported cell adhesion and proliferation. Increased focal adhesion kinase and promoted cell adhesion behavior were observed after an increase in Mg–CS content. In addition, the results indicate that the Mg–CS quantity in the composite is higher than 10%, and the quantity of cells and osteogenesis-related protein of hMSCs is stimulated by the Si ions released from the Mg–CS/PCL scaffolds when compared to PCL scaffolds. Our results proved that 3D Mg–CS/PCL scaffolds with such a specific ionic release and good degradability possessed the ability to promote osteogenetic differentiation of hMSCs, indicating that they might be promising biomaterials with potential for next-generation bone tissue engineering scaffolds.