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A Composite Biomaterial: Poly 2 (Hydropoxyethyl) Methacrylate / Alkaline Phosphatase Initiates Mineralization in Vitro

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A Composite Biomaterial: Poly 2 (Hydropoxyethyl) Methacrylate / Alkaline Phosphatase Initiates Mineralization in Vitro Cells and Materials Volume 6 Number 1 Numbers 1-3 Article 2 1996 A Composite Biomaterial: Poly 2 (Hydropoxyethyl) Methacrylate / Alkaline Phosphatase Initiates Mineralization In Vitro R. Filmon Université d'Angers D. Chappard Université d'Angers J. P. Monthéard Université d'Angers M. F. Baslé Université d'Angers Follow this and additional works at: https://digitalcommons.usu.edu/cellsandmaterials Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Filmon, R.; Chappard, D.; Monthéard, J. P.; and Baslé, M. F. (1996) "A Composite Biomaterial: Poly 2 (Hydropoxyethyl) Methacrylate / Alkaline Phosphatase Initiates Mineralization In Vitro," Cells and Materials: Vol. 6 : No. 1 , Article 2. Available at: https://digitalcommons.usu.edu/cellsandmaterials/vol6/iss1/2 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Cells and Materials by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Cells and Materials, Vol. 6, No. 1-3, 1996 (pages 11-20) 1051-6794/96$5.00 + .25 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA A COMPOSITE BIOMATERIAL: POLY 2(HYDROXYETHYL) METHACRYLATE I ALKALINE PHOSPHATASE INITIATES MINERALIZATION IN VITRO 1 2 1 3 1 2 R. Filmon • , D. Chappard , J.P. Montheard and M.F. Basle • " 1.aboratoire d'Histologie-Embryologie, Faculte de Medecine and CHU d'Angers, 49045 Angers Cedex, France 2I.aboratoire de Chirnie Macromoleculaire, Faculte des Sciences et Techniques, 42023 St Etienne Cedex, France 3Service Commun de Microscopie Electronique, Faculte de Medecine, 49045 Angers Cedex, France (Received for publication May 14, 1996 and in revised form November 21, 1996) Abstract Introduction Bone substitutes are nowadays largely used in ortho­ Polymers based on derivatives of methacrylic acid pedic surgery but they lack osteoinductive properties. have been extensively used as biomaterials with medical Poly (2-hydroxyethyl methacrylate) (pHEMA) has nu­ applications. These synthetic materials are characterized merous biomedical applications. Alkaline phosphatase by a high degree of resistance of their backbone against (AlkP), an ectoenzyme elaborated by osteoblasts, initi­ the hydrolytic properties of the body fluids (Kalal, ates bone mineralization by hydrolyzing organic phos­ 1984). Poly (2-hydroxyethyl methacrylate) - pHEMA­ phates before calcium-phosphorus deposition. We have has been proposed during the last decade for a number immobilized AlkP in pHEMA in a copolymerization of applications ranging from ocular lenses (contact or technic. Histochemical study revealed that AlkP has re­ intra ocular lenses) to contraceptive technics (Chappard tained its biological activity. Image analysis of sections et al., 1992; Montheard et al., 1992, 1996). Consider­ using a tessellation method showed a lognormal distribu­ able interest has been paid to pHEMA in the biomedical tion of the area of tessels around AlkP particles thus literature because the polymer possesses several attract­ confirming an homogeneous distribution of the enzyme ing properties: (a) it is highly biocompatible even in in the polymer. Pellets of pHEMA and pHEMA + contact with blood and does not activate the complement AlkP were incubated with synthetic body fluids contain­ system (Gobel et al., 1987); (b) it is insoluble in water ing either inorganic or organic phosphates (,6-glycero­ but can swell and form hydrogels due to the existence of phosphate). Mineral deposits with a round shape (calco­ pending primary alcohol groups ( -CH2-CH2-0H) spherites) were obtained on pHEMA + AlkP pellets (Wichterle and Lim, 1960). On the other hand, the incubated in the presence of organic phosphates. No monomer (HEMA) itself can be dissolved in water. deposits were observed on pHEMA in either incubating These properties have been used by pharmacologists for conditions or on pHEMA + AlkP incubated with the controlled release of drugs (Huglin and Sloan, 1993; inorganic phosphates. Calcospherites were observed by Robert et al., 1987), and for immobilization of cells, transmission and scanning electron microscopy. They enzymes and bacteria (Kumakura and Kaetsu, 1983). appeared composed of minute single tablets packed to­ When prepared from water-free and bulk-polymer­ gether. X-ray microanalysis showed a Ca/P ratio of ized monomer, pHEMA has an hardness comparable to 1.42 and X-ray diffraction identified hydroxylapatite. bone. Several preliminary studies have reported the use AlkP entrapped in an hydrogel is able to initiate min­ of pHEMA as a bone substitute when associated with or­ eralization in vitro by a mechanism that closely mimics ganic (collagen) or inorganic (,6 tri-calcium phosphate) the cartilage/bone mineralization in vivo . compounds (Smetana et al., 1992; Zavrel and Stol, 1993). Today, there is a growing demand for bone Key words: Alkaline phosphatase, poly (2-hydroxyethyl) grafting materials in orthopedic, neurologic, plastic and methacrylate, methacrylate, mineralization, image anal­ dental surgeries. Autograft is by far the best material ysis, bone biology, biomaterials, bone substitute, poly­ but suffers from increased surgical time, necessarily lim­ mer. ited volumes and possible residual pain at the site of har­ *Address for correspondence: vesting (Prolo and Rodrigo, 1985). Allografts have Michel Basle been extensively used during the last decade. However, I.aboratoire d' Histologie-Embryologie they necessitate the constitution of a bone bank; they are Faculte de Medecine, 49045 Angers Cedex -France not absolutely safe with respect to bacterial or viral Telephone: (33) 241 73 58 64 problems, and they use a non-delipidated material (Buck Fax: (33) 241 73 58 88 et al., 1989). Xenografts prepared from animal bones 11 R. Filmon, D. Chappard, J.P. Montheard, M.F. Basle can represent a good alternative when highly purified enzyme was homogenized by vortexing for 5 minutes. (Chappard et al., 1993). In the future, synthetic In parallel, tubes containing the same amount of distilled products will supersede natural grafts because it will be water were used to prepare controls. The polymeriza­ possible to obtain homogeneous and unlimited industrial tion initiator N-N dimethyl paratoluidine was added at a productions of active biomaterials. Immobilization of fmal concentration of 3% and the mixture was vortexed bioactive factors such as growth factors or bone matrix again for 5 minutes. The accelerated and initiated mix­ proteins in a biomaterial may promote and accelerate ture was poured into polyethylene tubes and polymerized bone healing (Brink et al., 1996). at +4°C for 24 hours. The cylinders of pHEMA were Alkaline phosphatase (an orthophosphoric-monoester collected and sectioned into pellets (1 mm in thickness) phosphohydrolase having an alkaline optimum - EC on an Isomet saw equipped with a diamond coated blade 3.1.3.1.). was found to preserve its mineralizing activity or into sections (3 J.!m thick) on a Leica (Rueil when chemically grafted onto collagen or dentine (Beert­ Malmaison, France) Polycut S microtome equipped with sen and van den Bos, 1982; van den Bos and Beertsen, tungsten carbide knives. 1994). In the present study, we have physically immo­ Histochemical controls of enzyme activity bilized alkaline phosphatase in a matrix made of A1kP was histochemically revealed by a simultane­ pHEMA. This enzyme is recognized to play a key role ous coupling reaction using Naphtol AS-BI phosphate as in the bone mineralization process (Whyte, 1989). We the substrate and Fast Blue BB as the diazonium salt. have tried to determine whether this chemical (organ­ The 3 J.!m thick sections were incubated for 30 minutes ic/synthetic) material can cause mineralization under in at room temperature, rinsed in distilled water and let dry vitro conditions. until mounting in an aqueous medium. Control histo­ chemical reactions were used by adding 0.25 mM of Materials and Methods NaF (a competitive AlkP inhibitor) in the staining fluid. The homogeneity of AlkP distribution within the poly­ The monomer (HEMA) mer was appreciated by image analysis on histochemi­ Commercial 2-hydroxyethyl methacrylate was pur­ cally stained sections using a Leica Quantimet Q570 chased from Aldrich Chemical (St Quentin Fallavier, image processor. After image acquisition and interactiv'.l France). The monomer is known to contain remnants of thresholding, the image was converted into a binary methacrylic acid and ethyleneglycol-dimethacrylate image (Fig. la). The size of each detected particle of which are made during the fabrication process. The AlkP was measured and values were stored in an array polymerization inhibitor 4-methoxyphenol (added by the and used to provide the histogram distribution frequen­ manufacturer before shipping at a concentration of 200 cy. The volume amount of A1kP in pHEMA was deter­ ppm) also needs to be removed. HEMA was purified mined as the ratio of the surface area of stained particles and distilled as follows: HEMA was shaken with 3% over the section area. The volume of polymer around (w/v) of NaHC03 in distilled water. NaHC03 makes each of the particles was determined by measuring their water-soluble complexes with methacrylic acid and 4- zone of influence. On the analyzer, partitioning of the metoxyphenol. Water was extracted with a large amount complementary image of the AlkP particles was automat­ of CHC1 3 and most of the solvent was removed by ically provided
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