An electron microscopic analysis of tricalcium phosphate hydroxyapatite and synthetic hydroxyapatite bioceramics for bone tissue engineering

Calcium and phosphate-based ceramics are reported to have osteoinductive properties. In this study, (1) tricalcium posphate hydroxyapatite, TCP/HA (resorbable) and (2) synthetic hydroxyapatite, HA (slow-resorbable) scaffold biomaterials, both composed of calcium and phosphate in varying compositions, were assessed as possible potential scaffold materials in bone tissue engineering. The present study used correlative light and electron microscopic analysis to investigate the surface morpholgy of human osteoprogenitor cells and its proliferation rate in response to alpha medium and differentiation medium at 1 week post-culture, effect of fibrin matrix inclusion into scaffold on cells 1 week postculture and interaction between human osteoprogenitor cells and physico-chemical characteristic of the scaffolds (TCP/HA and HA) studied in a three-week in vitro model. An in vivo study of a three months post-implantation tissue-engineered bone constructs in nude mice included the detection of fibroblast and inherent proteins of bone tissue, such as extracellular matrix protein (collagen type I) and non-collagenous proteins (osteopontin and bone sialoprotein) by scanning electron microscopic immunogold-silver labelling, elemental analysis and calcium phosphate elemental mapping determined by energy-dispersive X-ray (EDX) microanalysis and assessment of bone formation via light and electron microscopy, namely scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In the human osteoprogenitor cells culture study, a greater amount of bone nodules and more differentiated osteoprogenitor cells were observed in cell culture grown in one week osteogenic medium in comparison to alpha medium. To determine the effects of fibrin on cells, fibrin added to cell-biomaterial composite after one week showed a greater amount of cells were retained in calcium phosphate-fibrin composite but uneven cells distribution was seen in calcium phosphate-fibrin absent composite, resulted in cellular performance variability. Seeded cells seen along fibrin’s layer periphery displayed a spreading morphology but rounded within the fibrin layer, indicated that fibrin modulates the migration of cells. However, cells trapped within due to the fibrin structural properties (i.e. small pores, closely knitted thinner fibrin strands) may limit the cell migration ability. In in vivo study, TEM observation illustrated new bone formed interdigitally in the micropores of TCP/HA and HA grains; however, no bone formation was observed at the periphery of the TCP/HA and HA granules as revealed by light microscopy. A notable difference in the pattern of how bone formed in the comparison of the two composites, TCP/HA and HA, in which bone formed in random patches, coincide with occurrence of scaffold degradation in the former while the latter took on a more predicted form of bordering the HA struts with no signs of scaffold degradation. Elemental mapping and structural imaging using BSE grey-level and EDX analysis ascertain the similar bone formation patterns in the two composites. The expression of bone matrix proteins, bone sialoprotein and osteopontin and ECM deposition, which is predominantly collagen type I as determined by SEM immunolabelling has clearly verified that the tissue-engineered bone constructs possess inherent characteristics of a bone tissue. The role of bone substitute should be ideally replaced by autogenous bone regeneration with time, hence, the correlative microscopy study indicates that TCP/HA is much favourable than HA as bone substitute as it promotes new bone replacement in places where degradation was observed and moreover, the bone regeneration rate befitting to the resorption rate of the TCP/HA

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