B. Dupuy

Cellular biocompatibility and resistance to compression of macroporous β-tricalcium phosphate ceramics

The main problem for macroporous structures used as bone substitutes is their lower resistances when compared to that of cancellous bone. The present investigation aimed to improve the strength of ceramics with 65% porosities based on beta-TCP. The initial mixtures were rendered plastic by addition of non-ionic carbohydrate binders. Macropores were created using substances which were eliminated by heat. Mechanical tests indicated that the resistance of the ceramics depended more on the quantity than the nature of the binders. Porosity measurements were done with a mercury porosimeter, and cellular biocompatibility was evaluated by performing cellular attachment tests and observing the proliferation of differentiated cells.

Study of a (trimethylenecarbonate-co-ε-caprolactone) polymer-Part 2 : in vitro cytocompatibility analysis and in vivo ED1 cell response of a new nerve guide

Future surgical strategies to restore neurological function in peripheral nerve loss may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. Random copolymers of trimethylene carbonate and epsilon caprolactone (P(epsilonCL-TMC), 50: 50) have been synthesized by ring opening polymerization using rare earth alkoxides as initiator. Their potential use as nerve guide repairs has been assessed through indirect and direct in vitro biocompatibility tests and in vivo soft tissue response to EDI subclass macrophages. In vitro, we exposed monolayers of human skin fibroblasts and an established continuous cell line (Hela) to liquid extracts (either pure or diluted in the culture medium) of epsilonCL-TMC copolymer including positive (phenol) and negative controls. Then, colorimetric assays (Neutral red and MTT) were performed. The extracts of epsilonCL-TMC induced no significant cytotoxic effect. We also exposed in vitro Schwann cells to pieces of P(epsilonCL-TMC) and P(LA-GA) copolymers. We evaluated cell attachment at 1 and 3 h by measuring the activity of the lysosomal enzyme (N-acetyl-beta-hexosaminidase) and cell proliferation at 1, 3, 6 and 9 days by measuring the cell metabolic activity (MTT assay). Values for attachment slightly decreased between 1 and 3 h but were significantly higher than on agars (negative control). Cells plated on epsilonCL-TMC showed a rate of proliferation comparable with that of normalized controls and higher than on PGA-PLA at day 9. Finally, we evaluated in vivo the soft tissue response after implantation of cylindrical tubes of P(epsilonCL-TMC) and P(LA-GA) copolymers with an immunohistochemistry staining procedure for the newly recruited ED1 macrophages. An image analysis system automatically measured the optical density of labelled positive ED1 cells at 9, 21 and 60 days after implantation. epsilonCL-TMC copolymer showed a mild soft tissue reaction with no adverse chronic inflammatory reaction. These data allowed us to consider this conduit as a potential effective substitute in nerve repair. El sevier Science Ltd. All rights reserved.

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

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.

FT-IR of membranes made with alginate/polylysine complexes. Variations with the mannuronic or guluronic content of the polysaccharides

FT-IR spectra of polylysine/alginate membranes made with alginate containing various contents of mannuronic or guluronic acid residues have been recorded. The interpretation of the more important absorptions related to functional groups engaged in the complexes have been proposed and discussed using comparisons with spectra of cellulosic films and other published results. Mannuronnic rich alginate seemed to link stronger than guluronic rich alginate to the polylysine molecules which is illustrated by the continuum in absorption between 3000 cm-1 and 2000 cm-1. However, the analysis of the 2000-1000 cm-1 region prompted us to believe that the polymers were engaged in the same basic sort of molecular complexes. Therefore it is necessary that other parameters (either physical, as toughness, porosity, ...) other than variations in molecular structures are studied in order that the biological differences of the membranes may be explained.

Agarose encapsulation of islets of Langerhans: reduced toxicity in vitro

The physical and chemical properties of agarose have been exploited for encapsulation of islets of Langerhans. Formation of microcapsules by extrusion of a hydrophilic polymer (agarose) into a hydrophobic solution created an interface to which an immunoprotective membrane could be polymerized. The substances and procedure used were devoid of cellular toxicity. Islet function in vitro was found to be normal.

Evolution of the local calcium content around irradiated β-tricalcium phosphate ceramic implants: in vivo study in the rabbit

To evaluate whether dissolved calcium from tricalcium phosphate implants contributes to osseous wound healing in bone defects, the authors used nuclear radioactivated materials. Six months after irradiation, the calcium was still radioactive. Samples of the material were prepared and placed in rabbit condyles for 1, 3 and 9 months. Over time the condyles were retrieved and treated for histology or radiocounting. Measurements of the radioactivity of the slices and histomorphometry of the implants and surrounding tissues were performed. The authors observed that the radioactivity decreased regularly. Connective tissue had penetrated the pores and totally invaded the implants, first at the periphery of the implants, then inside the pores. Comparison of the results of radioactivity and histomorphometry suggest that part of the calcium from the implants was re-used specifically in the new osseous tissue.

Lack of responsiveness to glucose of microencapsulated islets of Langerhans after three weeks' implantation in the rat—influence of the complement

Microencapsulated islets of Langerhans retrieved from peritoneal cavity of rats three weeks after implantation had a reduced hormonal secretion and were no longer responsive to alterations in glucose levels. Activation of complement could not completely account for the observed fibrosis which was thought to be responsible for the loss of responsiveness of the encapsulated cells.

Resorption of corals implanted in diffusion chambers

Natural coral is a resorbable bone substitute currently used in osseous surgery. The action of cellular and interstitial fluids has been incriminated as a possible agent for coral resorption but it has not been possible to discriminate the importance of either factor. The aim of this study was to compare the resorption speed of the coral implant in contact only with biological fluids (coral dishes placed inside diffusion chambers closed with two filter membranes 1.2 μm pore size) or in contact with biological fluids and cells (coral dishes placed inside diffusion chambers closed with the above filters but with holes made with a 22 G needle or coral dishes in direct contact with soft tissue). Qualitative (SEM) and quantitative (gravimetric) results showed that the implants in contact with cells were resorbed faster than those in contact only with biological fluids. The cells in contact with the implant were mainly multinucleated giant cells and some were Trap +. TEM showed multinucleated cells with a ruffled border but without a clear zone or intracytoplasmic inclusions distinguishing them from osteoclasts. With only biological fluids, the latter intervened to a moderate extent in the resorption of coral implants in which the cellular action appears to be dominant. However, this action does not seem to be attributable to osteoclasts.

A two‐step embedding process for better preservation of soft tissue surrounding coral implants

Infiltration of paraffin or embedding polymers proceeds more quickly in soft than in mineralized tissue specimens (bone or biomaterial). The proposed method takes advantage of this difference to protect soft tissue from the action of decalcifying agents. After embedding a bone-soft-tissue sample in a hydrophobic polymer, it is cleared of the resin on one of its sides to permit access of the decalcifying solution to the mineral component. A second infiltration with another polymerizable solution that cures in the empty pores created by the dissolution makes it possible to obtain a homogeneous hard block, make thin slices, and perform enzyme histochemistry and immunostaining on well-preserved soft tissue with most of its antigenic and enzymatic properties intact.

Rationale for the design of biomaterials and the evaluation of their biocompatibility

The biocompatibility of a material can be considered as the ideally expectable result of its interactions with living tissues with which it is interfaced. This property determines the ability of devices involving this material in their constitution, to correctly assume their ascribed function; reciprocally a bad fitting, between devices and their intended use, coming from a non-optimized design or from an inappropriate prescription, may alter the original biocompatibility of constitutive materials. Accordingly, the actual biocompatibility of a biomaterial depends upon both its intrinsic properties and the application in which it is involved. Such considerations must be taken into account by specialists who try to design more performant biomaterials, or new assist devices, should they be implantable or not; but they draw also methodological guidelines for the evaluation of the biocompatibility of these biomedical products.