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Biphasic calcium phosphate coated with poly-d,l-lactide-co-glycolide biomaterial as a bone substitute

Biphasic calcium phosphate coated with poly-d,l-lactide-co-glycolide biomaterial as a bone substitute
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  Biphasic Calcium Phosphate/Poly-(DL-Lactide-co-Glycolide) Biocomposite as Filler and Blocks for Reparation of Bone Tissue N. Ignjatovi ć 1,a , P. Ninkov 2,b , Z. Ajdukovi ć 3,c , V. Konstantinovi ć 4,d  and D. Uskokovi ć 1,e 1 Institute of Technical Sciences of SASA, Belgrade, Serbia and Montenegro  2 Faculty of Medicine, Clinic of Stomatology, University of Novi Sad, Novi Sad  3 Faculty of Medicine, Clinic of Stomatology, University of Niš, Niš 4 Clinic for Maxillofacial Surgery, Faculty of Stomatology, Belgrade, Serbia and Montenegro a, b dujoni@ptt.yu, c zoricaa@eunet.yu, d vskvita@eunet.yu, e Keywords:  Bone Repair, Calcium Phosphate, Composite, Cytotoxicity, In Vitro, In Vivo.   Abstract. Composite biomaterials, like calciumphosphate/bioresorbable polymer, offer excellent potential for reconstruction and reparation of bone tissue defects induced by different sources. In this paper synthesis of calciumphosphate/poly-DL-lactide-co-glycolide (BCP/DLPLG) composite biomaterial formed as filler and blocks was studied. BCP/DLPLG composite biomaterial was produced in the form of spherical granules of BCP covered by a DLPLG layer, average diameter of 150-250 µ m. By cold and hot pressing of granules at up to 10000 kg/cm 2 , blocks with fine distribution of phases and porosity up to 3% were obtained. Characterization was performed by wide-angle X-ray structural analysis (WAXS), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), infrared spectroscopy (IR), and mechanical properties by defining the compressive strength.  In vitro citotoxicity research was carried out on cellular cultures of fibroblasts of human (MRC5) and mouse (L929).  In vivo  research was performed in two steps. Reparatory ability of BCP/DLPLG in mice was examined in the first step, and then bone tissue reconstruction possibilities on 10 patients in the next step.  In vitro  tests showed very good fibroblast adhesion and non-citotoxicity of the composite. A material is considered non-cytotoxic if the cell survival is above 50 %, and in our case it was 90%.  In vivo  research on mice indicated high level of reparatory ability of this composite with formation of new bone and vascular tissue six weeks after reparation. Application of this composite for healing infrabone defects of patients showed a high level of osseous regeneration. Introduction Ceramics/polymer composites play a significant role in bone tissue reparation, as their properties are very close to the natural bone tissue [1]. Calcium hydroxyapatite/bioresorbable polymer composite biomaterials belong to this group of composites, and due to their osteoconductive and biocompatible properties they can be successfully implemented in bone tissue reparation [2-4]. The structure and properties of this kind of composites depend on the polymer molecular weight, crystalline/amorphous ratio, porosity, etc. [5, 6]. Poly-DL-lactide-co-glicolide (DLPLG) polymer is also a biodegradable material that unites biocompatible properties of poly-l-lactide and polyglicol [7], which is preferable in some applications. Reparation processes in organism depend on interactions of organism cells with implanted biomaterial. Human osteoblast adhesion is better on DLPLG polymer surfaces than on other bioresorbable polymers; therefore, it is to be expected that the formation of human osteoblasts is also more intensive on the surface of DLPLG [8]. It is also important to emphasize that adhesion and osteoblast production phenomena in this case depend on the structure and type of the polymer but not on time, provided that the advantage is given to DLPLG over other bioresorbable polymers. Mixed with calcium phosphates, DLPLG polymer  Materials Science Forum Vol. 494 (2005) pp. 519-524online at © 2005 Trans Tech Publications, Switzerland  Licensed to Ignjatovic ( - Institute of Technical Sciences of SASA - Serbia-Montenegro All rights reserved. No part of the contents of this paper may be reproduced or transmitted in any form or by any means without thewritten permission of the publisher: Trans Tech Publications Ltd, Switzerland, (ID:,10:05:31)  realizes more intensive activity of alkaline phosphatase, which is important for differentiation of osteoblasts that dictate regeneration processes within the organism [9]. Addition of biphasic calcium phosphate (BCP) to polymers can largely increase the polymer bioactivity [10]. In this paper we report on a biphasic calciumphosphate/poly-DL-lactide-co-glicolide (BCP/DLPLG) composite biomaterial synthesized in the form of granules of desired shape and dimensions. Possibilities of producing blocks by cold and hot pressing at different pressures were examined. Citotoxicity was studied by in vitro tests on cellular cultures of mouse (L-929) and human (MRC-5). Reparability was examined in in vivo  conditions by reconstruction of mice mandible. Preliminary study of the possibility to compensate bone tissue with BCP/DLPLG composite was examined by reparation of infrabone defects of patients. Material and Method Calcium phosphate was produced by precipitation of calcium nitrate and ammonium phosphate [2]. Poly-DL-lactide-co-glicolide (DLPLG) (Sigma Chemical Company, USA) was used as a polymer component. Granules of calcium phosphate were added into completely dissolved polymer. Addition of non-dissolvent (methanol) into a 3-component system of solvent-polymer-calciumphosphate causes its thermodynamic destabilization. This induces sedimentation of the polymer onto calciumphosphate granules and their coverage by the polymer. The granules of calcium phosphate/DLPLG composite biomaterial of sizes 150-250 µ m were sterilized by γ   rays (25kGy) before use. Composite biomaterials were characterized by wide-angle X-ray structural analysis (WAXS), differential scanning calorimetry (DSC),   scanning electron microscopy (SEM), and infrared spectroscopy (IR). Blocks, which were the object of density and compressive hardness testing, were produced by cold and hot pressing of granules at a temperature of 324 K and pressure of 1000-10000 kg/cm 2 . Standard cellular cultures of mouse (L-929) and human (MRC-5) were   examined  in vitro . Cellular cultures were planted on BCP/DLPLG disks, 500 µ m high and 13 mm in diameter obtained by cold pressing at 200 KPa. Cells were analyzed after 24h by SEM. Citotoxicity test was conducted according to ISO 10993-5 [11].  In vivo  testing was done on white rats of Singen type Sprague Dolly, female, aged 6-8 weeks. Animals were divided into two groups: experimental group A (24 animals) and control group B (8 animals). Osteoporosis in animals of group A was induced by cortico-preparations (methylprednisoloni succinat natrium and dexamethason-natrium-phosphate to achieve osteoporosive bone tissue, prone to continuity breaks). In thus induced osteoporosive tissue between medial line and foramena mentale on the left side of the mandible, a defect was made 1.4 mm in diameter and 1.6 mm deep, where BCP/DLPLG samples were implanted. Group B was a control group. Animals were sacrificed after test and 24 weeks of implantation. Bone sample of lower jaw, between medial line and foramena mentale, was extracted. Histological sections 2-4 µ m thick were colored by routine hematoxilienosis (HA) and PAS method, and histologically analyzed. Infrabone defects of 10 patients were filled with BCP/DLPLG composite biomaterial granules. Under local anesthetic, a cut was made on the vestibular side. After filling the infrabone defect with the composite biomaterial, and reposition, stitches were fixed with 4/0 Silk Sutures thread. Orthopan tomography (OPJ X-ray) was used for postoperative period follow-up. Results and Discussion After production of powder and granules of calcium phosphate according to the previously given procedure, phosphate phase was analyzed by WAXS. Figure 1 shows a diffractogram of the obtained powder. As evident from the figure, the obtained calcium phosphate powder is highly crystalline. The most intense peaks at 2 θ =29 o  and 31.8° srcinate from calcium hydroxyapatite (HAp) and that at 2 θ =31 o  from calciumphosphate ( β -TCP). Current Research in Advanced Materials and Processes520   Fig. 1 WAXS of calcium phosphate granules. Fig. 2 SEM image of BCP/DLPLG granules.   Based on earlier described methodology [12], mass contents of HAp and β -TCP, 80% and 20%, respectively, were calculated. Thereby, this calcium phosphate is also called biphasic calcium phosphate (BCP) and is used for the BCP/ DLPLG composite biomaterial production. Composite production procedure via solvent - non-solvent system realizes covering of BCP particles with DLPLG polymer. Using sieves, a fraction of particles, sized 150-250 µ m, was separated. Figure 2 shows a SEM image of BCP/DLPLG composite particles. IR analysis of granules, shown in Fig. 3, reveals the existence of BCP and DLPLG, as expected. BCP is identified within the spectrum by a doublet with maxima at 1052 and 1087 cm -1 , which are the most intense and srcinate from phosphate groups, and by a triplet with maxima at somewhat lower frequencies of 571 and 602 cm -1 , arising from the PO 43-  group vibrations, and at 632 cm -1 , assigned to the hydroxyl group vibrations appearing also at 3567 cm -1  [6]. DLPLG is characterized by an absorption band at 1756 cm -1  corresponding to the C=O group vibrations and two smaller maxima at 2996 and 2944 cm -1  ascribed to C-H group vibrations. Absorption maximum at 1449 cm -1  srcinates from the CH 3  group [13]. DSC analysis established the existence of only one distinguished peak with a maximum at 324 K, which was attributed to the temperature of polymer glazing [14]. Non-existence of phase transition, characteristic of melting, indicates amorphousness of the polymer. Amorphousness of the polymer is an important parameter in its short-term bioresorption compared to other biodegradable polymers, which is generally 4-8 weeks [7]. The 3-dimensional blocks were produced by cold and hot pressing of BCP/DLPLG granules. By increasing the pressure and pressing temperature, the block density and compressive hardness increase, while their porosity decreases. Theoretical density of compact and poreless block is 2.48 g/cm 3 . Blocks with average density of 2.39 g/cm 3 were produced by cold pressing at 10000 kg/cm 2  pressure, while blocks with average density of 2.41 g/cm 3 were produced by hot pressing at the polymer glass transition temperature (T g =324 K). Fig. 3   IR spectrum of BCP/DLPLG composite Fig. 4 Compressive strength dependence on biomaterial. processing pressure and temperature. 0246810304050607080 cold pressing pressing at 324 K    C  o  m  p  r  e  s  s  s   i  v  e  s   t  r  e  n  g   t   h   [   M   P  a   ] pressing pressure [t/cm 2 ]4000350030002500200015001000500050001000015000C-OPO 43- OH - C-HC-HPO 43- CH 3 C=OOH -    I  n   t  e  n  s   i   t  y   [  a .  u   ] wavenumber [cm -1 ] 102030405060708090100 02000400060008000   HAp, 29 o   TCP, 31 o   HAp, 31.8 o    I  n   t  e  n  s   i   t  y   [  a .  u .   ] 2 θ  [ o ] Materials Science Forum Vol. 494521  Fig. 5   In vitro tests; a) body of fibroblast (MRC-5) on the BCP/DLPLG surface; b) peripheral part of the body of fibroblast (MRC-5); c) part of fibroblast (L-929) on the BCP/DLPLG surface. Figure 4 shows the values of compressive strength of the blocks pressed at different pressures and two temperatures: 291 K for cold and 324 K for hot pressing. Maximum value of compressive strength (82 MPa) was achieved when blocks were pressed at 10000 kg/cm 2  and pressing temperature of 324 K, while at the same pressure but different temperature (291 K), the obtained block had a lower value of compressive strength (73 MPa). Figure 5 shows SEM images obtained during in vitro  research on cellular cultures of human (MRC-5) and mouse (L-929). In Fig. 5a), part of fibroblast body from the MRC5 cellular culture, grown on the surface of the BCP/DLPLG disk for 24h, can be seen. Bases of ribbon extensions on the fibroblast body that give it a stellar shape are present. The particles of the material present at the surface of the fibroblast body can be seen detached from the base. Part of the body and fibroblast extensions are well adhered to the base. At higher magnification (Fig. 5b), the peripheral part of fibroblast with citoplasmatic extensions is noticeable, reaching towards the base and adhering to it. Fibroblast from the cellular culture L929 developed for 24h is evident in Fig. 5c. Citoplasmatic inclusions are seen on the fibroblast body, which like vacuoles imitate bubbly appearance, and also plasmatic extensions well adhered, like the body, to the base. Examination of citotoxicity on MRC-5 cellular culture has shown that the mortality percentage of the cells is 10.13%. High survival percentage of 89.87 indicates the absence of inhibition of this kind of composite on cellular culture of human fibroblast [15]. Figure 6 shows the results achieved during in vivo  research. Figure 6a illustrates surgical intervention itself and the mouse alveolar bone reconstruction. On histological preparations of alveolar bone where BCP/DLPLG was implanted, shown in Fig. 6b and 6c, new-formed bone tissue is evident, as well as spaces filled with mature bone. After 6 weeks (Fig. 6b), there are spaces with insignificant amount of BCP/DLPLG residue imbued with fibroblasts. Polymer matrix on its surface provides good adhesion of osteoprogenitive cells, so that they induce intensive development of osteogenesis. Results obtained 24 weeks after implantation are shown in Fig. 6c. In the 24 th  week, formation of new bone tissue is evident, which intensely overgrows the compact as well as the spongious part of mandible that directly and positively influences remodeling of damaged alveolar bone. Current Research in Advanced Materials and Processes522  Fig. 6   In vivo test on mouse: a) reconstruction on mandibule; b) histological preparation after 6 weeks of BCP/DLPLG implantation; c) after 24 weeks.   Figure 7 shows one of ten cases of a pilot-study of BCP/DLPLG composite biomaterial applicability for the reconstruction of alveolar bone defects. Figure 7a shows a bone defect around the root of the upper lateral incisor. After raising the mucoperiosteal flap, curettage of the granulations and cleaning of the defect to the healthy tissue, defect was filled with BCP/DLPLGA composite (Fig. 7b). After reconstruction, mucopaeriosteal flap was sutured with 4.0 silk sutures (Fig. 7c). In retroalveolar X-ray image made 12 months after intervention, the formed bone and complete permeation of implanted material with new-formed bone around the root, is obvious. Fig. 7 Filling infrabone defects with BCP/DLPLG: a) infrabone defects; b) applied BCP/DLPLG; c) sutured reconstruction; d) X-ray after 12 months. Conclusion BCP/DLPLG composite biomaterial was synthesized in the form of spherical granules, 150-200 µ m in diameter; each BCP particle is coated with amorphous DLPLG polymer. The composite is suitable for application as filler in reparation of osteoporosive bone tissue. Calcium phosphate present in the composite is in the form of biphasic calcium phosphate consisting of 80% calcium hydroxyapatite and 20% tricalciumphosphate. Blocks of 97% theoretical density and compressive strength of 82 MPa were produced by cold and hot pressing at the polymer glass transition temperature (324K).  In vitro  research on cellular cultures of human (MRC-5) and mouse (L-929) has shown good adherence of fibroblast cells of both cellular cultures to the composite biomaterial surface. Materials Science Forum Vol. 494523
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