Biomaterials 21 (2000) 813}820
Flow cytometry analysis of the e!ects of pre-immersion on the biocompatibility of glass-reinforced hydroxyapatite plasma-sprayed coatings M.P. Ferraz!,", J.C. Knowles#, I. Olsen$,*, F.J. Monteiro!,", J.D. Santos!," !Instituto de Engenharia Biome& dica (INEB), Laboratorio de Biomateriais, Rua do Campo Alegre, 823, 4150 Porto, Portugal "Department of Metallurgical 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 $Department of Periodontology, Eastman Dental Institute, University College London, Room RL 16, 256 Gray+s Inn Road, London, WC1X 8LD, UK Received 17 February 1999; accepted 7 October 1999
Abstract Multilayered coatings composed of mixtures of HA and P O -based bioactive glasses are of potential clinical bene"t in 2 5 orthopaedic and dental surgery. Pre-immersion of these materials has been reported to further enhance their e$cacy in vivo, although the precise biological e!ects of this treatment are not yet known. In this study we have therefore prepared double-layer plasmasprayed coatings and evaluated the e!ects of pre-immersion on the growth and function of human osteosarcoma cells in vitro, using the MTT assay and #ow cytometry analysis, respectively. The results showed that the increase in numbers of viable cells was the same or elevated following incubation on the pre-immersed HA and glass-reinforced HA coatings compared with the non-immersed materials. In addition, the expression of bone sialoprotein and "bronectin, two key connective tissue antigens, was up-regulated in cultures grown on the pre-immersed surfaces compared with the non-treated materials. Moreover, cell numbers and antigen expression both improved as the proportion of glass increased, particularly in the pre-immersed samples. Our "ndings thus suggest that the immersion treatment of these materials appeared to improve the response of these bone-like cells. ( 2000 Elsevier Science Ltd. All rights reserved. Keywords: Biocompatibility; Flow cytometry; Pre-immersion; Glass-reinforced hydroxyapatite
1. Introduction Calcium phosphate ceramics have been widely used as bone replacement materials because of their ability to bond directly to bone [1}3]. The use of one of these, hydroxyapatite (HA), which has many crystallographic features similar to those of the natural apatite present in bone, is limited to low-load applications because of its poor mechanical strength . In addition to apatite, however, the inorganic part of natural bone also contains b-tricalcium phosphate and several ions, including Na`, Mg2`, K` and F~ [4}6], and glasses within the P O }CaO}Na O system have been considered to have 2 5 2 good potential as biomaterials [7}9] because of these
* Corresponding author. Tel./Fax: 00-44-171/915-1254. E-mail address: [email protected]
inorganic constituents. Thus, glass-reinforced HA composites have been developed by incorporating phosphate-based glasses into the microstructure of HA through a simple liquid-phase process [10}12]. Such composites have a higher fracture toughness than sintered HA, and a comparative study of the formation of an apatite layer on the two surfaces have suggested that the composites exhibit an enhanced rate of bone bonding . A number of studies have shown that ceramic plasmasprayed coatings applied onto inert metal substrates (e.g., stainless steel, aluminium, titanium alloy) are also useful as implant materials in orthopaedic and dental applications because of their high level of biocompatibility as well as their mechanical strength [14}21]. In addition, in vitro studies studies suggested that these coated materials are more e!ective than either polished titanium or dense HA alone [21}24]. Moreover, the surface modi"cations of ceramic and glass}ceramic materials induced by
0142-9612/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 4 9 - 5
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pre-immersion in a physiological #uid, and presumably involving the formation of a calcium phosphate-rich layer, have been reported to further enhance their bioactivity [25,26]. However, the potential bene"t of these materials for human use is limited because they have hitherto not been accurately evaluated. In vitro measurements are therefore crucial for determining possible cytotoxic responses and, most importantly, for identifying even relatively small functional e!ects since these are likely to have an important in#uence on wound healing and tissue rebuilding processes in vivo. For this purpose, the present study has employed the technique of #ow cytometry (FCM). FCM has become well-established in a number of biomedical and clinical areas [27}29]. In this procedure, cells #owing in a #uid stream past a laser beam scatter light at small angles (forward scatter; FSC), which is considered proportional to cell size, and at orthogonal angles (side scatter; SSC), which is proportional to the granularity of the cells. In addition, #uorescent dyes and #uorochrome-linked polyclonal and monoclonal antibodies (mAbs) have been developed which serve as highly speci"c biological &tags' for identifying and measuring the relative levels of macromolecular components produced by the cells, such as DNA and many types of intracellular and cell surface antigens. The use of FCM has thus become an exceptionally powerful tool for investigating characteristic features of individual cells in a heterogeneous population without the need of physical separation. The ability to determine multiple parameters of cell phenotype and function has consequently been utilised in a wide range of applications [30}33], including studies in our own laboratory of the response of host cells to various types of tissue implant and regenerative materials [34,35]. As noted above, previous studies have suggested that pre-immersion of certain materials may improve their biocompatibility [25,26], although the precise e!ects of this treatment are not yet known. In the present study we have therefore examined, "rstly, whether pre-immersed and non-treated HA plasma-sprayed materials a!ect the growth of a human osteoblast-like cell line. FCM was then carried out to measure the speci"c e!ects of preimmersion of these materials on the expression of bone sialoprotein (BSP) and "bronectin (FN), two key connective tissue antigens which have a major role in bone integrity, di!erentiation and function [36,37].
was prepared as previously described [11}13]. This was added to HA (Plasma Biotal; Tideswell, UK) at "nal concentrations of 2 and 4% (w/w) (HA-2 and HA-4, respectively) and the composite powders dried, isostatically pressed at 200 MPa and sintered. Samples were milled and sieved to provide a particle size distribution between 53 and 150 lm, suitable for plasma spraying. A commercially available titanium alloy (Ti}6A1}4V; Thyssen; Dusseldorf, Germany) was used as substrate and 3 mm-thick discs having a diameter of 14 mm were prepared. These were plasma-sprayed with the HA powder to a coating thickness of 120 lm. For the composites, the discs were sprayed with a double layer, the "rst of 60 lm of HA and the second of the HA/2% and HA/4% glass composites (HA-2 and HA-4, respectively). For the immersion treatment, the discs were washed twice with distilled water and sterilised in a dry atmosphere for 1 h at 1803C. They were then placed in 1.5 ml of complete culture medium (see below) at 373C in a humidi"ed atmosphere of 5% CO in air for 5 days, since it 2 has been reported that there are no further surface changes after this period . The medium was then removed and the discs used for tissue culture as described below. For the control plastic surfaces, the culture dishes were also pre-incubated for 5 days with 1.5 ml of the complete medium, which was removed and discarded prior to addition of the cells. 2.2. Cell culture MG63 cells, derived from a human osteocarcoma, express a number of features characteristic of osteoblasts  and were used in these experiments. They were cultured at 373C in a humidi"ed atmosphere of a 5% CO in air, in 75 cm2 #asks containing 10 ml of alpha2 minimum essential medium (a-MEM) (Gibco; Paisley, Scotland), 10% foetal calf serum (FCS) (PAA Laboratories GmbH; Linz, Austria), 2 mM L-glutamine (Gibco), 50 IU/ml penicillin (Gibco) and 50 lg/ml steptomycin (Gibco). The medium was 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 to detach the cells. The e!ect of trypsin was then inhibited by adding the complete medium at room temperature, the cells washed twice by centrifugation and resuspended in complete medium for re-seeding and growing in new culture #asks.
2. Materials and methods
2.3. Cell proliferation
2.1. Preparation of HA substrates
The MTT assay was used as a measure of relative cell proliferation. MTT is a pale yellow substrate (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrasodium bromide) which is reduced, by living cells, to a dark blue formazan
A phosphate-based glass containing 35, 35, 20 and 10 mol% of P O , CaO, Na O and K O, respectively, 2 5 2 2
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reaction product. This process requires active mitochondria and is thus an accurate measure of the number of viable cells in a culture. The assay is therefore often used to measure cytoxicity, and is also widely employed to determine the growth of a culture based on the increase in the number of viable cells. To carry out the MTT assay, the 14 mm non-treated and pre-immersed discs were placed into 12-well culture plates (Becton Dickinson; Cowley, England), seeded with approximately 103 cells in 0.1 ml of medium and incubated at 373C for 6 h to allow the cells to attach, after which the discs were placed into 24-well culture plates with 1.5 ml of medium. In control cultures the cells were placed directly into the 24-well plates at the same density as placed onto the discs, the medium again being replaced after 6 h. A low initial cell density was used in these experiments in order to allow the cells to increase over a period of 5 days of culture without becoming excessively dense. After 6 h (day 0 control cells) and 1,3 and 5 days, cell proliferation was evaluated using the MTT assay (Kit CT020) (Chemicon; Harrow, England), in which 0.01 ml of MTT (5 mg/ml) was added to each well and incubated at 373C for 4 h. At the end of the assay, the blue formazan reaction product was dissolved by addition 0.1 ml of isopropanol/0.04 N HCl and transferred to a 96-well plate. The absorbance was measured at 570 nm (A570) using a Multiskan Plus spectrophotometer (Titerteek; Helsinki, Finland). The background absorbance produced by wells containing no cells was subtracted from all samples, and the results are presented as the growth of each culture at each time period relative to the initial number of cells on the disc (cells which adhered to the disc after 6 h; day 0 control cells, as noted above). This procedure thus accounts for possible di!erences in adhesion of the cells to the di!erent surfaces. 2.4. Immunoyuorescent staining Replicate 14 mm non-treated and pre-immersed discs were placed into 12-well culture plates (Becton Dickinson) and seeded with 104 cells on each disc. As described above, the cells were allowed to attach for 6 h and the discs then transferred to 24-well culture plates with 1.5 ml of medium. Control cultures again comprised cells placed directly into the 24-well plates at the same density as on the discs. In these experiments the cells were seeded at a relatively high initial cell density in order to obtain a similar and large number of cells for FCM at the end of the culture period, avoiding possible growth-dependent di!erences in antigen expression. After 5 days of incubation, by which time exponential cell growth in these high-density cultures had ceased, the cells were washed twice with PBS and detached using 20 mM EDTA only (i.e. with no trypsin), for 10 min at 373C. They were centrifuged at 400]g for 7 min, the
pellet resuspended, centrifuged again and the cells "xed in 1% (w/v) paraformaldehyde in PBS for 30 min. After centrifugation, the cells were resuspended in a washing bu!er containing 2% FCS and 0.05% sodium azide in PBS. The expression of bone sialoprotein (BSP) and "bronectin (FN) was measured by FCM as follows. Aliquots of 105 "xed cells were permeabilized, to facilitate the entry of antibodies, by treating for 10 min with the washing bu!er containing 0.1% (w/v) saponin (Sigma; Poole, UK). After centrifugation, the cells were resuspended in washing bu!er and incubated for 60 min at room temperature with rabbit polyclonal antibodies against human BSP and FN (from Dako; High Wycombe, UK), diluted 1 : 100. Cells without the primary antibody were used as negative controls. The cells were washed, centrifuged and resuspended again in washing bu!er with 0.1% saponin. The secondary antibody, #uorescein isothiocynate (FITC)-conjugated swine antirabbit IgG (Dako; diluted 1 : 20) was added for 30 min at room temperature. The cells were washed again and resuspended in 0.5 ml of washing bu!er and analysed as described below. 2.5. FCM analysis The light scattering properties and the #uorescence of cells stained with FITC were measured on a FACScan #ow cytometer (Becton Dickinson, Cowley, UK). The excitation source was an argon-ion laser emitting a 488 nm beam at 15 mW. Analyses were performed on 5000 cells. The FSC and SSC were measured on linear scales of 1024 channels, while green #uorescence (FITC) emission was detected on a logarithmic scale of four decades of log. For the analysis of antigen levels, the #uorescence signals corresponding to debris and cell aggregates were "rst gated out by using the FSC and SSC display. Data were collected, stored and analysed with CELLQuest Software (Becton Dickison). 2.6. Statistical analysis Triplicate culture experiments were performed. The results are shown as the arithmetic means$the standard deviation ($SD). Analysis of the results was carried out using the Student's t-test, with a signi"cance level of P(0.05.
3. Results 3.1. Ewects of materials on cell proliferation As described in the materials and methods, the growth of the MG63 cells was evaluated using the MTT assay, in which the absorbance values obtained are directly
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Fig. 1. E!ects of pre-immersion on the growth of MG63 cells. MG63 cells were grown on non-treated (open bars) and pre-immersed (closed bars) plastic (control), HA, HA-2 and HA-4. The MTT assay was carried out after 1 day (panel A), 3 days (panel B) and 5 days (panel C) of culture, as described in the materials and methods. The results are shown as the average increase in the number of viable cells on each day relative to the number present initially on day 0, which is de"ned as 1.0. The vertical lines are the $SD, obtained in three separate experiments. *Denotes statistically signi"cant di!erence compared with the non-immersed materials (P(0.05).
proportional to the number of viable cells. In the present experiments, few if any cells were observed on the surface of the culture dishes after the discs were removed and there were no non-adherent cells in the culture media, indicating that virtually all the cells had initially adhered to the discs. The change in the numbers of cells on the discs was therefore an accurate measurement of the increase in the number of viable cells compared with the number of cells which had originally adhered to the disc. The results in Fig. 1 show that the cells incubated on the control plastic culture dishes proliferated over 5 days, the number of cells progressively increasing over this period compared with their initial number. Panel A shows that, on day 1, growth on the non-treated HA and particularly on HA-2 was slightly lower compared with the plastic-grown MG63 cells, although the proliferation of the cells on HA-4 was found to be more similar to that of the control cells. Growth on the pre-immersed materials showed very similar pro"les to that on the respective non-treated materials. By day 3, however, although the non-treated HA culture had relatively many fewer cells than the control culture, the increase in cell number was greater in the HA-2 and especially in HA-4-grown culture (panel B). Notably, the increase in the number of MG63 cells grown on each of the pre-immersed materials was markedly elevated compared with the respective non-treated materials, the culture incubated on the preimmersed HA-4 again showing growth at nearly control levels. Similarly, on day 5, the relative cell growth was again reduced in the non-treated HA culture and was
higher in the HA-4 culture (panel C). As on day 3, incubation of the cells on the pre-immersed coatings increased the relative level of cell proliferation on all HA materials, as shown in Fig. 1, panel C. Statistical analysis of the data showed that the growth of the cells on all of the non-treated HA coatings was signi"cantly lower than that of the control culture on days 3 and 5 (P(0.05). Growth on all pre-immersed coatings was also lower than the control cultures, except on day 3 where the relative increase in the number of MG63 cells grown on HA-4 was statistically the same as that of the control, plastic-grown cells. However, at days 3 and 5, growth on all the HA-coated materials was greater when the materials had been pre-immersed compared with the non-treated materials, and these relative di!erences were statistically signi"cant for HA, HA-2 and HA-4. Thus, treatment of the glass-reinforced HA composites by pre-immersion notably reduced the inhibitory e!ect of the plasma-sprayed coated materials on the relative increase in the number of viable bone-like cells. 3.2. Ewects of pre-immersion on cell size and granularity FCM analysis of the size (FSC) and granularity (SSC) of the cells was carried out to determine whether the materials had any e!ect on the physical characteristics of the cells. The representative dot plots shown in Fig. 2 indicate that MG63 cells grown on the control, plastic surface are larger and less granular than the same cells grown on the non-treated HA. The results in Table 1
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Table 1 FCM analysis of the e!ects of pre-immersion on the relative size and granularity of MG63 cells Materials
Relative size (FSC)!
Relative granularity (SSC)!
Non-treated Pre-immersed Non-treated Pre-immersed HA HA-2 HA-4
58.7$2.1 67.9$1.8 86.1$1.9
94.2$2.1 94.9$2.3 95.3$2.1
155.8$3.0 137.9$2.8 102.7$2.6
101.5$2.2 101.2$2.1 100.2$2.2
!The size and granularity of the cells are the average values ($SD) obtained in three separate experiments and are shown as a % of the values obtained for the control cells grown on the plastic surface. These had an average size and granularity of 273$3.4 and 263$3.9, respectively.
Fig. 2. Representative FCM dot plot pro"les of 10 000 individual MG63 cells grown on the surface of plastic (control) and HA for 5 days, showing the size (FSC) and granularity (SSC) distribution of the cells. The cross bar has been placed at arbitrary values of 400 in order to facilitate comparison of the two growth conditions.
present a summary of 3 separate FCM analyses and show that the average size of the cells grown on the non-treated HA, HA-2 and HA-4 composites were reduced compared with the plastic-grown cells, whereas their granularity (SSC$SD) was found to be increased. Moreover, as with the proliferation of the cells, as the percentage of glass increased, the size and the granularity of the cells became more similar to those of the control cells. Most importantly, however, the size and granularity of the MG63 cells were almost entirely una!ected by growth on the pre-immersed coatings, as shown in Table 1. 3.3. Ewects of pre-immersion on antigen expression In order to assess the possible functional e!ects of immersion of the HA and HA composite coatings. FCM was used to measure the expression of two antigens which are considered to have a key role in bone structure and integrity. In each experiment, the light scattering of the cells in each culture was determined and this FSC/SSC display used to eliminate cell debris and cell aggregates from the subsequent #uorescence analysis, as described above. The remaining &events', usually more than 90% of the total, comprised intact, single cells and
were analysed for the green (FITC) #uorescence signals resulting from immunostaining of the cells with the antibodies against BSP and FN. For each cell culture a negative control was also included, which was the background #uorescence signal generated by cells treated with no primary antibody. This was subtracted from the histogram of each of the test samples of cells treated with speci"c polyclonal antibody. Fig. 3 shows representative #uorescence pro"les of the expression of BSP and FN by MG63 cells cultured on the control plastic dishes and HA. These histograms indicate that growth on the non-treated HA down-regulated antigen expression, whereas both BSP and FN levels in MG63 cells cultured on the pre-immersed HA were higher than in plastic-grown cells. The average antigen levels measured in three replicate experiments is shown in Fig. 4, relative to the levels expressed by the plasticgrown cells. These data show, "rstly, that BSP and FN were both signi"cantly down-regulated during growth on the non-treated HA and HA glass coatings compared with the control cultures (P(0.05). Moreover, this decrease was more pronounced on the HA than on the glass-reinforced composites, particularly for BSP, the levels in the HA-4-grown cells more closely approaching that of the control cells. Thus, an increasing proportion of glass in the coating partially restored antigen expression to control levels. Secondly, the results in Fig. 4 also show that, in marked contrast to the non-treated materials, growth of the cells on the pre-immersed coatings substantially up-regulated BSP and FN levels. Thus, the levels of the these two antigens on the HA coating was approximately 125% greater than in control cultures and increased to approximately 150% of control on the HA-4 composite (Fig. 4). In all cases, the relative levels of BSP and FN were very notably elevated in the cells grown on the pre-immersed compared with the non-treated materials, and all of these di!erences were statistically signi"cant (P(0.05).
M.P. Ferraz et al. / Biomaterials 21 (2000) 813}820
Fig. 3. Representative #uorescence intensity pro"les of BSP and FN expression by the MG63 cells described in Fig. 2. Antigen levels were measured by FCM after 5 days of growth on plastic (control) and on non-treated HA (dotted lines) and pre-immersed HA (solid lines). The vertical lines show the position of the average #uorescence intensity (AFI) of the control plastic culture, for comparison purposes. The pro"les of the cells grown on the pre-treated plastic dishes were identical to the non-treated control cultures and are not shown.
Fig. 4. E!ects of pre-immersion on BSP and FN levels in MG63 cells. The cells were grown for 5 days on non-treated plastic, HA, HA-2 and HA-4 (open bars) and on replicate surfaces which had been pre-immersed (closed bars). The bars show the average of the AFI values ($SD) for the HA, HA-2 and HA-4 cultures in three separate experiments, relative to the AFI of the control culture in each experiment, which is de"ned as 100%.
4. Discussion The aim of this work was to examine whether immersion of HA- and HA-glass-coated materials in culture
medium a!ected the growth and functional response of MG63 cells. This cell line has previously been used in biocompatibility studies because it exhibits a number of features which are similar to those of normal human
M.P. Ferraz et al. / Biomaterials 21 (2000) 813}820
osteoblasts . In the present study it was found that there was good attachment and increase in numbers of viable cells on all the materials, none appearing to elicit any major deleterious or cytotoxic responses. However, the increase in the relative growth of the cells on the non-immersed coatings was nevertheless delayed compared with the same cells grown on plastic. In addition, the inhibition was more pronounced on the HA alone than on the glass composites, the growth response becoming more similar to &control' levels as the percentage of glass increased. This e!ect was even more marked when the cells were grown on the pre-immersed composites, indicating that these discs, and especially the HA-4 composite, present more appropriate surface characteristics for cell proliferation. The e!ects of such surfaces are presumably exerted via the cell cycle of the MG63 cells, as we have previously reported for the di!erential response of these cells to HA and glass-reinforced HA , although the e!ects of pre-immersion on the speci"c phases and progression of the MG63 cell cycle have not yet been investigated. The present study also showed, using FCM analysis, that culture of the cells on HA alone clearly reduced their size and increased their granularity. Although the precise relevance of such changes to cell function are not clear, they are sometimes associated with the early stages of apoptosis in some types of cell, although terminal apoptotic cells are almost always less granular as well as smaller . It is notable, however, that the addition of increasing amounts of glass composite again at least partially restored these physical parameters to control levels. Moreover, when the materials were pre-immersed, they were found to have little if any e!ect on the size and granularity of the cells, which remained the same as when they were cultured on the control plastic surface. By using FCM it was also possible to assess e!ects of the materials on the functional activity of the cells. Thus, both BSP and FN, two essential components of bone ECM [36,37], were found to be reduced when grown on the non-immersed compared with the pre-immersed HA materials. However, as with the other parameters of biocompatibility examined in this study, increasing the percentage of glass in the composite appeared to have a bene"cial e!ect, at least on the expression of BSP. In addition, the expression of both BSP and FN was signi"cantly elevated in cultures grown on the HA and HAglass composites, in all cases being higher than in control cells grown on the plastic surface. Our "ndings thus show, "rstly, that HA composites, particularly HA-4, provide better features for cell proliferation and function compared with HA alone and, secondly, that pre-immersion enhances the growth of osteoblast-like cells and the production of connective tissue components in vitro. Since these antigens have a major role in the structure and integrity of hard connective tissue, our "ndings suggest that the pre-immersion-treated composites are more
likely to elicit a more e!ective tissue rebuilding process in vivo and thus represent improved materials for clinical application. The present study has not delineated the surface changes which are responsible for the apparently bene"cial e!ects of pre-immersion, although previous reports have suggested that enhancement or augmentation of a calcium phosphate-rich layer is likely to improve biocompatibility [25,26]. These surface changes are likely to depend on the molecular and ionic interactions between the material and the aqueous environment, including release of certain ionic species, local modi"cation of pH and also adsorption of biologically active molecules from the medium onto the surface. Our results suggest that such changes have a profound in#uence on cell growth and function, although further studies are required to clarify the precise relationship between particular components of the immersion medium, the surface characteristics of the plasma-sprayed HA-glasses and their resultant biological activity. It is notable, however, that despite the clear in#uence of pre-immersion on cell growth and function, this treatment nevertheless had little if any in#uence on the relative e!ects of di!erent amounts of glass in the HA material used in this in vitro model of biocompatibility, which improves as the proportion of glass increases.
Acknowledgements The authors are grateful for the "nancial support of JNICT (project ref.ref.C/CTM/1890/95) and Praxis XXI (grant ref. XXI/BD/9716/96).
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