Construction of Microfluidic Chip Structure for Cell Migration Studies in Bioactive Ceramics.

Cell migration is an essential bioactive ceramics property and critical for bone induction, clinical application, and mechanism research. Standardized cell migration detection methods have many limitations, including a lack of dynamic fluid circulation and the inability to simulate cell behavior in vivo. Microfluidic chip technology, which mimics the human microenvironment and provides controlled dynamic fluid cycling, has the potential to solve these questions and generate reliable models of cell migration in vitro. In this study, a microfluidic chip is reconstructed to integrate the bioactive ceramic into the microfluidic chip structure to constitute a ceramic microbridge microfluidic chip system. Migration differences in the chip system are measured. By combining conventional detection methods with new biotechnology to analyze the causes of cell migration differences, it is found that the concentration gradients of ions and proteins adsorbed on the microbridge materials are directly related to the occurrence of cell migration behavior, which is consistent with previous reports and demonstrates the effectiveness of the microfluidic chip model. This model provides in vivo environment simulation and controllability of input and output conditions superior to standardized cell migration detection methods. The microfluidic chip system provides a new approach to studying and evaluating bioactive ceramics.

Strategies of strengthening mechanical properties in the osteoinductive calcium phosphate bioceramics.

Calcium phosphate (CaP) bioceramics are widely applied in the bone repairing field attributing to their excellent biological properties, especially osteoinductivity. However, their applications in load-bearing or segmental bone defects are severely restricted by the poor mechanical properties. It is generally considered that it is challenging to improve mechanical and biological properties of CaP bioceramics simultaneously. Up to now, various strategies have been developed to enhance mechanical strengths of CaP ceramics, the achievements in recent researches need to be urgently summarized. In this review, the effective and current means of enhancing mechanical properties of CaP ceramics were comprehensively summarized from the perspectives of fine-grain strengthening, second phase strengthening, and sintering process optimization. What's more, the further improvement of mechanical properties for CaP ceramics was prospectively proposed including heat treatment and biomimetic. Therefore, this review put forward the direction about how to compatibly improve mechanical properties of CaP ceramics, which can provide data and ideas for expanding the range of their clinical applications.

Construction of Vascularized Tissue Engineered Bone with nHA-Coated BCP Bioceramics Loaded with Peripheral Blood-Derived MSC and EPC to Repair Large Segmental Femoral Bone Defect.

The regenerative repair of segmental bone defect (SBD) is an urgent problem in the field of orthopedics. Rapid induction of angiogenesis and osteoinductivity after implantation of scaffold is critical. In this study, a unique tissue engineering strategy with mixture of peripheral blood-derived mesenchymal stem cells (PBMSC) and endothelial progenitor cells (PBEPC) was applied in a 3D-printed biphasic calcium phosphate (BCP) scaffold with highly bioactive nano hydroxyapatite (nHA) coating (nHA/BCP) to construct a novel vascularized tissue engineered bone (VTEB) for rabbit femoral SBD repair. The 2D coculture of PBMSC and PBEPC showed that they could promote the osteogenic or angiogenic differentiation of the cells from each other, especially in the group of PBEPC/PBMSC = 75:25. Besides, the 3D coculture results exhibited that the nHA coating could further promote PBEPC/PBMSC adhesion, proliferation, and osteogenic and angiogenic differentiation on the BCP scaffold. In vivo experiments showed that among the four groups (BCP, BCP-PBEPC/PBMSC, nHA/BCP, and nHA/BCP-PBEPC/PBMSC), the nHA/BCP-PBEPC/PBMSC group induced the best formation of blood vessels and new bone and, thus, the good repair of SBD. It revealed the synergistic effect of nHA and PBEPC/PBMSC on the angiogenesis and osteogenesis of the BCP scaffold. Therefore, the construction of VTEB in this study could provide a possibility for the regenerative repair of SBD.

Dopamine/DOPAC-assisted immobilization of bone morphogenetic protein-2 loaded Heparin/PEI nanogels onto three-dimentional printed calcium phosphate ceramics for enhanced osteoinductivity and osteogenicity.

Nowadays, the three-dimensional (3D) printed calcium phosphate (CaP) ceramics have well-designed geometric structure, but suffer from relative weak osteoinductivity. Surface modification by incorporating bone morphogenetic protein-2 (BMP2) onto scaffolds is considered as an efficient approach to improve their bioactivity. However, high dose and uncontrolled burst release of BMP2 may cause undesired side effect. In the present study, porous BCP ceramics with inverse face-centred cube structure prepared by digital light processing (DLP)-based 3D printing technique were used as the substrates. BMP2 proteins were loaded in the self-assembled Heparin/PEI nanogels (NP/BMP2), and then immobilized onto BCP substrates through the intermediate mussel-derived bioactive dopamine and dihydroxyphenylacetic acid (DA/DOPAC) coating layers to construct functional BCP/layer/NP/BMP2 scaffolds. Our results showed that Heparin/PEI nanogel was a potent delivery system for BMP2, and BCP/layer/NP/BMP2 scaffolds exhibited the high loading capacity, controlled release rate, and sustained local delivery of BMP2. In vitro cell experiments with bone marrow stromal cells (BMSCs) found that BCP/layer/NP/BMP2 could promote cell proliferation, facilitate cell spreading, accelerate cell migration, up-regulate expression of osteogenic genes, and improve synthesis of osteoblast-related proteins. Moreover, the murine intramuscular implantation model suggested that BCP/layer/NP/BMP2 had a superior osteoinductive capacity, and the rat femoral condyle defect repair model showed that BCP/layer/NP/BMP2 could enhance in situ bone repair and regeneration. These findings demonstrate that the incorporation of BMP2 loaded Heparin/PEI nanogels to 3D printed scaffolds holds great promise in fabricating bone graft with a superior biological performance for orthopedic application.

Fabrication and biological evaluation of 3D-printed calcium phosphate ceramic scaffolds with distinct macroporous geometries through digital light processing technology.

Digital light processing (DLP)-based 3D printing technique holds promise in fabricating scaffolds with high precision. Here raw calcium phosphate (CaP) powders were modified by 5.5% monoalcohol ethoxylate phosphate (MAEP) to ensure high solid loading and low viscosity. The rheological tests found that photocurable slurries composed of 50 wt% modified CaP powders and 2 wt% toners were suitable for DLP printing. Based on geometric models designed by computer-aided design (CAD) system, three printed CaP ceramics with distinct macroporous structures were prepared, including simple cube, octet-truss and inverse face-centered cube (fcc), which presented the similar phase composition and microstructure, but the different macropore geometries. Inverse fcc group showed the highest porosity and compressive strength. The in vitro and in vivo biological evaluations were performed to compare the bioactivity of three printed CaP ceramics, and the traditional foamed ceramic was used as control. It suggested that all CaP ceramics exhibited good biocompatibility, as evidence by an even bone-like apatite layer formation on the surface, and the good cell proliferation and spreading. A mouse intramuscular implantation model found that all of CaP ceramics could induce ectopic bone formation, and foam group had the strongest osteoinduction, followed by inverse fcc, while cube and octet-truss had the weakest one. It indicated that macropore geometry was of great importance to affect the osteoinductivity of scaffolds, and spherical, concave macropores facilitated osteogenesis. These findings provide a strategy to design and fabricate high-performance orthopedic grafts with proper pore geometry and desired biological performance via DLP-based 3D printing technique.

Effect of Hydrothermal Media on the in-situ Whisker Growth on Biphasic Calcium Phosphate Ceramics.

BACKGROUND: There is still a big challenge to achieve a balance between mechanical characteristics and biological properties in biphasic calcium phosphate (BCP) ceramics. PURPOSE: The present study focused on the in-situ whisker growth on BCP ceramics via different hydrothermal treatments and investigated the influences of these whiskers on the mechanical property and biological performance of the ceramics. METHODS: Five kinds of BCP ceramics with in-situ whisker growth, ie, BCP-C, BCP-HNO3, BCP-Citric, BCP-NaOH, BCP-CaCl2 and BCP-Na3PO4 were fabricated by different hydrothermal treatments. The phase compositions, morphologies, crystal structures and mechanical strengths of the obtained BCP ceramics were firstly characterized. Then, the in vitro cell adhesion, proliferation and alkaline  phosphatase (ALP) activity of bone marrow stromal cells (BMSCs) on the BCP ceramics were evaluated. Lastly, the effects of in-situ whisker growth on the bone-like apatite formation abilities of BCP ceramics were also investigated by immersing them in simulated body fluid (SBF). RESULTS: The results demonstrated that the hydrothermal conditions, especially the hydrothermal media, were crucial to determine the phase composition and morphology of the in-situ whisker. Especially among the five media used (HNO3, Citric, NaOH, CaCl2 and Na3PO4), the Na3PO4 treatment resulted in the shortest whisker with a unique hollow structure, and kept the original biphasic composition. All five kinds of whiskers increased the mechanical strength of BCP ceramics to some extent, and showed the good ability of bone-like apatite formation. The in vitro cell study demonstrated that the in-situ whisker growth had no adverse but even positive effect on the adhesion, proliferation and ALP activity of BMSCs. CONCLUSION: Due to the growth of in-situ whiskers, the mechanical property and biological performance of the obtained BCP ceramics could increase simultaneously. Therefore, in-situ whiskers growth offers a promising strategy for the expanded application of BCP ceramics to meet the requirements of regenerative medicine.

Stereolithography-Based Additive Manufacturing of High-Performance Osteoinductive Calcium Phosphate Ceramics by a Digital Light-Processing System.

Digital light processing (DLP) is one of the additive manufacturing (AM) technologies suitable for preparation of high-performance ceramics. The present study provided an optimized formula to fabricate osteoinductive calcium phosphate (CaP) ceramics with high precision and controllable three-dimensional (3D) structure. Among the four surfactants, monoalcohol ethoxylate phosphate was the best one to modify the CaP powders for preparing the photocurable slurry with high solid loading and good spreading ability. By testing the photopolymerization property of the 60 wt % solid loading slurry, the appropriate processing parameters including the slice thickness (50 µm), exposure intensity (10.14 mW/cm2), and exposure time (8 s) were set to perform the 3D printing of the ceramic green body in the DLP system. After the debinding and sintering, the final CaP ceramics were acquired. The stereomicroscope and SEM observation confirmed the high precision of the ceramics. The average compressive strength of the ceramics with 64.5% porosity reached 9.03 MPa. On only soaking in simulated body fluid for 1 day, an even layer of apatite formed on the ceramic surface. The cell culture confirmed that the ceramics could allow the good attachment, growth, and proliferation of murine bone marrow mesenchymal stem cells. After implantation into the dorsal muscles of beagle dogs for 3 months, abundant blood vessels and obvious ectopic bone formation were observed clearly by the histological evaluation. Therefore, with good bioactivity and osteoinductivity as well as high precision and adjustable mechanical strength, the 3D printed CaP ceramics in the DLP system could have good potential in customized bone-repairing applications.