Biocompatibility of some materials used in dental implantology: histological study

Collolds and Surfaces B. Biointerfaces, 1 (1993) 323-329 0927-7765/93/$06.00 ,c 1993- Elsevier Sctence Publishers

323 B.V. All rights reserved

Biocompatibility of some materials used in dental implantology: histological study” A. Allabouch”,

J. Colat-Parros

b** ,

R . Salmon”, S. Naim”, J.M. Meunierb

“Hopital Militaire d’lnstruction, Mohamed V, Rabat, Morocco bUniversitk de Bordeaux II, 16, Cows de la Marne. F 33082 Bordeaux Cedex, France ‘Laboratoire de Chimie du Solide du C.N.R.S., 351, Cows de la Libkration, F 33405 Talence Cedex, France (Received 21 April 1993; accepted

26 April 1993)

Abstract This study deals with bioreacttve materials for potential applications as endosseus dental implants. Currently used dental implants are made of dental alloys but due to cytotoxic problems, high strength ceramic materials appear as an Interesting alternattve to these alloys. In order to combme good mechanical properttes and promotton of the osteomtegratton process, a ceramic composite maternal composed of 10 wt ‘% tricalcium phosphate (TCP) mixed with partially stabihzed ztrconia has been elaborated. Such a ceramic has been implanted into albino rats, the sites of Implantation being the adrenal gland for the toxictty evaluatton and the sphenotd bone to estimate the osteogenesis potential These results have been compared to those obtained with implants made of alumina (AI,03). zirconia (ZrO,) or poly(ethylene terephthalate) (Dacron !‘). In all cases no rejectton effect was observed. The histological study indtcated that ZrO,-TCP ceramics Induced a marginal and subjacent cell disorganization. However, the extstence of an irregular cellular band Indicated a cellular colonization process on the TCP particles. The microscopy study of the Implants after removal confirmed the bioresorption of TCP. Keq’ words' Bioceramics;

Biocompatibility;

Dental implant;

Histology;

Osteointegration

Introduction

or hydroxyapatite in order to facilitate osteointegration. Experimental studies and a clinical survey

In recent years dental implantology techniques have been improved with the osteointegration development of strengthening implants [ 11. High strength metal alloys such as cobalt-

showed

chromium, widely used in orthopedic surgery, have been implanted in the maxillary bone, but metal alloys which release metallic ions are likely to cause toxicity and implant rejection. More recently, Brdnemark and Zarb [2] used titanium metal, in some cases sprayed with alumina *Corresponding author. “The preliminary form of this paper was presented at the 7th International Conference on Colloid and Surface Science held in Compttgne, France, 7-13 July 1991, and was coordmated for publication by Professors D. Muller and D. Labarre.

that these implants

are well tolerated

the organism. However, some new medical investigation niques such as NMR exclude metal implants may cause artefacts; for these reasons materials have been largely studied. Alumina ceramics have been used mechanical properties seem unable to stress of chewing [3,4], while organic (PMMA,

PTFE)

can deteriorate

by

techwhich

non-metal but their resist the polymers

and their toxicity

will inhibit cellular activity [S]. The purpose of this study was the research and development of high strength ceramic materials exhibiting no biological toxicity and capable of osteointegration. For these reasons, we have

A. Allahouh

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et ul :Collotds

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selected a zirconia-based ceramic mixed with tricalcium phosphate (TCP), which is well known for its biological properties as an osteoinducer [668].

tubes), the same sort of structures which can also be found in gingiva. An oblique latero-dorsal median incision from

Experimental

made to open the muscular

the iliac spine to the lower edge of the thorax procedure

It is not easy to find among laboratory animals species exhibiting maxillar histo-physiology and anatomo-morphology similar to humans. For this study we have selected 58 in bred male albino rats of the same origin (Wistar). The animals were between 2.5 and 3.5 months old and weighed between 165 and 400 g. During the whole experiment. they were kept at 24426°C with 12 h of light per day. In order to test this implant (ZrO,-TCP) two anatomical sites have been used for animal experimentation. The toxicity has been evaluated by implanting the material in the adrenal gland whereas the osteointegration effect was evaluated by implantation in the sphenoid bone.

Muterials:

sample

preparation

The starting materials used in this study were reagent-grade chemical powders: ZrO, (Tosoh TZ3YB), Al,O, (Baikalox CR129 and p-tricalcium phosphate (TCP) (Rectapur). Powders were dried before compacting in roundtip cylinders (l-2 mm diameter, 2 mm length) and then the compacts were sintered in air according to the specifications given by the manufacturer. In the case of the mixed ceramic ZrO,-TCP, powders were carefully mixed at the desired ratio before forming. Poly(ethylene terephthalate) (Dacron “) specimens used were of surgical grade, cut into sections of the same size. In all cases the implant was sterilized before implantation. Methods Impluntation On the kidney.

both

connective

sites and surgical

The adrenal and epithelial

bed. The kidney was excised by pulling out the adipose and connective tissues of the lower pole. The adrenal gland was lifted, a 3 mm long incision made (on the upper third part and the back and dorsal parts of the organ), then the capsule was unlined from the renal parenchyma to the lower third part. An area of about 6 mm’ was cleared on which the implant was inserted. The elasticity of the capsule enables it to reclose. The kidney was laid again in its bed which is first stuck on the muscular plane. then on the cutaneous plane. Four days were needed for the capsule to heal but only 2 days for the skin to heal. On the sphenoid

Materials and methods

techniques

used

gland is made of structures (renal

was

plane and the kidney

hone.

Due to the thinness

of

the rat maxillary, implantation cannot be performed in this site. We have selected the sphenoid bone as implantation site, a spongy bone the diploe of which is similar to the maxillary bone. On the inside part, on the sella turcica, the thickness of the sphenoid bone allows the insertion of the implant. A median incision made from the chin symphysis to the upper extremity of the sternum opens the salivary glands. A lateral reclination of the glands allows a longitudinal incision of the muscular plane which opens the trachea. Then a laterotracheal and subthyroid parting plane is made by pulling out the recurrent nerve and the blood vessels. Subsequently the spheno-occipital suture is reached on which a partial trepanation is made and a cylindrical hole 3 or 4 mm wide is manually drilled. After being positioned, the implant was covered with a gelatinous formalin sponge. Two continuous sutures were successively made, one on the peritracheal muscular plane, the second on the cutaneous plane.

A. Allahouch et al./Collotds Surfaces B Biointerjhces

Removal of the kidneys and of the sphenoids The animals were sacrificed 30 days after the day of implantation.

The kidneys

by median laparotomy. In the case of the sphenoid

were removed

bone, the sella turcica

was removed through either the implantation or the occipital route.

325

1 (19931 323-329

gland and in the sphenoid bone did not stop the growth of animals and the average gain of weight was about

40% after 30 days.

Histological

observations

route

Histological preparation techniques The protocol used for optical microscopy observation was as follows. (1) Fixation of the samples with picric acid as fixing agent; (2) dehydration; (3) impregnation with paraffin, using leuckart bars used for inclusion; (4) seven micrometer microtome sections were colored with trichrome stain solution (Masson). Kidneys were dissected whereas sphenoid bones were decalcified. Implants were removed and the experimental samples were successively dipped into alcohol solutions. Study of the toxicity in the kidney. The material to be tested (ZrO,-TCP) was implanted in the left kidney adrenal gland, whereas the right kidney was reserved for sample material implantation (A1203, ZrO,, TCP, Dacron”‘). Thus the animals were their own controls.

The optical magnification tissue dissection

microscopy 40-500 x

histological studies at a were performed after

in implanted

sites.

Renal observation ZrOz and A120, implants. In both cases the clean boundary between the implant and the surrounding tissues was more comparable with an encystment than with a cyst (see Fig. l(a) representing the Al,O, implant). The continuity of the border line and the regularity of the marginal cell arrangement show an intimate contact between the implant interface and the soft tissue without any superficial tissue thickening, proving that encystment is not reactogenic. DacrortR’ implant. The adrenal gland was retained and its inner side was firmly attached to the implant. The same result was observed for the

Study of sphenoid bone osteointegration. In this case one material was on the uneven symmetrical

parenchyma the surface of which was irregularly depressed in the areas in contact with the implant (Fig. l(b)). At a larger magnification a direct join between the interstices of the mesh material and the renal

site and the osteoinduction evaluation of the ZrO,-TCP composite was studied in comparison with Al,O, and stabilized zirconia (PSZ) samples

tubula could be observed (Fig. l(c)). The undamaged walls of the renal tube confirm that the Dacron” implant is not cytoxic.

implanted

in rats of the same origin.

Mortality and e$ect on growth

The implant was partially TCP implant. resorbed and some TCP scraps remained, either free or attached to the irregular surface of the renal parenchyma (Fig. 2(a)).

The implantation of the ZrO,-TCP ceramic, or of Al,O,, ZrO,, TCP or Dacron’“’ did not cause any animal death after 30 days, i.e. the implantation sites (kidney or sphenoid) had no effect on the death rate. The implantation of materials in the adrenal

ZrO,-TCP implant. A part of the implant has been eliminated. Connective cells moved from the renal parenchyma, the epithelial structures of which were undamaged, and renal tubes could be observed (Fig. 2(b)). At a larger magnification (500 x ) it could be

Results

A. Allabouch et al.:‘Colloids Surface3 B Blointerfhces I

seen that material

particles

( 1993) 313-3-79

have undergone

phago-

cytosis by connective cells and have been incorporated between renal tubulae (Fig. 2(c)). Neither inflammation nor signs of cytotoxicity appeared. Sphenoid bone observation Composite implants (ZrO,-TCP) have been tested and compared with Al,O, and Dacron” implants. No inflammation from this Alumina implant. implant, inserted after trepanning the external table of the sphenoid bone (Fig. 3(a)), was observed. No osteogenesis could be seen in the depth of the sphenoid bone.

(b)

ZrO,-TCP implant. At a small magnification (40 x ) the diploe spongy bone of the sphenoid appeared normal. Around the implant area there was a moderate cellular reaction. No osteogenesis reaction appeared and no osteointegration process seemed to be induced (Fig. 3(b)). At a larger magnification (250 x ), no peripheral cell reaction appeared and implant scraps were seen in the medullar space (Fig. 3(c)). A large amount of hematopoietic tissue and an important megacaryocytar reaction were seen but this reaction may be not related to the ZrO,-TCP implant insertion (Fig. 3(d)). Discussion The purpose

of this study was the evaluation

of

(cl

the biological potential of a high strength ceramic material used for strengthening dental implants

Fig. 1. Histological study of the implantation of (a) Alz03 ceramic and (be) Dacron” m the adrenal gland. (a) The sharp outhnes of the tissue/implant interface mdicate that the A1,03 implant Inserted on the adrenal gland was very well tolerated (bar, 500 pm). (b) In the case of a Dacron” sample, the inner face of the adrenal gland appears strongly attached to the inserted material, the regular structure of the implant appears as printed on the renal parenchyma (bar, 500 urn). (c) Dacron” implant meshes are clearly connected to the undamaged renal tubes (bar, 200 pm).

C9,lOl. The kidney is a vital organ, highly fragile and reactogenic. The least toxicity in the epithelial structure and even more so in the connective tissue would have caused an inflammatory process, and the expulsion of the implant resulting in fatal uremia. The clean break between ZrO, or A1,09

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implants and the surrounding tissues is to be compared with a well-tolerated inert encystment. The continuity of the frontier line as well as the regularity of the marginal cell arrangement mean that implant and tissue are closely attached together. In the case of ZrO,-TCP implants the frontier line turned out to be irregular and crenalated but the implant contact line is still continuous. As a consequence an underlying marginal disorganization and concurrently irregular cell areas can be observed on the surface of the implant, giving evidence of a cellular colonization of the TCP particles. The newly formed tissues settled on the surface of the implant in the microporosities resulting from the partial bioabsorption of the TCP particles. The study of the osteoinclusion and of the osteointegration processes in the sphenoid bone did not indicate intense bioreactivity. In all cases, implants were fully covered with bone tissue, closing up the trepanning orifice and no sign of cytotoxicity was noticed [l 11. The reactivity in this site is less intense and between the Zr02-TCP implant and kidney, fibroblastic and collagenic reactions have been noticed, usually preceding a fibroblastic invasion. The evaluation period (30 days) may not last enough but the histomorphological differences between the sphenoid bone and the maxillary bone must be taken into consideration. The TCP particles were of small size (lo-20 urn) whereas sizes of 150 urn appear necessary for the connective cells to migrate to, and settle in, the porosities [6,12].

(cl Fig. 2. Histological study of the Implantation effect of (a) TCP and (b,c) ZrO,-TCP ceramic m the adrenal gland. (a) TCP free scraps are bonded to the renal parenchyma showing an irregular surface (bar, 200 urn). (b) After partial resorption of the ZrO,-TCP implant, connecttve cells moved from the renal parenchyma, the epithelial structure of which was undamaged (bar, 100 urn). (c) TCP particles located on the surface of the ceramic maternal have undergone phagocytosis and have become inserted between the renal tubes m a Malpighi corpuscule (bar, 50 urn).

Conclusion

The growth and death rates of the test animals did not vary significantly among the different groups, proving that the implanted materials are not as a rule toxic. This result showed that Zr02-TCP implants have been tolerated completely by the soft tissues

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(b)

(4 Fig. 3. Histological studies of the implantation effect of (a) Al,O, and (b--d) ZrO,-TCP ceramic m the sphenord bone. (a) Netther inflammation reactton nor osteogenesis appeared after msertion of an A&O, Implant m the outer part of the sphenoid bone (bar, 300 urn). (b) Moderate cellular reaction wtth netther osteointegratlon nor reactive osteogenesrs 1s observed (bar, 500 urn). (c) Extstence of some implant scraps Inserted in the medullar spaces near the trepannatton area is observed (bar. 100 urn) td) A large amount of hematopoietic ttssue and a megacaryocytar reaction can be observed, but these observatrons may not be correlated to the presence of the implant (bar, IO0 urn)

(kidney) as well as by the hard tissues (sphenoid bone). The renal modifications observed indicate that ZrO,-TCP has a high bioreactive potential. A more important osteointegration certainly needs larger porosity areas and osteoblasts should deeply resorb TCP so that the implant can be firmly rooted to the bone. What is needed is a porous material combining the high mechanical properties of stabilized zirconia ceramics and the bioreactive and osteogenic potentials of tricalcium phosphate (TCP).

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