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In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite

In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite JOURNAL OF MATE RIALS SCIENCE: M ATERI ALS IN ME DI CINE 8 (1997) 775 Ð 779 In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite J. HU A N G*,L.DI SILVIOs,M. WANG*,K.E.TANNER*,W.BONFIELD* *IRC in Biomedical Materials, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK, sIRC in Biomedical Materials, Institute of Orthopaedics, Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK In vitro performance of hydroxyapatite (HA)-reinforced polyethylene (PE) composite TM (HAPEX ) has been characterized from both mechanical and biological aspects. The TM mechanical properties of HAPEX , such as tensile strength and Young’s modulus, showed little change after immersion in a physiological solution at 37 and 70 ¡C for various periods. In addition, the biological response of primary human osteoblast-like (HOB) cells in vitro on TM HAPEX was assessed by measuring DNA synthesis and osteoblast phenotype expression. TM Cell proliferation rate on HAPEX was demonstrated by an increase in DNA content with time. A high tritiated thymidine ([ H]-TdR) incorporation/DNA rate was observed on day TM 1 for HAPEX , indicating a stimulatory effect on cell proliferation. The alkaline phosphatase TM (ALP) activity was expressed earlier on HAPEX than on unfilled PE and increased with time, indicating that HOB cells had commenced differentiation. Furthermore, it was found that the HA particles in the composite provided favourable sites for cell attachment. It appears TM that the presence of HA particles in HAPEX may have the advantage of acting as microanchors for bone bonding in vivo. 1. Introduction HAPEXTM has been shown to encourage bone Bioactive ceramics with their bone-bonding ability formation in vivo [14], but the complexities associated have revolutionized the concept of biomaterials. How- with in vivo studies make it di¦cult to examine the ever, their relatively poor mechanical properties have speciÞc interactions of various cells with the implant. In restricted potential clinical applications [1]. A vitro cell culture models, allowing the biological assess- composite material, incorporating bioactive hydroxy- ment of materials at a cellular level, are helpful in apatite (HA) ceramic particles into high-density evaluating speciÞc cell response. Several models using polyethylene (HDPE) matrix, HAPEXTM, has been animal or human osteoblast-like cells or stromal cells developed with optimal sti¤ness, toughness and bioac- have been developed for studying the in vitro response tivity [2, 3]. This type of composite is able to fulÞl the of the cells to various biomaterials [15Ð19]. These cell mechanical and biological requirements for an im- culture models, with their limited variables, allow more direct observation of the cellÐbiomaterial interaction. plant material, which make it a successful orbital ßoor implant [4, 5]. In this work, a primary human osteoblast-like (HOB) The e¤ects of various HA Þllers and PE matrices and cell model, a representative of the cell type that the di¤erent processing routes on the properties of the material surface will contact in vivo, has been used to composite have been investigated [6Ð10]. As an im- study the cell response to HAPEXTM. The knowledge plant material, the composite will contact physiological obtained will further aid in designing second generation ßuids in service. Thus the changes in the composite in implant materials to promote bone bonding. a physiological solution at 37 ¡C need to be evaluated. An in vitro simulation of mechanical properties of the composite has been established. As water absorption is 2. Materials and methods a major deterioratingfactoronthe properties of acom- HAPEXTM was produced by incorporation of HA posite in solution [11Ð13], accelerated ageing tests have (Plasma Biotal, P88) particles into HDPE (BP been performed at 70 ¡C for various periods to predict Rigidex) through twin-screw extrusion and compres- the long-term stability of the composite. sion moulding [10]. The microstructure has been *Author to whom all correspondence should be addressed. Selected paper from the 13th European Conference on Biomaterials, Go¬ teborg, Sweden. 0957Ð4530 ( 1997 Chapman & Hall 775 where ¼ and ¼ are the weight of the specimen 1 2 before and after incubation, respectively. 2.2. Tensile testing Tensile testing of HAPEXTM was conducted on an Instron 6025 computer-controlled screw-driven mech- anical testing machine at a crosshead speed of 0.5 mm min~1. An extensometer was attached to the specimen gauge length to measure YoungÕs modulus, and the tensile strength and strain to failure were also deter- mined. The YoungÕs modulus was calculated from the initial linear region of the stressÐstrain curve. The fracture surfaces of the composite were preserved and Þxed on specimen holders by conductive carbon ce- ment, and sputtered with gold before examination Figure 1 The uniform dispersion of HA particles (white) in the PE with a Jeol 6300 scanning electron microscope. matrix (dark) on the polished surface of HAPEXTM. 2.3. In vitro cell culture Primary human osteoblast-like (HOB) cells, isolated from trabecular bone fragments of the femoral heads of patients undergoing hip surgery [19], were used. HOB cells were cultured in DulbeccoÕs ModiÞed EagleÕs Medium (DMEM), supplemented with 10% foetal calf serum (FCS), 1% non-essential amino acids, L-ascorbic acid (150 g ml~1), 0.02 M L-glutamine, 0.01 M HEPES, 100 units ml~1 penicillin and 100 lg ml~1 streptomycin. Thermanox (TMX) and unÞlled PE were used as controls. HOB cells were seeded on the test materials at a density of 1]106 cells ml~1 with great care to avoid unwanted cell attachment to the surrounding surface of culture dish, and were allowed to attach for 1 h prior to ßooding with 1 ml DMEM medium. The cultures were incubated at Figure 2 The accumulation of crystallites within individual HA 37 ¡C in humidiÞed air with 5% CO for 1, 4, 7, 14 and particles from cryofracture surface, indicating the increase in surface 21 days. The culture medium was replaced carefully, area of HAPEXTM with the presence of HA particles. at appropriate time intervals, in order to minimize disturbance of the culture conditions. characterized by X-ray di¤raction, infrared spectro- scopy and electron microscopy (Figs 1 and 2). Tensile test specimens (ISO 527) were annealed at 80 ¡C for 2.4. Cell growth and proliferation 24 h before testing to eliminate the e¤ects of thermal The growth and proliferation of the HOB cells on the history. Specimens of 40 vol% HAPEXTM (HAPEX) materials were determined by measuring tritiated were sterilized by gamma irradiation at a dose of 2.5 thymidine ([3H]-TdR) incorporation and total DNA Mrad (Isotron, UK) using standard procedures for content. At each time point, the cells were incubated in medical devices. Simulated body ßuid (SBF K9) was the presence of 1 lCi ml~1 of [3H]-TdR (Amersham used to immerse the specimens, as it closely resembles International, UK) for the Þnal 16 h culture. Then the the inorganic ions concentrations in blood plasma cells were enzymatically lysed using a 0.1% papain [20]. The specimens were immersed at 37 and 70 ¡C digest solution (Sigma, UK). Total cellular DNA was for 1 and 3 months. measured using the Hoechst method [21]. Hoechst 33285, a DNA speciÞc dye, was reacted with the papain-digested cell suspension or DNA standard with concentrations of 0, 0.31, 0.62, 1.25, 2.5, 5, 10 and 2.1. Water absorption 20 lgml~1 in saline sodium citrate bu¤er (pH"7.0). The water absorption of HAPEXTM in SBF was esti- The ßuorescence was measured on a Fluoroskan mated by measuring weight changes. Samples were ßuorimeter at an excitation wavelength of 355 nm and removed from the solution, excess water was wiped an emission wavelength of 450 nm. The total DNA o¤ with a tissue and the weight was recorded using content for each test specimen was calculated from the an analytical balance. The weight change *¼%, is standard DNA curve. given by [3H]-TdR incorporation was measured by tri- ¼ !¼ chloroacetic acid (TCA) precipitation of the cell digests. 2 1 *¼%" ]100 (1) ¼ The precipitate was vacuum Þltered on to a porous 776 membrane. Unprecipitated lysate containing the ex- cess unbound radionucleotide was washed through the membrane with 10% TCA, the precipitate was then dissolved in 0.01M KOH solution. The amount of radiolabel incorporated was measured using a scintil- lation counter. 2.5. Osteoblast phenotype expression Alkaline phosphatase (ALP) activity of HOB cells, as an indicator of osteoblast phenotype activity, was determined biochemically. After the culture time, a freezeÐthaw method was used to lyse the cells and release their content. This procedure was performed by freezing at!70 ¡C for 15 min and thawing at 37 ¡C for 20 min three times. The ALP activity was deter- mined using a COBAS-BIO (Roche, UK) centrifugal analyser. P-nitro phenol phosphate in diethanolamine bu¤er (Merck, UK) was the substrate used. The en- zyme alkaline phosphatase, cleaves the phosphate group from p-nitro phenol phosphate to produce p-nitrophenol which is yellow at alkaline pH (9.8), and is monitored at the wavelength of 405 nm. 2.6. Cell morphology Test specimens were seeded with a density of approx- imately 8]105 HOB cells ml~1 and incubated at 37 ¡C in a humidiÞed air with 5% CO . After 24 h incubation, the cultures were Þxed with 2.5% glutar- aldehyde bu¤ered in 0.1 M sodium cacodylate, stained in 1% osmium tetroxide and 1% tannic acid bu¤er. The samples were dehydrated using a series of alcohol solutions, from 20%, 30%, 40%, 50%, 60% to 70%, then stained in 0.5% uranyl acetate and further dehy- drated in 90%, 96%, 100% ethyl alcohol (containing Na SO ), then dehydrated with hexamethyl-di- 2 4 silazane, and Þnally air dried overnight. The cultures were coated with a thin layer of gold before examina- tion under a Joel scanning electron microscope at an accelerating voltage of 15 keV. 3. Results 3.1. Water absorption No measurable dimensional change was seen for HAPEXTM after immersion in SBF. The weight of Figure 3 Comparison of the mechanical properties of HAPEXTM: HAPEXTM increased with incubation time and tem- (a) tensile strength (b) YoungÕs modulus and (c) strain to failure perature, but the increases were only 0.8% and 1.2% before and after immersion in SBF at 37 and 70 ¡C for 1 and after 3 months incubation at 37 and 70 ¡C, respective- 3 months. ly, indicating the low degree of water absorption by HAPEXTM. ature for 1 and 3 months. In addition, no evidence of degradation of HA particles was observed on the 3.2. Mechanical properties fracture surface. The mechanical properties of HAPEXTM following incubation at 37 and 70 ¡C for 1 and 3 months are shown in Fig. 3. It appeared that the tensile strength of 3.3. Biological assessment the composite decreased with immersion time and Continuous growth of HOB cells, expressed as the that the decrease at 70 ¡C was greater than that at total DNA content, increased with time on HAPEXTM 37 ¡C, but neither decrease was statistically signiÞcant. and PE during 21 days culture, but varying degrees of The YoungÕs modulus and fracture strain of cell proliferation were observed. The highest rate of HAPEXTM did not signiÞcantly alter at either temper- [3H]-TdR incorporation was seen on day 1, when the 777 Figure 4 [3H]-TdR incorporation/DNA for HOB cells on the test materials: TMX control, HAPEXTM and unÞlled PE from days 1Ð21. The cell proliferation rate was highest on day 1, where the greatest increase was observed on HAPEXTM. Figure 5 The ALP activity of HOB cells cultured on the test Figure 6 (a) The surface of HAPEXTM covered by a HOB cell layer materials: TMX control, HAPEXTM and PE from days 1Ð21. after 24 h culture, and (b) the cell processes attached to HA particles. greatest proliferation of HOB cells was found on composite is in contact with body ßuid, need to be HAPEXTM (Fig. 4). Following this initial rise in proli- considered carefully, as the long-term stability of an feration rate, a fall in cell proliferation was observed. implant will determine its outcome, unless the implant As cell proliferation decreased, the ALP activity in- is speciÞcally designed to be biodegradable. The re- creased, indicating that the osteoblasts were in a state sults from this study show that by careful selection of of di¤erentiation. This process occurred more rapidly the HA Þller in the composite, the tensile strength, on HAPEXTM than on PE (Fig. 5). The peak ALP YoungÕs modulus and strain to fracture of HAPEXTM activity was seen on day 7 for TMX and at day 14 on remained unchanged following incubation at 37 ¡C HAPEXTM, while a continuous increase in the ALP and accelerated ageing at 70 ¡C. No degradation in the activity was observed on PE up to day 21. properties of HAPEXTM ensures its implantation suc- Fewer cells adhered to the surface of PE, indicating cess and makes it a highly suitable implant material. that PE surface was not a favourable surface for HOB Biocompatibility of a material is considered to be its cell attachment and proliferation. In comparison, ability to perform with an appropriate host response many cells were seen to attach on the surface of in a speciÞc application [22]. In vitro cell culture HAPEXTM, a cell layer was observed covering the studies using the ÔÔappropriate cellsÕÕ, such as the HOB entire surface and cells at various stages of division cells used in this study, allow the investigation of the were often observed (Fig. 6a). The cells appeared ßat- interfacial response of the implant material in relation tened and maintained the polygonal morphology of to its Þnal use. The biocompatibility of HAPEXTM was osteoblasts, with Þlapodia attached to HA particles demonstrated by the ability of the cells to proliferate (Fig. 6b). on the material. The rapid cell attachment was fol- lowed by proliferation and di¤erentiation of the cells. The high [3H]-TdR incorporation/DNA observed on 4. Discussion HAPEXTM on day 1 indicated an immediate stimula- Bioactivity can be retained in a composite which in- tory e¤ect. When considering osteoblast expression corporates a bioactive phase. However, the inßuence and hence ALP activity, a greater activity was ob- of the bioactive phase on the mechanical properties of served in those HOB cells on HAPEXTM as compared the composite, particularly, those properties when the to those on unÞlled PE. The increase in ALP activity 778 indicated that the cells had ceased to proliferate and 3. W. BONFIELD, in ÔÔAnnals of New York Academy of SciencesÕÕ, Vol. 523, edited by P. Ducheyne and J. E. Lemons had begun to di¤erentiate. This process occurred (New York Academy of Science, New York, 1988) p. 173. earlier on HAPEXTM than on PE. Another striking 4. R. N. DOWNES, S. VARDY, K. E. T ANNER and W . BON- feature frequently observed was that cell Þlapodia FIELD, Bioceramics 4 (1991) 239. were embedded within HA particles on the surface of 5. K. E . T ANNER, R. N. DOWNES and W. B ONF IELD, Br. HAPEXTM. It appeared that HAPEXTM provided Ceram. ¹rans. 93 (1994) 104. 6. J. ABRAM , J. BOWMAN, J. C. BEHIRI and W. BON- preferential sites for cell attachment and stimulated FIELD, Plast. Rubb. Process. Applic. 4 (1984) 261. the overall HOB cell activity. The stimulatory e¤ect 7. W. BONFI ELD, J . C . BE HI RI, C. DOYLE, J. BOWMA N might be caused by the presence of HA particles which and J. ABRAM, in ÔÔBiomaterials and BiomechanicsÕÕ, edited provide a large surface area (Fig. 2) for the absorption by P. Ducheyne, G. Van der Perre and A. E. Aubert (Elsevier, of proteins and growth factors, which in turn may Amsterdam, 1983) p. 421. 8. G. P. EVA NS, J. C. BEHI RI, J. D. CURREY, W. BON- enhance cellular activity. Therefore, HA particles in FIELD, J. Mater. Sci. Mater. Med. 1 (1990) 38. HAPEXTM may act as microanchors for developing 9. K. HEMACHANDRA, PhD thesis, University of London direct bone bonding in situ. (1992). 10. M. WA NG, D. PORTER and W. BONFIELD, Br. Ceram. ¹rans. 93 (1994) 91. 11. P. S. T HEOCARIS, G. C. PAPA NOCOL AOU and E. A. 5. Conclusions KONTOU, J. Polym. Sci. 28 (1983) 3145. The mechanical properties of HAPEXTM were 12. G. C. PAPANOCOLAOU and R. MER COGL IANO, Plast. not altered by contact with physiological solution. Rubb. Process. Applic. 6 (1986) 229. Furthermore, HAPEXTM provided a favourable 13. J. SUWANPRATEEB, PhD thesis, University of London environment for HOB cell attachment, proliferation (1996). 14. W. BONFIELD, C. DOYLE and K. E. TANNER, in ÔÔBiolo- and di¤erentiation. A combination of these pro- gical and Biomechanical Performance of BiomaterialsÕÕ, edited perties will lead to HAPEXTM having the long life- by P. Cristel, A. Meunier and A. J. C. Lee (Elsevier, Amster- time required for permanent implants, which make dam, 1986) p. 153. it a highly suitable second generation implant 15. Y. I TAKURA, A. KOSUGI , H. SUDO, S. YAMAMOT O material. and M . KUMEGAWA, J. Biomed. Mater. Res. 22 (1988) 613. F. B. BAGAMBI SA and U. JOOS, Biomaterials 11 (1990) 50. 17. W. C. A. V ROUWENVE LDER, C. G. GROOT and K. DE GR OOT, J. Biomed. Mater. Res. 27 (1993) 465. Acknowledgements 18. C. FAUCHEUX, R. B AREILL E, F. ROUAI S, J. AMEDEE, The continuing support of EPSRC for the IRC core A. LIEBENDORFER and M. DARD, J. Mater. Sci. Mater. programme is gratefully acknowledged, together with Med. 5 (1994) 635. the assistance and useful discussion from Dr J. Suwan- 19. L. DI SI LVIO, PhD thesis, University of London (1995). prateeb, Mr M. Kayser, Mrs C. Cli¤ord and Mrs. 20. T. KOKUBO, H. KUSHITANI , S. SA KKA, T. KITSUGI and T. Y AMA MURO, J. Biomed. Mater. Res. 24 (1990) 721. N. Gurav. 21. R. RAGO, J. MITCHEN and G. WI LDING, Anal. Biochem. 191 (1990) 31. 22. D. F. WIL LIAMS, J. Biomed. Engng 11 (1989) 185. References 1. L. L . HENCH, J. Amer. Ceram. Soc. 74 (1991) 1487. 2. W. BONFIELD,M.D. GRYNPAS, A. E. TULLY, Received 5 May J. BOWM ANN and J. ABRAM, Biomaterials 2 (1981) 185. and accepted 11 May 1997 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Materials Science: Materials in Medicine Springer Journals

In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite

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Publisher
Springer Journals
Copyright
Copyright © 1997 by Chapman and Hall
Subject
Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
ISSN
0957-4530
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1573-4838
DOI
10.1023/A:1018516813604
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Abstract

JOURNAL OF MATE RIALS SCIENCE: M ATERI ALS IN ME DI CINE 8 (1997) 775 Ð 779 In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite J. HU A N G*,L.DI SILVIOs,M. WANG*,K.E.TANNER*,W.BONFIELD* *IRC in Biomedical Materials, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK, sIRC in Biomedical Materials, Institute of Orthopaedics, Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK In vitro performance of hydroxyapatite (HA)-reinforced polyethylene (PE) composite TM (HAPEX ) has been characterized from both mechanical and biological aspects. The TM mechanical properties of HAPEX , such as tensile strength and Young’s modulus, showed little change after immersion in a physiological solution at 37 and 70 ¡C for various periods. In addition, the biological response of primary human osteoblast-like (HOB) cells in vitro on TM HAPEX was assessed by measuring DNA synthesis and osteoblast phenotype expression. TM Cell proliferation rate on HAPEX was demonstrated by an increase in DNA content with time. A high tritiated thymidine ([ H]-TdR) incorporation/DNA rate was observed on day TM 1 for HAPEX , indicating a stimulatory effect on cell proliferation. The alkaline phosphatase TM (ALP) activity was expressed earlier on HAPEX than on unfilled PE and increased with time, indicating that HOB cells had commenced differentiation. Furthermore, it was found that the HA particles in the composite provided favourable sites for cell attachment. It appears TM that the presence of HA particles in HAPEX may have the advantage of acting as microanchors for bone bonding in vivo. 1. Introduction HAPEXTM has been shown to encourage bone Bioactive ceramics with their bone-bonding ability formation in vivo [14], but the complexities associated have revolutionized the concept of biomaterials. How- with in vivo studies make it di¦cult to examine the ever, their relatively poor mechanical properties have speciÞc interactions of various cells with the implant. In restricted potential clinical applications [1]. A vitro cell culture models, allowing the biological assess- composite material, incorporating bioactive hydroxy- ment of materials at a cellular level, are helpful in apatite (HA) ceramic particles into high-density evaluating speciÞc cell response. Several models using polyethylene (HDPE) matrix, HAPEXTM, has been animal or human osteoblast-like cells or stromal cells developed with optimal sti¤ness, toughness and bioac- have been developed for studying the in vitro response tivity [2, 3]. This type of composite is able to fulÞl the of the cells to various biomaterials [15Ð19]. These cell mechanical and biological requirements for an im- culture models, with their limited variables, allow more direct observation of the cellÐbiomaterial interaction. plant material, which make it a successful orbital ßoor implant [4, 5]. In this work, a primary human osteoblast-like (HOB) The e¤ects of various HA Þllers and PE matrices and cell model, a representative of the cell type that the di¤erent processing routes on the properties of the material surface will contact in vivo, has been used to composite have been investigated [6Ð10]. As an im- study the cell response to HAPEXTM. The knowledge plant material, the composite will contact physiological obtained will further aid in designing second generation ßuids in service. Thus the changes in the composite in implant materials to promote bone bonding. a physiological solution at 37 ¡C need to be evaluated. An in vitro simulation of mechanical properties of the composite has been established. As water absorption is 2. Materials and methods a major deterioratingfactoronthe properties of acom- HAPEXTM was produced by incorporation of HA posite in solution [11Ð13], accelerated ageing tests have (Plasma Biotal, P88) particles into HDPE (BP been performed at 70 ¡C for various periods to predict Rigidex) through twin-screw extrusion and compres- the long-term stability of the composite. sion moulding [10]. The microstructure has been *Author to whom all correspondence should be addressed. Selected paper from the 13th European Conference on Biomaterials, Go¬ teborg, Sweden. 0957Ð4530 ( 1997 Chapman & Hall 775 where ¼ and ¼ are the weight of the specimen 1 2 before and after incubation, respectively. 2.2. Tensile testing Tensile testing of HAPEXTM was conducted on an Instron 6025 computer-controlled screw-driven mech- anical testing machine at a crosshead speed of 0.5 mm min~1. An extensometer was attached to the specimen gauge length to measure YoungÕs modulus, and the tensile strength and strain to failure were also deter- mined. The YoungÕs modulus was calculated from the initial linear region of the stressÐstrain curve. The fracture surfaces of the composite were preserved and Þxed on specimen holders by conductive carbon ce- ment, and sputtered with gold before examination Figure 1 The uniform dispersion of HA particles (white) in the PE with a Jeol 6300 scanning electron microscope. matrix (dark) on the polished surface of HAPEXTM. 2.3. In vitro cell culture Primary human osteoblast-like (HOB) cells, isolated from trabecular bone fragments of the femoral heads of patients undergoing hip surgery [19], were used. HOB cells were cultured in DulbeccoÕs ModiÞed EagleÕs Medium (DMEM), supplemented with 10% foetal calf serum (FCS), 1% non-essential amino acids, L-ascorbic acid (150 g ml~1), 0.02 M L-glutamine, 0.01 M HEPES, 100 units ml~1 penicillin and 100 lg ml~1 streptomycin. Thermanox (TMX) and unÞlled PE were used as controls. HOB cells were seeded on the test materials at a density of 1]106 cells ml~1 with great care to avoid unwanted cell attachment to the surrounding surface of culture dish, and were allowed to attach for 1 h prior to ßooding with 1 ml DMEM medium. The cultures were incubated at Figure 2 The accumulation of crystallites within individual HA 37 ¡C in humidiÞed air with 5% CO for 1, 4, 7, 14 and particles from cryofracture surface, indicating the increase in surface 21 days. The culture medium was replaced carefully, area of HAPEXTM with the presence of HA particles. at appropriate time intervals, in order to minimize disturbance of the culture conditions. characterized by X-ray di¤raction, infrared spectro- scopy and electron microscopy (Figs 1 and 2). Tensile test specimens (ISO 527) were annealed at 80 ¡C for 2.4. Cell growth and proliferation 24 h before testing to eliminate the e¤ects of thermal The growth and proliferation of the HOB cells on the history. Specimens of 40 vol% HAPEXTM (HAPEX) materials were determined by measuring tritiated were sterilized by gamma irradiation at a dose of 2.5 thymidine ([3H]-TdR) incorporation and total DNA Mrad (Isotron, UK) using standard procedures for content. At each time point, the cells were incubated in medical devices. Simulated body ßuid (SBF K9) was the presence of 1 lCi ml~1 of [3H]-TdR (Amersham used to immerse the specimens, as it closely resembles International, UK) for the Þnal 16 h culture. Then the the inorganic ions concentrations in blood plasma cells were enzymatically lysed using a 0.1% papain [20]. The specimens were immersed at 37 and 70 ¡C digest solution (Sigma, UK). Total cellular DNA was for 1 and 3 months. measured using the Hoechst method [21]. Hoechst 33285, a DNA speciÞc dye, was reacted with the papain-digested cell suspension or DNA standard with concentrations of 0, 0.31, 0.62, 1.25, 2.5, 5, 10 and 2.1. Water absorption 20 lgml~1 in saline sodium citrate bu¤er (pH"7.0). The water absorption of HAPEXTM in SBF was esti- The ßuorescence was measured on a Fluoroskan mated by measuring weight changes. Samples were ßuorimeter at an excitation wavelength of 355 nm and removed from the solution, excess water was wiped an emission wavelength of 450 nm. The total DNA o¤ with a tissue and the weight was recorded using content for each test specimen was calculated from the an analytical balance. The weight change *¼%, is standard DNA curve. given by [3H]-TdR incorporation was measured by tri- ¼ !¼ chloroacetic acid (TCA) precipitation of the cell digests. 2 1 *¼%" ]100 (1) ¼ The precipitate was vacuum Þltered on to a porous 776 membrane. Unprecipitated lysate containing the ex- cess unbound radionucleotide was washed through the membrane with 10% TCA, the precipitate was then dissolved in 0.01M KOH solution. The amount of radiolabel incorporated was measured using a scintil- lation counter. 2.5. Osteoblast phenotype expression Alkaline phosphatase (ALP) activity of HOB cells, as an indicator of osteoblast phenotype activity, was determined biochemically. After the culture time, a freezeÐthaw method was used to lyse the cells and release their content. This procedure was performed by freezing at!70 ¡C for 15 min and thawing at 37 ¡C for 20 min three times. The ALP activity was deter- mined using a COBAS-BIO (Roche, UK) centrifugal analyser. P-nitro phenol phosphate in diethanolamine bu¤er (Merck, UK) was the substrate used. The en- zyme alkaline phosphatase, cleaves the phosphate group from p-nitro phenol phosphate to produce p-nitrophenol which is yellow at alkaline pH (9.8), and is monitored at the wavelength of 405 nm. 2.6. Cell morphology Test specimens were seeded with a density of approx- imately 8]105 HOB cells ml~1 and incubated at 37 ¡C in a humidiÞed air with 5% CO . After 24 h incubation, the cultures were Þxed with 2.5% glutar- aldehyde bu¤ered in 0.1 M sodium cacodylate, stained in 1% osmium tetroxide and 1% tannic acid bu¤er. The samples were dehydrated using a series of alcohol solutions, from 20%, 30%, 40%, 50%, 60% to 70%, then stained in 0.5% uranyl acetate and further dehy- drated in 90%, 96%, 100% ethyl alcohol (containing Na SO ), then dehydrated with hexamethyl-di- 2 4 silazane, and Þnally air dried overnight. The cultures were coated with a thin layer of gold before examina- tion under a Joel scanning electron microscope at an accelerating voltage of 15 keV. 3. Results 3.1. Water absorption No measurable dimensional change was seen for HAPEXTM after immersion in SBF. The weight of Figure 3 Comparison of the mechanical properties of HAPEXTM: HAPEXTM increased with incubation time and tem- (a) tensile strength (b) YoungÕs modulus and (c) strain to failure perature, but the increases were only 0.8% and 1.2% before and after immersion in SBF at 37 and 70 ¡C for 1 and after 3 months incubation at 37 and 70 ¡C, respective- 3 months. ly, indicating the low degree of water absorption by HAPEXTM. ature for 1 and 3 months. In addition, no evidence of degradation of HA particles was observed on the 3.2. Mechanical properties fracture surface. The mechanical properties of HAPEXTM following incubation at 37 and 70 ¡C for 1 and 3 months are shown in Fig. 3. It appeared that the tensile strength of 3.3. Biological assessment the composite decreased with immersion time and Continuous growth of HOB cells, expressed as the that the decrease at 70 ¡C was greater than that at total DNA content, increased with time on HAPEXTM 37 ¡C, but neither decrease was statistically signiÞcant. and PE during 21 days culture, but varying degrees of The YoungÕs modulus and fracture strain of cell proliferation were observed. The highest rate of HAPEXTM did not signiÞcantly alter at either temper- [3H]-TdR incorporation was seen on day 1, when the 777 Figure 4 [3H]-TdR incorporation/DNA for HOB cells on the test materials: TMX control, HAPEXTM and unÞlled PE from days 1Ð21. The cell proliferation rate was highest on day 1, where the greatest increase was observed on HAPEXTM. Figure 5 The ALP activity of HOB cells cultured on the test Figure 6 (a) The surface of HAPEXTM covered by a HOB cell layer materials: TMX control, HAPEXTM and PE from days 1Ð21. after 24 h culture, and (b) the cell processes attached to HA particles. greatest proliferation of HOB cells was found on composite is in contact with body ßuid, need to be HAPEXTM (Fig. 4). Following this initial rise in proli- considered carefully, as the long-term stability of an feration rate, a fall in cell proliferation was observed. implant will determine its outcome, unless the implant As cell proliferation decreased, the ALP activity in- is speciÞcally designed to be biodegradable. The re- creased, indicating that the osteoblasts were in a state sults from this study show that by careful selection of of di¤erentiation. This process occurred more rapidly the HA Þller in the composite, the tensile strength, on HAPEXTM than on PE (Fig. 5). The peak ALP YoungÕs modulus and strain to fracture of HAPEXTM activity was seen on day 7 for TMX and at day 14 on remained unchanged following incubation at 37 ¡C HAPEXTM, while a continuous increase in the ALP and accelerated ageing at 70 ¡C. No degradation in the activity was observed on PE up to day 21. properties of HAPEXTM ensures its implantation suc- Fewer cells adhered to the surface of PE, indicating cess and makes it a highly suitable implant material. that PE surface was not a favourable surface for HOB Biocompatibility of a material is considered to be its cell attachment and proliferation. In comparison, ability to perform with an appropriate host response many cells were seen to attach on the surface of in a speciÞc application [22]. In vitro cell culture HAPEXTM, a cell layer was observed covering the studies using the ÔÔappropriate cellsÕÕ, such as the HOB entire surface and cells at various stages of division cells used in this study, allow the investigation of the were often observed (Fig. 6a). The cells appeared ßat- interfacial response of the implant material in relation tened and maintained the polygonal morphology of to its Þnal use. The biocompatibility of HAPEXTM was osteoblasts, with Þlapodia attached to HA particles demonstrated by the ability of the cells to proliferate (Fig. 6b). on the material. The rapid cell attachment was fol- lowed by proliferation and di¤erentiation of the cells. The high [3H]-TdR incorporation/DNA observed on 4. Discussion HAPEXTM on day 1 indicated an immediate stimula- Bioactivity can be retained in a composite which in- tory e¤ect. When considering osteoblast expression corporates a bioactive phase. However, the inßuence and hence ALP activity, a greater activity was ob- of the bioactive phase on the mechanical properties of served in those HOB cells on HAPEXTM as compared the composite, particularly, those properties when the to those on unÞlled PE. The increase in ALP activity 778 indicated that the cells had ceased to proliferate and 3. W. BONFIELD, in ÔÔAnnals of New York Academy of SciencesÕÕ, Vol. 523, edited by P. Ducheyne and J. E. Lemons had begun to di¤erentiate. This process occurred (New York Academy of Science, New York, 1988) p. 173. earlier on HAPEXTM than on PE. Another striking 4. R. N. DOWNES, S. VARDY, K. E. T ANNER and W . BON- feature frequently observed was that cell Þlapodia FIELD, Bioceramics 4 (1991) 239. were embedded within HA particles on the surface of 5. K. E . T ANNER, R. N. DOWNES and W. B ONF IELD, Br. HAPEXTM. It appeared that HAPEXTM provided Ceram. ¹rans. 93 (1994) 104. 6. J. ABRAM , J. BOWMAN, J. C. BEHIRI and W. BON- preferential sites for cell attachment and stimulated FIELD, Plast. Rubb. Process. Applic. 4 (1984) 261. the overall HOB cell activity. The stimulatory e¤ect 7. W. BONFI ELD, J . C . BE HI RI, C. DOYLE, J. BOWMA N might be caused by the presence of HA particles which and J. ABRAM, in ÔÔBiomaterials and BiomechanicsÕÕ, edited provide a large surface area (Fig. 2) for the absorption by P. Ducheyne, G. Van der Perre and A. E. Aubert (Elsevier, of proteins and growth factors, which in turn may Amsterdam, 1983) p. 421. 8. G. P. EVA NS, J. C. BEHI RI, J. D. CURREY, W. BON- enhance cellular activity. Therefore, HA particles in FIELD, J. Mater. Sci. Mater. Med. 1 (1990) 38. HAPEXTM may act as microanchors for developing 9. K. HEMACHANDRA, PhD thesis, University of London direct bone bonding in situ. (1992). 10. M. WA NG, D. PORTER and W. BONFIELD, Br. Ceram. ¹rans. 93 (1994) 91. 11. P. S. T HEOCARIS, G. C. PAPA NOCOL AOU and E. A. 5. Conclusions KONTOU, J. Polym. Sci. 28 (1983) 3145. The mechanical properties of HAPEXTM were 12. G. C. PAPANOCOLAOU and R. MER COGL IANO, Plast. not altered by contact with physiological solution. Rubb. Process. Applic. 6 (1986) 229. Furthermore, HAPEXTM provided a favourable 13. J. SUWANPRATEEB, PhD thesis, University of London environment for HOB cell attachment, proliferation (1996). 14. W. BONFIELD, C. DOYLE and K. E. TANNER, in ÔÔBiolo- and di¤erentiation. A combination of these pro- gical and Biomechanical Performance of BiomaterialsÕÕ, edited perties will lead to HAPEXTM having the long life- by P. Cristel, A. Meunier and A. J. C. Lee (Elsevier, Amster- time required for permanent implants, which make dam, 1986) p. 153. it a highly suitable second generation implant 15. Y. I TAKURA, A. KOSUGI , H. SUDO, S. YAMAMOT O material. and M . KUMEGAWA, J. Biomed. Mater. Res. 22 (1988) 613. F. B. BAGAMBI SA and U. JOOS, Biomaterials 11 (1990) 50. 17. W. C. A. V ROUWENVE LDER, C. G. GROOT and K. DE GR OOT, J. Biomed. Mater. Res. 27 (1993) 465. Acknowledgements 18. C. FAUCHEUX, R. B AREILL E, F. ROUAI S, J. AMEDEE, The continuing support of EPSRC for the IRC core A. LIEBENDORFER and M. DARD, J. Mater. Sci. Mater. programme is gratefully acknowledged, together with Med. 5 (1994) 635. the assistance and useful discussion from Dr J. Suwan- 19. L. DI SI LVIO, PhD thesis, University of London (1995). prateeb, Mr M. Kayser, Mrs C. Cli¤ord and Mrs. 20. T. KOKUBO, H. KUSHITANI , S. SA KKA, T. KITSUGI and T. Y AMA MURO, J. Biomed. Mater. Res. 24 (1990) 721. N. Gurav. 21. R. RAGO, J. MITCHEN and G. WI LDING, Anal. Biochem. 191 (1990) 31. 22. D. F. WIL LIAMS, J. Biomed. Engng 11 (1989) 185. References 1. L. L . HENCH, J. Amer. Ceram. Soc. 74 (1991) 1487. 2. W. BONFIELD,M.D. GRYNPAS, A. E. TULLY, Received 5 May J. BOWM ANN and J. ABRAM, Biomaterials 2 (1981) 185. and accepted 11 May 1997

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Journal of Materials Science: Materials in Medicine – Springer Journals

Published: Sep 12, 2004

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