HEMA, pHEMA, poly (2-hydroxyethyl) methacrylate, alkaline phosphatase, mineralisation, bone mineralization, calcification, image analysis, bone biomaterial, bone substitutes, bone graft, bone grafting, poly (2-hydroxyethyl) methacrylate, biomateriau, tissu osseux, polyméthacrylique, HEMA, pHEMA, poly (2-hydroxyéthyl) methacrylate, substitut osseux, matériau hybride, phosphatase alcaline, alkaline phosphatase, minéralisation, bone mineralization, calcification, image analysis, bone biomaterial, bone substitue, bone graft, bone grafting, poly (2-hydroxyethyl) methacrylate, biomatériau, tissu osseux, polyméthacrylique, HEMA, pHEMA, poly (2-hydroxyethyl) méthacrylate, substitut osseux, matériau hybride, biomimetism, biomimetic material, phosphatase alcaline, alkaline phosphatase, minéralisation, bone mineralization, calcification, image analysis, bone biomaterial, bone substitutes, bone graft, bone grafting, poly (2-hydroxyethyl) methacrylate.

French version:

A biocompatible polymer p(HEMA)

by Daniel CHAPPARD

GEROM-LHEA, CHU Angers           Updated: 20 january 2014

  

Polymer biomaterials as Bone substitutes 

At the present time, numerous biomaterials are available as bone substitutes. They are used either in orthopedic surgery (fillings of bone defects, hip prostheses, osteotomy…), in neurosurgery (vertebral fusion), in reconstructive surgery, in dental surgery (and especially in parodontology and implantology). If one excepts the use of bone autograft, (whose volume is necessarily limited, with additional problems of harvesting), none of the biomaterials available have true osseoinduction capacities (i.e., the capacity to induce locally the differention of mesenchymal stem cells into actively bone-forming osteoblasts). Moreover, only purified bone allografts and bone xenografts have a sufficient mechanical resistance that allow them to be used in load-bearing areas. These materials are osteoconductive and allow a guided bone regeneration supported by the material architecture and the physicochemical states of bone surfaces. According to their biochemical nature, these materials are more or less resorbable. Nowadays, it appears clearly that an improvement of bone substitutes should be done to induce:

  1. an increased adherence of osteoblasts onto the material surface,

  2. an in situ differentiation of osteoblast progenitor cells into active osteoblasts,

  3. an increased capacity for osteoblasts to produce larger amounts of bone matrix.

Their is an increasing demand for the development of new synthetic biomaterials. Polymers can represent a very good answer to this challenge.

 


Alkaline Phosphatase in ROS 17/2.8 osteoblast-like cells
(confocal microscopy )

Poly (2-hydroxyethyl) methacrylate

(2-hydroxyethyl) methacrylate (HEMA) is an hydrosoluble monomer, which can polymerize (under various circumstances) at low temperatures (from -20°C to + 10°C), It can be used to prepare various hydrogels, to immobilize proteins or cells. It is widely used in medicine as a biomaterial as reviewed in several papers from our group [1-2]. The hydroxyethyl pending species of the polymer confer a high hydrophilicity, a good biocompatibility, and these groups can be used (after chemical modification or grafting) to complex various types of molecules or ions. The hardness of the polymer, obtained by bulk polymerization, depends upon the amount of water used to prepare the hydrogel: with a reduced amount of water, the mechanical resistance of the polymer is very similar to bone (or even higher). There are questions in the literature about the possibility for cells of the monocyte/macrophage lineage to degrade and resorb pHEMA after in vivo implantations (see below).

Immobilization of  proteins in the pHEMA

Immobilization of proteins can be done at low temperature to preserve the structure and the biological activity of a protein. Polymerization can be done at low temperature under strictly controlled conditions by a redox system. Alkaline phosphatase (AlkP)(EC 3.1.3.1.) is a phosphohydrolase which uses orthophosphoric mono-esters as substrates at high pH (pH between 7.6 and 8.5). AlkP is synthesized by osteoblasts and chondrocytes and is implied in the mineralization process of bone and cartilage. With our technique, we have found that the spatial repartition of the protein within the polymer was homogenous as shown by image analysis (9-12). When pellets of pHEMA-AlkP are incubated in a synthetic body fluid (mimicking the ionic composition of the human extracellular fluid), calcium-phosphate deposits can be observed onto the surface and inside the pellet. On the contrary, pellets of pHEMA alone do not induce calcification. An EDX microanalysis has confirmed that calcium/phosphate crystals (with a Ca/P ratio of 1.47) develop down to 30mm under the surface of the pellet. The crystals gradually grow and continue to develop on the surface of the material, giving calcified globules (calcospherites) containing hydrated octacalcium phosphate Ca8H2(PO4)6 , 5H2O, hydrated tri-calcium phosphate Ca3(PO4)2, xH2O and hydroxyapatite Ca10(PO4)6(OH)2. These calcospherites have the same characteristics and size than those observed in the human fetal growth cartilage and in rapidly growing bone (woven bone) observed in fracture callus.
Homogenous distribution of AlkP particles embedded in pHEMA (image analysis)


Calcospherites in human fetal growth plate (SEM)


Calcospherites developed on pHEMA-AlkP polymer (SEM)
 

 

Bisphosphonates, calcification and  pHEMA-AlkP

The pHEMA-AlkP biomaterial is a cell-free system for the study of calcification. It can also be used to investigate the direct effect of pharmacological and environmental factors on the growth of hydroxyapatite crystals (independently of their cellular effects). Bisphosphonates are pharmacological compounds characterized by a P-C-P backbone, closely related to the P-O-P structure of pyrophosphate, a widely distributed calcification inhibition in the extra cellular body fluids. Pyrophosphate can easily be hydrolyzed by numerous enzymes (pyrophosphatase, alkaline phosphatases) but bisphosphonates are extremely resistant to hydrolysis. We have studied the influence of 3 bisphosphonates (etidronate, alendronate and tiludronate) on the calcification process by using this cell free polymer system. The experiment was conducted in parallel on earth and in weightless ness conditions during the STS80 flight of the US space shuttle (December 1996). Pellets of pHEMA – AlkP were incubated in specially designed vials (DMDA, Dual Materials Dispersion Apparatus, ITA : Instrumentation Technology Associated Inc., Exton, PA) during a 17 day flight [12].

The hydrolytic activity of alkaline phosphatase is totally abolished with a 10-2 to 10-2 M concentration of any bisphosphonates. Such inhibition has been related to the ability of these compounds to chelate Zn2+(Zinc acts as an enzyme cofactor). Etidronate and alendronate completely inhibited the formation of calcospherites by this mechanism [13].

On the other hand, tiludronate had a less potent effect on calcium-phosphate crystallization at similar concentrations. In microgravity conditions, during the STS-80 flight of the space shuttle, the polymer calcification was markedly impaired by etidronate and abnormally large Ca-P crystals were obtained when tiludronate was added in the incubation medium. However, these results were obtained in non-physiologic conditions (the temperature was about 20°C during the space flight). It is likely that our pHEMA – AlkP material will represent a good system to investigate the effects of other pharmacological drugs (or other environmental factors) on the mineralization of bone. This system is by far superior to the classical calcium-oxalate model; it is an acellular model that mimics the physiological calcification process occurring in human bone and cartilage. We welcome pharmacological proposals.

 


 sodium etidronate

Amount of calcium deposited on pHEMA in presence of
various concentration of 3 bisphosphonates.

 

 

The carboxymethylated pHEMA biomaterial and mineralization in the presence of metal ions and proteins. 

Carboxymethylation of pHEMA surface confers the property to induce calcification in the absence of cells and proteins. Grafting carboxymethyl groups on the hydroxyl function of the polymer induces mineralization after immersion in a synthetic body fluid [15] . Mineralization of pHEMA mimics the calcification of human woven bone with the formation of calcospherites. This model allowed us to understand the action of different metal ions or molecules during calcification.

The role of iron : In vitro, Fe2+ ions can be incorported in tthe HAP crystals of the bone matrix and they induce an inhibition of crystal growth leading to an altered crystallinity and mechanical properties of the crystal [26]. In human pathology, iron is hyper-absorbed during hemochromatosis which is associated with osteoporosis. Clinically, patients have iron deposits inside the bone matrix which can be identified histologically by Perls staining.

The role of Co2+ , Ni2+ and Cr3+ : these metals are widely used in orthopedic implants, these ions have been placed in the body fluid during the incubation with pHEMA pellets [27]. In the presence of these ions, the calcospherite size was found greatly increased when compared to controls, crystallinity of the mineral formed was increased similarly. The lattice parameters of the HAP crystal have also undergone changes indicating a possible alteration of bone mineral.

The role of strontium: Strontium Sr2+ is an ion that has been used as a treatment for osteoporosis; it can be affixed to the hydroxyapatite crystal. We showed that this binding is weak and can be easily released [29].

The role of aluminum: Aluminum enters the composition of many alloys such as TA6V but also alumina ceramics used as biomaterials. Al is an ion which is can be fixed to hydroxyapatite crystals by complexing phosphate . Clinically, patients with chronic renal insufficiency who were dialyzed with water containing large amounts of aluminum exhibited mineralizationdefect (osteomalacia) with aluminum deposits in the bone matrix (this was first observed in the years 1970-1980). Currently , Al can cause aluminic osteoporosis in patients with digestive disorders when they consume a lot of gastro-protectors containing alumina. Carboxymethylated pHEMA pellets have shown that very small quantities of aluminum ion, and even the addition of commercial aluminum foils in the incubation medium, resulted in a profound inhibition of calcification [30] .

This model of calcification based on functionalized pHEMA also permits the study of adsorption of non-collagenous bony proteins on the surface of the formed mineral. In the presence of osteocalcin, calcospherites double in size while the Ca/P and Ca and PO4 concentrations report does not change. In the presence of fetuins, calcospherites are smaller, the Ca/P ratio increases and concentrations of Ca and PO4 decrease. Protein adsorption was performed in Raman spectroscopy [28].

 

aluminum

A) Control calcospherites B) after incubation with 40g/L d'ion Al3+, C )after incubation with 6 pieces of aluminum foil (10 x 20 mm) (MEB).
 

The pHEMA-AlkP biomaterial and osteoblast-cell adherence  

Because of the high hydrophylicity of pHEMA, osteoblasts like cells have a low tendency to spread and flatten on the polymer surface. When pHEMA-AlkP pellets have been incubated in body fluid, they are covered with calcospherites. Osteoblast-like cells seeded on such Ca-P nodules exhibit a preferential adherence and attachment on to the nodules. They flatten and anchor via long pseudopods extending to the calcospherites while thin and short filopods are directed from the cell body to the polymer surface. The number of filopods and pseudopods was measured by image analysis on scanning electron microscopy images. The number of filopods decreases and the number of pseudopods increases on cells seeded on calcospherite-covered polymer pellets [14].

Osteoblast-like cells adherence was found to be mediated by Bone Sialoprotein (BSP) and fibronectin adsorbed at the surface of elementary hydroxyapatite crystals forming the calcospherites. Both molecules were identified by confocal microscopy with fluorescent-labelled antibodies or by the immunogold technique in transmission electron microscopy. BSP and fibronectin are molecular glues that are also synthesized and released by the cells themselves to facilitate adherence. Both proteins readily adsorbed on the hydrophobic hydroxyapatite crystals but not on the polymer itself.

 

 


A ROS 17/2.8 osteoblast-like cell spread onto calcospherites that have developed on the surface of pHEMA-AlkP polymer.
 

A ROS 17/2.8 osteoblast-like cell spread onto calcospherites that have developed on the surface of pHEMA-AlkP polymer, image analysis: filopods in yellow, pseudopods in green, calcospherites pink and orange.
The pHEMA-AlkP biomaterial and macroporosity

It is actually largely admitted that a biomaterial usable as a bone substitute must be porous. This ensures invasion by bone cells and vascular sprouts rapidly to the center of the graft. It has been demonstrated that the pore size should be larger than 100µm in diameter and that pore interconnectedness is also a very important factor. Few techniques are now available in the literature to prepare interconnected porous polymer blocks. We have developed a technique to prepare porous blocks of pHEMA with large and interconnected canals. Porosity and 3D morphology of these porous blocks was measured by X-ray microtomography on a Skyscan 1072 system (this is the first published example of 3D analysis of a biomaterial by this technique) [16].

Interconnectedness of the pores was measured by image analysis algorithms previously described by our group for bone histomorphometry. Diffusion of fluids inside large blocks of polymer was evidenced by a histochemical method that detected carboxylic groups grafted on the polymer surface |15].

 


A block of pHEMA with an interconnected porosity. X-Ray microtomography. The polymer is figured in blue with a semi-transparency effect, the green channels are empty holes created after dissolution of the porogen.

A block of pHEMA with a non-interconnected porosity. X-Ray microtomography. The polymer is figured in violin with a semi-transparency effect, the yellow spheres are empty holes created after dissolution of the porogen.
  The pHEMA-AlkP biomaterial and  phosphate pending groups

The possibility that pending phosphate groups on the surface of a polymer could increase mineralization was investigated. In the living world, calcification of organic matrices (bone, tooth, egg shell…) necessitates the presence of specialized phosphoproteins. We have prepared copolymers with monomers containing phosphate groups. Methacryloyloxyethyl phosphate (MOEP) was copolymerized with 1-vinyl-2-pyrrolidinone (VP) or (diethylamino) ethylmethacrylate (DEAEMA). The reactivity ratios of MOEP with VP and DEAEMA have been calculated to provide suitable biomaterials. When incubated in synthetic body fluid, polymers containing MOEP were heavily calcified. However, the Ca-P deposits were in the form of large tablets and calcospherites were never observed [17]. Granules of the copolymer (MOEP-VP) were implanted in large calvaria defects of the rat. After 12 weeks, polymer particles were calcified (histochemical and scanning electron microscopy identification with BSE). However there was no osteoconduction of the material [18]. Optimal conditions of copolymerization of HEMA and MOEP have been reviewed to prepare microbeads[25].

 

 Calcification developed on a block of MOEP-co-VP
 

Is pHEMA resorbable in vivo and in vitro by macrophages ?

The in vivo biodegradability of polymers is of the utmost interest. Biodegradation can be interesting in some circumstances when the material must replace a tissue temporarily (e.g. as a bone substitute). Otherwise,  degradation is undesirable when  the biomaterial is used to replace a physiological function definitely or in long term periods (e.g., an intra-ocular lens).

We have recently found that small amounts of a cross-linking reagent (ethylene glycol dimethacrylate) added during polymerization, can dramatically modify the hydrophylicity of the polymer (data obtained with atomic force microscopy - AFM).

We have also studied the in vitro capacity of the macrophage J774.2 cell line to be activated when cells are in direct contact with polymer beads. The activation occurs in contact with the polymer surface, whether it is linear or cross-linked. With linear polymer, the peripheral layer of the material is highly hydrated and this favors resorption by macrophage cells. Phagocytosis of polymer fragments have been observed by transmission electron microscopy [19]. So, pHEMA can be eroded by macrophages specially when polymerized without cross-linking agents.

 

 AFM views of the surface of a dried pellet of linear pHEMA (top)
and an hydrated linear pHEMA pellet (bottom).
  Surface topography of dry and hydrated pHEMA

The surface topography of an implantable material is a significant parameter in the appreciation of biocompatibility. The surface roughness, for example, can notably influence the adherence of certain cellular types (fibroblasts, osteoblasts…). The surface roughness a biomaterial can be visualized by different methods like scanning electron microscopy (SEM). However, it is often of importance to have roughness measurements; methods like contact profilometry, optical profilometry or atomic force microscopy (AFM) can provide average roughness (Ra, fractal dimension…). However, these methods are not easily applicable to strongly hydrated hydrogels (dehydration is imperative for SEM, the probe is "limed" in the highly swollen layers of the material in AFM). New technologies such as environmental microscopy can allow visualization but do not give roughness quantification.

We showed that texture analysis applied to the images of the surface of hydrophilic polymers constituted a simple method for roughness appreciation. The surface of a hydrophilic polymer is illuminated with an external fibre optic illuminator providing cold light to limit the evaporation of water. Direction of the light is positioned at a 30° angle to the surface of the disks to increase the details of the surface. In this way, details of the polymer surface are clearly highlighted. When the polymer is placed in an aqueous fluid, the relaxation of the surface chains smoothen these irregularities [20]. Addition of a cross-linker limits the swelling of polymer. When images of the polymer surface are taken at successive times of hydration, the measurement of the fractal dimension of these images is highly correlated with the swelling rate [21].

 

Aspect of the surface of a dry pHEMA pellet (A) and after 2hours immersion in saline (B)
 

Immobilization of bioactive molecules in pHEMA

Biomaterials, and especially polymers, can be used as carriers to deliver growth factors directly in bone defects. We have used pHEMA cylinders to immobilize basic fibroblast growth factor (FGF-2). Cylinders were grafted in the supra-critical defects created in the distal femoral epiphysis of rabbits and healing was followed at 2 and 3 months after surgery. Microcomputed tomography (microCT) was used to measure and observe the bone formation around the grafted area: bone trabeculae appeared as a thin lace formed at the surface of the polymer [22].

 

Aspect of the bone formed at the surface of a pHEMA cylinder with controlled release of FGF-2
  A new less toxic initiator/accelerator polymerization system

HEMA is often polymerized by radicalar methods using free radicals that are liberated during a redox reaction. In a redox reaction, a polymerization accelerator (usually benzoyl peroxide) is decomposed by an initiator (usually a substituted amine as NN-dimethyl aniline or NN dimethyl paratoluidine) and this release free radicals. Free radicals induce the aperture of the double bond of a HEMA molecule and a radical is transmitted to this unstable monomeric molecule which can in turn react (and be grafted on) with a new monomer molecule. However the use of substituted amines generates brownish side reaction products that are trapped inside the polymer and have been shown to be irritative. Furthermore, the benzoyl peroxide/amine system generates a large amount of heat during polymerization which can proceed very rapidly. We have propose to use a new redox system in which benzoyl peroxide is decomposed by ascorbic acid (vitamin C). The polymerization kinetics were studied by Raman microspectroscopy which allows a quantitative study of the disappearance of the C=C double bond of the monomer upon time. In addition, Raman analysis showed that -COOH residues appear on the polymer and could be due to the fragmentation of ascorbic acid [23].

Polymerization of pHEMA with benzoyl peroxide and ascorbic acid studied by Raman microspectroscopy.
   

 

Preparation of charged pHEMA microbeads

pHEMA can be polymerized in suspension to prepare microbeads. We have prepared charged microbeads with either positive groups (obtained by copolymerization with 2-(methacryloyloxy) ethyl acetoacetate -MOEAA) or negative groups (by copolymerization with diallyldimethylammonium chloride -DADMAC).

A fluorescent dye (Nile red) was incorporated inside the microbeads at the time of polymerization. Fluorescent microbeads were seeded on endothelial cells and were rapidly internalized.

 

 

Analysis by transmission electron microscopy and confocal microcopy evidenced that both types of beads were internalized by the endothelial cells but negatively charged appeared more easily phagocytized. Such microbeads can be used to analyze tumoral angiogenesis because they can accumulate inside tissues by the EPR effect EPR (Enhanced permeability retention) [24].

 Confocal Microscopy and 3D reconstruction of EA.hy 926 endothelial cells (green pseudo color) cocultured with fluorescent pHEMA microbeads (red pseudocolor). A) only the extracellular beads are visible; B) cell cytoplasms have been made semi transparent and internalized beads are now visible.
Short list of references of our research group INSERM Unit 922 and GEROM on pHEMA-based biomaterials
  1. Chappard D, Laurent JL, Camps M, Montheard JP. A simple and reliable method for purifying glycol methacrylate for histopathological studies. Acta Histochem. 71, 95-102, 1982.

  2. Chappard D, Alexandre C, Palle S, Montheard JP, Riffat G. Improved stability of a purified glycol methacrylate preparation: comments. Stain Technol. 61, 185-186,1986

  3. Chappard D, Alexandre C, Camps M, Montheard JP, Riffat G. Embedding iliac bone biopsies at low temperature using glycol and methyl methacrylates. Stain Technol. 58, 299-308, 1983.

  4. CHAPPARD D., ALEXANDRE C., RIFFAT G. Orientation and handling of large blocks during embedding in Glycol-methacrylate (GMA) for High Performance Optical Microscopy. Mikroskopie, 41; 1-3, 1984.

  5. CHAPPARD D., BENASCAR Z., ALEXANDRE C., MONTHEARD J.P. Practical investigation of radical polymerization of glycolmethacrylate as an embedding medium. J. Histotechnol. 12, 89-94, 1989.

  6. CHAPPARD D., MONTHEARD ;J.P., CHATZOPOULOS M., ALEXANDRE C., Utilisations biomédicales d'une résine hydrophile : le 2-hydroxy éthylméthacrylate. ITBM Innovation et Technologie en Biologie et Médecine 13, 322-340, 1992.

  7. MONTHEARD J.P., CHATZOPOULOS M., CHAPPARD D., 2-hydroxyethyl methacrylate (HEMA): chemical properties and applications in biomedical fields. J. Macromol. Sci., Macromol. Rev. 32, 1-34, 1992.

  8. MONTHEARD J.P., KAHOVEC J., CHAPPARD D., Homopolymers and copolymers of 2-hydroxyethyl Methacrylate for biomedical applications. In «Desk reference of functional polymers; Syntheses and applications» Arshady R., Ed. American Chemical Society, Washington DC, Chapter 5.3, 699-717, 1997.

  9. FILMON R., CHAPPARD D., MONTHEARD J.P., BASLE MF. A composite biomaterial: poly 2(hydroxyethyl) methacrylate / Alkaline phosphatase (pHEMA / AlkP) initiates mineralization in vitro. Cells Mater. 6, 11-20, 1996.

  10. FILMON R., CHAPPARD D., BASLE MF. Scanning and Transmission Electron microscopy of poly 2(hydroxyethyl) methacrylate-based biomaterials. J. Histotechnol., 20, 343-346, 1997.

  11. Chappard D, Gaborit-Retailleau N, Montheard JP, BaslE MF. Photo polymerized 2-hydroxyethyl methacrylate is a mounting medium preserving immunocytochemical reaction and nuclear counterstain. Biotech. Histochem. 74, 135-140, 1999.

  12. Filmon R, Basle MF, Barbier A, Chappard D. Etude in vitro de l'influence des bisphosphonates sur la mineralisation induite par un materiau composite : le poly (2-hydroxyethyl) methacrylate couplé à la phosphatase alcaline. Morphologie, 84, 23-33, 2000.

  13. Filmon R, Basle MF, Barbier A, Chappard D. Poly 2(hydroxyethyl) methacrylate- alkaline phosphatase: a composite biomaterial allowing in vitro studies of bisphosphonates on the mineralization process. J. Biomater. Sci. Polym. Ed., 11, 849-868, 2000.

  14. Filmon R, Basle MF, Atmani H, Chappard D. Preferential adherence of osteoblast-like cells on calcospherites developed on poly (2-hydroxyethyl) methacrylate-AlkP biomaterial. Bone 30,152-158, 2002.

  15. Filmon R, Grizon F, Basle MF, Chappard D. Effects of negatively charged groups (carboxymethyl) on the calcification of poly (2-hydroxyethyl) methacrylate Biomaterials, 23, 3053-3059, 2002.

  16. Filmon R, Retailleau-Gaborit N, Grizon F, Galloyer M, Cincu C, Basle MF, Chappard D Non-connected versus inter-connected macroporosity in poly (2 hydroxyethyl methacrylate) polymers. A X-ray microtomographic and histomorphometric study. J. Biomater. Sci. Polym. Ed. 13, 1105-1117, 2002.

  17. STANCU I.C., FILMON R., CINCU C., MARCULESCU B., ZAHARIA C., TOURMEN Y., BASLÉ M.F., CHAPPARD D. Synthesis of methacryloyloxyethyl phosphate copolymers and in vitro calcification capacity. Biomaterials 25,205-213, 2003.

  18. Stancu IC, Filmon R, Grizon F, Zaharia C, Cincu C, Basle MF, Chappard D. The in vivo calcification capacity of a copolymer, based on methacryloyloxyethyl phosphate, does not favor osteoconduction. J Biomed. Mater. Res. 69A, 584-589,

  19. Mabilleau G, Moreau MF, Filmon R, Basle MF, Chappard D. Biodegradability of poly (2-hydroxyethyl methacrylate) in the presence of the J774.2 macrophagic cell line. Biomaterials 25, 5155-6512, 4.

  20. Mabilleau G., Stancu I. C., Honoré T., Legeay G., Cincu C., BaslE M.F., CHAPPARD D. Effects of the chain length of cross-linkers on poly(2-hydroxyethyl methacrylate) (pHEMA) swelling and biomechanical properties J. Biomed. Mater. Res A., 77: 35-42, 2006.

  21. Mabilleau G., BaslE M.F., CHAPPARD D. Evaluation of surface roughness of hydrogels by fractal texture analysis during swelling. Langmuir, 22, 4843-4845, 2006.

  22. Mabilleau G., Aguado E., Stancu I.C.CINCU C., BaslE M.F., Chappard D. Effects of FGF-2 release from a hydrogel polymer on bone mass and microarchitecture. Biomaterials, 29, 1593 – 1600, 2008.

  23. Mabilleau G., Cincu C., BASLE M.F., CHAPPARD D. Polymerization of 2- (hydroxyethyl) methacrylate by two different initiator / accelerator systems: a Raman spectroscopic monitoring. J. Raman Spectroscopy, 39, 767–771, 2008.

  24. NYANGOGA H., ZECHERU T., FILMON R., BASLÉ M.F., CINCU C., CHAPPARD D. Synthesis and use of pHEMA microbeads with human EA.hy 926 endothelial cells.J Biomed Mater Res Part B: Appl. Biomater. 89, 501-507, 2009.

  25. ZECHERU T., SĂLĂGEANU A., CINCU C., CHAPPARD D., ZERROUKHI A. Poly(HEMA-co-MOEP) microparticles: Optimisation of the preparation method and in vitro tests. UPB Scientific Bul., Series B: Chem. Mater. Sci. 70, 45-54, 2008.

  26. GUGGENBUHL P., FILMON R., MABILLEAU G., M.F. BASLE., CHAPPARD D. Iron inhibits hydroxyapatite crystal growth in vitro. Metabolism. 57, 903-910, 2008.

  27. MABILLEAU G., FILMON R., PETROV P.K., BASLE M.F., SABOKBAR A., CHAPPARD D. Cobalt, chromium and nickel affect hydroxyapatite crystal growth in vitro. Acta Biomater. 6, 1555-1560, 2010

  28. LIBOUBAN H., FILMON R., MAUREAC A., BASLE M.F., CHAPPARD D. Fetuin and osteocalcin interact with calcospherite formation during the calcification process of poly (2-hydroxyethylmethacrylate) in vitro. J. Raman Spectrosc., 40, 1234-1239, 2009.

  29. BEUVELOT J., FILMON R., MAURAS Y., BASLE M.F., CHAPPARD D. Adsorption and release of strontium from hydroxyapatite crystals developed in Simulated Body Fluid (SBF) on poly (2-hydroxyethyl) methacrylate substrates. Dig. J. Nanomater Biostruct. 8, 207 - 217, 2013.

  30. DEGERATU C.N., MABILLEAU G., CINCU C., CHAPPARD D. Aluminum inhibits the growth of hydroxyapatite crystals developed on a biomimic methacrylic polymer. J Trace Elem Med Biol,4, 3, 346-351, 2013.

 

 
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