Development of a Scaffold-on-a-Chip Platform to Evaluate Cell Infiltration and Osteogenesis on the 3D-Printed Scaffold for Bone Regeneration.

Developing a scaffold for efficient and functional bone regeneration remains challenging. To accomplish this goal, a "scaffold-on-a-chip" device was developed as a platform to aid with the evaluation process. The device mimics a microenvironment experienced by a transplanted bone scaffold. The device contains a circular space at the center for scaffold insert and microfluidic channel that encloses the space. Such a design allows for monitoring of cell behavior at the blood-scaffold interphase. MC3T3-E1 cells were cultured with three different types of scaffold inserts to test its capability as an evaluation platform. Cellular behaviors, including migration, morphology, and osteogenesis with each scaffold, were analyzed through fluorescence images of live/dead assay and immunocytochemistry. Cellular behaviors, such as migration, morphology, and osteogenesis, were evaluated. The results revealed that our platform could effectively evaluate the osteoconductivity and osteoinductivity of scaffolds with various properties. In conclusion, our proposed platform is expected to replace current in vivo animal models as a highly relevant in vitro platform and can contribute to the fundamental study of bone regeneration.

Enhanced Osteogenesis of Dental Pulp Stem Cells In Vitro Induced by Chitosan-PEG-Incorporated Calcium Phosphate Cement.

The use of bone graft materials is required for the treatment of bone defects damaged beyond the critical defect; therefore, injectable calcium phosphate cement (CPC) is actively used after surgery. The application of various polymers to improve injectability, mechanical strength, and biological function of injection-type CPC is encouraged. We previously developed a chitosan-PEG conjugate (CS/PEG) by a sulfur (VI) fluoride exchange reaction, and the resulting chitosan derivative showed high solubility at a neutral pH. We have demonstrated the CPC incorporated with a poly (ethylene glycol) (PEG)-grafted chitosan (CS/PEG) and developed CS/PEG CPC. The characterization of CS/PEG CPC was conducted using Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The initial properties of CS/PEG CPCs, such as the pH, porosity, mechanical strength, zeta potential, and in vitro biocompatibility using the WST-1 assay, were also investigated. Moreover, osteocompatibility of CS/PEG CPCs was carried out via Alizarin Red S staining, immunocytochemistry, and Western blot analysis. CS/PEG CPC has enhanced mechanical strength compared to CPC, and the cohesion test also demonstrated in vivo stability. Furthermore, we determined whether CS/PEG CPC is a suitable candidate for promoting the osteogenic ability of Dental Pulp Stem Cells (DPSC). The elution of CS/PEG CPC entraps more calcium ion than CPC, as confirmed through the zeta potential test. Accordingly, the ion trapping effect of CS/PEG is considered to have played a role in promoting osteogenic differentiation of DPSCs. The results strongly suggested that CS/PEG could be used as suitable additives for improving osteogenic induction of bone substitute materials.

3D-Printed Poly(ε-Caprolactone)/Hydroxyapatite Scaffolds Modified with Alkaline Hydrolysis Enhance Osteogenesis In Vitro.

The 3D-printed bioactive ceramic incorporated Poly(ε-caprolactone) (PCL) scaffolds show great promise as synthetic bone graft substitutes. However, 3D-printed scaffolds still lack adequate surface properties for cells to be attached to them. In this study, we modified the surface characteristics of 3D-printed poly(ε-caprolactone)/hydroxyapatite scaffolds using O2 plasma and sodium hydroxide. The surface property of the alkaline hydrolyzed and O2 plasma-treated PCL/HA scaffolds were evaluated using field-emission scanning microscopy (FE-SEM), Alizarin Red S (ARS) staining, and water contact angle analysis, respectively. The in vitro behavior of the scaffolds was investigated using human dental pulp-derived stem cells (hDPSCs). Cell proliferation of hDPSCs on the scaffolds was evaluated via immunocytochemistry (ICC) and water-soluble tetrazolium salt (WST-1) assay. Osteogenic differentiation of hDPSCs on the scaffolds was further investigated using ARS staining and Western blot analysis. The result of this study shows that alkaline treatment is beneficial for exposing hydroxyapatite particles embedded in the scaffolds compared to O2 plasma treatment, which promotes cell proliferation and differentiation of hDPSCs.

Sulfur(VI) Fluoride Exchange (SuFEx)-Mediated Synthesis of the Chitosan-PEG Conjugate and Its Supramolecular Hydrogels for Protein Delivery.

Supramolecular hydrogels are considered promising drug carriers in the tissue engineering field due to their versatile nature. Chitosan hydrogels without chemical cross-linkers have low cytotoxicity and good delivery capacity; however, they have lower mechanical properties for injectable hydrogel usage. In this study, we developed novel chitosan derivatives via click chemistry for fabricating supramolecular hydrogels with higher mechanical strength under mild conditions. The chitosan derivative was successfully synthesized by a sulfur fluoride exchange reaction, and the synthesized chitosan-mPEG/Pluronic-F127 (CS-mPEG/F127) interacted with α-cyclodextrin (α-CD) to form a supramolecular hydrogel via a host-guest reaction. The gelation dynamics, hydrogel properties, and bovine serum albumin (BSA) release could be modulated by the concentration ratio of chitosan-mPEG and F127. This supramolecular hydrogel is a promising protein releasing carrier candidate for long term regeneration therapy.