Evaluation of proliferation and protein expression of human bone marrow cells cultured on coral crystallized in the aragonite or calcite form

Abstract

The two crystalline forms of CaCO3, aragonite (from natural coral) and calcite (from natural limestone), have been used with success as bone graft substitutes. However, natural coral transformed into calcite by heating has never been tested. The objective of this work was to study the proliferation and alkaline phosphatase, osteonectin, and osteocalcin expression of human bone marrow cells cultured on CaCO3 crystallized both in the aragonite form (natural coral) and in the calcite form (natural coral modified by heating). The methods used to characterize calcite obtained from the coral were volumic porosimetry, scanning electron microscopy (SEM) and X-ray diffraction. Cell colonization of the material was assessed by SEM performed on days 1, 7, 20, and 30 and [3H]thymidine incorporation was performed on days 3, 7, 12, 18, 25, and 32. Phenotypic expression was assessed by using in situ cytochemistry (alkaline phosphatase), immunocytochemistry (osteonectin and osteocalcin), and hybridization (osteocalcin, beta-actin, and alkaline phosphatase mRNA). Results showed the transformation of aragonite into calcite after heating, the conservation of macroporosity, and a modification of the surface. Calcite appeared to have a smoother and more uniform surface than aragonite crystals. As for [3H]thymidine there was an increase incorporation from days 3 to 18, a stabilization from days 18 to 25, and a decrease from days 25 to 32. After 20 days of culture, immunological studies using monoclonal antibodies to osteocalcin, osteonectin, cytochemical analysis of alkaline phosphatase activity, and in situ hybridization using osteocalcin, beta-actin, and alkaline phosphatase cDNA indicated that the cells had not lost their osteoblastic phenotype. These experiments demonstrate that coral crystallized in the aragonite or calcite form present a similar degree of specific cytocompatibility.

Calcite as a bone substitute. Comparison with hydroxyapatite and tricalcium phosphate with regard to the osteoblastic activity

“…In all cases, the number of cells counted after 3-or 6-day culture is always higher than the cell number initially seeded, which shows a positive cell growth on the test substrate. Nevertheless, β-TCP and calcite induce the relatively lower proliferation rates, respectively 40% and 30% after 6-day culture, which are anyway consistent with the results, which are already known in literature [20][21][22]. Obviously, certain cytotoxicity of β-TCP and calcite materials to MC3T3-E1 osteoblasts, even slight, can be questioned from such finding.…”

“…With the similar non-cytotoxicity found from the cell viability test for all ceramics and controls, these lower cell growth rates may arise from some other factors related to the renewal rate of nutrients in in vitro culture environment, or the surface morphology, chemistry etc., which are difficult to be determined on the macroporous materials and also be considered as the cause of the lower proliferation rates by other authors working on phosphocalcic ceramics and coral [20,21]. Anyhow, the similar proliferation rates for both β-TCP and calcite at least confirm the similar interest of calcite material to the widely-used β-TCP material.…”

Effects of humidity on calcite block fabrication using calcium hydroxide compact

“…Marine coral, which contains CaCO 3 as aragonite, has been used as an artificial bone substitute [6][7][8][9][10][11][12][13]; however, usage of coral as a bone substitute can lead to the destruction of natural environments and can cause serious medical complications, including an inflammatory response. The limitations of natural aragonite's use as a biomaterial are thought to be caused by the impurities from coral [9].…”

Calcite Biohybrids as Microenvironment for Stem Cells

“…A calcite polymorph of calcium carbonate from natural limestone was used as a bone substitute in vivo in rabbits [17]. Calcite, transformed by heating aragonite from natural coral, has been tested in vitro where it was shown to be osteogenic [18].…”