BGHA block was powdered and mixed with KBr powder in a weight ratio of approximately 1:100 and pressed into pellets.
BGHA was powdered, dispersed in acetone and a drop of the suspension was placed on copper support film (former/carbon) grids of 300 mesh type and viewed under TEM (H-600) at an accelerating voltage of 75 kV.
When the cells attained confluency, the sterilized BGHA blocks were placed in direct contact with the cells and incubated for 48 h under the same condition.
To study the cell adhesion behaviour of the BGHA block, osteoblast cells (Human osteoblast cell line--HOS) were seeded on the surface of conditioned BGHA block (pre-incubated for 24 h in the medium) and maintained in Minimal Essential Medium supplemented with 10 % fetal bovine serum and 100 units/ml of penicillin and 100 [micro]g/ml streptomycin, respectively and incubated at 37 [degrees]C in humid atmosphere and 5 % C[O.sub.2] for 48 h.
The cells seeded BGHA block was fixed in 3 % gluteraldehyde in phosphate buffer (pH 7.4), rinsed with PBS, and dehydrated in a graded acetone series.
The cells seeded BGHA block was fixed in 10 % neutral buffered formalin, rinsed with PBS, and dehydrated in graded acetone series.
Fourier transform infrared spectroscopy (FTIR): The IR spectrum of HA and BGHA granules were recorded using a Nicolet Impact 410 FT-IR spectroscopy, using the KBr pellet technique.
An in vitro model comprising of Human Osteosarcoma Cell line (HOS) was used to check the toxicity of HA and BGHA granules.
Test on extract (Based on ISO 10993-5, 1999): Extracts of porous HA and BGHA, were prepared by incubating the materials in saline at 37[degrees]C for 24 h at an extraction ratio of 0.2 g/ml.100 % extracts were diluted to get concentrations of 50 % and 25 % with MEM supplemented with Foetal Bovine Serum.
BGHA granules (test material) were implanted in distal and proximal critical size defects in the right femur of the animal.
SEM (Fig.1.a & Fig.2.a) depicted BGHA and HA granules to be of size 300 to 500 [micro]m with well-defined pores of approximately 100 [micro]m with interconnections which is ideal for osteoblast invasion and proliferation.
The EDAX spectrum of BGHA showed silica peaks apart from Ca and P peak and the elemental composition in the material is Si = 6.27 %, P = 27.11 % and Ca = 66.61 % (Fig.2.b).