the biocompatibility testing of some dental amalgams in vivo

The biocompatibility amalgams in vivo

Graham Ellender* Sophie A. Feik* Claudia Gaviria*

Key words: Amalgam (dental), biocompatibility, material testing.

Abstract The biological responses to some dental amalgams were determined in vivo and compared with those of dental porcelain. The technique of implantation employed in the study addressed some of the vagaries of the Recommended Standard Practices for Biological Evaluation of Dental Materials (RSP) and considered both cellular responses (inflamma- tion, infiltration and fibrogenic cell activity) and the organizational status of the resultant encapsulation. The implantation sites for both the experimental and control were biogeometrically similar, unlike those currently recommended in RSP. At the end of the test period, all the dental amalgams tested caused minor responses reflected by the formation of thin capsules with an acceptable matrix organization. The Australian manufactured dental amalgams - Permite C”, Lojic, F4OO0, New Ultrafine@ and GS8Oo all produced even capsules with quiescent cells. By one hundred days, the capsule around Dispersalloyo, although generally well formed, showed some areas of cellular activity and matrix variability. The biological responses to all the dental amalgams examined were mild and considered to be acceptable for clinical usage. The matrix organization of enveloping capsules must be considered in

testing of some dental

*Restorative Dentistry, and Preventive and Community Dentistry Sections, School of Dental Science, University of Melbourne.

the determination of the biocompatibility of a dental restorative material.

(Received for publication October 1988. Revised June 1989. Accepted October 1989.)

Introduction All materials used in clinical situations must be

free of adverse biological effects both locally and systemically. To ensure the biocompatibility of dental materials various levels of testing are recom-

Initially, materials are tested in v i m to elicit overt toxic effects. Once materials with extreme responses are eliminated, those remaining are tested in the in vivo situation in experimental models at appropriate implantation sites, such as muscle, bone and subcutaneous tissues. When their safety at this level is established clinical testing can be undertaken.

The Recommended Standard Practices for Biological Evaluation of Dental Materials (RSP)’ forms the basis for current testing at all three levels. The in vivo subcutaneous test which is recommended in this protocol involves the implantation of Teflon tubes (polytetrafluorethylene) of 1.3 mm internal diameter as vehicles for the material under test into multiple sites in the same animal. The lateral surface of the tube acts as a control and the response to this surface is compared with that of the material placed in the ends of the tube.2 Concern has already been expressed by others regarding the standardized testing for dental restorative material^.^

This study reports on the biocompatibility of several dental amalgams manufactured in Australia and an imported brand of dental amalgam often

Australian Dental Journal 1990;35(6):497-504. 497

Table 1. The dental amalgam alloys: types and manufacturers Name Type of alloy Manufacturer

Permite Non-gamma admix SDI, Australia Lojic Non-gamma admix SDI, Australia GS80 Non-gamma admix SDI, Australia New Ultrafine Fine grain conventional SDI, Australia F400 Micrograin conventional SDI, Australia Dispersalloy Non-gamma admix J&J> USA

used for comparison in dental research. Dental porcelain was used as the control. The testing technique is based upon the RSP with certain modifications to overcome perceived deficiencies of the technique.

Materials and methods Implant materials

Dental amalgam implants were constructed from the alloy-mercury capsules listed in Table 1.

Porcelain implants were made from VMK shade 530,t obtained from the Royal Dental Hospital of Melbourne Ceramics Laboratory.

Implant dimensions Both test and control implants were constructed

in the form of flat discs of 4.0 mm diameter with a thickness of 2.0 mm.

Implant construction Dental amalgam

The alloy and mercury were triturated according to the manufacturer's specifications. Amalgam was condensed into a mould consisting of a hollow Teflon cylinder of 10 mm external diameter, 4 mm internal diameter containing a tight fitting stainless steel insert 2 mm shorter than the cylinder. Condensation was undertaken in a clinical manner with small increments using suitable pluggers. Moulds were overfilled and carved flush with the top. After five minutes, the discs so formed were ejected from the mould using the stainless steel insert and implanted.

Control Discs of the same size and shape as those for the

test implants were constructed from dental porcelain. Discs of a silicone rubber3 formed in the

tVita Fabriken. H. Rauter GmbH & Co. KG, Bad Sackingen, West Germany. SXantopren. Bayer Dental, Leverkusen, West Germany.

Teflon moulds were flush embedded in HTV indus- trial investment.$ When set, the silicone replicas were removed and the investment soaked in distilled water. Porcelain powder, acid washed in 25 per cent nitric acid, was vibrated into the line angles of the investment mould and baked under vacuum at 950 "C. Successive increments were made and baked until the mould was slightly overfilled. The porcelain discs were removed from the investment and sandblasted. Porcelain flashes were removed with appropriate stones and the surfaces lapped on graded silicone carbide papers to 1200 grit. Discs were cleaned ultrasonically in water for 15 minutes, washed in 25 per cent nitric acid for 24 hours and glazed at 920°C to produce a medium bisque surface texture.

Animal implantation technique Male Sprague-Dawley rats of 110-130 g were

used for the study. Prior to and throughout the study period the rats were maintained on the standard laboratory diet 11 and allowed water ad libitum. The rats were housed in plastic cages with a maximum of five rats per cage.

The site of implantation was the loose connective tissue of the suprascapular region. One day prior to implantation the incision site was cleared of hair with clippers. For implantation, the rats were anaesthetized with chloral hydrate [4 mg1lOO g body mass] administered via the intraperitoneal route. An incision 10 mm wide was made over the inferior border of each scapula and a 10 mm square pocket was created in the subcutaneous tissue by blunt dissection. A single implant was placed in each pocket with the test surface formed against the stainless steel insert adjacent to the superficial tissue. The wound was closed with 410 black silk sutures.(

At 2, 4, 7, 14, 28, 50 and 100 days the rats were killed with an overdose of Nembutal** and blocks of tissue containing the implant excised and fixed in neutral buffered formalin. After fixation, the implants were removed through an incision across the deeper surface of the block. Tissues were processed for histological examination using a standard protocol. Sections, 5 pm thick, were stained with haematoxylin and eosin to examine cellular detail, and with Masson's trichrome and picrosirius red (Pic.R)4 for connective tissue organization.

§Whipmix Corporation, Louisville, USA. Baristoc, Melbourne, Victoria.

(Ethnor Pty Ltd, Sydney, NSW. "Abbott Laboratories, Sydney, NSW.

498 Australian Dental Journal 1990;35:6.

Fig. la-g. - Representative early capsules (C) developing at the interface between the implants (I) and the enveloping connective tissue (E) seven days after implantation of disks of: a, Control (porcelain); b, Permite; c, Lojic; d, GS80; e, New Ultrafine;

f, F400, g, Dispersalloy. H&E, x 600.

Australian Dental Journal 1990;35:6. 499

Fig. 2a-g.-Representative capsules (C) at one hundred days after implantation of disks of: a, Control (porcelain); b, Permite; c, Lojic; d, GS80; e, New Ultrafine; f, F400; g, Dispersalloy. Picrosirius red under polarizing conditions, x 700. (e-g opposite page.)

Tissue reaction was studied essentially on the superficial aspect of the discs, which in the case of amalgam was the region adjacent to the test surface. The reactions were subjectively graded for cellular activity, as based on that of the RSP, and for connective tissue organization of the capsule.

Results Control

Two days after implantation, the porcelain disc was surrounded by an active tissue displaying few acute inflammatory cells, an occasional fibroblast and little oedema. By four days, significant numbers of small round cells were present at the interface, but few persisted in the tissue enveloping the implant. Active fibroblasts were aligned parallel to the implant and some new collagen, detected under polarized light with the Pic.R stain, had formed adjacent to the implant surface. The enveloping loose connective tissues appeared normal by this time. A thin capsule had formed by seven days and

consisted of loose but orderly arranged collagen and aligned plump fibroblasts (Fig. 1 a-g). Further development occurred until at 28 days the implant was enveloped by a narrow fibrocollagenous capsule which was characterized by fibrocytes within a dense matrix of aligned collagen. No inflammatory cells resided within the capsule. By 100 days the capsule around the porcelain was dense, compact, and contained only a few elongated fibrocytes.

Reaction to dental amalgam Two days after implantation, Dispersalloy, Lojic,

and New Ultrafine were surrounded by a wide zone of reaction characterized by distinct oedema and an acute inflammatory response. Reactions to Permite C, F400 and GS80 displayed localized inflammatory lesions which more closely approximated that of the control.

By four days after implantation, the tissue around Dispersalloy and Lojic showed a diffuse mixed inflammatory response with active fibroblasts at the


Table 2. Tabular representation of the active cellular reaction to dental amalgam*

Table 3. Tabular representation of the capsule grading

Days 2 4 7 14 28 50 100

Control 2 1 1 1 0 0 0 Dispersalloy 3 3 3 2 2 2 2 Lojic 3 3 3 2 1 3 1 1 1 Perrnite C 2 2 2 2 1 1 1 New Ultrafine 3 2 2 2 1 1 0 F400 2 2 2 2 1 1 0 GS80 2 2 2 2 2 1 0

*Gradings: 0 = none, 1 =slight, 2 =mild, 3 =moderate.

periphery. New Ultrafine induced a more mild but still d i f ise response with some collagen formation. Reactions to Permite C, F400 and GS80 were characterized by discrete and aligned active granulation tissue.

Seven days after implantation the tissues surrounding all the amalgams showed early capsule formation; the capsules around Dispersalloy and Lojic were irregularly organized whilst the matrix around the remainder were aligned parallel to the surface of the implants.

Days 2 4 7 14 28 50 100

Control - 3 t 3 3 1 1 1 Dispersalloy - - 4 t 3 3 3 312$ Lojic - - 4 t 413 2 2 2 Perrnite C - - 3 7 2 2 2 2 New Ultrafine - 47 3 3 2 2 2 F400 - - 3 t 3 2 2 2 GS80 - - 3 7 3 3 2 2

'Gradings represent a subjective assessment of the arrangement and organization of the fibro-collagenous capsules. - =No organization around the implant; 1 =capsule with compact arrangement; 2 =capsule with well ordered arrangement; 3 =capsule with slight disordered arrangement; 4 = indistinct or immature capsule with disordered arrangement. tRepresents the stage at which collagen is first detected by the picrosirius red stain. tRepresents variability in capsule structure.

Fourteen days after implantation, the capsule around Lojic was still poorly formed and infiltrated by a number of small round cells. The reactions around Dispersalloy, F400, New Ultrafine and GS80 were similar with the formation of distinct but relatively unorganized capsules. The well

Australian Dental Journal 1990:35:6 501

E

c -

Fig. 3a.-The tube implant used in the RSP is enclosed in the enveloping connective tissue (CT). The procedure employs biogeometrically differing surfaces for the control (C), expressed as an area of resolution (R), and the test (T), expressed as an area of granulation tissue (GT). The restricted region available for the histological study of the test material is complicated by the edge effect of the tube (arrow), the interface between the tube and the test material (*). b, In this modified test the histological response is determined for both the amalgam (T) and control porcelain (C) at comparable sites with a substantial

area available for examination.

ordered capsule around Permite C contained involuting fibroblasts.

At 28 days maturation of the capsules denoted by the presence of few active fibroblasts and well aligned collagen was evident around Lojic, Permite C, New Ultrafine and F400. Capsules around Dispersalloy and GS80 were still immature and contained active fibroblasts and a few inflammatory cells.

By 50 days, dense inactive connective tissue capsules surrounded the implants of Lojic, Permite C, F400, GS80 and New Ultrafine. The collagen around the latter was less abundant. Dispersalloy showed some variability in the organization of the capsule characterized by some active fibroblasts, a few small round cells and an occasional vessel.

One hundred days after implantation (Fig. 2a-g) the implants of Lojic, Permite C, New Ultrafine, F400 and GS80 possessed compact, evenly ordered capsules which appeared quiescent and avascular. The capsule around Dispersalloy was generally well

ordered, but a few regions maintained a few small round cells, plump fibroblasts and vessels.

The relative cellular responses are presented in Table 2 and the organization of the capsules in Table 3.

Discussion The biocompatibility testing used in this study

was based on the Recommended Standard Practices for Biological Evaluation of Dental Materials2 modified to render it more appropriate for the testing of dental amalgam and other restorative materials.

The area of the specimen exposed to the tissue was enlarged from the stipulated 1.3 mm diameter (1.3 mm2) to 4.0 mm (12.6 mm2) which afforded a greatly increased area for more valid comparison. In addition, the tissue responses using the RSP method are complicated by the influence of ends of the implant’ in the Teflon tube, and further compounded with the interfaces with the material being tested. These complications are minimized


by the use of a larger implant without the Teflon tube.

The control used in the RSP method is represented by the tissue response at the middle section of the Teflon tube (Fig. 3a) - 'this response serves as a control and shall be compared to the reaction at the end of the tube'? The authors believe this practice is erroneous as this site does not represent a site biologically comparable with that at the end of the tube in which the material is inserted. The biological geometry of the system differs in the two sites as the arrangement of the implant in the tissues results in dilation of the tissues at the ends of the tube which must heal by secondary intention (granulation) compounded by the tissue response of the material, whereas, the lateral surfaces used in the recommended standard tests for the control heal largely by primary intention (resolution) (Fig. 3a).

In the present study, the tissue reaction around the dental amalgam under review was compared with that around porcelain implants of the same size and shape at an equivalent site. It is thus a true control (Fig. 3b). Dental porcelain has been shown to be a reliable control6 and results in a mild tissue response and permits repair which conforms to established parameter^.',^

The site chosen in this study was bilaterally symmetrical and standard for all implants. This overcomes a minor flaw in the RSP which stipu- lates that implants are placed in a ring configuration in the back. This arrangement subjects implants to a relative variability to movements in the differing anatomical sites of implantation and so can influence the biological response which is e l i~i ted.~ Bilateral placement of single implants does, however, require the use of more animals.

Three distinct phases were seen in the response to the implants. The early response - resulting from the immediate surgery compounded by the presence of the implant. The repair phase - in which the granulation response results in the gener- ation of a capsule. The maturation phase - during which the capsule and the associated tissues become stabilized. Neither the repair phase nor the maturation phase are likely to reflect variables in the surgical technique. Thus the more reliable predictors of biocompatibility are seen in the repair and maturation phases and not in the inflammatory phase.

Two factors which have not been considered adequately in reports are the persistence of blood vessels in the capsule and the arrangement of the matrix in the capsule. The former factor implies the persistence of an active, albeit, a minimal granu-

lation response indicative of protracted repair. The latter represents perturbation caused by the foreign material and thus is indicative of disturbed biocompatibility.

Examination of the connective tissue matrix organization of the capsule and the enveloping connective tissue has largely been ignored and biocompatibility judged by inflammatory responsesL,2 and by capsule thickness.'O Only recently has connective tissue matrix maturity been considered in biological testing of materials." In this current study, tissues were examined using the Masson trichrome stain under transmitted light and by the Pic.R stain under polarized light. Masson's stain may reflect the proteoglycan status of the capsule,'* whereas, the Pic.R stain displays the arrangement of the collagen fibrils. The Pic.R stain was found to be of particular value in detecting the early secretion of collagen to form the presumptive capsule.

Capsule thickness is often taken as a quantitative measure of biological activity, hence thin capsules are considered to represent greater biocompatibility. Various complex scoring systems based on thickness measurements have been devised. l3 In many instances it is a reliable predictor of biocompatibility; in a recent study," sulphonated polystyrene was considered to be a nearly ideal biocompatible material and stimulated no discernible encapsulation, the sole response was limited to the interface between the material and the capsule where there was a region of disturbed matrix for 20 pm. This material may have the potential to act as an ideal control for the RSP if capable of being moulded to the appropriate shape.

Factors such as the arrangement and compactness of the connective tissue matrix must be considered as an indicator of biocompatibility. Poor tissue organization probably reflects a disturbance in the biosynthesis and maturation of collagen and other components comprising the capsule. Thus capsule organization is a better indicator of biocompatibility than is capsule thickness.

When the test system used in this study was applied to the commercial dental amalgams, all were found to be biocompatible. The best organization occurred with F400, GS80 and Ultrafine, closely followed by Permite and Lojic. The least well organized was Dispersalloy which showed a range of variability. This variability, which may reflect the heterogeneous composition of the admixed components in Dispersalloy, was not seen in the other admix amalgams. Both Permite C and GS80 comprise dispersion phases which could function as dental amalgam in their own right, whereas,

Australian Dental Journal 1990;35:6. 503

Dispersalloy is composed of a conventional dental amalgam alloy and a spherical silverkopper eutectic. This latter phase, which reacts slowly with mercury, tends to oxidize and separate with storage” and alter its electrochemical properties with may explain the heterogeneous biological response. The variability in the capsule is unlikely to have been discerned with the Teflon tube system.

This study of various dental amalgams, using a system suited to the histological testing of the biocompatibility of dental amalgam, showed that the Australian produced dental amalgams rated well when compared with Dispersalloy in the short and the long terms. They were equal to or more compat- ible than the one brand considered to be a benchmark against which new products are often compared. The Australian produced dental amalgams show no protracted inflammatory infiltration and the fibrocollagenous capsules which form around them mature normally and are well organized.

Conclusion A. Any revision of IS0 TR7405, published as

FDI Technical Report No. 9, must consider relevance to dental amalgam and other restorative materials. The system must be validly controlled above all other considerations.

B. The Australian dental amalgams compared well with the competitor and the profession should be confident in their biological acceptability when used correctly.

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Address for correspondenceheprints: Restorative Dentistry Section,

School of Dental Science, University of Melbourne,

71 1 Elizabeth Street, Melbourne, Victoria, 3000.


the biocompatibility testing of some dental amalgams in vivo

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