Preparation of laponite bioceramics for potential bone tissue engineering applications

Abstract

We report a facile approach to preparing laponite (LAP) bioceramics via sintering LAP powder compacts for bone tissue engineering applications. The sintering behavior and mechanical properties of LAP compacts under different temperatures, heating rates, and soaking times were investigated. We show that LAP bioceramic with a smooth and porous surface can be formed at 800°C with a heating rate of 5°C/h for 6 h under air. The formed LAP bioceramic was systematically characterized via different methods. Our results reveal that the LAP bioceramic possesses an excellent surface hydrophilicity and serum absorption capacity, and good cytocompatibility and hemocompatibility as demonstrated by resazurin reduction assay of rat mesenchymal stem cells (rMSCs) and hemolytic assay of pig red blood cells, respectively. The potential bone tissue engineering applicability of LAP bioceramic was explored by studying the surface mineralization behavior via soaking in simulated body fluid (SBF), as well as the surface cellular response of rMSCs. Our results suggest that LAP bioceramic is able to induce hydroxyapatite deposition on its surface when soaked in SBF and rMSCs can proliferate well on the LAP bioceramic surface. Most strikingly, alkaline phosphatase activity together with alizarin red staining results reveal that the produced LAP bioceramic is able to induce osteoblast differentiation of rMSCs in growth medium without any inducing factors. Finally, in vivo animal implantation, acute systemic toxicity test and hematoxylin and eosin (H&E)-staining data demonstrate that the prepared LAP bioceramic displays an excellent biosafety and is able to heal the bone defect. Findings from this study suggest that the developed LAP bioceramic holds a great promise for treating bone defects in bone tissue engineering.

Figure 1. The SEM surface morphologies of the LAP compact.

The LAP compact (a) before and (b–e) after sintered. (b) Sample #2, (c) Sample #3, (d) Sample #5, and (e) Sample #4. See Table 1 for sample information.

Figure 2. Mechanical properties of the LAP bioceramics.

(a) Hardness–displacement, (b) elastic modulus–displacement, (c) harmonic contact stiffness-displacement, and (d) load-displacement curves of LAP compact before and after sintered under different conditions. Inset in (a) shows the hardness change in the range of 20–600 nm.

Figure 3. XRD patterns of LAP compact before and after sintering under different conditions.

Figure 4. Hemolytic assay data of LAP bioceramics.

(a) Hemolytic percentage (%) of pRBCs after treatment with LAP before and after sintered under different conditions for 2 h (mean ± SD, n = 3). (b) shows the photograph of rRBC suspensions after treatment with different LAPs (shown in (a)), followed by centrifugation.

Figure 5. Biomineralization onto the surface of LAP ceramic.

(a) and (c) show the SEM surface morphology of LAP ceramic before and after soaking in SBF solution for 7 days, respectively. (b) and (d) show the EDS analysis of the LAP ceramic before and after soaking in SBF solution for 7 days, respectively.

Figure 6. Protein adsorption and metabolic activity assay of rMSCs.

(a) The adsorption of protein onto TCP and LAP ceramic (mean ± S.D., n = 3). (b) and (c) show the SEM micrographs of the TCP and LAP ceramic with protein adsorption, respectively. (d) shows the metabolic activity assay of rMSCs cultured onto TCP and LAP ceramic (mean ± S.D., n = 3). (e) shows the micrograph of rMSCs proliferated onto the LAP bioceramic for 14 days. (f) is the magnified image of (e).

Figure 7. Biosafety test of LAP ceramic extract.

(a) Body temperature and normalized body temperature curves of SD rat treated with saline or LAP ceramic extract. (b), (c), (e), and (f) show the results of acute toxicity on normal skin of SD rat immediately after injection or 1 d, 2 d or 3 d post injection of saline (dots1–6), LAP ceramic extracts (dots 7–9), and alcohol (dots 10–12), respectively.

Figure 8. Histological examination.

H&E-stained tissue sections of major organs, including the heart, liver, spleen, lung, and kidney from mice treated with saline or LAP ceramic extract for 14 days.

Figure 9. In vitro osteogenic differentiation of rMSCs and in vivo bone repair evaluation.

(a) ALP activity (normalized for the DNA content, nmol of transformed substrate per unit of time and per mass of DNA) of rMSCs cultured onto different substrates in growth medium after 14 days culture. Insert of (a) shows the picture of alizarin red staining of rMSCs cultured onto TCP (left) and LAP ceramic (right) in growth medium without any inducing factors on day 14. (b–f) show the macroscopic appearance of bone defects (A 2-mm bone defect was created in the middle of the tibia, which was implanted with laponite ceramic as shown in (d)). (b) and (e) show the macroscopic appearance of defects without and with implantation for 24 weeks, a trace of laponite ceramic residual is observed in (e) as pointed by an arrow. (c) and (f) show the radiographic images of bone defects without and with LAP implantation after 24 weeks.