Immunohistochemical study of the soft tissue around long-term skinpenetrating titanium implants

Biomoteriols 0

16 (1995)

611-616

1995

Elsevier Science Limited in Great Britain. All rights reserved 0142-9612/95/$10.00

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Immunohistochemical study of the soft tissue around long-term skinpenetrating titanium implants KM. Holgers

* +,

P. Thomsen*,

A. Tjellstriim+and L.M. Bjursten’

‘Department of Anatomy and Cell Biology, University of GGteborg, Medicinaregatan 3, Giiteborg, S-413 90 Sweden; +ENT Department, Sahigren’s Hospital, University of Giiteborg, Medicinaregafan 3, Giiteborg, S-413 90 Sweden; tDepartment of Experimental Research, University of Lund, Ma/m6 Genera/ Hospital, Malmd, Sweden Bone-anchored

percutaneous

with a low incidence barrier

to exogenous

the distribution from

patients

penetrating patients clinical

titanium

of adverse pathogens.

of lymphocyte

there was an increased

tion. The data suggest function at these

that there

implants

study,

a well-established through

monoclonal

in the soft tissue

The number

compared

become passage

skin, five from

analysed.

with skin penetration irritation

In the present

irritated

were

have

However,

subpopulations

with clinically implants

implants

reactions.

with

antibodies around

non-irritated of immune

patients

without

and that an antibody-mediated

response

in the

used to investigate Eight

skin without

was increased

compared

compensation

procedure

to a breach

implants.

from

skin penetration.

level of B-lymphocytes

is an immunological

were

such

and eight cells

clinical

the skin leads

biopsies skin-

in the group

of

In the group with

with those without irrita-

for the mechanical is present

at clinical

loss in barrier signs of

irritation. Keywords:

lmmunohistochemistry,

Received 20 June

1994; accepted

titanium, 15 September

Bone-anchored skin-penetrating implants are used for the anchorage of external epistheses and hearing aids in the head and neck region. As these implants penetrate the skin they cause a breach in the defence barrier to the outside. Clinically favourable results of this type of implant indicate, however, that the mechanical breach is compensated by other defence mechanisms. Thus, a previous morphological study of the soft tissue shows that the number of inflammatory cells is increased close to the implants’. Normal skin has a low number of Tcells around the vessels and the cells have a 1:l CD4 CD8 immune phenotype ratioza3. In skin with delayed hypersensitivity reaction T-cells are selectively increased with a significant increase in the T-helper (CD4+) population’. B-cells and plasma cells are not detected in normal skin3, nor in skin with a delayed hypersensitivity reaction’ and the T-cytotoxiclsuppressor subpopulation (CDs+) remains low4. The gingiva represents another situation where there is a discontinuity in the epithelium and a continuous confrontation with potential pathogenic agents. The junctional epithelium is, however, not only adapted to adhere to tooth enamel but also to dental implants via hemidesmosomes and a basal lamina5.6. In clinically healthy gingiva low numbers of inflammatory cells are present7-‘. In stable parodontitis the inflammatory Correspondence

percutaneous

implants

1994

response is increased and characterized by a predominance of T-lymphocytes”. In advanced parodontitis Blymphocytes and plasma cells are the predominant cell types”-13. Seymour et a1.14 reported some degree of inflammation in the gingiva around titanium implant a.m. Branemark, even in clinically healthy specimens. The response is characterized by the presence of T- and Blymphocytes and the proportion of these subsets is the same with or without signs of gingivitis. In order to understand possible immunological mechanisms protecting the skin-penetrating site from severe adverse reaction15’16, we made the present study. We have analysed skin biopsies adjacent to percutaneous bone-anchored titanium implants from both clinically irritated skin and non-irritated skin and also compared these findings with those of continuous skin from the same area.

MATERIALS AND METHODS Surgical procedure The surgical installation procedure for the implants has been described by Tjellstr6m’7. In brief, the installation of the implants is made in a two-stage procedure. A screw-shaped titanium implant (the fixture) is installed in the temporal bone behind the

to Dr K.M. Holgers. 611

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ear canal in a first operation. Profuse saline irrigation is made during drilling to reduce surgical trauma by preventing generation of excess heat and keeping the drill free from bone fragments. The fixtures are then inserted in prethreaded holes in the bone. After suturing the skin the fixtures are left unloaded for a period of 3-4 months, and the skin-penetrating abutments are put in place in a second operation. To get a close contact between the skin and underlying bone, it is generally necessary to surgically reduce the thickness of the subcutaneous tissue. Often it is also necessary to place a thin hairless skin graft at the site of the implant. After 3-4 weeks the healing process has generally reached the stage when it is possible to start fabrication of the epistheses or to give the patient the hearing aid.

Patients Thirteen biopsies from 11 patients with titanium implants were used (Table I). Seven of the patients (three women and four men; biopsies from skin at eight implants) had a clinical skin irritation around the implants at biopsy (skin irritation group (IG)). Four of the biopsies were from skin around implants for attachment of hearing aids and four from ear epistheses. The duration of skin penetration was between 20 and 115 months (average 54 months). The remaining five biopsies were from five patients (four women and one man) who had no clinical skin irritation (non-irritated group (NIG)). In this group two of the patients had hearing aids and three ear epistheses. The skin penetration had lasted between 7 and 55 months (average 31 months). All patients had subcutaneous tissue reduction at the time for installation of the skin-penetrating unit. None of the patients had been treated with radiotherapy. Biopsies from eight patients (four female and four male) who had a titanium fixture installed in the bone, but no skin-penetrating abutment, were also obtained and used as controls (control group (CG)).

Table 1

Patients with respect to diagnosis: EO=external CM0 = chronic otitis; M = malignancy; otitis; media TA = traumatic CD = cochlear defect; OS = otosclerosis; ablatio; CA=congenital aplasia of the auricle. Type: H = hearing aid; A = auricular prosthesis: 0 = orbita prosthesis; SP = skin-penetration time (months) Patient

Sex

Non-irritated 1 2 3 4 5

group (N/G) F 55 F 23 F 16 M 7 F 52

Irritated 3 6 7 8 9 10 10 11

group (IG) F F M F M M M M

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Age

16 79 79 65 44 28 28 13

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Diagnosis

Type

Time

M CD CA CA EO

A H A A H

62 31 52 6 51

CA OS CM0 TA CM0 CA, CM0 CA, CM0 CA

A H H A H A A H

52 46 115 71 30 49 49 20

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Biopsy and tissue preparation The biopsies were obtained after careful removal of the skin-penetrating part of the implant. It was not possible to get the soft tissue biopsy together with the implant since the implants did not macroscopically seem to be attached to the soft tissue. A circular incision down to the underlying bone was made around the titaniumtissue interface. The biopsies were shaped as a ring with the inner side of the ring having tissue facing the titanium surface. The abutments slid smoothly out through the soft tissue and were macroscopically clean from organic soft tissue. The biopsies were pinned to cork plates to facilitate orientation. The tissue was embedded in Tissue-Tek (OCT compound, Histolab Products AB, Gbteborg, Sweden], quick-frozen for 1 min in 2-methylbutane (isopentane, Kebo Lab AB, Stockholm, Sweden) in liquid nitrogen and kept at -70 “C until cryostat sectioning. The tissue was sectioned to a thickness of 6pm in a cryostat (Leitz, Germany). Finally, the sections were placed on microscope slides, air-dried for 30 min and fixed in cold pure acetone (Lab Kemi, Stockholm, Sweden), and stored at -70 “C. Five slides were processed to display human leucocyte antigen HLA-DR, CD5 (T-lymphocytes), CD8 (T-cytotoxic/suppressor), CD4 (T-helper/inducer) and CD22 (B-lymphocytes) (Table 2). The tissue sections were air-dried for 20 min, and washed in phosphatebuffered saline (PBS), pH 7.2 for 15 min. Endogenous peroxidase activity was blocked by incubation in 0.3% H202 for 15 min. The sections were then washed in PBS three times for 15 min and incubated for 20 min. All incubations were made in a humid chamber with horse serum (Vector Lab. Inc., Burlingame, CA, USA) diluted in volume proportions of 3:200 in PBS. The sections were then incubated for 30 min with the monoclonal antibodies anti-Leu-1 (1:30), anti-Leu-2a (l:lO), anti-Leu-3a (1:15), anti-leu14 (1:lO) or anti-HLA-DR (1:30) (Becton Dickinson, Mountain View, CA 94039, USA). The sections were then washed in PBS and incubated for 30 min with biotinylated goat-anti-mouse IgG (BA-2001, Vector Lab. Inc.) diluted 1:200 in PBS. The sections were washed in PBS for 15 min, and incubated for 45 min with Vectastain solutions A and B (peroxidase-labelled avidin, Vector Lab. Inc.) diluted in volume proportions of 1:l:lOO in PBS. The sections were then washed with PBS for 10 min and developed for 2 min in a mixture of 3,3-diaminobenzidine (DAB) in PBS (1 mg DAB in 1 ml PBS) and 0.02% HzOz in 1:l volume proportions. Positive

Table 2 tion

The monoclonal

antibodies

and their antigen

distribu-

CD

Specificity

Clone

CD4 CD5 CD8 CD22 HLA-DR

T-lymphocytes (helper/inducer) T-lymphocytes T-lymphocytes (cytotoxic/suppressor) B-lymphocytes Human B-lymphocytes, monocytes/ macrophages, activated T-cells and Langerhans cells in epidermis

Leu-3a Leu-1 Leu-2a Leu-14

lmmunohistochemical

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controls with the monoclonals used were performed on sections from human tonsils. Negative controls consisted of omission of primary monoclonal antibowith unrelated incubation dies; alternatively, monoclonal was performed (insulin; Immunotech, Paris, France). Sections were washed with PBS four times for 20 min and counterstained with haematoxylin for 7 min, rinsed in water for 5 min and washed in PBS. The sections were dehydrated in a graded series of ethanol and mounted in Pertex@ (Histolab Prod., Goteborg, Sweden).

Tissue evaluation The inflammatory response was quantified in a Nikon FXA microscope. In each specimen from skin around skin-penetrating implants five areas (0.1 mm2 each) were chosen for morphometry, and in biopsies of skin without skin penetration two areas were chosen. The total number of cells and the number of cells positive to the specific antibody were counted within By using a squared grid. cross-points (point counting)l* within the squared grid the lack of tissue was determined and taken into account when calculating the cell density. The location of areas used in the quantitative evaluation is shown in Figure 2. Areas A, B, C and D were situated close to the (removed) implant, directly under the epithelium. Area E was located 1.66 mm from the interface zone. Two areas were measured in the biopsies from skin without permanent skin penetration. Area A was directly below the epidermis and area B directly below area A. Comparisons between the groups with and without skin penetration were made using the mean value of A-D in the group with skin penetration and the mean value of A-B in the group without skin penetration.

Statistics The Kruskall-Wallis and Mann-Whitney tests were used for statistical analysis on independent samples and the Wilcoxon sign rank test was used on the related sampleslg. The statistical significance level was set to P-co.05. Corrections due to multiple comparisons were done with the sequentially rejective Bonferroni test”. Thus, the rejection level corresponding to a

Figure 1 Schematic showing (A-E) used for the quantitative

the location analysis.

of

the

areas

implants:

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single comparison varies depending on the number comparisons performed in each analysis.

of

RESULTS General distribution of cells Figure 2 shows a representative micrograph from a patient without clinical signs of irritation. The cell number from the skin-penetrating and control groups is given in Figure 3 (median value). Mean values of areas A-D in the skin-penetrating group and of areas A-B in the non-skin-penetrating group were compared statistically. The mean value of the total number of cells in the respective areas was calculated based on the total number of cells in all sections for each patient. When comparing the control group, the skinpenetration group with clinical irritation and the skinpenetrating group without irritation, statistical analysis revealed a significant difference with respect to HLADR expressing cells, total number of cells, T-lymphocytes, T-helper cells, T-suppressor cells and B-lymphocytes (P
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skin barrier is broken. When comparing skin with clinical irritation at the implant with skin without irritation there were more B-cells in the irritated group than in the non-irritated group. However, the differences between these groups with regard to the total number of HLA-DR expressing cells were not statistically significant (P-c 0.052). In our present study there were no alterations in the TH/TS ratio in the different groups. This study thus does not confirm our earlier study” where we reported a decreased TH/TS ratio in patients with clinical skin irritation. When analysing the spatial distribution of cells, there was an increased level of HLA-DR expressing cells close to the titaniumtissue interface compared with a more distant area (R). 450 400 350 300 250 200 150 100 50 0 Totcell

Figure 2 a, Micrograph from tissue adjacent to the implant from soft tissue without clinical inflammation. The darker stained cells are expressing CD5 (T-lymphocytes). The arrows show some of the positive cells (bar=32 pm). b, Micrograph from the same area as a, but the positive cells cells) CD8 (T-cytotoxic/suppressor expressing are (bar = 30 pm).

lymphocytes (PC 0.139, P
DR

CD5

CD8

CD4

CD22

Figure 3 Numbers of cells (median values shown) per area. A-D for the groups with (IG) and without (NIG) clinical soft tissue irritation, and A-B for the group without skin penetration (CG) (m, IG; 0, NIG; II, CG).

TH/TS 10 9 8 7 6 5

DISCUSSION Analysis of the subpopulations of lymphocytes has been used as a means to characterize the immunological events in various inflammatory condition?. In the present study this methodology was used to study the number and distribution of immune cells in the tissue around skin-penetrating titanium implants. The study shows that the total number of cells was elevated in skin with percutaneous implants even in the absence of clinical signs of inflammation. An increased level of immune cells close to the skinpenetration site could be expected since the soft tissue is more exposed to exogenous agents when the Biomaterials

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4 3 2 1 0

IG

NIG

CG

Figure 4 The T-helper and T-cytotoxic/suppressor cell (TH/ TS) ratio (median value) in area A-D for the groups with (IG) and without (NIG) clinical soft tissue irritation, and A-B for the group without skin penetration (CG).

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ACKNOWLEDGEMENTS No benefits in any form have been received from any commercial party. This study has been supported by grants from the Gothenburg Medical Society, the Hjalmar Svensson Research Fund, the King Gustav V 80-year Fund, The Swedish National Board for Technical Development (NUTEK) and the Swedish Medical Research Council. We express our thanks to MS Christina Gretzer for technical assistance.

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DR

CD5

CD8

CD4

CD22

Figure

5 The numbers of cells (median values) in area AD (close to the implant, W) and area E for groups with skin penetration (El), both the group with skin irritation and the group without skin irritation taken together (IG and NIG).

The small number of samples does not allow for detection of minor differences in subpopulations of cells. There is, however, an increased level of cells expressing HLA-DR, T-lymphocytes subsets (T-helper and T-cytotoxic/suppressor) and B-lymphocytes at all skin-penetrating implants compared with normal skin. When comparing clinically irritated and non-irritated skin with skin penetration there was a significantly elevated level of B-lymphocytes in the irritated skin. This may be consistent with an antibody-mediated response which is often associated with bacterial infectionsZ3. This is further supported by our earlier findings of phagocytic cells in biopsies particularly from patients with skin irritation1 and the finding of S. aureus at the implant sites in clinically irritated skinz4. It is therefore possible that the presence of bacteria such as S. aureus and bacterial products at the site of skin penetration is an important stimulus for the chemotaxis of phagocytes and immune cells to the tissue and the induction of antibody productionZ5. Around intraoral implants an inflammation is demonstrated both in macroscopically inflamed and non-inflamed gingiva14. In a recent ultrastructural analysis of the soft tissue at percutaneous titanium implants’” no evidence for an attachment between the epithelium and titanium surface was revealed. Due to the suggested differences in epithelium-implant contact where probably the epithelial cells are more tightly adapted to the surface at the tooth and dental implant5*“, the exposure to exogenous antigens is more prominent in the percutaneous site than in the intraoral situation. In both clinically healthy and clinically irritated specimens there was an increased number of immune cells compared to biopsies from tissue without skin penetration. In most patients the increased exposure to exogenous antigens seems to be compensated by an increased antibody-mediated immune response as well as a T-cell-mediated response, keeping the soft tissue environment of the implant in sufficient health for clinical long-term function.

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