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Behaviour of fetal rat osteoblasts cultured in vitro on bioactive glass and nonreactive glasses
382
Behavior of fetal rat ost~oblasts cultured IkeV&D on bioactive glass and nonreactive glasses IAEA.
Vrouwenvelder, C.G. Groat* and K, de Groot
Department of Biomatefials and *Laboratory of Cell Biology and Histology, School of Medicine, University of L&den, The Netherlands We examined the behaviour of fetal rat osteoblasts cultured upon bioactive glass and nonreactive glasses, and the supposed stimulatory effects of bioactive glass on osteoblasts. Nonreactive glass cultures showed flattened ceils with afmost no dorsal ruffles. Bioactive glass cultures showed compact cells with dorsal ruffles and filapodia resulting in the formation of a denser cell layer. For confluent nonreactive glass cultures the osteoblast expression was mainly concentrated in the clustered cells which were formed upon the monolayer, whereas for confluent bioactive glass cultures the osteoblast expression was more generally distributed. The production of type I collagen, osteocalcin and an osteoblast-specific antigen was shown by immuno~~ochemistry for all cultures, although differences in distribution were observed. The bioactive layer of bioactive glass is responsible for a better osteoblast-like morphology, a higher proliferation rate and generally a better osteoblast expression. Ka~words: Received
Bioactive
3 February
glass, osreo~lasis,
1991; revised 2 May 1991; accepted
In the field of biomaterials, bioactive glasses are known to have Potential bone-bonding properties1-3. In contrast with more traditional biomaterials, which are inert and do not bond to bone directly because of fibrous encapsulation of the implant, bioglasses appeared to have good biocompatibility after implantation in animals4* 5. Hench and co-workers developed a series of soft glasses with a low silicon content and relatively high percentages of sodium and calcium, thereby stimulating the corrosion behaviour of these so-called bioactive glasses or bioglasses. These bioactive glasses were varied in their composition within certain limits and this resulted in differences in their capability to bond to bone. In spite of these differences, all bioactive glasses demonstrated a characteristic corrosion behaviour after exposure to bodyfluids: a preferential leaching of alkali ions resulting in the formation of a silica-rich gel layer and a calcium phosphate layer on the bioactive glass surfaces-g. Within a certain time, the calcium phosphate layer will recrystallize into hy~oxyapatite. This hydroxyapatite is thought to be responsible for the bone-bonding properties of bioactive glasses and is therefore called a ‘bioactive’ layer. The bone-bonding properties are designated as bioactive behaviour. From in viva experiments’s the optimal composition for bioactive glass, in terms of maximum Correspondence to Dr W.C.A. Vrouwenvelder, Department of Biomaterials, School of Medicine [Building No. 551, University of Leiden, Rijnsburgerweg 10, 2333 AA Leiden. The Netherlands Biomaterials 1992, Vol.
13 No. 6
ceil cu/fufe 20 June 1991
bonding to bone, was found to be: 45% SiO,, 24.5% Na,O, 24.5% CaO and 6% P,O, (in weight 7~).This composition was codedIm as 4555. As bioactive glass demonstrated better bonding to bone, it is thought that the bioactive layer might stimulate osteoblasts in their bone forming behaviour’1*12. In v&o, it appeared that a positive correlation existed between the formation of a bioactive layer and the appearance of collagen in the extracellular matrix13.14. An investigation into the behaviour of osteoblasts in close contact with bioactive glasses will, give more understanding of the bonding-to-bone properties of bioactive glasses15-‘g. To eliminate the complexities normally present in in viva studies4, 5, we chose a simple in vitro model using fetal rat osteoblasts. The purpose of our study was to examine the cell behaviour of fetal rat osteoblast&‘~ ” cultured upon bioactive glass and nonreactive glasses, and to investigate the supposed stimulatory effects of bioactive glass on osteoblasts. Three nonreactive glasses were chosen: a soda-silica-lime glass (like window glass), silica-quartz glass and microscopical coverslips (often used as cell culture substrates). We checked the ~producibility of the glass preparations with two independant analyses of the elemental compositions. The corrosion behaviour of the different glass types was investigated with routine scanning electron microscopy (SEM) techniques connected to a X-ray micro analysis system. Cell cultures were observed histologically after several culture periods by SEM and transmitted light microscopy as we11 as 0 1992 Bu~e~orth-~einemann Ltd 0142-9612/92/060382-11
Rat osteoblasts
cultured
on bioactive
and non reactive
glasses:
immunocytochemistry for type I and II collagen and osteocalcin in the extracellular matrix, and a membrane characterization for osteoblasts. The DNA-content and the total alkaline phosphatase activity (APA) of the cultures were determined biochemically”* 23. The distribution of APA in the cultures was visualized histochemicallyz4.
MATERIALS AND METHODS Preparation of glasses The theoretical compositions (in weight ala)of the glasses used in our experiments are presented in Table 1.
The bioactive glass was the 45S5 type according to Hench and co-workers1-3, prepared at our request by Yokogawa-Electrofact BV (Amersfoort, The Netherlands)25. High grade Na,CO,, CaCO,, SiO, and (NH,),HPO, were thoroughly mixed in a alumina ball mill for 1 h. The mixed powders were preheated at llOO*C in a platinum crucible to eliminate chemically bound carbon dioxide and water. The mixture was held at the reaction temperature (I,) of 135O’C for 2-3 h. To obtain a homogeneous reaction product the mixture was stirred with a platinum spoon. After another 30 min at T, the mixture was cast in preheated graphite moulds. The glass rods obtained, approximately 20 cm length, were annealed at 5OO’C for 6 h to release potential internal physicochemical stresses due to fast cooling. Slow cooling of the rods after annealing took another 10 h. The rods were sawn transversely with an oil-cooled diamond saw. The resulting slides (0.6 mm thickness) were fixed with paraffin wax on a plane parallel dish (diameter approximately 14 cm and containing about 60 slides) and ground with watercooled silicon carbide (fine grit 500 and 800), followed by polishing with cerium oxide paste (gritsize 2 pm: 1650 Cerox, Rhone-Poulenc, France). During polishing water-ethanol mixtures, from 50-50% up to pure ethanol, were used to prevent the bioactive glass from undesirable corrosion by water. All glass samples were cleaned first by rinsing with xylene, followed by a 50-50% acetone-ethanol mixture and then three times with pure ethanol. We obtained thin translucent slides with a smooth and clean surface (diameter 6 mm and 0.4 mm thickness). Sawing, grinding and polishing was done by the Leidse Instrumentmakers School (Leiden, The Netherlands).
Table 1
Theoretical
compositions SiO,
Na,O
of glasses in weight %. CaO
p2°5
Other oxides
Bioactive glass 4585 Normal glass (soda-silica-lime glass) Quartz glass Coverslips ‘125%(Na,O SbO,
45
24.5
24.5
6
-
72
16
12
-
-
_
_
_
t
>99.9 65
+ K,O), 3%A1,03,6.5%Zn0,5.5%TiOp,
7.5% B,O,and2.5%
W.C.A. Vrouwenvelder
et al.
383
Normal glass Preparation of this glass (Yokogawa-Electrofact BV) was comparable with the procedure for bioactive glass, except for a different reaction temperature (Tr = 155O*C) and annealing temperature (T, = 55O’C). Sawing, grinding, polishing and cleaning were performed as described above, resulting in slides with the same dimensions. Quartz glass Quartz glass slides (Heraeus, Nijmegen, with purity >99.9 wt% SiO, were procedure described above.
The Netherlands) obtained by the
Commercially available coverslips [diameter 10 mm and 0.1 mm thickness) were only ethanol cleaned. All samples were sterilized by dry heat in a furnace 150% for 3 h before culturing.
at
Cell culture method Osteoblasts were isolated according to the method of Boonekamp” with some modifications. Briefly, calvaria from 20-day-old rat embryos (n = 10-15) were excised aseptically. The endo- and extracranial periostea were mechanically removed. The calvaria were incubated for 2 X 10 min at 37°C with 4 mM EDTA in phosphate buffered saline (PBS). After rinsing the calvaria for 3 X 5 min with PBS, they were incubated for 10 min with collagenase (1 mglml PBS) at 37°C. The cell suspension obtained was discarded as it contained periosteal fibroblasts, still present after removal of the periostea. Then osteoblasts were isolated by further collagenase treatment (2 X 30 min). The supernatant was centrifuged for 5 min at 1400 r.p.m. (275g). The pellet obtained was resuspended in culture medium: minimum essential medium (a-MEM), containing 5% inactivated fetal calf serum (Gibco), 1 mg/ml glucose and 90 pglml gentamycin. The sterilized glass samples were placed in 24-well culture dishes (Costar]. Samples for morphological studies (SEM and light microscopy) were seeded with a cell density of approximately 5 X lo5 cells/ml. The often observed spontaneous detachment of the cell layer in nonreactive glass cultures was minimized, in the culture periods used, at this cell density. Samples for the DNA content and APA determinations were seeded with a higher cell density of approximately 1 X 10” cells/ml to get a well defined signal with the spectroscopical techniques used. In earlier investigations it was established that the chosen cell densities had no influence on osteoblast expression. On every glass sample, 50 ~1 cell suspension was applied with great care, to avoid unwanted cell attachment to the surrounding surface of the well, so that cells were only allowed to attach for 2 h to the underlying glass substrate, then 800~1 culture medium was also added carefully. During culturing the multi-well dishes were kept in an incubator (with 95% air humidity and 5% CO,) at 37’C. During the first 6 d, half of the medium was replaced every 72 h, thereafter it was changed every 48 h. Biomaterials 1992, Vol. 13 No. 6
Rat osteoblasts
384
Preparation
cultured
on bioactive
for SEM
Osteoblast morphology After 2, 6 and 12 d cultures were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH = 7.4) for 2 h, rinsed with PBS (3 X 5 min) and dehydrated in a graded ethanol series. Critical point drying of the samples (Balzer) was followed by gold sputtering (Balzer). The preparations were examined at 15 kV in a Philips SEM 525 M.
Surface corrosion behaviour To observe morphological differences in the glass surfaces caused by corrosion, samples without cells were placed in culture medium for 2, 6, and 12 d, fixed and further treated as routine SEM samples.
X-ray micro analysis To investigate the surface corrosion behaviour of the glasses, samples were exposed to the culture medium for 2, 6, and 12 d and compared with untreated samples. Bioactive glass samples were also examined at an interval of 20 d of exposure. All samples were critical point dried and carbon-sputter coated. We used the Philips SEM 525 M connected to a Tracor/Noran Voyager X-ray micro analysis system. All samples were analysed at 15 kV. The microscopical conditions selected remained constant during all measurements. The results from the X-ray micro analysis were used to obtain semi-quantitative data. To obtain these data a pRZ correction method was performed. Changes in the mutual percentages of the oxides present in the samples were used to determine the surface corrosion behaviour.
Transmitted
light microscopy
Cultures were fixed after 2,6 and 12 d with glutaraldehyde as for SEM and stained with hematoxylin and eosin.
Fluorescence
microscopy
and non reactive
glasses:
W.C.A. Vrouwenvelder et al.
Immunocytochemistry was performed according to standard procedures. The samples were observed with an Olympus BHRFL fluorescence microscope.
DNA content and alkaline
phosphatase
activity
DNA con tent After 0.25, 3, 6 and 8 d the cell cultures were rinsed with PBS and stored at -20°C. The samples collected were trypsinized (1 mg trypsin [Sigma, USA]/ml tyrode buffer) according to the method of Karsten and Wollenberge?. The cell membranes were sonified to release the DNA content. Heparin solution 1 ml, (Tromboliquine (Organon 5000 units, diluted 1:600)/ml PBS) and 0.5 ml RNase solution (ribonuclease-A (Sigma, USA) 0.05 mg/ml PBS) were added to 0.5 ml trypsinized suspension. After incubation at room temperature (0.5-l h) we added 0.5 ml ethidium bromide (0.025 mg/ml PBS). All samples, including a series of standardized DNA samples, were measured on a Perkin-Elmer 1000 Fluorescence Spectrometer at 590 nm. A correction was performed for the larger area of the coverslips to correspond with the other glass types.
Alkaline phosphatase
activity (APA)
After 0.25, 3, 6 and 8 d the cell cultures were rinsed with PBS and stored at -20°C. The samples collected were treatedz3 with 1 ml PBS with 0.05% triton (X-100) and then sonified. To 100 ~1 sonified solution, 100 ~1 substrate (20 mM paranitrophenylphosphate (Sigma, USA) in 1 M diethanol-amine and 1 mM MgCl,, (pH = 9.8) was added. The mixture was incubated at 37’C. The reaction was stopped with 1 ml 0.1 M NaOH when the colour of the mixture was comparable to the colour of a standardized series of paranitrophenol samples within 15-30 min. All samples including a series of standards were measured on a Gilford N-300 Micro-sample Spectrophotometer at 410 nm. A correction was performed for the larger area of the coverslips to correspond with the other glass types.
Type I and II collagen
APA/DNA
Type I collagen (as a marker for osteoblast-like character) and type II collagen (as a marker for possible contamination with chondrocytes) were located immunohistochemically after 12 d culture with a goat-antiserum and a sheep-anti-goat/FITC conjugate [Sigma, USA). The cultures were fixed in pure acetone for 2 min and air dried.
The determination of the ratio APA/DNA gives information about the mean APA per cell. Both methods described above were performed on separate cultures (at each time interval] because specific conditions were required for each method. Therefore, we attempted to use one series in which both determinations were performed on the same cultures: to the remaining 9OOpl from the APA determination, 1 mg/lOO ,h1 trypsin solution was added whereby the total trypsin concentration was kept at 1 mg/ml as for normal DNA determinations. After sonification once more, the DNA determination procedure was as described above. As the APA/DNA is expressed as the amount of APA per cell no correction for the larger area of the coverslips was needed.
Osteocalcin Other cultures were screened for the presence of osteocalcin, after 1% paraformaldehyde (in 0.1 M sodium cacodylate buffer] fixation, with a goat-antiserum (Sanbio) and a rabbit-anti-goat/FITC conjugate (Sigma].
Osteoblast antigenicity We used monoclonal antibodies directed against a membrane associated antigen of rat osteoblasts as an exclusive marker for osteoblast expression. The monoclonal antibody was located with a goatanti-mouse/FITC conjugate (Sigma) after 12 d culture. These cultures were also fixed with paraformaldehyde. Biomaterials
1992. Vol. 13 No. 6
Histochemical
staining of APA
To visualize the distribution of the APA in the cultures histochemical stainingz4 was used after 3, 6 and 8 d corresponding with the periods for the biochemical determinations.
Rat osteoblasts
cultured
on bioactive
and non reactive
alasses:
W.C.A. Vrouwenvelder
et al.
385
Statistical analysis Mean values and standard deviations for the DNA content and APA were computed for six determinations at each time-interval. The Student’s t-test was used to establish the significance of differences between the mean values.
Starting from 4 d a partial detachment of the cell layer was sometimes observed for nonreactive glass cultures with an increasing incidence when confluency was reached (in about 6 days). As a general observation a lot of nonreactive glass cultures showed spontaneous detachment of the cell layer within 12 d.
Elemental analyses Two independant analytical determinations were used. The procedure of the first analytical method is described briefly. First, the ICP-AES (Elephant Industries BV, Hoorn, The Netherlands) was carried out as follows: a sample of 0.1 g powdered glass was dissolved in a Teflon flask containing 4 ml HCl(3770), 2 ml HNO, (65%) and 2 ml HF (40%). The mixture was placed in a CEM microwave oven (type MDS 61 D). After dissolution, 3 g H,BO, in 80 ml distilled water was added to complex the HF. This solution was transferred into a 500 ml TPX measuring flask and the volume was adjusted with distilled water to 500 ml. For the final analysis we used an ICAP 61 (Therm0 Jarrell Ash), a simultaneous emission spectrometer with an inductively coupled Argon plasma excitation source. The second determination included three analytical techniques. For SiO, determination a gravimetrical method was used. Na,O and CaO were determined by means of atomic absorption spectroscopy, and the P,05 was determined photometrically. These determinations were carried out by TNO, Apeldoorn, The Netherlands.
Surface corrosion morphology Figure 3a shows the appearance of the bioactive glass surface after exposure to the culture medium for 12 d. The irregular cracks are caused by the corrosion process. The typical shrinkage of the corroded layer is due to dehydration during critical point drying. With X-ray micro analysis we confirmed this to be the calcium phosphate enriched layer. The irregularities at the calcium phosphate enriched surface are caused by incorporated and/or precipitated serum proteins from the culture medium. Figure 3b shows the appearance of the surface of quartz glass after exposure to the culture medium for 12 d. The irregular spots on the surface are also caused by precipitation of serum proteins. No corrosion was observed for any of the nonreactive glass types.
RESULTS Scanning
electron
microscopy
Osteoblast morphology Figure 1 shows the morphology of osteoblasts cultured on bioactive glass and quartz glass for 2 d. Cells cultured on bioactive glass are compact, have dorsal ruffles and filapodia (with a fibre like attachment to the substrate surface). Cell divisions are relatively often seen. Notice the presence of the corroded top layer on bioactive glass (Figure la and b). Cells on quartz glass have a flattened morphology with a smooth dorsal surface on which dorsal ruffles and filapodia are hardly seen (Figure lc and d). No surface corrosion was observed. Figure 2a and b show the morphology of osteoblasts cultured on bioactive glass for 12 d. On top of the monolayer individual cells are observed as well as clustered cells which have a compact structure with many dorsal ruffles. At higher magnification, fibres (possibly of collagen) connecting the cells are seen in the extracellular matrix (Figure zb). Figure zc and d show the morphology of an intact quartz glass culture after 12 d. Clusters of cells are seen on top of the existing monolayer, although they are small in size. At higher magnification it can be seen that the clustered cells show a poor morphology often with numerous blebs on the dorsal membranes which indicates poor condition (Figure Zd). Fibres are also observed in the extracellular matrix.
X-ray micro analysis The corrosion processes of the samples are expressed as the mutual variations of the sum of elemental oxide concentrations. Nonreactive glasses did not show any changes in composition. Bioactive glass showed the expected corrosion behaviour by the development of a calcium phosphate-rich top layer during time. In Figure 4 the variations in the concentrations of the elemental oxides versus time are presented. Within 2 d a strong decrease in sodium oxide and an increase in calcium and phosphorous oxides are observed. During the first 12 d the calcium and phosphorous oxides increase rapidly, which indicates that an (amorphous) calcium phosphate layer is formed on top of the bioactive glass. After 12 d the corrosion process of the bioactive glass slows down.
Transmitted
light microscopy
All cultures showed an irregular cell pattern after 2 d. After 6 and 12 d a confluent cell layer was seen for all (intact) cultures. Clustered cells as well as individual cells were seen on top of the existing monolayer. These findings are in agreement with our SEM observations. Forbioactive glass cultures the morphology was difficult to interpret because bioactive glass samples lost their translucency due to the corrosion. Within 2 d these cultures could only be observed at low magnification (X40).
Fluorescence
microscopy
Type I and II collagen The osteoblast cultures showed strong synthesis of type I collagen on all substrates, whereas type II collagen was hardly produced in any of the cultures. Figures 5a and b show the presence of type I collagen in the extracellular matrix after 12 d of culture on bioactive glass and on quartz glass respectively. Upon bioactive glass the type I collagen is mainly concentrated in the clusters of cells Biomaterials
1992. Vol. 13 No. 6
386
Rat osteoblasts
cultured
on bioactive
and non reactive glasses: W.C.A. Vrouwe~weidef
b
et al
I
Fiiure 1 Scanning electron microscopy images showing osteoblasts after 2 d: a, osteoblasts cultured on bioactive glass for 2 d, note the corroded layer (cl); b, magnification of marked area shows filapodia (f) and dorsal ruffles (r); c, shows flattened and spread osteoblasts with almost no dorsal ruffles on quartz glass, no surface corrosion (ns) is seen; d, the flattened cell structure and the lack of dorsal ruffles are shown in more detail. Comparison with a and b shows the striking difference in osteoblast morphology. Biomaterials 1992, Vol. 13 No. 6
Rat osteoblasts cultured on bioactive and non reactive ~--_ ___..~~-._._..____.~._...
glasses:
W.C.A. Vrouwenvelder
et al.
387
Figure 2 Scanning electron microscopy showing the morphology of osteoblasts on bioactive glass and quartz glass after 12 ii of culture: a, on top of the confluent monolayer (ml) large clusters of cells (C) as well as individual cells (i) are seen on bioactive glass; b, detail of marked area shows fiiapodia or fibres (f) and dorsal ruffles (r) and in the extracellular matrix collagenous fibers (cf); c, on quartz glass smaller-sized clusters of cells on top of the monolayer; d, detail of c shows dorsal ruffles on osteoblasts and collagenous fibres in the extracellular matrix. The numerous blebs (b) indicate the poor condition of the osteoblasts. Biomaterials 1992, Voi. 13 No. 6
Rat osteoblasts
388
cultured
on bioactive
and
non reactive glasses: W.C.A. ~~ouwenYe/~eret at.
F@ms 3 Scanning electron microscopy showing the difference in corrosion behaviour of bioactive glass and quartz glass after 12 d of exposure to the culture medium: a, the surface of bioactive glass shows irregular cracks fc), the amorphous calcium phosphate layer (cpl) contains incorporated or precipitated serumproteins (i/p) and minerals originating from the culture medium: b, no surface corrosion is observed as illustrated by an accidental scratch at the quartz glass surface, the irregularities at the surface are caused by serumprotein deposition from the culture medium.
quartz glass the major activity of the antibody is mainly concentrated in the clusters (Figure 54. The clustered cells have spherical as well as polygonal shapes.
DNA content and alkaline phosphatase
Time
(d)
Figure 4 The results of the X-ray micro analysis for bioactive glass. Untreated samples were compared with samples after 2, 612 and 20 d exposure to the culture medium. The corrosion process is expressed as the mutual variations of the sum of elemental oxide concentrations in percentages %: 0, SiOz; 0, CaO; n t Na,O; A, P,O,.
but also diffusely distributed over the monolayer. However, it seems that the type I collagen still remains in the cytoplasm (Figure 5s). Upon nonreactive glass the type I collagen is localized in the cytoplasm and also in the extracellular matrix in a network of fibres thereby showing the cell shapes (Figure 5b).
activity
DNA content The results of the DNA content determinations are presented in Figure 6. For all substrates the proliferative activity of the osteoblasts results in an increase in the DNA content after 3, 6 and 8 d. For bioactive glass, the increase of the DNA content becomes significantly higher (P < 0.001, n = 6) after 6 and 8 d when compared with nonreactive glasses. The DNA content of all nonreactive glass cultures is lower and they are of the same order, Alkaline phosphatase activity The biochemical determinations of the APA are summarized in Figure 7. Up to 6 d the APA is very low, but after 6 d the APA increases considerably for all substrates due to the differentiation of the osteoblasts. For the bioactive glass, the increase is significantly higher after 6 d and 8 d (P < 0.001, R = 6) in comparison with the nonreactive glasses.
Osteocalcin Osteocalcin antigenicity was also observed in all cultures. A very weak signal was obtained after 6 and 12 d. However, after 16 d all cultures showed osteocalcin antigenicity located diffusely over the monolayer and mainly in the cytoplasm of the clustered cells.
APA/lXVA Both APA and DNA were dete~~ned for four samples at each time interval and compared with the data from the separate determinations. Our results indicate that the combined and the separate DNA and APA determinations can be used to express the APAlDNA ratio. Figure 8 shows the ratio APA/DNA according to the combined procedure. It can be seen that no significant differences (P > 0.05, n = 41 in the ratio APA/DNA were found for the different glass types after 6 and 8 d.
Usteoblast an t&e&city All cultures showed a positive anti-osteoblast reaction after 12 d of culture. Upon bioactive glass the osteoblast antigen is diffusely located in the cell membranes of the monolayer: it has a stronger presence in the clustered cells with a typical polygonal shape which is a characteristic of osteoblast morphology (Figure 5~). For
His~uchemical staining of APA The histochemical staining showed an obvious increase in APA after 3, 6 and 8 d for all cultures. A more even staining was seen for bioactive glass in comparison with nonreactive glasses at all three time intervals but most clearly after 6 d. The APA staining for bioactive glass and quartz glass after confluency (2 6 d) are presented in
Biomaterials
1992,
Vol.
13
No.6
Rat osteoblasts
cultured
on bioactive
and non reactive
glasses:
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et al.
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Figure 5
lmmunofluorescence images showing the distribution of a-collagen type I antibodies and a-osteoblast antigens for bioactive glass and quartz glass after 12 d culture: a, is focussed on the clusters (C, arrow) showing a higher local concentration of antibodies and a diffuse distribution over the monolayer (M) for bioactive glass; b, on quartz glass the antibodies are located in the extracellular matrix (em) where the type I collagen is organized in (collagenous) fibres (cf), also in thecytoplasm (Cyt) probably due to precursors of type I collagen (with the exclusion of the nucleus (n)). Location of a-osteoblast antigen on quartz glass after 12 d; c, focussing on clusters: polygonal (p) shaped cells are observed in the clusters for bioactive glass; d, spherical and, in lesser extent, polygonal shaped cells are seen in the clusters for quartz glass. All parts same magnification.
Figure 9a-d. After 8 d the APA on bioactive glass is evenly distributed over the monolayer but more concentrated in the clusters of cells (Figure 9c). On all nonreactive glasses the clusters of cells are very intensely stained after 8 d whereas the monolayer was, on the average, more weakly stained (Figure 9d).
-2 t
/
6
/’ t
i’
Time
(d)
Figure 7
0
I
I
I
3
6
9
Time
(d)
Figure 6 Results of the DNA content determinations. The increase of the DNA-content is significantly higher (P < 0.001) in confluent cultures for bioactive glass after 6 and 6 d when compared with the nonreactive glasses: 0, bioactive glass 4585; 0, normal glass (soda-silica-lime glass); A, quartz glass; A, coverslips. Note: a correction has been made for the data found because of the larger area of the coverslips.
Results of the alkaline phosphatase activity (APA) determinations. A significantly higher APA (P < 0.001) is observed for bioactive glass in comparison with the nonreactive glasses after 6 and 8 d: 0, bioactive glass 4585; 0, normal glass (soda-silica-lime glass); A, quartz glass; A, coverslips. Note: a correction has been made for the data found because of the larger area of the coverslips.
Elemental analysis We confirmed with both elemental analyses that the compositions of both glass types were in agreement with the theoretical compositions, [see also Table 2). Biomaterials
1992. Vol.
13 No. 6
Rat osteoblasts cultured on bioactive and non reactive alasses: W.C.A. Vrouweffvelder et a/.
390
T
5
I 0
I
I
1
3
6
9
Time
(d)
Figure8 The ratio APAIDNA. No significant differences (P < 0.05) werefound in the mean APA per cell after 6 and 6 d: 0, bioactive glass 4585; 0, normal glass (soda-silica-lime glass); A, quartz glass; A, coverslips. Note: a correction has been made for the data found because of the larger area of the coverslips.
DISCUSSION Many in vim studieP* 26have been performed regarding the bone bonding behaviour of bioactive glasses. However, the fundamental mechanisms concerning the
interfacial reactions between bioactive glass and bone are poorly understood. Cell culture models have become a more important instrumenP *’ to investigate interface reactions and might be used as a first screening for biomaterials. In our in vitro model we have tried to investigate the behaviour of bone cells cultured upon a bioactive glass which forms a calcium phosphate layer at the surface after exposure to bod~uids. The calcium phosphate enriched layer creates a micro-environment, thereby approaching the in viva situation of osteoblasts, but without unwanted in viva complexities. Using nonreactive glasses as a control we investigated the unique properties of bioactive glass in its supposed stimulatory effects on osteoblasts. Our results showed differences in morphological and biochemical parameters between osteoblasts cultured upon bioactive glass and nonreactive glasses. We were able reproducibly to obtain homogeneous bioactive glass slides with a composition almost equal to the theoretical 45S5 composition, After exposure to the culture medium an amorphous calcium phosphate rich film was formed on top of the glass surface as was established with the microprobe analyses. This is in agreement with previous work6-‘* 26, Small aberrations in the microprobe analyses are possibly caused by the influence of the high energetic electron beam on the sample, which may lead to the volatilization of sodium oxide causing a shift in the mutual percentages of the other elemental oxides, A more reasonable
Figure 9 Light microscopy images of histochemical staining for the alkaline phosphatase activity (APA) after 6 and 8 d culture for bioactive glass, a,c, and quartz glass b,d respectively. After 8 d the difference in APA between bioactive glass and quartz glass is very clear. Larger clusters (C) are seen on bioactive glass (c) after 8 d in comparison with quartz glass, although clusters on quartz glass (d) are very intensely stained. The APA-staining is on average higher in the monolayer (M) for bioactive glass in comparison with the nonreactive glasses at all time intervals: all parts equally magnified. Biomaterials 1992, Vol. 13 No. 6
Rat osteoblasts
cultured
on bioactive
and non reactive
glasses:
Table 2 Experimental elemental analyses compared with the theoretical composition of bioactive glass 4585 and normal glass (soda-silica-limeglass): A, ICP-AES method; B, combined gravimetry, atomic absorption spectroscopy, and photometry. SiO,
Na,O
CaO
P&5
Other oxides
Bioactive glass 4585 A B
45.0
24.5
24.5
6.0
0.0
44.9 45.0+
23.6 24.6*
25.3 24.4*
5.7 5.969
-
Normal glass (soda-silica-lime glass) A B
72.0
16.0
12.0
0.0
0.0
72.5 72.2+
15.2 16.5*
12.3 11.5*
0.0 0.05
-
l
* and 0.15% AI,O,, 0.025% MgO, 0.006% Fe,O, +gravimetric method *atomic absorption spectroscopy method §photometric method. explanation for this is the formation of discrete nucleation foci with a higher local calcium phosphate content. The appearance of calcium phosphate nucleation foci possibly may lead to the formation of crystalline hydroxyapatite in the course of time. We observed morphological differences between osteoblasts cultured upon bioactive glass and nonreactive glasses which corresponds with earlier work”’ lg. The process of cell spreading is influenced by the nature of the underlying substrateZ7-“. Previous studies showed that osteoblasts cultured on positive or negative surface charges adopted a ‘stand-off’ morphologyz7. The appearance of the bioactive layer containing calcium and highly negatively charged phosphate groups and creating a high local pH (7 < pH < 9)“-’ certainly influences the behaviour and morphology of the osteoblasts cultured on bioactive glass. We observed compact cells with dorsal ruffles and filapodia resulting in the formation of a compact cell layer with a higher cell density. On uncharged surfaces osteoblasts exhibited a flattened morphologyz7 as was also observed in our nonreactive glass cultures. Confluent cultures showed clustered cells upon bioactive glass as well as upon nonreactive glass although the cell layer detached fmm the underlying substrate in a lot of the nonreactive glass cultures. It is known that confluent cultures detach from the underlying substrate which is often caused by manipulating the culture dishes. Confluent bioactive glass cultures did not show any detachment suggesting a better attachment to the underlying substrate. The appearance of fibres in the extracellular matrix was seen with SEM for all cultures”3 lg. These fibres are probably responsible for the positive reaction with the a-collagen type I antibodies. For the nonreactive glass cultures these fibres are located in the cytoplasm and the extracellular matrix thus depicting the more flattened shape of the osteoblasts. However, the appearance of collagenous fibres in the extracellular matrix of osteoblasts cultured on nonreactive glasses is in contrast with the results of Matsuda and Davies who stated that no significant production of extracellular matrix was seen on quartz samples”. For bioactive glass, a -collagen type I antibodies seem more diffusely located in and around the cells. The denser cell layer in which the cells are more tightly organized, could prevent good visualization of
W.C.A. Vrouwenvelder
et al.
391
collagenous fibres in the underlying extracellular matrix because of insufficient penetration of the antibodies. Another assumption might be a higher concentration of type I collagen in the cytoplasm and the extracellular matrix due to the more compact cell layer causing a higher [and more diffuse) overall fluorescence signal. Osteocalcin antigenicity was observed mainly after 16 d for all cultures. The synthesis of the bone-related protein osteocalcin is a phenotypic expression of the osteoblast-like behaviour which becomes sufficiently apparent after 16 d in all cultures. Differences in the distribution of osteocalcin between bioactive glass and nonreactive glass were hardly found, possibly because of the longer culture period. Although all cultures showed antigenicity for the a-osteoblast antibodies, we observed differences in the morphology of the clustered cells. On bioactive glass the clustered cells have a polygonal shape which is a characteristic of osteoblast-like behaviour. The sphericalshaped cells in the clusters on nonreactive glasses indicate poor condition. The distribution of the a-osteoblast antibodies in confluent nonreactive glass culture osteoblasts was different from bioactive glass cultures. In nonreactive glass cultures the a-osteoblast antibodies were mainly detected in the clusters of cells whereas for bioactive glass a more general distribution was seen over the cells. From the biochemical determinations we observed a significantly higher DNA-content (P < 0.001, R = 6) after 6 and 8 d for bioactive glass compared with the other substrates, which can be translated as more cells per unit area. This correlates with our SEM-observations discussed above. Before reaching confluency (within 6 d) the APA is very low, because the cells proliferate. At confluency the behaviour of osteoblasts expressed by the APA [a cell membrane associated enzyme) becomes significantly higher for bioactive glass (P < 0.001, R = 6). The ratio of APA and DNA (the mean activity per cell) is not significantly different (P > 0.08, R = 4) for all substrates after 6 and 8 d. Therefore, it can be deduced that the APA in the clusters on nonreactive glass must be much higher than on bioactive glass. However, the total APA is higher for bioactive glass probably because a better overall differentiation was observed. The histochemical staining demonstrated a general APA distribution on bioactive glass, but on nonreactive glass the APA was mainly concentrated in the clusters, which is a strong confirmation of our earlier discussed findings. The differences in the distribution of type I collagen, a -osteoblast antibodies and the APA can be explained as follows: the better overall differentiation of the cells is certainly due to the presence of the bioactive layer upon bioactive glass and which refers to the ‘normal’ in viva situation in living bone. Osteoblasts cultured upon nonreactive glasses need to create a favourable microenvironment which only occurs in the clusters.
CONCLUSIONS The results lead to the following conclusions. Initially, osteoblasts show better osteoblast-like behaviour which leads to a higher cell density during culture and a generally better osteoblast expression on bioactive glass Biomaterials
1992, Vol. 13 No. 6
392
Rat osteoblasts cultured on bioactive and non reactive glasses: i4f.C.A. Vrouwenvel~er
as can be deduced from enzyme cytochemical, biochemical and immunocytochemi~al parameters. Therefore, osteoblasts attach, proliferate and differentiate better upon bioactive glass than upon nonreactive glass types. ACKNOWLEDGEMENTS The authors want to thank: Mr L. Zwiers, Mr H, de Jong and Mr J. Bunschoten (Yokogawa-Electrofact BV); Mr B. Kret and Mr P. Junger [Leids Instrumentmakers School) for participating in the processing of our glasses; Mr -A.A. Driessen [Elephant Industries BV) for his participation in the ICP-AES analyses: also, Mr D. Schakelaar for his contribution in the other analyses (TNO). Special thanks to Mr G. van Amsterdam for his valuable suggestions and help. Antisera against type I and type II collagen were a generous gift of Dr G. Rucklidge, Rowett Res. Inst., Aberdeen, UK, Monoclonal antibodies against osteoblasts were a generous gift of Mrs A. ~ette~ald, University of Bern, Switzerland.
13
14
15
16
17
18
19
REFERENCES 20 1
2
3
4
5
6 7 8
9
10
11
12
Hench, L.L., Splinter, R.J., Allen, W.C. and Greenlee, T.K., Bonding mechanisms at the interface of ceramic prosthetic materials, _f.Biomed. Mater. Res. Symp. 1971, tr, 117-141 Hench, L.L. and Paschall, H.A., Direct bonding of bioactive glass-ceramic materials to bone and muscle, J, Biomed. Mater. Res. Symp. 1973, 4, 25-42 Hench, L.L. and Paschall, H.A., Histochemical responses at a biomaterial’s interface,f. Homed. Mater. Res. Symp. 1974, 5, 49-64 Piotrowski, G., Hench, L.L., Allen, W.C. and Miller, G. J,, Mechanical studies of the bone biogiass interfacial bond, J, Biomed. Mater. Res. Symp. 2975, i&47-61 Hench, L.L., Pantano, C.G. Jr, Buscemi, P.J. and Greenspan, DC., Analysis of bioglass fixation of hip prostheses, J. Biomed. Mater. Res. 1977, 11,267-282 Sanders, D.M. and Hench, L.L., Mechanisms of glass corrosion, 1. Am. Ceram. Sot. 1973,56, 373-377 Hench, L.L., Characterization of glass corrosion and durability, J. Non-Cryst. Solids 1973, 19, 27-39 Ogino, M., Ohuchi, F. and Hench, L.L., Compositional dependance of the formation of calcium phosphate films on bioglass, J. Boomed. Mater. Res. 1980, 14, 55-64 Ogino, M. and Hench, L.L., Formation of calcium phosphate films on silicate glasses, J. Non-Cryst. Solids 1980, 38/S@, 673-678 Walker, M.M., An investigation into the bonding mechanism of bioglass, MSThesis, University of Florida, USA, 1977 Gross, U.M. and Strunz, V., The anchoring of glass ceramics of different solubility in the femur of the rat, J. Biomed. Mater. Res. 1980, 14, 607-618 Jones, S.J. and Boyde, A., The migration of osteoblasts, Cell Tbs. Res. 1977, 184, 179-193
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1992, Vol. 13 No. 6
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Gross, U.M., Brandes, J., Strunz, V., Bab, I. and Sela, J., The ultrastructure of the interface between a glass ceramic and bone, J Biomed. Mater. Res. 1981, 15, 291-305 Gross, U.M. and Strunz, V., The interface of various glasses and glass ceramics with a bony implantation bed, J. Biomed. Mater. Res. 1985, 19,251-271 Tarrant, SF. and Davies, J.E., Interactions between primary bone cell cultures and biomaterials. Part 2: osteoblast behaviour. In Biomaterials and Clinical Applications, (Eds. A. Pizzoferrato, P.G. Marchinetti, A. Ravaglioli and A.J.C. Lee) Elsevier, Amsterdam, The Netherlands, 1987 pp 585-590 Ito, G., Matsuda, T., Inoue, N. and Kamegai, T., A histological comparison of the tissue interface of bioglass and silica glass, J. Biomed. Mater. Res. 1987,21,485-497 Davies, J.E., Tarrant, S.F. and Matsuda, T,, Interaction between primary bone cell cultures and biomaterials. Part 1: method: the in vitro and in vivo stages. In Biomaterials and Clinical Applications, (Eds. A. Pizzoferrato, P.G. Marchinetti, A, Ravaglioli and A.J.C. Lee) Elsevier, Amsterdam, The Netherlands, 1987 pp 579-584 Matsuda, T. and Davies, J.E., The in vitro response of osteoblasts to bioactive glass, ~iomaterja~s 1987, 8, 275-284 Davies, J.E. and Matsuda, T., Extracellular matrix production by osteoblasts on bioactive substrata in vitro, Scanning Microscopy 1988, 2,1445-1452 Boonekamp, P., Hormone responsiveness of bone and bone cells: effects of culture conditions and specific cations. Thesis (1982). Leiden, The Netherlands Cohn, D.V. and Wong, G.L., Isolated bone cells. In Skeletal Research (Eds D.J. Simmons and A.S. Kunin), Academic Press, NY, USA, 1979, pp 3-20 Karsten, U. and Wollenberger, A., Improvements in the ethidium bromide method for direct fluorometric estimation of DNA and RNA in cell and tissue homogenates, Anal. Biocbem. 1977, 77, 484-470 Bessey, O-A., Lowry, O.H. and Breck, M.J., A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum, J. Eiol. Chem. 1948, 164, 321-326 Burnstone, MS., Histochemical comparison of naphthol AS-phosphates for the demonstration of phosphatases, J. Nat. Cancer Inst. 1958, 28, 601-613 Fujui, T. and Ogino, M., Difference of bone-bonding behaviour among surface active glasses and sintered apatite, 1. Biomed. Mater. Res. 1984, 18, 645-859 Ducheyne, P., Bioglass coatings and bioglass composites as implant materials, J. Biomed. Mater. Res. 1985, 19, 273-291 Shelton, R.M., Whyte, I.M. and Davies, J.E., Intemction between primary bone cell cultures and biomate~als. Part 4: colonization of charged polymer surfaces. In Biomaterials and Clinical Applications (Eds. A. Pizzoferrato, P.G. Marchinetti, A. Ravaglioli and A.J.C. Lee) Elsevier, Amsterdam, The Netherlands, 1987, pp 597-602 Davies, J.E., Causton, B., Bovell, Y., Davy, K. and Sturt, C.S., The migration of osteoblasts over substrata of discrete surface charge, Biomaterials 1988, 7, 231-233 Maroudas, N.G., Adhesion and spreading of cells on charged surfaces, J. Theor. Biol. 1975, 49,417-424
Behavior of fetal rat ost~oblasts cultured IkeV&D on bioactive glass and nonreactive glasses IAEA.
Vrouwenvelder, C.G. Groat* and K, de Groot
Department of Biomatefials and *Laboratory of Cell Biology and Histology, School of Medicine, University of L&den, The Netherlands We examined the behaviour of fetal rat osteoblasts cultured upon bioactive glass and nonreactive glasses, and the supposed stimulatory effects of bioactive glass on osteoblasts. Nonreactive glass cultures showed flattened ceils with afmost no dorsal ruffles. Bioactive glass cultures showed compact cells with dorsal ruffles and filapodia resulting in the formation of a denser cell layer. For confluent nonreactive glass cultures the osteoblast expression was mainly concentrated in the clustered cells which were formed upon the monolayer, whereas for confluent bioactive glass cultures the osteoblast expression was more generally distributed. The production of type I collagen, osteocalcin and an osteoblast-specific antigen was shown by immuno~~ochemistry for all cultures, although differences in distribution were observed. The bioactive layer of bioactive glass is responsible for a better osteoblast-like morphology, a higher proliferation rate and generally a better osteoblast expression. Ka~words: Received
Bioactive
3 February
glass, osreo~lasis,
1991; revised 2 May 1991; accepted
In the field of biomaterials, bioactive glasses are known to have Potential bone-bonding properties1-3. In contrast with more traditional biomaterials, which are inert and do not bond to bone directly because of fibrous encapsulation of the implant, bioglasses appeared to have good biocompatibility after implantation in animals4* 5. Hench and co-workers developed a series of soft glasses with a low silicon content and relatively high percentages of sodium and calcium, thereby stimulating the corrosion behaviour of these so-called bioactive glasses or bioglasses. These bioactive glasses were varied in their composition within certain limits and this resulted in differences in their capability to bond to bone. In spite of these differences, all bioactive glasses demonstrated a characteristic corrosion behaviour after exposure to bodyfluids: a preferential leaching of alkali ions resulting in the formation of a silica-rich gel layer and a calcium phosphate layer on the bioactive glass surfaces-g. Within a certain time, the calcium phosphate layer will recrystallize into hy~oxyapatite. This hydroxyapatite is thought to be responsible for the bone-bonding properties of bioactive glasses and is therefore called a ‘bioactive’ layer. The bone-bonding properties are designated as bioactive behaviour. From in viva experiments’s the optimal composition for bioactive glass, in terms of maximum Correspondence to Dr W.C.A. Vrouwenvelder, Department of Biomaterials, School of Medicine [Building No. 551, University of Leiden, Rijnsburgerweg 10, 2333 AA Leiden. The Netherlands Biomaterials 1992, Vol.
13 No. 6
ceil cu/fufe 20 June 1991
bonding to bone, was found to be: 45% SiO,, 24.5% Na,O, 24.5% CaO and 6% P,O, (in weight 7~).This composition was codedIm as 4555. As bioactive glass demonstrated better bonding to bone, it is thought that the bioactive layer might stimulate osteoblasts in their bone forming behaviour’1*12. In v&o, it appeared that a positive correlation existed between the formation of a bioactive layer and the appearance of collagen in the extracellular matrix13.14. An investigation into the behaviour of osteoblasts in close contact with bioactive glasses will, give more understanding of the bonding-to-bone properties of bioactive glasses15-‘g. To eliminate the complexities normally present in in viva studies4, 5, we chose a simple in vitro model using fetal rat osteoblasts. The purpose of our study was to examine the cell behaviour of fetal rat osteoblast&‘~ ” cultured upon bioactive glass and nonreactive glasses, and to investigate the supposed stimulatory effects of bioactive glass on osteoblasts. Three nonreactive glasses were chosen: a soda-silica-lime glass (like window glass), silica-quartz glass and microscopical coverslips (often used as cell culture substrates). We checked the ~producibility of the glass preparations with two independant analyses of the elemental compositions. The corrosion behaviour of the different glass types was investigated with routine scanning electron microscopy (SEM) techniques connected to a X-ray micro analysis system. Cell cultures were observed histologically after several culture periods by SEM and transmitted light microscopy as we11 as 0 1992 Bu~e~orth-~einemann Ltd 0142-9612/92/060382-11
Rat osteoblasts
cultured
on bioactive
and non reactive
glasses:
immunocytochemistry for type I and II collagen and osteocalcin in the extracellular matrix, and a membrane characterization for osteoblasts. The DNA-content and the total alkaline phosphatase activity (APA) of the cultures were determined biochemically”* 23. The distribution of APA in the cultures was visualized histochemicallyz4.
MATERIALS AND METHODS Preparation of glasses The theoretical compositions (in weight ala)of the glasses used in our experiments are presented in Table 1.
The bioactive glass was the 45S5 type according to Hench and co-workers1-3, prepared at our request by Yokogawa-Electrofact BV (Amersfoort, The Netherlands)25. High grade Na,CO,, CaCO,, SiO, and (NH,),HPO, were thoroughly mixed in a alumina ball mill for 1 h. The mixed powders were preheated at llOO*C in a platinum crucible to eliminate chemically bound carbon dioxide and water. The mixture was held at the reaction temperature (I,) of 135O’C for 2-3 h. To obtain a homogeneous reaction product the mixture was stirred with a platinum spoon. After another 30 min at T, the mixture was cast in preheated graphite moulds. The glass rods obtained, approximately 20 cm length, were annealed at 5OO’C for 6 h to release potential internal physicochemical stresses due to fast cooling. Slow cooling of the rods after annealing took another 10 h. The rods were sawn transversely with an oil-cooled diamond saw. The resulting slides (0.6 mm thickness) were fixed with paraffin wax on a plane parallel dish (diameter approximately 14 cm and containing about 60 slides) and ground with watercooled silicon carbide (fine grit 500 and 800), followed by polishing with cerium oxide paste (gritsize 2 pm: 1650 Cerox, Rhone-Poulenc, France). During polishing water-ethanol mixtures, from 50-50% up to pure ethanol, were used to prevent the bioactive glass from undesirable corrosion by water. All glass samples were cleaned first by rinsing with xylene, followed by a 50-50% acetone-ethanol mixture and then three times with pure ethanol. We obtained thin translucent slides with a smooth and clean surface (diameter 6 mm and 0.4 mm thickness). Sawing, grinding and polishing was done by the Leidse Instrumentmakers School (Leiden, The Netherlands).
Table 1
Theoretical
compositions SiO,
Na,O
of glasses in weight %. CaO
p2°5
Other oxides
Bioactive glass 4585 Normal glass (soda-silica-lime glass) Quartz glass Coverslips ‘125%(Na,O SbO,
45
24.5
24.5
6
-
72
16
12
-
-
_
_
_
t
>99.9 65
+ K,O), 3%A1,03,6.5%Zn0,5.5%TiOp,
7.5% B,O,and2.5%
W.C.A. Vrouwenvelder
et al.
383
Normal glass Preparation of this glass (Yokogawa-Electrofact BV) was comparable with the procedure for bioactive glass, except for a different reaction temperature (Tr = 155O*C) and annealing temperature (T, = 55O’C). Sawing, grinding, polishing and cleaning were performed as described above, resulting in slides with the same dimensions. Quartz glass Quartz glass slides (Heraeus, Nijmegen, with purity >99.9 wt% SiO, were procedure described above.
The Netherlands) obtained by the
Commercially available coverslips [diameter 10 mm and 0.1 mm thickness) were only ethanol cleaned. All samples were sterilized by dry heat in a furnace 150% for 3 h before culturing.
at
Cell culture method Osteoblasts were isolated according to the method of Boonekamp” with some modifications. Briefly, calvaria from 20-day-old rat embryos (n = 10-15) were excised aseptically. The endo- and extracranial periostea were mechanically removed. The calvaria were incubated for 2 X 10 min at 37°C with 4 mM EDTA in phosphate buffered saline (PBS). After rinsing the calvaria for 3 X 5 min with PBS, they were incubated for 10 min with collagenase (1 mglml PBS) at 37°C. The cell suspension obtained was discarded as it contained periosteal fibroblasts, still present after removal of the periostea. Then osteoblasts were isolated by further collagenase treatment (2 X 30 min). The supernatant was centrifuged for 5 min at 1400 r.p.m. (275g). The pellet obtained was resuspended in culture medium: minimum essential medium (a-MEM), containing 5% inactivated fetal calf serum (Gibco), 1 mg/ml glucose and 90 pglml gentamycin. The sterilized glass samples were placed in 24-well culture dishes (Costar]. Samples for morphological studies (SEM and light microscopy) were seeded with a cell density of approximately 5 X lo5 cells/ml. The often observed spontaneous detachment of the cell layer in nonreactive glass cultures was minimized, in the culture periods used, at this cell density. Samples for the DNA content and APA determinations were seeded with a higher cell density of approximately 1 X 10” cells/ml to get a well defined signal with the spectroscopical techniques used. In earlier investigations it was established that the chosen cell densities had no influence on osteoblast expression. On every glass sample, 50 ~1 cell suspension was applied with great care, to avoid unwanted cell attachment to the surrounding surface of the well, so that cells were only allowed to attach for 2 h to the underlying glass substrate, then 800~1 culture medium was also added carefully. During culturing the multi-well dishes were kept in an incubator (with 95% air humidity and 5% CO,) at 37’C. During the first 6 d, half of the medium was replaced every 72 h, thereafter it was changed every 48 h. Biomaterials 1992, Vol. 13 No. 6
Rat osteoblasts
384
Preparation
cultured
on bioactive
for SEM
Osteoblast morphology After 2, 6 and 12 d cultures were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH = 7.4) for 2 h, rinsed with PBS (3 X 5 min) and dehydrated in a graded ethanol series. Critical point drying of the samples (Balzer) was followed by gold sputtering (Balzer). The preparations were examined at 15 kV in a Philips SEM 525 M.
Surface corrosion behaviour To observe morphological differences in the glass surfaces caused by corrosion, samples without cells were placed in culture medium for 2, 6, and 12 d, fixed and further treated as routine SEM samples.
X-ray micro analysis To investigate the surface corrosion behaviour of the glasses, samples were exposed to the culture medium for 2, 6, and 12 d and compared with untreated samples. Bioactive glass samples were also examined at an interval of 20 d of exposure. All samples were critical point dried and carbon-sputter coated. We used the Philips SEM 525 M connected to a Tracor/Noran Voyager X-ray micro analysis system. All samples were analysed at 15 kV. The microscopical conditions selected remained constant during all measurements. The results from the X-ray micro analysis were used to obtain semi-quantitative data. To obtain these data a pRZ correction method was performed. Changes in the mutual percentages of the oxides present in the samples were used to determine the surface corrosion behaviour.
Transmitted
light microscopy
Cultures were fixed after 2,6 and 12 d with glutaraldehyde as for SEM and stained with hematoxylin and eosin.
Fluorescence
microscopy
and non reactive
glasses:
W.C.A. Vrouwenvelder et al.
Immunocytochemistry was performed according to standard procedures. The samples were observed with an Olympus BHRFL fluorescence microscope.
DNA content and alkaline
phosphatase
activity
DNA con tent After 0.25, 3, 6 and 8 d the cell cultures were rinsed with PBS and stored at -20°C. The samples collected were trypsinized (1 mg trypsin [Sigma, USA]/ml tyrode buffer) according to the method of Karsten and Wollenberge?. The cell membranes were sonified to release the DNA content. Heparin solution 1 ml, (Tromboliquine (Organon 5000 units, diluted 1:600)/ml PBS) and 0.5 ml RNase solution (ribonuclease-A (Sigma, USA) 0.05 mg/ml PBS) were added to 0.5 ml trypsinized suspension. After incubation at room temperature (0.5-l h) we added 0.5 ml ethidium bromide (0.025 mg/ml PBS). All samples, including a series of standardized DNA samples, were measured on a Perkin-Elmer 1000 Fluorescence Spectrometer at 590 nm. A correction was performed for the larger area of the coverslips to correspond with the other glass types.
Alkaline phosphatase
activity (APA)
After 0.25, 3, 6 and 8 d the cell cultures were rinsed with PBS and stored at -20°C. The samples collected were treatedz3 with 1 ml PBS with 0.05% triton (X-100) and then sonified. To 100 ~1 sonified solution, 100 ~1 substrate (20 mM paranitrophenylphosphate (Sigma, USA) in 1 M diethanol-amine and 1 mM MgCl,, (pH = 9.8) was added. The mixture was incubated at 37’C. The reaction was stopped with 1 ml 0.1 M NaOH when the colour of the mixture was comparable to the colour of a standardized series of paranitrophenol samples within 15-30 min. All samples including a series of standards were measured on a Gilford N-300 Micro-sample Spectrophotometer at 410 nm. A correction was performed for the larger area of the coverslips to correspond with the other glass types.
Type I and II collagen
APA/DNA
Type I collagen (as a marker for osteoblast-like character) and type II collagen (as a marker for possible contamination with chondrocytes) were located immunohistochemically after 12 d culture with a goat-antiserum and a sheep-anti-goat/FITC conjugate [Sigma, USA). The cultures were fixed in pure acetone for 2 min and air dried.
The determination of the ratio APA/DNA gives information about the mean APA per cell. Both methods described above were performed on separate cultures (at each time interval] because specific conditions were required for each method. Therefore, we attempted to use one series in which both determinations were performed on the same cultures: to the remaining 9OOpl from the APA determination, 1 mg/lOO ,h1 trypsin solution was added whereby the total trypsin concentration was kept at 1 mg/ml as for normal DNA determinations. After sonification once more, the DNA determination procedure was as described above. As the APA/DNA is expressed as the amount of APA per cell no correction for the larger area of the coverslips was needed.
Osteocalcin Other cultures were screened for the presence of osteocalcin, after 1% paraformaldehyde (in 0.1 M sodium cacodylate buffer] fixation, with a goat-antiserum (Sanbio) and a rabbit-anti-goat/FITC conjugate (Sigma].
Osteoblast antigenicity We used monoclonal antibodies directed against a membrane associated antigen of rat osteoblasts as an exclusive marker for osteoblast expression. The monoclonal antibody was located with a goatanti-mouse/FITC conjugate (Sigma) after 12 d culture. These cultures were also fixed with paraformaldehyde. Biomaterials
1992. Vol. 13 No. 6
Histochemical
staining of APA
To visualize the distribution of the APA in the cultures histochemical stainingz4 was used after 3, 6 and 8 d corresponding with the periods for the biochemical determinations.
Rat osteoblasts
cultured
on bioactive
and non reactive
alasses:
W.C.A. Vrouwenvelder
et al.
385
Statistical analysis Mean values and standard deviations for the DNA content and APA were computed for six determinations at each time-interval. The Student’s t-test was used to establish the significance of differences between the mean values.
Starting from 4 d a partial detachment of the cell layer was sometimes observed for nonreactive glass cultures with an increasing incidence when confluency was reached (in about 6 days). As a general observation a lot of nonreactive glass cultures showed spontaneous detachment of the cell layer within 12 d.
Elemental analyses Two independant analytical determinations were used. The procedure of the first analytical method is described briefly. First, the ICP-AES (Elephant Industries BV, Hoorn, The Netherlands) was carried out as follows: a sample of 0.1 g powdered glass was dissolved in a Teflon flask containing 4 ml HCl(3770), 2 ml HNO, (65%) and 2 ml HF (40%). The mixture was placed in a CEM microwave oven (type MDS 61 D). After dissolution, 3 g H,BO, in 80 ml distilled water was added to complex the HF. This solution was transferred into a 500 ml TPX measuring flask and the volume was adjusted with distilled water to 500 ml. For the final analysis we used an ICAP 61 (Therm0 Jarrell Ash), a simultaneous emission spectrometer with an inductively coupled Argon plasma excitation source. The second determination included three analytical techniques. For SiO, determination a gravimetrical method was used. Na,O and CaO were determined by means of atomic absorption spectroscopy, and the P,05 was determined photometrically. These determinations were carried out by TNO, Apeldoorn, The Netherlands.
Surface corrosion morphology Figure 3a shows the appearance of the bioactive glass surface after exposure to the culture medium for 12 d. The irregular cracks are caused by the corrosion process. The typical shrinkage of the corroded layer is due to dehydration during critical point drying. With X-ray micro analysis we confirmed this to be the calcium phosphate enriched layer. The irregularities at the calcium phosphate enriched surface are caused by incorporated and/or precipitated serum proteins from the culture medium. Figure 3b shows the appearance of the surface of quartz glass after exposure to the culture medium for 12 d. The irregular spots on the surface are also caused by precipitation of serum proteins. No corrosion was observed for any of the nonreactive glass types.
RESULTS Scanning
electron
microscopy
Osteoblast morphology Figure 1 shows the morphology of osteoblasts cultured on bioactive glass and quartz glass for 2 d. Cells cultured on bioactive glass are compact, have dorsal ruffles and filapodia (with a fibre like attachment to the substrate surface). Cell divisions are relatively often seen. Notice the presence of the corroded top layer on bioactive glass (Figure la and b). Cells on quartz glass have a flattened morphology with a smooth dorsal surface on which dorsal ruffles and filapodia are hardly seen (Figure lc and d). No surface corrosion was observed. Figure 2a and b show the morphology of osteoblasts cultured on bioactive glass for 12 d. On top of the monolayer individual cells are observed as well as clustered cells which have a compact structure with many dorsal ruffles. At higher magnification, fibres (possibly of collagen) connecting the cells are seen in the extracellular matrix (Figure zb). Figure zc and d show the morphology of an intact quartz glass culture after 12 d. Clusters of cells are seen on top of the existing monolayer, although they are small in size. At higher magnification it can be seen that the clustered cells show a poor morphology often with numerous blebs on the dorsal membranes which indicates poor condition (Figure Zd). Fibres are also observed in the extracellular matrix.
X-ray micro analysis The corrosion processes of the samples are expressed as the mutual variations of the sum of elemental oxide concentrations. Nonreactive glasses did not show any changes in composition. Bioactive glass showed the expected corrosion behaviour by the development of a calcium phosphate-rich top layer during time. In Figure 4 the variations in the concentrations of the elemental oxides versus time are presented. Within 2 d a strong decrease in sodium oxide and an increase in calcium and phosphorous oxides are observed. During the first 12 d the calcium and phosphorous oxides increase rapidly, which indicates that an (amorphous) calcium phosphate layer is formed on top of the bioactive glass. After 12 d the corrosion process of the bioactive glass slows down.
Transmitted
light microscopy
All cultures showed an irregular cell pattern after 2 d. After 6 and 12 d a confluent cell layer was seen for all (intact) cultures. Clustered cells as well as individual cells were seen on top of the existing monolayer. These findings are in agreement with our SEM observations. Forbioactive glass cultures the morphology was difficult to interpret because bioactive glass samples lost their translucency due to the corrosion. Within 2 d these cultures could only be observed at low magnification (X40).
Fluorescence
microscopy
Type I and II collagen The osteoblast cultures showed strong synthesis of type I collagen on all substrates, whereas type II collagen was hardly produced in any of the cultures. Figures 5a and b show the presence of type I collagen in the extracellular matrix after 12 d of culture on bioactive glass and on quartz glass respectively. Upon bioactive glass the type I collagen is mainly concentrated in the clusters of cells Biomaterials
1992. Vol. 13 No. 6
386
Rat osteoblasts
cultured
on bioactive
and non reactive glasses: W.C.A. Vrouwe~weidef
b
et al
I
Fiiure 1 Scanning electron microscopy images showing osteoblasts after 2 d: a, osteoblasts cultured on bioactive glass for 2 d, note the corroded layer (cl); b, magnification of marked area shows filapodia (f) and dorsal ruffles (r); c, shows flattened and spread osteoblasts with almost no dorsal ruffles on quartz glass, no surface corrosion (ns) is seen; d, the flattened cell structure and the lack of dorsal ruffles are shown in more detail. Comparison with a and b shows the striking difference in osteoblast morphology. Biomaterials 1992, Vol. 13 No. 6
Rat osteoblasts cultured on bioactive and non reactive ~--_ ___..~~-._._..____.~._...
glasses:
W.C.A. Vrouwenvelder
et al.
387
Figure 2 Scanning electron microscopy showing the morphology of osteoblasts on bioactive glass and quartz glass after 12 ii of culture: a, on top of the confluent monolayer (ml) large clusters of cells (C) as well as individual cells (i) are seen on bioactive glass; b, detail of marked area shows fiiapodia or fibres (f) and dorsal ruffles (r) and in the extracellular matrix collagenous fibers (cf); c, on quartz glass smaller-sized clusters of cells on top of the monolayer; d, detail of c shows dorsal ruffles on osteoblasts and collagenous fibres in the extracellular matrix. The numerous blebs (b) indicate the poor condition of the osteoblasts. Biomaterials 1992, Voi. 13 No. 6
Rat osteoblasts
388
cultured
on bioactive
and
non reactive glasses: W.C.A. ~~ouwenYe/~eret at.
F@ms 3 Scanning electron microscopy showing the difference in corrosion behaviour of bioactive glass and quartz glass after 12 d of exposure to the culture medium: a, the surface of bioactive glass shows irregular cracks fc), the amorphous calcium phosphate layer (cpl) contains incorporated or precipitated serumproteins (i/p) and minerals originating from the culture medium: b, no surface corrosion is observed as illustrated by an accidental scratch at the quartz glass surface, the irregularities at the surface are caused by serumprotein deposition from the culture medium.
quartz glass the major activity of the antibody is mainly concentrated in the clusters (Figure 54. The clustered cells have spherical as well as polygonal shapes.
DNA content and alkaline phosphatase
Time
(d)
Figure 4 The results of the X-ray micro analysis for bioactive glass. Untreated samples were compared with samples after 2, 612 and 20 d exposure to the culture medium. The corrosion process is expressed as the mutual variations of the sum of elemental oxide concentrations in percentages %: 0, SiOz; 0, CaO; n t Na,O; A, P,O,.
but also diffusely distributed over the monolayer. However, it seems that the type I collagen still remains in the cytoplasm (Figure 5s). Upon nonreactive glass the type I collagen is localized in the cytoplasm and also in the extracellular matrix in a network of fibres thereby showing the cell shapes (Figure 5b).
activity
DNA content The results of the DNA content determinations are presented in Figure 6. For all substrates the proliferative activity of the osteoblasts results in an increase in the DNA content after 3, 6 and 8 d. For bioactive glass, the increase of the DNA content becomes significantly higher (P < 0.001, n = 6) after 6 and 8 d when compared with nonreactive glasses. The DNA content of all nonreactive glass cultures is lower and they are of the same order, Alkaline phosphatase activity The biochemical determinations of the APA are summarized in Figure 7. Up to 6 d the APA is very low, but after 6 d the APA increases considerably for all substrates due to the differentiation of the osteoblasts. For the bioactive glass, the increase is significantly higher after 6 d and 8 d (P < 0.001, R = 6) in comparison with the nonreactive glasses.
Osteocalcin Osteocalcin antigenicity was also observed in all cultures. A very weak signal was obtained after 6 and 12 d. However, after 16 d all cultures showed osteocalcin antigenicity located diffusely over the monolayer and mainly in the cytoplasm of the clustered cells.
APA/lXVA Both APA and DNA were dete~~ned for four samples at each time interval and compared with the data from the separate determinations. Our results indicate that the combined and the separate DNA and APA determinations can be used to express the APAlDNA ratio. Figure 8 shows the ratio APA/DNA according to the combined procedure. It can be seen that no significant differences (P > 0.05, n = 41 in the ratio APA/DNA were found for the different glass types after 6 and 8 d.
Usteoblast an t&e&city All cultures showed a positive anti-osteoblast reaction after 12 d of culture. Upon bioactive glass the osteoblast antigen is diffusely located in the cell membranes of the monolayer: it has a stronger presence in the clustered cells with a typical polygonal shape which is a characteristic of osteoblast morphology (Figure 5~). For
His~uchemical staining of APA The histochemical staining showed an obvious increase in APA after 3, 6 and 8 d for all cultures. A more even staining was seen for bioactive glass in comparison with nonreactive glasses at all three time intervals but most clearly after 6 d. The APA staining for bioactive glass and quartz glass after confluency (2 6 d) are presented in
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Figure 5
lmmunofluorescence images showing the distribution of a-collagen type I antibodies and a-osteoblast antigens for bioactive glass and quartz glass after 12 d culture: a, is focussed on the clusters (C, arrow) showing a higher local concentration of antibodies and a diffuse distribution over the monolayer (M) for bioactive glass; b, on quartz glass the antibodies are located in the extracellular matrix (em) where the type I collagen is organized in (collagenous) fibres (cf), also in thecytoplasm (Cyt) probably due to precursors of type I collagen (with the exclusion of the nucleus (n)). Location of a-osteoblast antigen on quartz glass after 12 d; c, focussing on clusters: polygonal (p) shaped cells are observed in the clusters for bioactive glass; d, spherical and, in lesser extent, polygonal shaped cells are seen in the clusters for quartz glass. All parts same magnification.
Figure 9a-d. After 8 d the APA on bioactive glass is evenly distributed over the monolayer but more concentrated in the clusters of cells (Figure 9c). On all nonreactive glasses the clusters of cells are very intensely stained after 8 d whereas the monolayer was, on the average, more weakly stained (Figure 9d).
-2 t
/
6
/’ t
i’
Time
(d)
Figure 7
0
I
I
I
3
6
9
Time
(d)
Figure 6 Results of the DNA content determinations. The increase of the DNA-content is significantly higher (P < 0.001) in confluent cultures for bioactive glass after 6 and 6 d when compared with the nonreactive glasses: 0, bioactive glass 4585; 0, normal glass (soda-silica-lime glass); A, quartz glass; A, coverslips. Note: a correction has been made for the data found because of the larger area of the coverslips.
Results of the alkaline phosphatase activity (APA) determinations. A significantly higher APA (P < 0.001) is observed for bioactive glass in comparison with the nonreactive glasses after 6 and 8 d: 0, bioactive glass 4585; 0, normal glass (soda-silica-lime glass); A, quartz glass; A, coverslips. Note: a correction has been made for the data found because of the larger area of the coverslips.
Elemental analysis We confirmed with both elemental analyses that the compositions of both glass types were in agreement with the theoretical compositions, [see also Table 2). Biomaterials
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Rat osteoblasts cultured on bioactive and non reactive alasses: W.C.A. Vrouweffvelder et a/.
390
T
5
I 0
I
I
1
3
6
9
Time
(d)
Figure8 The ratio APAIDNA. No significant differences (P < 0.05) werefound in the mean APA per cell after 6 and 6 d: 0, bioactive glass 4585; 0, normal glass (soda-silica-lime glass); A, quartz glass; A, coverslips. Note: a correction has been made for the data found because of the larger area of the coverslips.
DISCUSSION Many in vim studieP* 26have been performed regarding the bone bonding behaviour of bioactive glasses. However, the fundamental mechanisms concerning the
interfacial reactions between bioactive glass and bone are poorly understood. Cell culture models have become a more important instrumenP *’ to investigate interface reactions and might be used as a first screening for biomaterials. In our in vitro model we have tried to investigate the behaviour of bone cells cultured upon a bioactive glass which forms a calcium phosphate layer at the surface after exposure to bod~uids. The calcium phosphate enriched layer creates a micro-environment, thereby approaching the in viva situation of osteoblasts, but without unwanted in viva complexities. Using nonreactive glasses as a control we investigated the unique properties of bioactive glass in its supposed stimulatory effects on osteoblasts. Our results showed differences in morphological and biochemical parameters between osteoblasts cultured upon bioactive glass and nonreactive glasses. We were able reproducibly to obtain homogeneous bioactive glass slides with a composition almost equal to the theoretical 45S5 composition, After exposure to the culture medium an amorphous calcium phosphate rich film was formed on top of the glass surface as was established with the microprobe analyses. This is in agreement with previous work6-‘* 26, Small aberrations in the microprobe analyses are possibly caused by the influence of the high energetic electron beam on the sample, which may lead to the volatilization of sodium oxide causing a shift in the mutual percentages of the other elemental oxides, A more reasonable
Figure 9 Light microscopy images of histochemical staining for the alkaline phosphatase activity (APA) after 6 and 8 d culture for bioactive glass, a,c, and quartz glass b,d respectively. After 8 d the difference in APA between bioactive glass and quartz glass is very clear. Larger clusters (C) are seen on bioactive glass (c) after 8 d in comparison with quartz glass, although clusters on quartz glass (d) are very intensely stained. The APA-staining is on average higher in the monolayer (M) for bioactive glass in comparison with the nonreactive glasses at all time intervals: all parts equally magnified. Biomaterials 1992, Vol. 13 No. 6
Rat osteoblasts
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Table 2 Experimental elemental analyses compared with the theoretical composition of bioactive glass 4585 and normal glass (soda-silica-limeglass): A, ICP-AES method; B, combined gravimetry, atomic absorption spectroscopy, and photometry. SiO,
Na,O
CaO
P&5
Other oxides
Bioactive glass 4585 A B
45.0
24.5
24.5
6.0
0.0
44.9 45.0+
23.6 24.6*
25.3 24.4*
5.7 5.969
-
Normal glass (soda-silica-lime glass) A B
72.0
16.0
12.0
0.0
0.0
72.5 72.2+
15.2 16.5*
12.3 11.5*
0.0 0.05
-
l
* and 0.15% AI,O,, 0.025% MgO, 0.006% Fe,O, +gravimetric method *atomic absorption spectroscopy method §photometric method. explanation for this is the formation of discrete nucleation foci with a higher local calcium phosphate content. The appearance of calcium phosphate nucleation foci possibly may lead to the formation of crystalline hydroxyapatite in the course of time. We observed morphological differences between osteoblasts cultured upon bioactive glass and nonreactive glasses which corresponds with earlier work”’ lg. The process of cell spreading is influenced by the nature of the underlying substrateZ7-“. Previous studies showed that osteoblasts cultured on positive or negative surface charges adopted a ‘stand-off’ morphologyz7. The appearance of the bioactive layer containing calcium and highly negatively charged phosphate groups and creating a high local pH (7 < pH < 9)“-’ certainly influences the behaviour and morphology of the osteoblasts cultured on bioactive glass. We observed compact cells with dorsal ruffles and filapodia resulting in the formation of a compact cell layer with a higher cell density. On uncharged surfaces osteoblasts exhibited a flattened morphologyz7 as was also observed in our nonreactive glass cultures. Confluent cultures showed clustered cells upon bioactive glass as well as upon nonreactive glass although the cell layer detached fmm the underlying substrate in a lot of the nonreactive glass cultures. It is known that confluent cultures detach from the underlying substrate which is often caused by manipulating the culture dishes. Confluent bioactive glass cultures did not show any detachment suggesting a better attachment to the underlying substrate. The appearance of fibres in the extracellular matrix was seen with SEM for all cultures”3 lg. These fibres are probably responsible for the positive reaction with the a-collagen type I antibodies. For the nonreactive glass cultures these fibres are located in the cytoplasm and the extracellular matrix thus depicting the more flattened shape of the osteoblasts. However, the appearance of collagenous fibres in the extracellular matrix of osteoblasts cultured on nonreactive glasses is in contrast with the results of Matsuda and Davies who stated that no significant production of extracellular matrix was seen on quartz samples”. For bioactive glass, a -collagen type I antibodies seem more diffusely located in and around the cells. The denser cell layer in which the cells are more tightly organized, could prevent good visualization of
W.C.A. Vrouwenvelder
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collagenous fibres in the underlying extracellular matrix because of insufficient penetration of the antibodies. Another assumption might be a higher concentration of type I collagen in the cytoplasm and the extracellular matrix due to the more compact cell layer causing a higher [and more diffuse) overall fluorescence signal. Osteocalcin antigenicity was observed mainly after 16 d for all cultures. The synthesis of the bone-related protein osteocalcin is a phenotypic expression of the osteoblast-like behaviour which becomes sufficiently apparent after 16 d in all cultures. Differences in the distribution of osteocalcin between bioactive glass and nonreactive glass were hardly found, possibly because of the longer culture period. Although all cultures showed antigenicity for the a-osteoblast antibodies, we observed differences in the morphology of the clustered cells. On bioactive glass the clustered cells have a polygonal shape which is a characteristic of osteoblast-like behaviour. The sphericalshaped cells in the clusters on nonreactive glasses indicate poor condition. The distribution of the a-osteoblast antibodies in confluent nonreactive glass culture osteoblasts was different from bioactive glass cultures. In nonreactive glass cultures the a-osteoblast antibodies were mainly detected in the clusters of cells whereas for bioactive glass a more general distribution was seen over the cells. From the biochemical determinations we observed a significantly higher DNA-content (P < 0.001, R = 6) after 6 and 8 d for bioactive glass compared with the other substrates, which can be translated as more cells per unit area. This correlates with our SEM-observations discussed above. Before reaching confluency (within 6 d) the APA is very low, because the cells proliferate. At confluency the behaviour of osteoblasts expressed by the APA [a cell membrane associated enzyme) becomes significantly higher for bioactive glass (P < 0.001, R = 6). The ratio of APA and DNA (the mean activity per cell) is not significantly different (P > 0.08, R = 4) for all substrates after 6 and 8 d. Therefore, it can be deduced that the APA in the clusters on nonreactive glass must be much higher than on bioactive glass. However, the total APA is higher for bioactive glass probably because a better overall differentiation was observed. The histochemical staining demonstrated a general APA distribution on bioactive glass, but on nonreactive glass the APA was mainly concentrated in the clusters, which is a strong confirmation of our earlier discussed findings. The differences in the distribution of type I collagen, a -osteoblast antibodies and the APA can be explained as follows: the better overall differentiation of the cells is certainly due to the presence of the bioactive layer upon bioactive glass and which refers to the ‘normal’ in viva situation in living bone. Osteoblasts cultured upon nonreactive glasses need to create a favourable microenvironment which only occurs in the clusters.
CONCLUSIONS The results lead to the following conclusions. Initially, osteoblasts show better osteoblast-like behaviour which leads to a higher cell density during culture and a generally better osteoblast expression on bioactive glass Biomaterials
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Rat osteoblasts cultured on bioactive and non reactive glasses: i4f.C.A. Vrouwenvel~er
as can be deduced from enzyme cytochemical, biochemical and immunocytochemi~al parameters. Therefore, osteoblasts attach, proliferate and differentiate better upon bioactive glass than upon nonreactive glass types. ACKNOWLEDGEMENTS The authors want to thank: Mr L. Zwiers, Mr H, de Jong and Mr J. Bunschoten (Yokogawa-Electrofact BV); Mr B. Kret and Mr P. Junger [Leids Instrumentmakers School) for participating in the processing of our glasses; Mr -A.A. Driessen [Elephant Industries BV) for his participation in the ICP-AES analyses: also, Mr D. Schakelaar for his contribution in the other analyses (TNO). Special thanks to Mr G. van Amsterdam for his valuable suggestions and help. Antisera against type I and type II collagen were a generous gift of Dr G. Rucklidge, Rowett Res. Inst., Aberdeen, UK, Monoclonal antibodies against osteoblasts were a generous gift of Mrs A. ~ette~ald, University of Bern, Switzerland.
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REFERENCES 20 1
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