toxicology and biocompatibility of bioglasses

Toxicology and biocompatibility of bioglasses

June Wilson* and G. H. Pigott Central Toxicology Laboratory, I.C.I. Lid., Alderley Park Macclesfield, United Kingdom F. J. Schoent Department of Pathology, College of Medicine, University of Florida, Gainesville, Florida 3261 1

L. L. Hench Department of Materials Science and Engineering, University of Florida, Gainesville, Florida 32611

Evidence for the lack of toxicity of various bioglass formulations has been deduced from studies carried out, both in vivo and in vitro, in several different centers. Recent studies of the authors, described here, include testing of solid bioglass implants in the soft tissues of rats and rabbits for time periods of up to eight weeks. Two new techniques are described for the toxicological testing of particulate biomaterials.

These tests, which involve rat peritoneal macrophages in culture and a mouse pulmonary biomaterial embolus model, indi- cate the biocompatibility of bioglass powders. Thus, the surface activity so critical in bone adhesion is without toxic effect in non- osseous tissues in contact with solid bioglass implants. Should wear occur and produce particulate bioglass, the material should be eliminated without consequence.

INTRODUCTION

The group of materials known as bioglass has been developed over a 12 year period to have specific surface activities which would ensure bonding with living tissues. -4

Biomaterials, whether designed to be inert (and as far as possible ignored by the host) or designed to produce a specific pharmacological effect, must be extensively tested to determine efficacy and compatibility. Above all it is essential that in the body its effects will not be in any way detrimental to the host. Regulations which govern this range of tests have been set down in detail by many federal and national a g e n ~ i e s . ~ - ~ The selection of tests appropriate to the bioglasses is not yet complete but will probably include those tests which are applied to materials for devices, with refinements to cover the peculiar properties of the bioglass systems. These have been used in the past several years in many different animal models and information on toxicity can be derived from these experiments despite the fact that to date, no sys-

* Present address:

t Present address: Peter Bent Brigham Hospital, Harvard Medical School, Boston, MA.

Department of Materials Science and Engineering, University of Florida, Gainesville, FL

Journal of Biomedical Materials Research, Vol. 15,805-817 (1981) 0 1981 John Wiley & Sons, Inc. CCC 0021-9304/81/060805-13$01.30

806 WILSON ET AL.

tematic toxicity program has been applied to them. In this article we sum- marize the available data.

MATERIALS AND M E T H O D S

Bioglasses have usually been tested in two different formulations known as 45S5 and 52S4.6. These two are close together in the bone-bonding region3t9 with the difference in chemistry primarily in the percentage of SiOz (Table I). The purpose of a second formulation was related to the adhesion to inert substrates which are used to compensate for certain mechanical deficiencies in the glass system^.^,^ A third formulation (45S5F) which substitutes calcium fluoride for some of the calcium oxide used in the manufacture of the glass is currently being evaluated for use in dental applications. Tests on this composition are not quite complete but as yet no difference in toxicology has been detected.

The test systems to be discussed and the forms in which the materials were used are shown in Table 11. (Standard protocols have been used whenever possible and are detailed in the references cited.) Some additional experimental information is available where the tests were done by the authors (see Table 11). This appears with the appropriate test section.

In all tests the bioglass samples were prepared from reagent grade sodium carbonate, calcium carbonate, phosphorous pentoxide and silica. Premixed batches were melted in covered platinum crucibles in a temperature range of 1250-1350 "C. Fibers were pulled directly from the crucibles. Powders were dry milled and mechanically sieved to the appropriate size range. Solid samples were polished to 600 grit Sic paper followed by cleaning with elec- tronic grade acetone in an ultrasonic cleaner.

IN VITRO TESTING

Rat bone cells1*

Bone cells derived from embryo rat calvaria grew normally and divided on bioglass substrates. In en face preparations they were seen to be spreading and dividing at time intervals up to 30 days in culture (Fig. 1).

TABLE I Composition of Bioglasses in mol%'

SiOz h.05 CaO CaF2 Na20

4555 46.1 2.6 26.9 24.4 52S4.6 52.1 2.6 23.8 21.5 455F 46.1 2.6 13.4 13.5 24.4

a See Ref. 9 for a discussion of the designation of 45S5, etc., mol vs. wt % formulas, and effect of composition on mechanism of phosphate film formation on bioglasses.

TOXICOLOGY AND BIOCOMPATIBILITY 807

TABLE I1 Model Systems

Model No. Material System Species Form Reference (in text)

In Vitro 1 bone cells rat solid 2 3 4 5 6 7 8 a

9

10 1 l a 12 1 3a 14

1 5a

fibroblasts fibroblasts fibroblasts fibroblasts lymphocytes macrophages macrophages

In Vivo bone bone bone subcutis muscle muscle peritoneal

cavity peritoneal

cavity

rat chick human hamster human mouse rat

rat

primates rat rat rabbit rat

rat

dog

solid solid solid solid solid/extract solidiextract powder

solid solid solid solid/powder solid fibers solid

powder

1 6a lung mouse powder

10 10 11 11 12 10 10 13

( 1 ~ 3 15 16,17,18,19 23 2 3 24 23,25

23

27

a These tests were done by the authors of this paper.

Rat fibroblastslO

Fibroblasts derived from rat skin grew equally well on bioglass and on control substrates at times up to 30 days.

Chick fibroblastsll

Chick fibroblasts were maintained under physiological conditions for two and three weeks on bioglass substrates. Cells attached to the substrates and multiplied normally.

Human fibroblastsll

Human fibroblasts in culture behaved in the same way as chick.

Hamster fibroblasts1*

Fibroblasts derived from Chinese hamster ovary and N.I.L. cells (also hamster fibroblasts) have been grown and maintained on bioglass substrates for up to seven days. They grew slowly but could be removed from the substrate and would then resume their normal growth pattern in subculture without evidence of toxic effect.

808 WILSON ET AL.

Figure 1.

Human 1ymphocyteslO

Bone cells on bioglass substrate (29 days).

In tests using cultured human thymus-dependent lymphocytes the increased RNA synthesis accompanying cell division was assayed. Incubation with solid bioglass substrates or with an extract prepared from them produced results comparable with control materials. Addition of toxic additives to control materials usually reduced the synthesis of RNA to zero.

Mouse macrophageslo

Mouse peritoneal macrophages were exposed to both solid bioglass substrates and to extracts prepared from them and no reduction of their phagocytic ability was observed. Incorporation of toxic additives to control materials reduced the phagocytic activity to less than 5% of control values.

Rat macrophagesl3

Rat peritoneal macrophages in primary culture have been used to determine the toxicity of various particulate materials. Toxicity is defined as the loss of ability of the macrophage to exclude Trypan blue after ingestion of test ma- t e r i a l ~ . ~ ~ Tests with cultured macrophages differ from the other in vitro tests cited in that effects within the cell are measured rather than those at the cell surface. This test has previously been used1* to predict the behavior of test materials in vivo. Dust of bioglass in this model was almost without effect, the number of cells with ingested material which continued to behave normally in culture was greater than with any other material previously tested in this


system, including materials considered "inert." Quartz powder is used as a reference standard and causes around 50% toxicity to cells. Bioglass powders produced 2% and 5% toxicity in replicate experiments. A plug of cells obtained by centrifuging a sample suspension from this test was fixed in glutaraldehyde and examined in the transmission electron microscope. Particles of material were seen inside and outside macrophages. Analysis of these particles showed a high silicon content which is presumptive evidence of their derivation from bioglass (Fig. 2).

IN-VIVO TESTING

Bone bonding1t3

The bonding of bioglass to bone is well do~umented. l -~ Insertion of a solid implant into a cortical defect drilled in either tibia or femur in rats has resulted in bonding with bone within 10 days. Bonding has been shown in rats with a wide range of age and weight and has been examined histologically after time intervals of up to 2.5 yr. In no case has there been evidence of a toxic effect. Bioglass has been implanted in bone in several other species and there has been no evidence of toxicity. These include dogs,15 monkeys,16J7 baboons,18 and mice.20 Bioglass had no adverse effect on bone m a r r ~ w . ~ J ~ Investigations at present underway into the use of bioglass to replace ossicles in the middle ear in the mouse show no signs of toxicity after several months in situ and no exacerbation of preexisting low levels of otitis media.*O

Figure 2. EM of macrophage with contained bioglass.

810 WILSON ET AL.

Dental implants in baboonsl8Pz1

Different formulations of bioglass have been implanted as replicate tooth replacements in the maxillae and mandibles of baboons. No significant number of inflammatory cells was associated with any formulation after six months implantation. The bioglasses all appeared biologically acceptable in terms of nontoxic manifestations after a six month period. Longer term studies showed that after two years implantation there was no infection and little or no inflammation in the tissues. There was healthy ankylosed bone on the implan ts.2’

Soft tissues in rats

Subcutaneous implantation of disks was made into the dorsal subcutaneous tissue of Wistar derived albino rats.22,23 Two similar disks were implanted in each animal and there were sixteen animals, 32 disks in all. Concurrent experiments used silicone rubber disks as control material. These were considered to provide sufficient control data for this e ~ p e r i r n e n t . ~ ~ The difficulty of processing hard glassy implant material in soft tissue was recognized particularly where the area of interest was the interface between the two. Dif- ferent histological techniques were used to prepare sections in this experiment to find out which combination would generate the most useful information. Four animals were killed after two weeks to determine the short term response. At autopsy the implant and surrounding tissue from one side of each rat was fixed whole in 10% formal saline. After fixation, the disks were removed carefully from the capsule so as not to disturb the growth of cells on the surface. Disks were stained with either hematoxylin and eosin or by the technique of Papadimitrow (a modification of Giemsa’s stain). These disks were mounted on microscope slides before examination of the upper surface under the light microscope. The associated capsules were embedded in paraffin and processed routinely then stained with hematoxylin and eosin. Implants and capsules from the remaining implants were taken at autopsy and immediately frozen in liquid nitrogen. They were then thawed in warm 10% formal saline so that thermal shock would fracture the residual glass from the reactive interface. Processing was then routine. At autopsy, capsules from which disks were removed were thin and translucent except one which showed evidence of infection and therefore was not further examined. Microscopic examination showed thin collagenous capsules, in one case some edema was noted. The disks removed from these capsules had good growth of cells on the surface; fibroblasts, macrophages, foreign body giant cells, erythrocytes, and mast cells were all identified.

Identification of the different cell types seen on the surface was by size, shape and staining reactions. Fibroblasts had a characteristic spindle shape and were associated with collagen fibrils, themselves identified by their ap- pearance under polarized light. Fibroblasts growing on the surface of Bioglass in culture have been shown to take up a peculiar stretched shape.12 In en face

TOXICOLOGY AND BIOCOMPATIBILITY 81 1

preparations such as these, macrophages are stellate and can sometimes be seen to contain particulate matter and, in these specimens, occasional erythrocytes. Giant cells could be identified by their size and the disposition of their multiple nuclei. Mast cells, some of which had discharged their granules, are stained by the Giemsa technique. Erythrocytes and polymorphonuclear leukocytes (PMNs, neutrophils) can be distinguished by size and, in the case of PMNs, their nuclear shape. Cells on the surface of biocompatible disks prepared in this way are well preserved and easily identified. A problem with bioglass disks develops as the optical properties of the surface change with the pro- duction of the surface active layers on the glass, after two weeks the cells are increasingly difficult to see in detail, but may still be seen to be viable. These means of identifying cell types were used on all disks retrieved from con- nective tissues and also on those removed from the peritoneal cavity. The disk from the infected side had very large numbers of PMNs on its surface.

Remaining samples were removed after seven weeks and were treated similarly except that eight samples were put directly into fixative and then decalcified in a solution of 20% formic acid in formalin before sectioning.

At autopsy, disks were within thin translucent capsules (Fig. 3) and those which were removed were often removed with difficulty due to adhesion. Disks had sheets of cells on the surface; these included fibroblasts, macrophages, and giant cells. Some dividing cells were seen and adherent collagen fibrils were apparent, particularly at the edges of the disks. Capsules associated with these disks were thin and made up of few layers of collagen fibrils lying parallel with the surface of the implant. The inner layers were torn during removal of the disks. There was a granular basophilic material at the interface and some associated macrophage activity. Capsules which were decalcified before processing were similar but there was preservation of the interface layer. Amorphous material and occasionally cells could be seen lining the capsule and adhering to residual glass fragments (Fig. 4). Generally there was little cellular activity and evidence for direct adhesion of collagen fibrils to the glass

Figure 3. Disk in subcutis (macroscopic).

812 WILSON ET AL.

Figure 4. Disk in subcutis after decalcification (microscopic).

surface could be seen. The surface layer appeared as a basophilic gel material.

Samples which were thermally shocked had similar capsules and similar basophilic gel at the interface.

The bioglass samples produced no toxic effect after either two or seven weeks. There was no macroscopic inflammation associated with the materials and even at two weeks there was evidence of adhesion between implant and capsule. The thin capsules which were forcibly separated from the disks before cutting showed tearing within the capsule which suggests that the adhesion between disk and capsule was mechanically stronger than between the fibrils in the capsule. In decalcified samples, where remnants of glass could still be seen, there was adhesion of collagen fibers to these pieces and the basophilic gel was present at the interface.

Intramuscular implantation in rats

Intramuscular implantation of disks of solid material in rat muscle included 45S5 bioglass together with more reactive formulation^.^,^ There was no adverse effect associated with this type of glass, after six weeks only thin elon- gated fibroblasts and intercellular mature collagen was seen with a single row of macrophages at the interface.

Intramuscular implants in rabbits

The protocol followed was that described by A ~ t i a n . ~ ~ No evidence of untoward reaction was seen grossly after either 9-12 days or six weeks.

Reaction to bioglass after 9-12 days was indistinguishable from that to the control polyethylene implants. Relatively sharply demarcated from the surrounding muscle bed, there was a cellular capsule of variable thickness. It was well-vascularized fibrous tissue with variable amounts of macrophages


at the implant surface. Eosinophils were present focally, but virtually no lymphocytes or PMNs were seen. Moderate collagenization was seen in many of the specimens. Occasionally, at the interface between implant and tissue either a thin layer of fibrinoid material or small flakes of glasslike material were noted. There was minimal evidence of surrounding muscle degeneration in all specimens (Fig. 5). Giant cells were occasionally noted away from the implant at the capsule/muscle interface in both glass and control specimens and appeared to be a nonspecific response to focal muscle damage secondary to implantation trauma. The positive control material, polyvinyl chloride with 3% organotin added as stabilizer, at 9-12 days caused extensive necrosis and acute inflammation with thick capsule formation and considerable surrounding muscle damage, dystrophic calcification and giant cell reaction.

At six weeks, all implant sites (test and both positive and negative control implants) were characterized by an extremely thin compact, relatively acellular, fibrous capsule oriented parallel to the implant surface. The implant/tissue interface was sharply defined and smooth. Inflammatory cells were rarely noted. In the several specimens for which decalcification of the tissue and implant in situ was carried out, there were glass fragments which suggested attachment to the tissue interface. There was a fine capillary network at the capsule/muscle interface. At the six-week interval, tissue reaction to the positive controls did not differ substantially from that to negative controls of bioglass test specimens. Clearly the small amount of toxic leachable in the fiber implant did not have a permanent damaging effect on the muscle.

Intraperitoneal implantation

Bioglass disks were inserted into the peritoneal cavity as described by Cal- nanZ5 and Wilson et al.23 Two samples were retrieved after three weeks. At autopsy the disks were lying freely in the abdominal cavity with no sign of adhesion or peritonitis. Previous experiments with more active polymeric

Figure 5. Implant in muscle (microscopic).

814 WILSON ET AL.

materials produced both of these.23 The disks were fixed briefly in 10% formal saline and stained with hematoxylin and eosin. After three days there were cells on the surface; erythrocytes, PMNs, and macrophages could be identified. Occasionally, dividing cells could be seen. After three weeks in situ the de- velopment of the surface active layer on the glass and its fragmentation during dehydration made it difficult to see the cells; however, those visible appeared viable and there was some collagen present.

Intraperitoneal implantation in rats

Powdered material has been implanted into the peritoneal cavity of Wistar derived rats. Approximately 500 g of dust (max. size 40-km) was sterilized by ethylene oxide exposure and suspended in 5-ml sterile saline. 1 ml of this suspension was injected into the peritoneal cavity of four animals and left for two weeks (for 45S5) and one week (45S5F). At autopsy there was no peritonitis or adhesion and all major organs appeared normal. The preferred route of excretion from the peritoneal cavity is via the subserous lymphatics of the diaphragm to the right lymphatic d ~ ~ t ~ ~ , ~ ~ and the sternal lymph nodes will often contain trapped material. In these experiments examination of these lymph nodes showed macrophages which contained eosinophil material. This may represent ingested bioglass, in any case there was no associated fibrosis or reaction. Very small white specks were sometimes seen in the peritoneal fat but after histological preparation only normal peritoneum was seen. Similar experiments with particulate quartz have produced lymph node fibrosis.

Mouse lung studiesz7

Particulates injected intravenously into the mouse tail vein embolize to the lung in a somewhat uniform distribution. Most arrive in the lung as indi- vidual particles, although small aggregates are commonly present. Because the site of introduction of the implants into the animal is spatially disassociated from the site of reaction, the tissue reaction may be observed at intervals generally considered too brief to assess tissue / biomaterials interactions.

Reaction to the bioglass particles was observed as early as 12-h and consisted of a thin rim of PMNs closely opposed to the implant. The reaction appeared to reach peak volume at approximately 48-h at which polymorphonuclear cells were predominant but some mononuclear cells (mostly macrophages) were apparent. By 4-8 days the reaction had subsided in intensity and most particles were surrounded by a layer of mononuclear cells. Thereafter there was pro- gressive diminution of the reaction volume, leading ultimately to a stable encapsulation with an exceedingly thin band of collagen surrounding. No foreign body giant cells were seen in any lesions at any time periods.

When the particle did not completely occlude the lumen of the vessel in which it resided, the reaction appeared to differ in several respects. First, the reaction appeared to be diminished in activity relative to that for occluding


particles at similar times in the sequence. Also, in such cases the particles did not induce complete local thrombosis of the arteriole. Rather a thin layer of fibrin covered the blood-contacting surface of the implant and the remainder of the vessel was patent. At longer times this layer appeared to organize into a thin collagen band with a cellular coating not distinguishable from endo- thelium (Fig. 6).

DISCUSSION

Microscopic evidence for a toxic effect of a solid material in tissue can manifest itself in several ways. The most obvious is in the presence of dead and dying tissue, especially if this response increases with exposure. Where a toxic leachable is present in the material the effects may be more subtle but are generally seen as a diffuse inflammatory response especially notable in tissues such as muscle. The most sensitive tests of all are those which involve cells in culture; indeed tissue culture experiments are considered by many to be too sensitive if used alone since failure in these tests would eliminate many useful materials which are presently in use. Effects on division, growth, and function of cells in culture can be easily and quantitatively estimated and such tests can be considered “failsafe,” that is any material which shows no toxic effect in culture is unlikely to have one in use, yet a material which displays some toxic signs in culture may yet prove acceptable because of the capacity of the whole animal to buffer such effects. These manifestations of toxicity are only a part of the wider range of possible effects, and eventually consid- eration of possible sensitization and longer-term effects will require specific testing, but nevertheless, ”a clean sheet” in the type of experiments described here is a requirement for a new material at an early stage. Establishment of appropriate test regimes for toxicity testing of biomaterials in general have been the subject of many reviews; a particularly comprehensive one being that of Autian.28

Figure 6. scopic).

Bioglass particulate vessel wall from mouse lung studies (micro-

816 WILSON ET AL.

In soft tissues the problem of relative movement between implant and capsule may sometimes allow macrophage activity to be more marked in association with hard materials like bioglasses. There is no evidence that particulate material derived from bioglass has an effect either in macrophages or as emboli. The experiments which examined the elimination of particulate material from the peritoneal cavity support the contention that wear particles from devices using bioglass will not provide a toxicological hazard. It was interesting to note the variable staining reaction of the bioglass. Reasons for this are discussed in ref. 29.

In conclusion, we feel that any material which allows close contact of living cells at its surface, which does not contain leachables which produce inflammation and which does not prevent growth and division of cells in culture, can be considered biocompatible. Such materials are the bioglasses used in the experiments described here.

The authors would particularly like to thank Dr. R. Grant of Howmedica, Inc., and Dr. K. Noonan, Dr. G. Merwin, and Dr. H. Stanley of the University of Florida for permission to quote prematurely from their research programs, and Dr. Iona S. Pratt of ICI Ltd. for the electron micrograph. During a portion of this study F.J.S. was a Clinical Training Fellow of the American Lung Association. The support of NIGMS grant GM 21065 is gratefully acknowledged by L.L.H.

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Received April 4, 1980 Accepted March 27, 1981

toxicology and biocompatibility of bioglasses

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