Robert Filmon

Osteoclastic resorption of Ca-P biomaterials implanted in rabbit bone.

SummaryThe nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium-phosphate biomaterials grafted into rabbit bone was studied using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, scanning, and electron microscopy. Samples were taken at days 7, 14, and 21 after implantation. As early as day 7, osteogenesis and resorption were observed at the surface of the biomaterials, inside the macropores. Resorption of both newly formed bone and calcium-phosphate biomaterials was associated with two types of multinucleated cells. Giant multinucleated cells were found only at the surface of the biomaterials; they showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of mineral crystals from the biomaterials. Osteoclasts exhibited TRAP positivity and well-defined ruffled border. They were observed at the surface of both newly formed bone and biomaterials, around the implant, and inside the macropores. In contact with the biomaterials, infoldings of their ruffled border were observed between the mineral crystals, deeply inside the microporosity. The microporosity of the biomaterials (i.e., the noncrystalline spaces inside the biomaterials) increased underneath this type of cell as compared with underneath giant cells or to the depth of the biomaterials. These observations demonstrate that macroporous calcium-phosphate biomaterials implanted in bone elicit osteogenesis and the recruitment of a double multinucleated cell population having resorbing activity: giant multinucleated cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials.

Poly(2-hydroxy ethyl methacrylate)-alkaline phosphatase: a composite biomaterial allowing in vitro studies of bisphosphonates on the mineralization process.

We have immobilized the mineralizing agent alkaline phosphatase (AlkP) in a hydrophilic polymer: poly(2-hydroxy ethyl methacrylate) - (pHEMA) - in a copolymerization technique. Histochemical study on polymer sections revealed that AlkP has retained its enzymic activity. The image analysis of sections using a tessellation method showed a lognormal distribution of the area of the tiles surrounding AlkP particles, thus confirming a homogeneous distribution of the enzyme in the polymer. Pellets of pHEMA-AlkP were incubated with a synthetic body fluid containing organic phosphates (β-glycerophosphate). Mineral deposits with a rounded shape (calcospherites) were obtained in about 17 days. We have investigated the effects of three bisphosphonic pharmacological compounds (etidronate, alendronate and tiludronate) on this system which mimics the mineralization process of cartilage and woven bone. Bisphosphonates at a concentration of 10-2 M totally inhibited AlkP in solution at a concentration of 10-4 mg/ml. Inhibition has been reported being due to the chelation of a metal cofactor (Zn2+). Etidronate and alendronate appeared to similarly inhibit the calcospherite deposition onto the pHEMA-AlkP material. Both bisphosphonates possess three sites for the mineral complexion by Ca chemisorbtion. On the other hand, tiludronate having only two sites, was associated with a reduced inhibitory effect on mineralization but larger crystals were obtained. The pHEMA-AlkP material contains an immobilized enzyme in a hydrogel and mimics the physiological conditions of matrix vesicles entrapped within the cartilage(or bone) matrix. It provides an interesting method to study the effects of pharmacological compounds on the mineralization process in bone and cartilage in a non cellular and protein-free model.

Non-connected versus interconnected macroporosity in poly(2-hydroxyethyl methacrylate) polymers. An X-ray microtomographic and histomorphometric study.

Poly(2-hydroxyethyl methacrylate) (pHEMA) has potentially broad biomedical applications: it is biocompatible and has a hardness comparable to bone when bulk polymerized. Porous biomaterials allow bone integration to be increased, especially when the pores are interconnected. In this study, three types of porogens (sugar fibers, sucrose crystals, and urea beads) have been used to prepare macroporous pHEMA. The pore volume and interconnectivity parameters of the porosity were measured by X-ray microtomography and image analysis. Sucrose crystals, having a high volumetric mass, gave large pores that were located on the block sides. Urea beads and sugar fibers provided pores with the same star volume (2.65 ± 0.46 mm3 and 2.48 ± 0.52 mm3, respectively) but which differed in interconnectivity index, fractal dimension, and Euler–Poincarés number. Urea beads caused non-connected porosity, while sugar fibers created a dense labyrinth within the polymer. Interconnectivity was proved by carrying out surface treatment of the pHEMA (carboxymethylation in water), followed by von Kossà staining, which detected the carboxylic groups. Carboxymethylated surfaces were observed on the sides of the blocks and on the opened or interconnected pores. The disconnected pores were unstained. Macroporous polymers can be prepared with water-soluble porogens. X-ray microtomography appears a useful tool to measure porosity and interconnectedness.

Cellular response to calcium phosphate ceramics implanted in rabbit bone

Two hydroxyapatite ceramics, synthesized by sintering from bovine bone and from a mixture of phosphate tricalcium and natural hydroxyapatite, were implanted in bone sites in rabbits. From day 7 after implantation, osteoblast-like cells were visible with thin layers of new bone on both biomaterials. Histomorphometry showed progressive increase in volume and surface of newly formed bone. Signs of cell-dependent resorption were visible at the surface of biomaterials and newly formed bone. There was a progressive decrease in relative volume and trabecular thickness of the biomaterials. Resorption of biomaterials appears to involve two cell types: multinucleated giant cells and osteoclast-like cells. The multinucleated giant cells observed had neither tartrate resistant acid phosphatase activity (TRAP) nor a ruffled border. Vesicles and vacuoles containing crystals observed in these cells suggest phagocytosis of biomaterials. The number of these cells decreased after day 14 following implantation. The osteoclast-like cells were TRAP positive. The structured modification and the TRAP activity demonstrated in the subjacent biomaterial suggest that the dissolution of the implant may be associated to an extracellular enzymatic activity of these cells. Electron microscopy revealed a clear zone and cytoplasmic membrane infolding in these cells, suggesting a ruffled border differentiation. The number of these cells increased with delay after implantation. It was concluded that the implantation of calcium phosphate ceramics in bone leads to new bone formation as well as to resorption of the biomaterials. The mechanism of resorption appears to associate crystal endocytosis by multinucleated giant cells and more classical resorption by osteoclast-like cells.

Biomimetic potential of some methacrylate-based copolymers: A comparative study

Preparation of new biocompatible materials for bone recovery has consistently gained interest in the last few decades. Special attention was given to polymers that contain negatively charged groups, such as phosphate, carboxyl, and sulfonic groups toward calcification. This present paper work demonstrates that other functional groups present also potential application in bone pathology. New copolymers of 2-hydroxyethyl methacrylate with diallyldimethylammonium chloride (DADMAC), glycidyl methacrylate (GlyMA), methacrylic acid (MAA), 2-methacryloyloxymethyl acetoacetate (MOEAA), 2-methacryloyloxyethyltriethylammonium chloride (MOETAC), and tetrahydrofurfuryl methacrylate (THFMA) were obtained. The copolymers were characterized by FTIR, swelling potential, and they were submitted to in vitro tests for calcification and cytotoxicity evaluation. GlyMA and MOETAC-containing copolymers show promising results for further in vivo mineralization tests, as a potential alternative to the classical bone grafts, in bone tissue engineering.

Hereditary leukonychia totalis, acanthosis-nigricans-like lesions and hair dysplasia: a new syndrome?

Leukonychia is an ungueal discoloration or dyschromia. The hereditary form is rare. In the observations reported in the literature, leukonychia was total or sub-total, and was sometimes associated to other various symptoms. We report an original observation of hereditary leukonychia totalis in a father and two of his children, associated with acanthosis-nigricans-like lesions and hair dysplasia. These symptoms were also present in eight other members of the same family.