Journal of Cranio-Maxillo[bcial Surgery (1999) 27, 117 123 © 1999 European Association for Cranio-Maxillofacial Surgery

The behaviour of titanium as a biomaterial: microscopy study of plates and surrounding tissues in facial osteosynthesis

Julio Acero, 1 Javier Calderon, 1 Jos6 I. Salmeron, 1 Juan J. Verdaguer, 1 Carlos Concejo, I Maria L. Somacarrera 2

1Department of Maxillofacial Surgery (Chairman: Prof Dr C. Navarro), Gregorio Mara~6n University Hospital, C/Doctor Esquerdo 46, 28007 Madrid, Spain, 2Department of Stomatology, Complutense University Dental School, Madrid, Spain

SUMMARY. Titanium has become the biomaterial of choice for facial osteosynthesis. Titanium is considered a highly biocompatible and corrosion resistant material, although the ultrastructural behaviour of titanium in human tissues after bone fixation is not well documented.

A prospective scanning electron microscopy study was carried out on 37 commercially pure titanium miniplates which were removed from 23 patients who had undergone surgery for maxillofacial trauma or deformity. Twenty two cases were used as a control group. Implant-bone specimens were excised using tungsten burs and studied with a scanning electron microscope (Jeol JSM-T-300). Findings at the bone-titanium interface were analyzed, as well as the presence of contaminating bodies on the specimen surface. Biopsies were also obtained from the soft tissues adjacent to 20 miniplates, then sectioned and stained with Haematoxilin-Eosin for histological evaluation by light microscopy.

The results showed good ultrastructural osseointegration of the osteosynthesis material in most cases (81.8%). Mobility was found upon removal in 80% of plates which showed clinical complications. A significant correlation was found between the degree of microscopical osseointegration and macroscopic fixation of the plate. Microscopical contamination was found in 100% of the nine plates with intraoral exposure, while only 36% of the 22 miniplates of the control group had contaminating elements (P<0.001). Thirty-five point one percent of the plates showed hole-like substance loss images, whose size ranged from 10-25 g. Light microscopy showed granular deposits in soft tissues surrounding the plates in 80% of the 20 specimens investigated. Our findings suggest a higher development of corrosion in titanium than previously reported. These findings are not correlated, however with the clinical complications.

INTRODUCTION

Bone fixation systems with plates and metallic screws, both compressive and semirigid, have created radical changes in the treatment of facial fractures and fixa- tion of osteotomies. Titanium has become the bioma- terial of choice for these systems, since it meets the requirements of resistance, adaptability and biocom- patibility. Biomechanically, titanium has a high tensile strength (Weber et al., 1990) and a low modulus of elasticity that allows good contouring to the osseous surface of the facial skeleton (Lemond and Lucas, 1986 Marsh, 1989). Most reports rate titanium bio- compatibility as excellent. It is a biomaterial with a high superficial energy and after implantation it pro- vides a favourable body reaction that leads to direct apposition of minerals on the bone-titanium interface and titanium osseointegration (Carlsson et al., 1986). Titanium is protected by a strong and stable layer of titanium dioxide that forms spontaneously in contact with air. This layer regenerates after bending and makes titanium very resistant to corrosion. As far as we know there have been no reports of allergic

reactions associated with the use of titanium (Muster, 1987).

This information suggests that titanium plates and screws are non toxic and could be left in the body per- manently, thus avoiding a second procedure for removal (Disegi and Wyss, 1989). Nevertheless, some disagree- ment has been reported by authors such as Bos et al. (1990) or Kim et al. (1997). In spite of the widespread utilization with good results of titanium as an osteo- synthesis material in maxillofacial surgery, a question remains: need titanium material be removed? Kroon et al. (1991) report screw mobility on miniplate removal, and Williams describes some corrosion susceptibility in biomaterials such as titanium (1987). Bos et al. (1990) and Moran et al. (1991) consider that the behaviour of titanium as an osteosynthesis material has not been clearly investigated from the histological point of view. Titanium particles have been found in human tissues related to osteosynthesis material (Sehliephake et al., 1993; Jorgenson et al., 1997). The purpose of this paper is to analyse the behaviour of titanium as an osteosyn- thesis material in humans, when used in bone repair after facial'fractures or osteotomies.

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118 Journal of Cranio-Maxillofacial Surgery

MATERIAL AND METHODS

A prospective histological study was carried out on 37 commercially pure titanium miniplates, removed from 23 patients who had undergone surgery for maxillofa- cial traumatic injuries or deformities affecting the maxilla (40.5%) or the mandible (59.5%). Their mean age was 31.89 years. Twenty-five miniplates were removed from male patients and 12 from female. Fifteen miniplates were removed due to clinical com- plications, mainly intraoral exposure (nine cases) and pain (four cases). Other reasons were local inflamma- tory signs (1) and nasal exposure (1). Plate removal was performed between 4 and 160 weeks after surgery. The control group included 22 miniplates, removed 3 months after surgery from patients who did not show any complications.

Plate removal was always performed by intraoral surgery. Bone biopsies around the titanium material were taken in 22 cases, a bone cylinder being-obtained surrounding at least one of the plate's screws. Patient consent was obtained in each case. In 20 cases we also obtained a sample of soft tissue adjacent to the plate for histological evaluation by optical microscopy.

Scanning electron microscopy preparation

Titanium miniplates and screws, alone or with sur- rounding bone, were fixed in glutaraldehyde-millonig 1:4. Implant-bone specimens were obtained by sec- tioning with tungsten burs. Specimens consisting of miniplates alone or complexes of plate-screw and sur- rounding bone were dehydrated in increasing concen- trations of acetone and desiccated in a critical point device at a temperature of 35°C and a pressure of 72 Kg, using CO 2 as intermediate agent in a CPD-0.20 desiccant device. Every specimen was covered with gold by sputtering in an argon atmosphere (Ion Sputter J.KC.-1.100, Jeol Ltd) and analyzed with a scanning electron microscope (Jeol JSM-T-300), with an acceleration voltage of up to 30 Kv and magnifica- tion scale within 15 to 200.000x. Photographic images were taken from the most outstanding findings in every specimen, with special focus on the bone- titanium interface, microbial contamination and pos- sible morphological anomalies of the titanium.

Optical microscopy preparation

The 20 adjacent soft tissue specimens were fixed in 10% saline formaldehyde and embedded in paraffin. The resulting blocks were sectioned with a microtome (6 gm thick) and stained with Haematoxilin-Eosin for microscopy study.

Resulting data was stored and statistical analysis was carried out on a 486 PC using DBASE-III PLUS database software (Imprise Corporation, Scotts

Valley, CA, USA) and SPSS-PC statistical software (SPSS Institute, Chicago, IL, USA). Statistical analy- sis included descriptive statistics, nonparametric two- and k- independent sample tests (Mann-Whitney, Kruskal Wallis) and the Kendall Tau-B nonparamet- ric correlation test in order to anlalyse the possible relationship between the microscopic data and the macroscopic or clinical findings.

RESULTS

Macroscopic findings

We found good bone healing in 91.7% of the osseous fixations; evaluated results were poor in three cases (8.3%). Associated tissue macroscopically compatible with the surrounding fibrosis was seen in 78% of the plates assessed. Dark staining of surrounding tissue was observed in a high proportion of the plates inspected.

Surgical exploration revealed good miniplate fixa- tion to the underlying bone in 19 cases (51.4%), even with osseous overgrowth in seven of them. We found deficient plate fixation in 18 cases, with partial screw mobility in 12 cases and complete mobility of the plate in six. This deficient plate fixation to bone was more frequent in the group of miniplates presenting clinical complications than in the control group: mobility was found in 12 (80%) of the 15 plates which presented some clinical problems, while it appeared in only six (27%) of the 22 plates with no clinical compli- cations. The difference was statistically significant.

Scanning electron microscopy findings

Titanium-bone interface

We will describe first our findings after SEM analysis of the titanium-bone interface in the 22 cases with associated surrounding bone biopsy:

• Osseointegration, defined as direct attachment or connection of vital osseous tissue to the surface of a titanium screw or miniplate, with absence of intervening connective tissue, was excellent or good in 81.8% of the cases analysed (Fig. 1).

• Osseous overgrowth above the plate, verified by microscopy, was found in six cases (27.3%).

• We found connective tissue at the titanium-bone interface in four specimens (18.2%). In two cases, defined as medium osseointegration, this fibrous tissue was seen only in isolated areas. In the other two specimens with lack of integration this fibrosis was strong and clinical association with plate mobility was found (Fig. 2).

Statistical analysis showed a significant association between degree of osseointegration and macroscopic

The behaviour of titanium as a biomaterial 119

I d i ! i : ~ I . I !

Fig. 1 - Scanning electron microscopy (low magnification): bone- titanium screw interface showing good integration with absence of connective tissue (TI: titanium; B: bone).

Fig. 2 Lack of osseointegration. SEM image showing interposition of fibrous tissue between bone and titanium screw (B: bone; Ti: titanium; Fib: fibrous tissue).

Fig. 3 - Filamentous contamination compatible with fungi on the plate surface. Filamentous bodies with chonidiophora (Ti: titanium plate surface; Ch: image of chonidiophora).

Fig. 4 - Scanning electron microscopy preparation, high magnification (1 cm = 10 g). Unidentified bilanceolated contaminating body (arrow) on the plate's surface (Ti: titanium plate).

plate fixation to the surrounding bone; a statistically significant correlation was found between screw or plate mobility and ultrastructural finding of fibrous tissue in the bone-titanium interface (P<0.01). No relationship was found between osseointegration and the length of permanence of the biomaterial in the body. We found an association between poor micro- scopical osseointegration of plates and screws and microscopy findings of contaminating elements, but it was not significant.

Findings on plate surfaces

Microscopical contamination on the plate surface was found in 100% of the nine intra-orally exposed plates removed, while only 36% of the 22 miniplates in the control group were contaminated. This difference in contamination incidence was found to be statistically significant (P<0.001). Contaminating elements, defined as foreign bodies over the surface of the plates,

were classified according to the morphological find- ings as follows: fiJamentous elements compatible with fungi, lanceolated bodies (symmetrical or atypical) and polymorphic or mixed contamination. Fila- mentous elements showing hyphae, sometimes with spore formation or even conidiophores, were found over 11 plates (six of them associated with other cont- aminating bodies). These elements were considered morphologically as fungi (Fig. 3). We found 14 cases of bilanceolated elements contaminating the surface of plates, eight isolated and six associated with filamen- tous contamination as mentioned, with a morphology ranging from perfectly symmetrical to more irregular and a length from 60 to 110 g (Fig. 4). We could not identify these elements. Culture of the plates after these findings was not possible, because they were already fixed for histological analysis. Only one of the last specimens was Cultured before fixing. In this case we found candida albicans, with a correspondence in electron microscopy with mixed contamination including lanceolated elements (Fig. 5).

120 Journal of Cranio-Maxillofacial Surgery

Fig. 5 - SEM picture of a plate culture with candida albicans. Fig. 6 - Scanning electron microscopy. High magnification image showing possible formation of microholes approximately 10-25 g (arrows) on the surface of a titanium plate.

Fig. 7 - Modulation of SEM image. Erosion on the titanium plate surface (25 g diameter).

Fig. 8 - Light microscopy of soft tissue surrounding a titanium plate showing extra and intracellular granular deposits (HE x 200).

A statistically significant relationship was found between SEM findings of contamination and the macroscopic status of miniplates at the time of removal. Osteosynthesis material with good osseoin- tegration showed contamination in only 26.3% of the specimens, while those with deficient fixation were contaminated in 77.8% (P<0.005). No relationship was found between microscopical contamination and previous fracture or osteotomy, bone consolidation or duration of insertion in the organism.

SEM analysis of removed miniplates and screws revealed some cases with defects or irregularities on the biomaterial surface. Thirteen out of the 37 plates studied (35.1%) showed, on the surface, hole-like appearances, generally isolated, ranging in size from 10 to 25 g (Fig. 6). These anomalies were due to sub- stance loss in the titanium material, as we verified by modulating the electron beam (Fig. 7). We found no relationship between these surface defects and factors such as time of permanence in the body, micro- scopical contamination or deficient osseointegration.

Defects on plate surfaces were not more frequent statistically in intra-orally exposed plates than in unexposed ones.

O p t i c a l m i c r o s c o p y f i n d i n g s

Specimens from soft tissue surrounding 20 miniplates were investigated by optical microscopy. This tissue exhibited dark pigmented deposits in 80% of the spec- imens studied. These deposits were birefringent parti- cles with a dark brown colour and a size ranging from 15-20 g, and very probably consisted of both intra- and extracellular metallic deposits (Fig. 8).

Histological signs of fibrosis or poorly cellular connective tissue growth adjacent to osteosynthesis material were observed in 11 out of the 20 plates analysed. No statistical relationship was found between optical microscopy findings and clinical parameters such as plate exposure or other complica- tions related to osteosynthesis material.

The behaviour of titanium as a biomaterial 121

DISCUSSION

Clinical experience has shown advantages in osteosynthesis systems with titanium screws and mini- plates as compared with old bone fixation methods in the maxillofacial region (Breme et al., 1988). However, some reports suggest the necessity of further evalua- tion of titanium behaviour as a biomaterial. In our lit- erature review, we found a lack of ultrastructural studies on titanium osteosynthesis in humans. We have found some pathological analysis of titanium dental implants and the surrounding bone in humans (Ldpez Arranz et al., 1980; Krekeler et al., 1985; Gores et al., 1989). Nevertheless, screw plate systems used in fractures or osteotomies bear functional loadings that do not take place in the first weeks after dental implant placement. Kiilebo and Jacobson (1988), report in an experimental study on rabbits, how tita- nium implants attain osseointegration with cortical bone in 3 weeks. It is in this critical period when plates and screws fixing a fracture sustain the maximum loading and are responsible for the stability of the whole osteosynthesis system.

Scanning electron microscopy (SEM) is a useful method for studying materials placed in organic envi- ronments. The image we obtain with SEM has higher resolution and therefore reveals more realistic infor- mation than optical microscopy (Raveh et al., 1983; Katsikeris et al., 1987). Steflik et al. (1990a), state SEM advantages for analysis of biomaterials inserted in jaws: SEM avoids complex preparations for study- ing specimens, there is a wide magnification range that allows us to obtain true and 3-D quality images, and allows the study of larger specimens. Disadvantages include: some cellular types are sometimes more diffi- cult to identify and artifacts may appear after speci- men preparation. In our study, sectioning of specimens after fixation, and preparation for SEM study, was a simple but sufficient procedure for obtaining these bone and titanium specimens, as Katsikeris et al., stated in 1987.

Deficient plate fixation to bone was found at the time of removal in 80% of the cases in which compli- cations led to the removal of the osteosynthesis mate- rial and in 27% in the group of plates with no complications. Kroon et al. (1991) also found frequent plate mobility at the time of removal, but did not observe effects on fracture consolidation. Statistically significant correlation was found between macro- scopic plate fixation to the bone and the degree of microscopic osseointegration of the screws. Experi- mental studies in animals on non loaded titanium implants have revealed that failures of such implants were associated with SEM findings of connective tis- sue at the titanium-bone interface (Steflik et al., 1990a, 1990b), while successful implants had a clear interface. Factors affecting titanium biomaterial

osseointegration are: meticulous surgical technique avoiding heat excess (Carlsson et al., 1986; Kiilebo and Jacobson, 1988), correct preparation of biomaterial surface and absence of micromovement during the osseointegration period of the implant (Heck et al., 1986; Steflik et al., 1990a). Osteosynthesis materials may not fulfil this last condition due to functional loading.

We also found a statistically significant higher rate of microscopical contamination among plates with clinical mobility than in those with good fixation to the bone. Morphologically considered contamination was found over the plates surface in 36% of the cases presenting no complications, and in 100% of the plates removed following exposure (statistically signif- icant difference). A reasonable explanation of these results could be that plate contamination had an influence on ultimate plate exposure and mobility. Another explanation could be secondary plate contamination after exposure, but that would not explain why 36% of non-exposed plates became contaminated. The last possibility would be that plate contamination occurred during the surgical procedure for plate removal, but that would not explain the differences found, either. No differences in bio- material contamination was found by Nishioka et al. (1988) between plates inserted via the intraoral or extraoral route.

Observed polymorphism of contaminating ele- ments is in agreement with findings reported by Gristina and Costerton (1985). In their SEM study of 25 infected biomaterials they found an adherent layer on the surface in 76% of the cases. This fact explains the difficulty in obtaining representative cultures in infection of biomaterials and the difficulties found in eradicating that infection. Nishioka et al. (1988) and Marrie and Costerton (1983) report similar results. One of the problems with SEM might be the difficulty in identifying cellular types, as reported by Steflik et al. (1990a). Due to the morphological design of our study, fixing the specimens for their examination, we could only perform cultures in one of the last cases, in which we found candida albicans. We believe that most contaminating elements found may correspond to fungi, and we have even identified conidiophores and typical fungal filaments in some specimens. Symmetric bilanceolated elements were found in 13 cases, but they could not be identified since we did not find any refer- ence to that kind of element. Perhaps there is some dimorphism, as Deng et al. (1988) found for penicillin fungi due to time or temperature changes.

Before titanium miniplate insertion, SEM analysis of these plates shows regular edges and smooth sur- faces, with occasional crevices due to their industrial manufacture. In many of our study subjects, however, after removal we found marked defects on the plate surface, such as contour or surface fragmentation,

122 Journal of Cranio-Maxillofacial Surgery

We do not know whether such defects are due to material deterioration in an organic environment or to plate manipulation at the time of insertion or removal. Nevertheless, we found hole-like images ranging from 10-25 g, generally isolated, in 35.1% of the plates studied. We suppose that such anomalies may be corrosion effects in a biomaterial, with tita- nium particles released to the surrounding tissues. This would explain the optical microscopy images of deposits found in the tissues adjacent to plates. However, comparison with other studies is difficult due to the scarcity of similar reports. Wil l iams (1987) suggest the possibility of titanium developing corro- sion in a biological environment. H e c k et al. (1986) reported titanium deposits found in lymph nodes in dogs, and increased titanium content in organs such as the spleen, liver, lungs, serum and urine, although these levels were very low. B l a c k et al. (1990) reports failure of a titanium alloy hip prosthesis due to metal- losis and pain. Histological analysis of synovial tis- sues showed dark particles both intracellularly and inside the histiocytes M o r a n et al. (1991) found similar deposits in six cases of titanium dioxide lesions. In the maxillofacial region, Schliephake et al. (1993) investi- gated soft tissues surrounding titanium miniplates used in the osteosynthesis of mandibular fractures in 10 patients. He also reported groups of opaque parti- cles consisting of titanium surrounded by poorly cel- lular connective tissue. Most of them appeared to be located extracellularly with some exceptions located intracellularly, as we found in our study. Inflam- matory response was weak, thus such deposits seem to be apparently well tolerated. Schl iephake et al. (1993) suggest that one major cause of the liberation of tita- nium is fretting between the titanium plates and screws during the fracture healing period which pro- duces small shavings of titanium when the stabilized fracture is functionally loaded and micro-movements between plates and screws might occur. K a t o u et al. (1996) reported titanium particles found in tissues adjacent to miniplates in biopsies taken from 12 patients with mandibular fracture osteosynthesis; thus, he suggested systematic removal of every mini- plate once its function is accomplished. B l a c k et al. (1990) and M o r a n et al. (1991) succeeded in identify- ing these granular deposits with certainty as titanium by diffraction analysis, that we could not make. It is worth emphasizing the coincidence between the approximate size of the extracellular minor granules found with optical microscopy and the possibly devel- oping holes detected on the surface of some plates by electron microscopy, 15-30 p. wide.

The consequence of our findings might be, that deterioration of titanium exposed to the biological environment in bone healing situations could be more important than previously described. On the other hand, no correlation was found between the images of

morphologic alteration of the biomaterial and other SEM findings in our study, like biological contamina- tion or poor osseointegration of the implanted mater- ial. No association was found, either, with clinical complications related to the biomaterial. All these facts suggest that the described findings of titanium deterioration, even though observed at a higher rate than previously reported, are of no clinical impor- tance, according to our results.

In conclusion, microscopical osseointegration of the titanium osteosynthesis material is good in most cases. Fibrosis on the bone-titanium interface is associated with mobility of the screw-plate system. Findings of contamination on scanning electron microscopy are related to clinical complications of the plates. Defects on the surface of the biomaterial and pigmented deposits in soft tissues surrounding the plates suggest a higher development of corrosion in titanium than previously reported. Finally, bone con- solidation was not influenced by these findings.

Acknowledgement

The authors would like to thank To Prof. Emilio Alvarez, Head of Department of Pathology, Gregorio Marafidn University Hospital, for his support in the microscopy study we have carried out in this paper.

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Dr Julio Aeero Assistant Professor in Surgery C/Velazquez 27, 2 ° izq 28001 Madrid Spain

Tel: +914319913 Fax: +915761950

Paper received 6 July 1998 Accepted 25 January 1999