Acellular mineral deposition in collagen-based biomaterials incubated in cell culture media.

Rapid developments in tissue engineering have renewed interest in biodegradable three-dimensional structures such as collagen-based biomaterials. Collagen matrices seeded in vitro with fibroblasts, osteoblasts, and chondrocytes can form tissues resembling skin, bone, and cartilage that could be used as functional substitutes for damaged tissues. Collagen is associated with both dystrophic calcification of collagenous implants and bone mineralization. We report here the calcification properties of collagen sponges incubated in cell-free media. Mineral deposited in sponges was identified by X-ray and electron diffraction, Fourier transform infrared spectroscopy, and the molar ratio of calcium:phosphorus (Ca:P) as a poorly crystalline apatite similar to bone. The degree of calcification increased with length of incubation and the Ca and P content of the media, with 10-15% Ca (dry weight) after 21 days' incubation in media containing 1.6-3 mM Ca and a Ca x P molar product of 2-3 mM(2), but only 2% Ca after incubation in medium with 1.33 mM Ca and a 1.7 mM(2) Ca x P molar product. Mineral deposition was completely inhibited in sponges that were washed extensively and initially contained less than 0.01% P. Readdition of phosphate in these sponges and subsequent freeze drying and sterilization restore their mineralization capacity, suggesting that collagen per se cannot initiate calcification and that the inorganic phosphate content associated with the collagen preparation process is in the solid state a potential nucleator. Addition of chondroitin 4-sulfate to the sponges partially or totally inhibited mineral deposition, even though 80-90% of the compound was released within 24 hours. These results indicate that acellular calcification of collagen-based biomaterials can occur under the culture conditions currently used in tissue engineering.

Activation of fibroblast metabolism in a dermal and skin equivalent model: a screening test for activity of peptides.

Skin firmness, elasticity and tone are gradually lost with age. These changes originate in the dermis and correspond to a decrease in the ability of cells, particularly the fibroblasts, to regenerate the molecules which make up the extracellular matrix. Skin ageing is also characterized by a reduction of the epidermal thickness and by a flattening of the basal membrane. The recent development of two 3-dimensional culture systems, in which the cells develop within a porous structure reproducing the extracellular matrix of the human dermis, is a way of reproducing in vivo conditions and demonstrating the biological effects of anti-ageing compounds. The dermal equivalent model used in this study is composed of a dermal matrix made of collagen-chitosan-glycosaminoglycans populated by normal human fibroblasts which synthesized their own extracellular matrix. A skin equivalent model is obtained by the cell culture of normal human keratinocytes onto a dermal equivalent elevated at the air-liquid interface. Such models were used to prove anti-ageing activity of promising compounds. Cosmetic Science has used many protein hydrolysates in order to fight skin ageing, but up to now, these natural peptides were poorly studied, and their efficacy poorly demonstrated. Eight protein hydrolysates were screened in a proliferation study in monolayered cultures giving two selected polypeptides. A soya derived peptide was used for an efficiency study in 3-dimensional models. In the dermal equivalent model, this peptide increased fibroblast proliferation by 40% and led to a stimulation of collagen formation (165%) and elastin (116%) synthesis. The effect of this soya peptide on glycosaminoglycan synthesis was also significant, with increases of 36% for chondroitin-4-sulfate and 68% for hyaluronic acid. These results were confirmed using a skin equivalent model. In this model, the soya peptide increased the thickness of the epidermis.

Cellular activity of osteoblasts in the presence of hydroxyapatite: an in vitro experiment.

The use of synthetic calcium phosphate as bone substitute calls for the knowledge of the influence on adjacent cells. Effects on monocytes, macrophages, synovial cells and fibroblasts have been largely described in vivo and in vitro but few data are available as concerns osteoblast responses. The present experiments tested the activity of MC3T3-E1, ROS 17/2.8 and mouse calvaria cells cultured in the presence of hydroxyapatite powders. The three osteoblast-like cells were shown to phagocytoze the calcium phosphate particles. As a consequence, they exhibit reduced cell growth and alkaline phosphatase activity. This response was different when compared with other cell types. The osteogenetic function of osteoblastic cells could be involved in these specific effects of hydroxyapatite.

The influence of calcium phosphate biomaterials on human bone cell activities. An in vitro approach.

An in vitro method is described to assess the influence of synthetic calcium phosphate powders on osteoblast activities. Human osteoblast cell cultures were established from iliac crest. MC3T3-E1, an established osteogenic cell line, was employed as a control. Scanning and transmission electron microscopic observations clearly demonstrated the internalization of particles of calcium phosphate by the two osteoblast cell populations. As a consequence to the phagocytotic process, RNA transcription and protein synthesis were stimulated, as indicated by the measurements of labeled uridine, leucine and proline uptakes. From these data, it is proposed that such an in vitro model, using one of the specific cell types involved in the tissue responses to implants, could be useful to assess the biological response at the cell-biomaterial interaction.

Effects of synthetic calcium phosphates on the 3H-thymidine incorporation and alkaline phosphatase activity of human fibroblasts in culture.

Gingival fibroblasts were cultured with four different calcium phosphate minerals (hydroxyapatite, whitlockite, beta-tricalcium phosphate, and octocalcium phosphate). 3H-thymidine incorporation into DNA and alkaline phosphatase specific activity were determined after different incubation periods. As a consequence of the phagocytosis of calcium phosphates crystals, we pointed out, compared to control, a stimulation of the rate of 3H-thymidine incorporation and sharp decreases in alkaline phosphatase activity. The magnitude of the alkaline phosphatase activity inhibition was observed to be increased with the solubility of the materials. We propose that the effects of calcium phosphates on alkaline phosphatase and 3H-thymidine incorporation could be calcium-mediated events, resulting from intracellular dissolution of phagocytized materials. We suggest that in vitro determination of 3H-thymidine incorporation and alkaline phosphatase activity, which are highly sensitive tests, could be involved in evaluation procedures of calcium phosphates biomaterials.

Chemical changes in hydroxyapatite biomaterial under in vivo and in vitro biological conditions.

The introduction of a synthetic calcium phosphate into a biological environment is likely to result in surface-mediated chemical events. On the basis of such an assessment, we studied the chemical changes occurring in the mineral after exposure of a synthetic hydroxyapatite ceramic to both in vivo (implantation in human) and in vitro (cell culture) conditions. A small amount of the material was phagocytized but the major remaining part behaved as a secondary nucleator as evidenced by the appearance of a newly formed mineral. Morphologically, the newly formed mineral appeared as tiny crystals precipitated and grown from the surface of the initial synthetic crystals. The density of the additional mineral increased from the periphery to the core of each biomaterial aggregate. Chemically, it was identified by IR spectroscopy as a carbonated apatitic mineral. We propose that the adsorption of biomolecules could inhibit precipitation, accounting for the increasing amount of precipitate from the periphery to the core of the aggregates.

Hydroxyapatite biomaterial implanted in human periodontal defects: an histological and ultrastructural study.

The purpose of the present work was to study the response of human periodontium to hydroxyapatite biomaterial particles (180-200 microns). The biomaterial was implanted in two infra-osseous periodontal defects (two patients) after clearing of the granulation tissue. At two months post-surgery, biopsies were studied using light and electron microscopy. No sign of inflammation was observed, the biomaterial aggregates were surrounded either by typical fibroblasts or larger phagocytotic cells with phagocytosis vesicles containing biomaterial crystals. These intracellular crystals were noticeably smaller than the non-phagocytized ones. Some of the phagocytized crystals showed morphological signs of intracellular dissolution. The spaces between the crystals constitutive of the aggregates were filled with organic substance containing collagen fibers.

Solution-mediated transformation of octacalcium phosphate (OCP) to apatite.

OCP crystals were hydrolyzed in solutions containing Ca2+, Mg2+, HPO4(2-), CO3(2-), F-, citrate or P2O7 ions. Products of hydrolysis were analyzed using scanning (SEM) and transmission (TEM) electron microscopy, infrared spectroscopy and x-ray diffraction. Results demonstrated that the OCP to Apatite (AP) transformation is influenced by: (1) types of ions in solution: inhibited by Mg2+, citrate or P2O7(4-); facilitated by F-, CO3(2-), HPO4(2-) or Ca2+ ions; (2) ionic concentrations; (3) solution pH; (4) OCP crystal size. SEM showed needle-like micro-crystals on the surfaces and ends of OCP macrocrystals. TEM showed side-to-side and end-to-end arrangements and presence of central defects in the apatite crystals. IR spectra showed the incorporation of CO3, or HPO4, the HPO4 incorporation being least from F-containing solutions. These results suggest that OCP to AP transformation occurred by the process of dissolution of OCP and subsequent precipitation of Ca-deficient apatites, incorporating CO3(2-), HPO4(2-) or F- present in solution. These results indicate that the observed stability of OCP in pathological calcifications may be due to the presence of Mg2+, citrate and/or P2O7(4-) and/or low levels of CO3(2-), F-, Ca2+, HPO4(2-) ions in the biological fluids.

Scanning electron microscopy and electron probe microanalyses of the crystalline components of human and animal dental calculi.

A review of the use of scanning electron microscopy (SEM) and electron probe microanalyses in the study of dental calculus showed that such studies provided confirmatory and supplementary data on the morphological features of human dental calculi but gave only limited information on the identity of the crystalline or inorganic components. This study aimed to explore the potential of combined SEM and microanalyses in the identification of the crystalline components of the human and animal dental calculi. Human and animal calculi were analyzed. Identification of the crystalline components were made based on the combined information of the morphology (SEM) and Ca/P molar ratios of the crystals with the morphology and Ca/P molar ratio of synthetic calcium phosphates (brushite or DCPD; octacalcium phosphate, OCP; Mg-substituted whitlockite, beta-TCMP; CO3-substituted apatite, (CHA); and calcite. SEM showed similarities in morphological features of human and animal dental calculi but differences in the forms of crystals present. Microanalyses and crystal morphology data suggested the presence of CaCO3 (calcite) and CHA in the animal (cat, dog, tiger) and of OCP, beta-TCMP and CHA in human dental calculi. X-ray diffraction and infrared (IR) absorption analyses confirmed these results. This exploratory study demonstrated that by taking into consideration what is known about the crystalline components of human and animal dental calculi, combined SEM and microanalyses can provide qualitative identification.

Transmission electron microscopy of the morphological relationship between fibroblasts and pulp calcifications in temporary teeth.

Calcifications found in the coronal pulps of primary teeth extracted in an 8-year-old child were studied by TEM. Different types of relationship were observed between fibroblasts and pulp calcifications: extension of cell processes towards calcifications, modelling of the cells upon calcifications, internalizing process of calcifications. Fibroblasts proved to be able to enclose small pulp calcifications within intracytoplasmic vesicles. There was no evidence of any active role played by fibroblasts in the genesis of pulp calcifications. It was shown that collagen fibres could be involved or not in the mineralizing process. It is suggested that mitochondria might provide an adequate environment for initial mineralization. It is likely that the role played by the cytoskeleton in the internalizing process of calcifications is important.

In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior.

The effect of synthetic granular hydroxyapatite (HAP) on cultured fibroblastic cells (L929, human bone and gingiva cells) was studied. Phagocytosis of HAP particles and resulting morphological cell changes were demonstrated by microscopic examinations. Cell counts and [3H]thymidine uptake indicated significant increases in cell proliferation and DNA synthesis. These results could account for some of the alterations of the fibroblast behavior induced by changes in intracellular levels of calcium ions released from the material.