Bacterial cellulose-hydroxyapatite nanocomposites for bone regeneration

Abstract

The aim of this study was to develop and to evaluate the biological properties of bacterial cellulose-hydroxyapatite (BC-HA) nanocomposite membranes for bone regeneration. Nanocomposites were prepared from bacterial cellulose membranes sequentially incubated in solutions of CaCl(2) followed by Na(2)HPO(4). BC-HA membranes were evaluated in noncritical bone defects in rat tibiae at 1, 4, and 16 weeks. Thermogravimetric analyses showed that the amount of the mineral phase was 40%-50% of the total weight. Spectroscopy, electronic microscopy/energy dispersive X-ray analyses, and X-ray diffraction showed formation of HA crystals on BC nanofibres. Low crystallinity HA crystals presented Ca/P a molar ratio of 1.5 (calcium-deficient HA), similar to physiological bone. Fourier transformed infrared spectroscopy analysis showed bands assigned to phosphate and carbonate ions. In vivo tests showed no inflammatory reaction after 1 week. After 4 weeks, defects were observed to be completely filled in by new bone tissue. The BC-HA membranes were effective for bone regeneration.

Figure 1

(a) TG curves: (thick line) bacterial cellulose (BC), (solid line) BC-HA and (dashed line) BC-HA sterilized by 20 kGy gamma radiation (BC-HAγ); TG curve (solid line) and DTG curve (dashed line) of BC (b), BC-HA (c) and BC-HAγ (d).

Figure 2

X-ray diffraction patterns of the BC (a) and BC-HA nanocomposites (b). The characteristic HA peaks are indexed at the top for BC-HA.

Figure 3

FTIR spectra of the BC (a) and BC-HA nanocomposites (b). Characteristic hydroxyapatite bands correspond to PO₄ ³⁻ and CO₂ ³⁻ ions (inset figure).

Figure 4

SEM images of the BC-HA nanocomposites (a) and (b) at 30.000× and 60.000×, respectively, and SEM images of the BC membrane (c) at 30.000×. A 5 kV accelerating voltage was used for the BC sample, and a 25 kV voltage was used to obtain the image of the BC-HA nanocomposites. EDS spectrum was taken from a typical nanofibril with surrounding HA crystals (d).

Figure 5

Histological photomicrographs: (a) Control group: 1 week. Bone defect filled by fibrotic tissue (white star); inflammatory infiltrate (white arrow). Hematoxylin-eosin staining (HE), scale bar (500 μm); (b) Treated group: 1 week. Bone defect filled by newly formed bone, osteoids (om), medullary spaces with mesenchymal cells (ms), and several blood vessels (v); mature bone (B); BC-HA membrane (black star); inflammatory infiltrate (I) (HE), scale bar (500 μm); (c) Control group: 4 weeks. New formed bone tissue is observed with several osteocytes (black arrows), blood vessels (arrow heads), and medullary spaces (stars); bone defect is not filled completely (white star) (HE), scale bar (100 μm); (d) Treated group: 4 weeks. BC-HA membrane (star), periosteum (P), osteoblasts (white arrow), osteocytes (arrow heads), and bone matrix (blue arrow) (HE), scale bar (100 μm); (e) Control group: 16 weeks. Mature bone (HE), scale bar (200 μm). (f) Treated group: 16 weeks. BC-HA membrane (star); bone defect completely repaired by mature bone, osteocytes (black arrows), and blood vessels (arrow head) (HE), scale bar (200 μm).