Synthesis and Evaluation of a Chitosan-Silica-Based Bone Substitute for Tissue Engineering

Abstract

Bone defects have prompted the development of biomaterial-based bone substitutes for restoring the affected tissue completely. Although many biomaterials have been designed and evaluated, the combination of properties required in a biomaterial for bone tissue engineering still poses a challenge. In this study, a chitosan-silica-based biocomposite was synthetized, and its physicochemical characteristics and biocompatibility were characterized, with the aim of exploring the advantages and drawbacks of its use in bone tissue engineering. Dynamic light scattering measurements showed that the mean hydrodynamic size of solid silica particles (Sol-Si) was 482 ± 3 nm. Scanning electron microscopy of the biocomposite showed that Sol-Si were homogenously distributed within the chitosan (CS) matrix. The biocomposite swelled rapidly and was observed to have no cytotoxic effect on the [3T3] cell line within 24 h. Biocompatibility was also analyzed in vivo 14 days post-implant using a murine experimental model (Wistar rats). The biocomposite was implanted in the medullary compartment of both tibiae (n = 12). Histologically, no acute inflammatory infiltrate or multinucleated giant cells associated to the biocomposite were observed, indicating good biocompatibility. At the tissue-biocomposite interface, there was new formation of woven bone tissue in close contact with the biocomposite surface (osseointegration). The new bone formation may be attributed to the action of silica. Free silica particles originating from the biocomposite were observed at the tissue-biocomposite interface. According to our results, the biocomposite may act as a template for cellular interactions and extracellular matrix formation, providing a structural support for new bone tissue formation. The CS/Sol-Si biocomposite may act as a Si reservoir, promoting new bone formation. A scaffold with these properties is essential for cell differentiation and filling a bone defect.

Keywords: biocomposite; bone tissue engineering; chitosan; murine experimental model; silica.

Figure 1

(a) Representative SEM image of Sol-Si particles; (b) representative TEM image of Sol-Si particles; (c) representative SEM image of CS; (d,e) representative SEM image of the (CS/Sol-Si) biocomposite.

Figure 2

FT-IR IR Spectra. (A) Sol-Si particles, (B) CS/Sol-Si biocomposite, (C) CS.

Figure 3

(a) Percentage of swelling of CS and CS/Sol-Si over a period of 24 h in PBS buffer. (b) CS and CS/Sol-Si experimental data for water content and time plotted according to a second-order kinetic model. Data were obtained from triplicate results and are shown as mean ± SD.

Figure 4

Contact angle of CS and CS/Si-Sol biocomposite. ** (p < 0.0001) according to Student’s t-test.

Figure 5

Biodegradation of CS and CS/Si-Sol biocomposite over 6 days with 1 mg/mL of lysozyme in PBS buffer, * (p < 0.05) according to Student’s t-test.

Figure 6

Percentage of viability of [3T3] mouse fibroblasts after incubation with CS and CS/Sol-Si at 1 and 5 days, by the MTT assay. Data are expressed as percentage relative to control. Data are shown as mean ± SD of triplicate experiments.

Figure 7

Histological evaluation of CS/Sol-Si biocomposite and pure chitosan (CS) at 1 day post-implantation in the hematopoietic bone marrow compartment. (a–c) Show the biocomposite composed of chitosan (*) and the silica particles (►). (d) shows only CS. All cases show the clot covering the entire surface of the biomaterial. (a) Orig. Mag. ×50; (b) Orig. Mag. ×100; (c,d) Orig. Mag. ×400. H–E stain. CS/Sol-Si (a–c). CS (d).

Figure 8

Histological evaluation of CS at 14 days post-implantation. At the tissue–biomaterial interface there is reparative granulation tissue (#) and formation of woven bone tissue (♦), sometimes in close contact with the surface of the biomaterial (osseointegration ↑). Orig. Mag. ×400; H-E stain. * Pure chitosan (CS).

Figure 9

Histological evaluation of CS/Sol-Si biocomposite at 14 days post-implantation. (a,b) Show at the tissue–biomaterial interface, the replacement of the clot by reparative granulation tissue (#) and formation of woven bone tissue (♦). (c) Shows osseointegrated woven bone tissue (↑). (a–c) Orig. Mag. × 400; H-E stain. (*) chitosan; (►) silica particles.

Figure 10

Histological evaluation of CS/Sol-Si biocomposite at 14 days post-implantation. At the tissue–biomaterial interface, there is reparative granulation tissue (#), which is progressively replaced by woven bone tissue (♦). Figures (a,b) correspond to the same image, (a) stained with H-E and (b) stained with Masson’s trichrome. (c) Higher magnification of part of image (b). Blue clearly shows the osteoid tissue that progressively replaces the reparative granulation tissue (red) next to the biomaterial (*). (a,b) Orig. Mag. × 400. (c) Orig. Mag. ×1000. (a) H-E stain. (b,c) Masson’s trichrome.

Figure 11

Histological evaluation of CS/Sol-Si biocomposite (*) at 14 days post-implantation. Silica particles (►) detached from within the biomaterial are observed at the tissue–biomaterial interface. No inflammatory infiltrate or multinucleated giant cells associated with the particle clusters are observed. Orig. Mag. ×1000; H–E stain.

Figure 12

EDS analysis. (a) Area where analysis was performed at the level of the particles detached from the biomaterial. (b) EDS analysis of the particles showing the presence of Si. LG: light microscopy; SEM: scanning electron microscopy. Silica particles (►).

Figure 13

In vivo procedure: A laminar chitosan–silica biocomposite implant being placed through a hole in the tibial medullary compartment.