Direct and indirect effects of P2O5 glass reinforced-hydroxyapatite composites on the growth and function of osteoblast-like cells

Direct and indirect effects of P2O5 glass reinforced-hydroxyapatite composites on the growth and function of osteoblast-like cells

Biomaterials 21 (2000) 1165}1172 Direct and indirect e!ects of P O glass reinforced-hydroxyapatite   composites on the growth and function of osteo...

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Biomaterials 21 (2000) 1165}1172

Direct and indirect e!ects of P O glass reinforced-hydroxyapatite   composites on the growth and function of osteoblast-like cells M.A. Lopes , J.C. Knowles, J.D. Santos , F.J. Monteiro , I. Olsen * INEB*Instituto de Engenharia Biome& dica, Rua do Campo Alegre N 823, 4150 Porto, Portugal Department of Metallurgical and Materials Engineering, FEUP, University of Oporto, Rua dos Bragas, 4099 Porto, Portugal Department of Biomaterials, Eastman Dental Institute, University College London, 256 Gray+s Inn Road, London, WC1X 8LD, UK Department of Periodontology, Eastman Dental Institute, University College London, Room RL16, 256 Gray+s Inn Road, London, WC1X 8LD, UK Received 7 June 1999; accepted 16 December 1999

Abstract Human osteoblast-like cells were plated on hydroxyapatite and P O -glass reinforced hydroxyapatite composite discs. They were   also cultured in the presence of media obtained by incubating the discs in the absence of cells. The e!ects of these direct and indirect interactions were examined by measuring cell proliferation and the expression of certain key extracellular matrix antigens. One composite was found to initially delay cell growth, while the extract of a di!erent composite appeared to down-regulate DNA synthesis. Flow cytometry analysis showed that growth directly on the discs had little e!ect on collagen type I, but reduced "bronectin and osteocalcin levels. The extracts of the materials generally had less e!ect, although one extract obtained from the glass-reinforced hydroxyapatite signi"cantly down-regulated "bronectin. These in vitro studies thus suggest that there were only few di!erences overall in the growth of the cells directly on the glass-reinforced compared with the hydroxyapatite discs and also only relatively small e!ects of the extracts on the cells. However, the #ow cytometry results suggest that both the materials and the extracts may have a potentially important in#uence on connective tissue production, and that these e!ects are both material- and antigen-speci"c.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Biocompatibility; In vitro; Glass-reinforced hydroxyapatite; Flow cytometry; Bone cells

1. Introduction As as result of the time, e!ort, cost, increasing restrictions and lack of precise basic data derived from animal experimentation, in vitro testing has become an important, major tool for evaluating the compatibility of new and modi"ed materials for surgical application [1]. A main type of cell culture system widely used to evaluate biological responses involves placing &target' cells directly on the surface of the material to be tested [1,2]. This has the major advantage that it most closely mimics physiological conditions by establishing a concentration gradient of chemicals which di!uses away from the cells, as it would in intact tissues in situ. Moreover, it is clear that in studying such direct cell}surface interactions, the surface charge, surface topography and surface free energy play

* Corresponding author. Tel./fax: 00-44-171-915-1254. E-mail address: [email protected] (I. Olsen).

a fundamental part in cell adhesion, spreading, growth and utimately function of the cell [1,2]. A second type of approach for assessing biocompatibility has also been used, in which the the test materials are "rst &extracted' by immersion in a physiological solution which is subsequently placed into culture with the target cells [1,2]. This indirect method has the important and highly relevant advantage of being able to examine whether substances are &leached' from the test materials and exert a deleterious, or indeed bene"cial, e!ect. Only relatively few studies of biocompatibility have been carried out using primary cell cultures obtained directly from the tissues to be tested. This is partly because of the need to repeatedly obtain fresh sources of tissue and of maintaining the isolated cells for long periods in tissue culture, particularly under conditions in which their functional activities are not substantially altered [3,4]. To overcome this di$culty, we have recently used long-term cell lines &immortalized' by transduction with the SV40 large T antigen [5,6]. Generally,

0142-9612/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 0 0 7 - 7


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however, established cell lines have been used as in vitro models for testing biocompatibility because they are readily available in large numbers and their genetic uniformity of phenotype and function produces more reproducible assays and reduces variability within and between laboratories. The MG63 cell line was originally isolated from a human osteosarcoma and is now wellcharacterized, exhibiting a number of features closely associated with the osteoblast phenotype [7]. The present study has used these cells to compare the functional consequences of direct and indirect interactions with novel calcium phosphate materials that have been developed by using a P O glass system as a sintering aid   for hydroxyapatite (HA) [8]. The biocompatibility of these glass reinforced-hydroxyapatite (GR-HA) composites has been assessed by measuring their e!ects on cell proliferation and by using the #ow cytometry (FCM) technique to measure the expression of a number of key connective tissue antigens which are fundamental in the successful response to implant materials.

2. Materials and methods 2.1. Preparation of discs for direct assay

Table 1 The composition of the two glasses, designated B and D1, used to prepare the HA composites containing a "nal 4.0 mol% of each glass (designated 4B and 4D1)

B D1

2.2. Preparation of liquid extracts for indirect assay Powders were prepared by milling the disc samples of all materials and then sieved, particle sizes of between 150 and 300 lm being used for the preparation of the extract (surface area, BET*0.12$0.02 m/g). The powders were sterilised as above and 0.2 g was mixed with 15 ml of alpha-minimum essential medium (a-MEM) (Gibco; Paisley, Scotland) in sterile plastic containers, which were gently agitated on a mechanical roller at room temperature for 48 h. After centrifugation, the supernatants were removed and "ltered using 0.2 lm sterile inorganic membrane "lters (Anatop 25) (Whatman International Ltd.; Maidstone, England). This sterilisation procedure was carried out to remove any possible bacterial contamination which might interfere with the subsequent cell culture experiments and also removed all particulate materials above 0.2 lm in size. The extracts were stored at 43C and used instead of the a-MEM, described below, for indirect evaluation. 2.3. Cell culture

The chemical precursors phosphorous pentoxide, calcium hydrogen orthophosphate, calcium #uoride and magnesium hydrogen phosphate were used to generate two glasses (B and D1), having the compositions shown in Table 1. Composites were obtained by mixing 4.0% (w/w) of these glasses with HA (batch P120) (Plasma Biotal; Tideswell, UK), yielding the GR-HA glasses 4B and 4D1. A detailed procedure for the preparation of these materials has been reported previously [8]. Brie#y, the HA and HA composite powders were uniaxially pressed at 288 MPa in steel dies to produce 14 and 30 mm diameter discs using 4 g of powder per specimen. Discs were then placed on an alumina tile and "red to 13003C at a ramp rate of 43C/min. The set temperature was maintained for 1 h, followed by natural cooling inside the furnace. All disc specimens were mechanically polished to 1 lm "nish, ultrasonically de-greased and cleaned in ethanol followed by deionized water. Prior to


cell culture, the discs were sterilised in a dry atmosphere at 1803C for 60 min.

Composition (% of total) P O  




75.0 50.0

15.0 16.5

10.0 00.0

00.0 33.5

MG63 cells were cultured at 373C in a humidi"ed atmosphere of 5% CO in air, in 75 cm #asks contain ing 10 ml of the complete a-MEM medium, containing 10% foetal calf serum (FCS) (PAA Laboratories; Consett, England), 2 mM L-glutamine (Gibco), 50 IU/ml penicillin (Gibco) and 50 lg/ml streptomycin (Gibco). Media were changed every third day and for sub-culture the cell monolayer was washed twice with phosphate-bu!ered saline (PBS) (Gibco) and incubated with trypsin}EDTA solution (0.25% trypsin, 1 mM EDTA) (Gibco) for 10 min at 373C. The e!ect of trypsin was then inhibited by adding the complete medium at room temperature. The cells were washed twice by centrifugation and resuspended in complete medium for re-seeding and growing in new culture #asks. 2.4. Cell morphology The cells were seeded on the plastic and the discs at low density (5000 cells/cm), as described below. The morphology of the cells cultured directly on the plastic was examined by phase contrast microscopy. Cells cultured on the HA and GR-HA discs were examined by scanning electron microscopy (SEM) as previously described [9]. Brie#y, for SEM the specimens were "xed in 1.5% cacodylate-bu!ered glutaraldehyde for 3 h at room temperature, dehydrated through a series of graded alcohols and critical point dried. Microscopic observations were performed using a JEOL WINSEM 6300 series "eld

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emission scanning electron microscope (Jeol UK Ltd; Welwyn Garden City, England). 2.5. DNA synthesis and content For direct evaluation, the 14 mm HA and GR-HA discs were placed into 24-well Falcon culture plates (Becton Dickinson; Cowley, England). Aliquots of MG63 cells were plated directly onto the discs at a density of 5000 cells/cm. They were allowed to settle for 2 h in the incubator at 373C, after which 1.0 ml of complete medium was added. At the time periods indicated, DNA synthesis was assessed by incubating the cells for 4 h at 373C with 2 lCi/ml of tritiated thymidine ([H]-TdR) (Amersham International; Amersham, England), a radiolabelled precursor of DNA. The cells were then washed twice with cold PBS and treated for 24 h at 603C with 400 ll of a papain solution containing 1 ll/ml of papain (type III) (Sigma; Poole, England) in 5 mM cysteine and 5 mM EDTA in PBS, pH 5.7 [9]. A volume of 150 ll of the cell digest was added to 5 ml of a scintillation cocktail (Unisolve1) (NBS Biologicals; Edison, NJ, USA) and the amount of isotope incorporated measured using a Wallac 1409 liquid scintillation counter (Turku, Finland). A second aliquot of 150 ll of the digest was used to measure the total DNA by the DAPI method, as previously described [10]. Standards were prepared using calf thymus DNA (Sigma) and the #uorescence measured on an LS 50 luminescence spectrometer (Perkin Elmer; Beacons"eld, England). The results are shown as the counts per min (cpm) of [H]-TdR incorporated per lg DNA. For indirect evaluation of the e!ects of the material extracts, the cells were seeded directly into the plastic culture and incubated for 2 h at 373C, after which the medium was removed and replaced with the HA and GR-HA extracts. At the time periods indicated, digests of the cells were prepared and assayed using the DAPI method for DNA content, as described above. 2.6. Immunoyuorescent staining of antigens For direct evaluation, 30 mm HA and GR-HA discs were placed into 6-well Falcon culture plates. The cells were plated at 10 000/cm on the discs and on the control plastic dishes and allowed to settle for 2 h , as described above, after which 2.5 ml of complete medium was added. By 6 days of culture, the cells had ceased exponential growth and were visualized, on the plastic dishes, as a &con#uent' monolayer. The expression of collagen type I (COL I), "bronectin (FN) and osteocalcin (OC) were measured because these antigens have a major role in connective tissue integrity and function, as noted. The cells were washed twice with PBS and detached using 20 mM EDTA in PBS for approximately 5 min at 373C. Trypsin was not used to detach the cells in order to avoid


possible proteolytic loss of surface-associated antigen. The cells were centrifuged at 400g for 7 min and the pellet was resuspended and again centrifuged. They were "xed in 1% (w/v) paraformaldehyde in PBS for 30 min, then washed by centrifugation and resuspended in a washing bu!er containing 2% FCS and 0.05% sodium azide in PBS. They were then permeabilized for 10 min using washing bu!er containing 0.1% (w/v) saponin (Sigma), a non-ionic detergent, to facilitate antibody entry in order to detect intracellular antigen, then washed, centrifuged and resuspended in washing bu!er. Aliquots of 10 "xed, permeabilized cells were used to measure the level of each antigen by FCM, as follows. The cells were incubated for 60 min at room temperature in the following primary antibodies: mouse monoclonal antibodies (mAbs) against human COL I (Gibco) and FN (Sigma), diluted 1 : 100 and 1 : 10, respectively; rabbit polyclonal antibody against human OC (Biogenesis; Poole, England), diluted 1 : 100. Mouse IgG (Dako;  High Wycombe, England) and normal pre-immune rabbit serum were used as negative controls. Cells were then washed by centrifugation and resuspended again in washing bu!er with 0.1% saponin. Secondary antibodies, #uorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Dako) and FITC-conjugated swine anti-rabbit IgG (Dako), diluted 1 : 20, were added for 30 min at room temperature. Cells were washed again and resuspended in 400 ll of washing bu!er and analysed as below. For indirect evaluation, the cells were seeded directly into the plastic culture dishes at the same cell density as above and incubated for 2 h at 373C, after which the medium was removed and replaced with the HA and GR-HA extracts. After 6 days of culture, antigen expression was measured as described above. 2.7. FCM analysis The light scattering properties and the #uorescence of cells stained with FITC were measured on a FACScan #ow cytometer (Becton Dickinson). The excitation source was an argon-ion laser emitting a 488 nm beam at 15 mW. Analysis was performed on 10 000 individual cells. Light scattering was measured on a linear scale of 1024 channels, the forward light scattering (FSC) considered to be proportional to the size of the cells, while the side light scattering (SSC) is related to the granularity and intracellular complexity of the cells. The green #uorescence (FITC) emission was detected on a logarithmic scale of four decades of log and is proportional to the relative level of expression of an antigen using a particular antibody under speci"ed and constant conditions, i.e., no comparisons were made between #uorescence values obtained using di!erent antibodies or altered staining conditions. In addition, to accurately determine relative antigen levels expressed by cells stained with FITC, it


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was "rst necessary to eliminate cell debris and cell aggregates by 'gating' the FSC and SSC dot plot displays. Data were collected, stored and analysed with CELLQuest Software (Becton-Dickinson). 2.8. Statistical analysis Three separate, individual culture experiments of each type were performed in which all the materials, extracts and controls were measured at the same time. The results are shown as the arithmetic means$standard deviation (SD). For the measurement of relative antigen levels, the results are shown as the average #uorescence intensity (AFI) of 10 000 individual cells$SD. Statistical analysis of the results was performed using the Students' t-test, P(0.05 considered to be signi"cant.

3. Results 3.1. Cell morphology Fig. 1 is a representative SEM photomicrograph of MG63 cells grown at sub-con#uent cell density directly on the 4D1 composite. At day 1, the cells were wellattached and there were numerous rounded cell bodies (Fig. 1a). By day 3, the cells were even more #attened and spread, showing numerous, highly extended "lipodia (Fig. 1b) and rough dorsal surfaces characteristic of active cells, as shown in Fig. 1c. No obvious di!erences in morphology were noted between the cells grown on the GR-HA composites and the HA alone. In addition, the cells incubated on plastic with the extracts of the HA and HA-glasses had the same morphological appearance, by phase contrast microscopy, as did the control cells cultured with fresh medium only (data not shown). 3.2. DNA content and synthesis The total DNA content of cells grown directly on the HA and GR-HA composites was measured during 7 days of culture. The results in Fig. 2 show that there was a progressive and very similar increase in the DNA content of the HA and 4D1 composite cultures over this period. However, the increase in DNA content of the 4B glass-grown culture was notably delayed until day 4,

䉴 Fig. 1. SEM of MG63 cells cultured directly on the 4D1 composite. Note that, in these sub-con#uent cultures, (a) after 1 day of culture there were numerous rounded cell bodies (arrows), corresponding to actively dividing cells, while by 3 days (b) the cells were well-spread and had many extended cytoplasmic processes (arrows). In the higher magni"cation in (c), the rough dorsal surfaces of the cells are evident. The "gures show the accelerating voltage (KV) and the magni"cation, indicated by the length of the bar (lm) and X value.

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Fig. 2. Growth of MG63 cells directly on HA and GR-HA discs. The cells were seeded on the HA (open circles) and the 4B (closed triangles) and 4D1 (closed circles) glass composite discs and DNA content measured by the DAPI method, as described in the Materials and Methods. Total DNA content is shown as lg/150 ll. Note the lack of increase in DNA content between 2 and 4 days in the cells grown on the 4B discs.

after which the rate of increase became similar to the HA and 4D1 cultures. Thus, despite an initial delay in the growth of the cells on the #uoride-containing glass (4B), the HA and HA composites appeared to have little, long-term e!ect on the overall proliferation rate of the MG63 cells. Fig. 3 shows the synthesis of DNA by the MG63 cells cultured in the presence of extracts of HA and the 4B and 4D1 glass composites. These data indicate that while DNA synthesis in the 4B culture was intially slightly elevated (on day 2), [H]-TdR incorporation in the 4D1 culture was substantially reduced at day 4 and remained at a low level. By day 6, the synthesis of new DNA was low in all cultures.


Fig. 3. H-TdR incorporation by MG63 cells incubated in the presence of the material extracts. The MG63 cells were cultured in the presence of the extracts of the HA (open circles), 4B (closed triangles) and 4D1 (closed circles) discs. The rate of DNA synthesis was measured by the incorporation of H-TdR (cpm/lg DNA, ;10), as described in the Materials and Methods. Note the reduced rate of DNA synthesis in the presence of the 4D1 extract on day 4.

Table 2 Expression of connective tissue antigens by MG63 cells incubated directly on the HA and GR-HA discs Disc material

HA 4B 4D1

AFI (% of control) COL I



88$2 87$2 88$6

82 $9 60$3 67$7

90 $6 67$7 80 $10

The AFI values ($SD) of COL I, FN and OC are shown as the % of the AFI of the control cells incubated directly on plastic culture dishes. Signi"cantly di!erent from control cultures; P(0.05.

3.3. Expression of connective tissue antigens FCM was used to measure the relative cell-associated levels of COL I, FN and OC expression by the MG63 cells grown for 6 days directly on the discs and those incubated in the presence of extracts of the materials, as described in the Materials and Methods. Table 2 shows the direct e!ects of the materials on antigen expression, relative to the levels of each of the respective antigens in cells grown directly on the control plastic surface. These data indicate that the HA and GR-HA composites all down-regulated these proteins compared with control cultures. However, while HA reduced all three antigens by between 10 and 18%, the 4B composite caused a signi"cant reduction of the expression of both FN and OC expression, to only 60 and 67% of control levels, respectively (P(0.05). 4D1 signi"cantly down-regulated only FN (Table 2).

In contrast to the direct growth of the cells on the discs, the extracts of the materials generally had a much less pronounced e!ect on the expression of the connective tissue antigens. Thus, as shown in Table 3, both HA and 4B had no in#uence on COL I and OC and only HA reduced FN, to 85% of the control level. However, 4D1, which reduced the expression of OC by 20%, signi"cantly down-regulated FN to 45% of the control level (P(0.05).

4. Discussion The physico-chemical properties of underlying surfaces are considered to have a major in#uence on cellbiomaterial interactions [2,11}14]. Such e!ects are


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Table 3 Expression of connective tissue antigens by MG63 cells incubated with extracts of the HA and GR-HA discs Material extracts

HA 4B 4D1

AFI (% control) COL I



100$12 100$8 100$16

85 $7 100 $9 45$9

100$8 110$14 80$6

The AFI values ($SD) of COL I, FN and OC are shown as the % of the AFI of the control cells incubated with non-treated culture media. Signi"cantly di!erent from control cultures; P(0.05.

mediated, at least partly, by altered attachment and subsequent modulation of the intracellular signals which are generated. These lead eventually to changes in the regulation of many gene sequences, including those of the ECM. Thus, surface material parameters such as roughness, zeta potential, hydrophilic degree and chemical composition have a major impact on the viability and functional activity of anchorage-dependent cells such as osteoblasts. In the present study, all the materials used were identically polished, to minimize the surface roughness e!ect which is known to play a major part in the cellular response. The crystallinity as well as porosity of the materials used here were previously also found to be very similar [15,16]. However, wettability studies performed by measuring static contact angles using the sessile drop method have shown that the GR-HA composites are slightly more hydrophobic than HA [17], while zeta potential measurements have shown that composites are more negatively charged compared with HA [17]. These two important surface properties are thus likely to in#uence the biological behaviour of these biomaterials. The direct method for the bioevaluation of new and modi"ed materials is of particular importance in testing for potential biointegration, in which cells necessarily interact with the materials [18]. In the present study, when the MG63 osteoblast-like cells were cultured directly on the materials, they initially grew more slowly on the 4B composite. This material has been shown by quantitative phase analysis using the Rietveld method to contain, in addition to HA and b-tricalcium phosphate (b-TCP), an a-tricalcium phosphate (a-TCP) secondary phase in the microstructure as a result of the reaction between the P O glass and HA during the liquid sinter  ing process [15,16]. The presence of this a-TCP phase, which is more soluble than HA and b-TCP [19], results in the loss of more Ca> from the surface of the 4B disc. This may change the pH of the cellular environment and have a profound in#uence on cell proliferation. In addition, the glass-reinforced composites are known to be more hydrophobic and negatively charged than HA

alone, thereby also possibly reducing cell spreading and subsequent cell proliferation [20]. However, despite the initial delay in cell growth on glass 4B, in agreement with previous studies of the e!ects of glass composites on the progression of the cell cycle [21,22], the MG63 cells nevertheless appeared to recover and proliferated at a very similar rate as on the other disc materials. The technique of using extracts of the materials to indirectly evaluate their biological potential has the advantage that it provides a more clear indication of the possible e!ects of any ions released by the materials themselves [23], by-passing the additional complication of surface-associated modulation of cell proliferation and function. In the present experiments, phosphorous, calcium, #uoride and magnesium ions could have been leached from the materials and may have had a potent in#uence on the cells, as shown previously for example for the e!ects of #uoride ion on cell proliferation [24]. Although we have not measured possible changes in the chemical composition of the media supplemented with the extracts, these could have been partly responsible for the initial, small elevation of DNA synthesis observed to have been elicited by the 4B extract. Moreover, while the e!ect of this extract was transient, incubation in the presence of the 4D1 extract appeared to cause a continuing reduction in the level of DNA synthesis. The reason for this is not yet known, but this observation suggests the possibility that some deleterious substance(s) may have been leached from this particular composite. It is unlikely, however, that the e!ects of the extracts which we observed were caused by the presence of particulate materials in the extracts, at least larger in size than 0.2 lm, since the extracts were sterilized by "ltration through a 0.2 lm sieve prior to use in the cell culture experiments. The ECM antigens COL I, FN and OC are known to have major roles in connective tissue integrity, adhesion and bone structure and di!erentiation [9,25,26]. Although a large and variable amount of these antigens is normally secreted by cells in vitro and in vivo, a proportion of each antigen is nevertheless retained by the cell. The relative cellular level of each antigen comprising the total of both the intracellular and surface-associated components, can readily and very accurately be measured using the FCM technique, as we have reported previously [27}30]. Moreover, while the precise kinetics of expression of many antigens are known to vary during cell culture, in the present experiments we have compared the e!ects of the materials (and extracts) with that of the control cultures for each antigen at each particular time. Under these conditions, OC and particularly FN appeared to be somewhat adversely a!ected when the MG63 cells were incubated directly on the HA composite discs, particularly glass 4B. In marked contrast, however, incubation of the cells in the presence of the extract of this same glass had no deleterious a!ect on antigen

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expression, OC levels even increasing to some extent. Notably, however, FN was down-regulated by direct growth on 4D1 and was even more severely reduced by the extract of the same material. The reason for these apparently di!erential e!ects of the discs and the extracts on ECM antigen expression are not yet clear, but may be related to physico-chemical features of the materials which could selectively in#uence the relative distribution of cell- and matrix-associated antigen. It is also possible that the materials and extracts may profoundly in#uence gene expression directly, and thus the di!erentiation and functional activities of the &target' cells. The present study has measured certain biological responses of bone-like cells to biomaterial surfaces and biomaterial 'extracts' during the period between one day and one week in tissue culture. However, the cells must "rst attach, spread and begin to grow, and this early phase of cell}biomaterial interaction is thus of paramount importance in ultimately determining the successful bene"t of the material in a clinical application. In vitro studies are therefore now in progress to examine the initial morphological and molecular events which occur in response to HA and modi"ed HA glass composites. Acknowledgements The authors wish to acknowledge the "nancial support of ref. PBICT/CTM/1890/95 research project and ref. FMRH/BD/1355/94 grant "nanced by FCT (Fundac7 a o para a Cie( ncia e Tecnologia). References [1] Kirkpatrick CJ. A critical view of current and proposed methodologies for biocompatibility testing: cytoxicity in vitro. Regulatory A!airs 1992;4:13}32. [2] Hanson S, Lalor PA, Niemi SM, Ratner BD, et al. In: Ratner BD, Ho!man AS, editors. Biomaterials science. An introduction to materials in medicine. Basel: Karger, 1996. p. 215}42. [3] Bou-Gharios G, Osman J, Atherton A, Monoghan P, Vancheeswaran R, Black C, Olsen I. Expression of ectopeptidaes in scleroderma. Ann Rheum Dis 1995;54:111}6. [4] Goseki T, Shimizu N, Iwasawa T, Takiguchi H, Abiko Y. E!ects of in vitro cellular aging on alkaline phosphatase, cathepsin activities and collagen secretion of human periodontal ligamentderived cells. Mech Age Dev 1996;91:1761}83. [5] Parkar MH, Kuru L, O'Hare M, Newman HN, Hughes F, Olsen I. Retroviral transduction of periodontal cells with a temperature-sensitive SV40 large T antigen. Archs Oral Biol 1999; 44:823}34. [6] Simon LV, Beauchamp JR, O'Hare M, Olsen I. Establishment of long-term myogenic cultures from patients with Duchenne muscular dystrophy by retroviral transduction of a temperaturesensitive SV40 large T-antigen construct. Exp Cell Res 1996; 224:264}71. [7] Clover J, Gowen M. Are MG-63 and HOS TE 85 human osteosarcoma cell lines representative models of the osteoblastic phenotype. Bone 1994;15:585}91.


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