A histological and immunohistochemical study of tissue reactions to solid poly(ortho ester) in rabbits

Int. J. Oral Maxillofac. Surg. 2006; 35: 631–635 doi:10.1016/j.ijom.2006.01.029, available online at http://www.sciencedirect.com

Research Paper New Technology

A histological and immunohistochemical study of tissue reactions to solid poly(ortho ester) in rabbits M. Ekholm, P. Helander, J. Hietanen, C. Lindqvist, A. Salo, M. Kelloma¨ki, R. Suuronen: A histological and immunohistochemical study of tissue reactions to solid poly(ortho ester) in rabbits. Int. J. Oral Maxillofac. Surg. 2006; 35: 631–635. # 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. In many cases only the temporary presence of a biomaterial is needed in tissue support, augmentation or replacement. In such cases biodegradable materials are better alternatives than biostable ones. At present, biodegradable polymers are widely used in the field of maxillofacial surgery as sutures, fracture fixation devices and as absorbable membranes. The most often used polymers are aliphatic polyesters, such as polyglycolic acid (PGA) and polylactic acid (PLA). Poly(ortho ester) is a surface eroding polymer, which has been under development since 1970, but is used mostly in drug delivery systems in semisolid form. The aim of this study was to evaluate the tissue reactions of solid poly(ortho ester) (POE), histologically and immunohistochemically. Resorption times and the effect of 2 different sterilization methods (gamma radiation and ethylene oxide) upon resorption were also evaluated. Material was implanted into the tibia and subcutaneously into the mandibular ramus area of 24 rabbits. Follow-up times were 1–10, 14 and 24 weeks. Histological studies showed that POE induces a moderate inflammation in soft tissue and in bone. At 24 week follow-up, inflammation was mild in soft tissue and moderate in bone. In immunohistochemical studies, no highly fluorescent layer of tenascin or fibronectin was found adjacent to the implant. Resorption of gammasterilized rods was faster than ethylene oxide-sterilized rods. The total resorption time was more than 24 weeks in both groups. Clinically the healing was uneventful and the implants the well tolerated by the living tissue. This encourages these authors to continue studies with this interesting new material to search for the ideal material for bone filling and fracture fixation.

0901-5027/070631 + 05 $30.00/0

M. J. A. R.

Ekholm1,2, P. Helander1,2, Hietanen2,3, C. Lindqvist1,4, Salo1, M. Kelloma¨ki5, Suuronen6

1 Department of Oral and Maxillofacial Surgery, Helsinki University Central Hospital, Helsinki, Finland; 2Department of Oral Pathology, Institute of Dentistry, University of Helsinki, Helsinki, Finland; 3Oral Pathology Unit/HUSLAB, Helsinki University Central Hospital, Helsinki, Finland; 4Department of Oral and Maxillofacial Surgery, Institute of Dentistry, Helsinki University, Helsinki, Finland; 5Institute of Biomaterials, Tampere University of Technology, Tampere, Finland; 6 REGEA - Institute for Regenerative Medicine and Medical School, University and University Hospital of Tampere, Tampere, Finland

Key words: solid poly(ortho ester); bone substitute; foreign body reaction; tenascin; fibronectin. Accepted for publication 27 January 2006 Available online 15 March 2006

# 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

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Currently, biodegradable materials are widely used in surgery for fixation of fractures and osteotomies12, as sutures and as bioabsorbable membranes. Bioabsorbable polymer-based bone substitute materials are also commercially available1,18. The most often used polymers are aliphatic polyesters like polyglycolic acid (PGA) and polylactic acid (PLA). Bioresorbable fixation is a fascinating alternative for metallic fixation. By using bioresorbable materials it is possible to avoid disadvantages caused by the use of metallic implants, such as pain, infection, possible corrosion and even carcinogenity. If radiotherapy or postoperative imaging is needed, bioresorbable implants do not disturb it. When bioresorbable materials are used, a removal operation is not needed if total resorption is accomplished after tissue has healed. This makes it possible to reduce the hospital costs and morbidity of the patient. The implant material must however fulfil several basic demands. Good compatibility between host tissue and implant is, naturally, essential. Biocompatibility studies of the most often used absorbable polymers PGA and PLA with laboratory animals have shown that these materials induce only mild nonspecific inflammatory response. Clinical studies in humans have however revealed a relatively high incidence of inflammatory reactions in some studies with pure poly-Llactide (PLLA) and pure PGA. These reactions have been found some months and even some years after operation4,6. The aetiology of this reaction is still unknown, and further studies are ongoing. Poly(ortho esters) (POE) have been under development since 1970. They are synthetic, bioerodible polymers in which the bioerosion process is limited to the surface of the device. Devices made of POE can be formulated so that it undergoes surface erosion, e.g. the polymeric device degrades only at its surface and becomes thinner with time, rather than crumples8. POE hydrolyses in an aqueous environment. Hydrolysis of the polymer is not autocatalytic because the neutral hydrolysis products can diffuse away from the polymer before the carboxylic acid is formed9. An erosion process that is confined predominantly to the surface layers has some important consequences. As most of the hydrolysis occurs in the outer layers of the device, acidic hydrolysis products diffuse away from the device and do not accumulate in the bulk material. Thus, the interior of the matrix does not become highly acidic and cause autocatalytic degradation as is the case with bulk hydrolysing poly(lactide-co-glyco-

lide) copolymers or polylactides8. Therefore, this study hypothesized that the foreign body reactions might be minor compared to PLA or PGA. The wax-like POE has been used in experimental studies in the treatment of burns27 and it has also been studied in many surgical applications23,22,21,20,16, 24,19 . Minor inflammatory tissue reactions have been reported and the polymer has been well tolerated. In the present study, solid POE implants were implanted into bone and connective tissue. Tissue reactions were studied histologically and immunohistochemically. The influence of sterilization method upon the resorption time was also studied. Materials and methods Experimental animals and implant material

The study was approved by the ethics committee of the State Province Office of Southern Finland. Twenty-four adult female rabbits, weighing 3000–4400 g, were used as experimental animals. The solid POE was supplied by Dr Jorge Heller, Advanced Polymer Systems, Redwood City, California, USA. The molecular weight of the polymer was 46,460. The polymer had a composition of 90 mole% trans-cyclohexandimethanol and 10 mole% polyacetal. The synthesis of POE is described by HELLER et al.9 The polymer powder was ultrasonically moulded to 2mm-thick plates from which rods were sawn, with a method described earlier by KELLOMA¨KI et al.10 The rods had dimensions approximately 2 mm  2 mm  5 mm. The first group was gamma-irradiated with a minimum dose of 25 kGy and the second group was sterilized with ethylene oxide (EO). Implants were made in the Institute of Biomaterials, Tampere University of Technology, Tampere, Finland.

tion of a combination of medetomidine 0.3 mg/kg (Domitor1 1 mg/ml, La¨a¨kefarmos, Turku, Finland) and ketamine hydrochloride 50 mg/kg (Ketalar1 50 mg/ml, Parke-Davis, Barcelona, Spain). For infection prophylaxis animals received preoperatively 150,000 IU benzylpenicillin procaine and benzatine penicillin (Duplocillin LA, Gist-Brocades NV, Delt, The Netherlands) s.c. Postoperatively the animals were given buprenorphin 0.05 mg/kg (Temgesic1 0.3 mg/ml, Reckitt & Colman, Hull, U.K.) s.c. The lateral sides of both tibias were shaved and washed with an antiseptic chlorhexidine gluconate solution (Klorhexol1 5 mg/ml, Leiras, Finland). An incision was made laterally on the distal end of the tibia, and the periosteum was reflected down to the bone. A defect (diameter 3 mm) was made with a small cylindrical bur through cortical bone. The implants were placed so that a part of the implant was in the bone marrow and a part was in the cortical bone (Fig. 1). The gamma-sterilized rods were placed into the right tibia (group A), and the ethylene oxide-sterilized into the left tibia (group B). Incision was closed with absorbable sutures (Dexon1). The ramus area of the right mandible was shaved and washed with the same antiseptic solution. An incision was made on skin of the ramus area extraorally and a soft tissue pouch was created subcutaneously. A single gamma-sterilized rod was placed in the pouch. and the incision was closed with the same absorbable sutures. Postoperative procedure

Postoperatively the animals were given a single dose of buprenorphin 0.05 mg/kg (Temgesic1 0.3 mg/ml, Reckitt & Colman, Hull, U.K.) s.c. Animals were free to move in their cages and they were fed ad libitum. No postoperative complications were registered after the operation.

Operative procedure

Follow-up times and specimens

All animals were fed on standard laboratory food and water ad libitum. There was no preoperative fasting. Anaesthesia was induced with a subcutaneous (s.c.) injec-

The follow-up times were 1–10, 14 and 24 weeks (Table 1). After the follow-up time animals were killed in groups of 2 with overdose of pentobarbital (Mebunat1,

Fig. 1. Schematic drawing of the surgical technique in the tibia of rabbit.

A histological and immunohistochemical study of tissue reactions Table 1. Follow-up times and number of experimental animals Weeks Number of rabbits

1 2

2 2

3 2

4 2

Orion, Turku, Finland). Both tibias were exarticulated. In the ramus area of the mandible, the implant together with the connective tissue next to it was carefully dissected free. Histological studies, hard tissue

For histological studies the bony specimens were fixed in 70% alcohol and embedded in methylmetacrylate17. They were cut into 5-mm sections and stained by the MASSON-GOLDNER method7. The samples were evaluated by light microscopy (Olympus BH-2 microscope with attached Olympus D12 digital camera). Histological and immunohistochemical studies, soft tissue

For histological studies half of the sample was fixed in 10% neutral buffered formalin and embedded in paraffin. Five-micrometer sections were cut and stained using the haematoxylin–eosin method. The other half of the sample was snap frozen in precooled isopentane and stored at 70 8C for immunohistochemistry. The frozen sections were first fixed with acetone (20 8C) for 10 min, rinsed in Tris Buffered Saline (TBS) (pH 7.6) 3 times, incubated with mouse monoclonal antibodies (Mabs) (52 DH1 for Fibronectin and 100 EB2 for Tenascin) for 1 h, washed again, followed by incubation with goat anti-mouse antibody IgG (Dako, Glostrup, Denmark) and then washed again and incubated with the APAAP complex (Dako, Glostrup, Denmark). Then, naphtol AS-BI phosphate (Sigma Chemical Co, St Louis, MO, USA) was added to develop the colour reaction. Finally, sections were counterstained with Mayer’s haematoxylin. Immunohistochemical results were evaluated by 2 observers independently using 40 magnification by means of arbitrary scale (( ) no reactivity, (+) weak, (++) moderate, (+++) high reactivity).

5 2

6 2

7 2

8 2

9 2

10 2

14 2

24 2

(Fig. 2). At week 24 the inflammation was mild. The foreign body giant cells were present around the implants from week 2 to week 24 (Fig. 3). Some epithelioid cells were found at weeks 3, 4 and 5. The number of eosinophils was highest during the first 4 weeks. After that eosinophils were found only occasionally. Bone

Gamma-sterilized POE, group A: The material itself was not visible, but there was an empty, square-shaped space indicating the site of the implanted polymer. The site of implantation was clearly visible in all samples. The shape of the space was square-shaped from week 1 until week 5. After week 6 the implant started to erode from the surface, and the shape of the empty

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space started to become rounded (Fig. 4). At the end of the follow-up, week 24, the empty material space was still evident. Foreign body giant cells were observed from week 2 until the end of follow-up. No severe inflammatory reaction was seen; mostly the inflammation was graded as moderate. Weak bone formation was observed at the margin of the defect during the whole period of follow-up. Ethylene oxide-sterilized POE, group B: The material itself was not visible, but there was an empty, square-shaped space indicating the site of implanted polymer. The site of implantation was clearly visible in all samples during the entire follow-up time. The empty space was square-shaped (Fig. 5) from the beginning until the end of observation, week 24, and there were no signs of surface erosion. The area of implantation was surrounded by connective tissue capsule. New bone formation at the vicinity of the implanted material was noticed only during the last 2 follow-up times. Inflammation around the implant

Fig. 2. Soft tissue specimen at week 8 follow-up. Moderate inflammation is seen in soft tissue specimen around the implanted material. Foreign-body giant cells are seen near the material space. Original magnification 125.

Results Histological study

Soft tissue (gamma-sterilized POE)

The site of implantation was visible in all samples. The inflammatory cell infiltrate was strongest during first 4 weeks and after that (weeks 5–10, 14) the intensity of inflammatory infiltrate was moderate

Fig. 3. Implanted material (arrows) is seen inside a large foreign-body giant cell in a week 2 soft tissue specimen. Original magnification 400.

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Fig. 4. Week 14 follow-up, gamma-sterilized rod. The empty material space is still visible, but the surface erosion of the implant can be seen as the space has become smaller and rounded. Original magnification 100.

Fig. 5. Week 14 follow-up, ethylene oxide-sterilized rod. The empty material space is clearly visible, but there were no signs of surface erosion. Original magnification 100.

was moderate during the period of followup. Foreign body giant cells were found in all follow-up points. Immunohistochemical study

In immunohistochemical studies, both fibronectin (Fn) and tenascin (Tn) were found adjacent to the implant. Tn and Fn were detected from week 1. The amount of Tn and Fn immunoreactivity was clearly highest at week 3. From week 7 onwards, Fn immunoreactivity was down-regulated. Tn immunoreactivity as a restricted layer facing the implant was found up to week 24. Discussion

After using intraosseously-placed polyglycolide or polylactide-glycolide copolymers as absorbable fixation materials in

human patients, BOSTMAN et al. reported histologically-manifested non-specific foreign body reactions4. The average incidence of the manifest reactions was 5% for implants made of PGA. The tissue responses to PGA manifest themselves 11 weeks after surgery, whereas foreign body reactions to devices made of poly-Llactide can emerge as late as 4 or 5 years after the original fracture fixation operation4. The reasons for these tissue reactions are not yet completely known, but it has been suggested that the tissue response might be initiated by a change in the morphology of material. It has also been suggested that degradation kinetics of material depend on the site of implantation, local tissue tolerance and transport potential3,25. It is apparent that such biodegradable materials that will not undergo crystal formation and microfragmentation

as a part of their degradation process must be developed in order to avoid particledebris-induced late inflammatory reactions. In this study, it was observed that gamma-sterilized POE induces a moderate inflammation in soft tissue for 14 weeks, after which inflammation was classified as mild. In bone, the degradation of POE was faster with gamma-sterilized rods than with ethylene oxide-sterilized rods. After 6 weeks of follow-up gamma-sterilized rods started to erode from the surface, and the shape of the empty space started to become rounded, whereas ethylene oxide-sterilized rods showed no surface erosion. It is known in general, that gamma irradiation decreases molecular weights of polyesters. In a study of effects of gamma sterilization to semi-solid POE, MERKLI et al.15 showed that gamma sterilization did decrease the molecular weight of semi-solid POE. Reduction in molecular weight caused by gamma sterilization is probably the reason for shorter resorption times when compared to ethylene oxide-sterilized devices. Resorption time was longer than 24 weeks in both soft tissue and bone, independent of the sterilization method. No visible manifestations of inflammation (i.e. swelling) were seen in any specimens. Two extracellular matrix glycoproteins, tenascin and fibronectin, were also evaluated. Tenascin is absent from most normal adult tissues but is found during inflammatory processes and wound healing. During wound healing, tenascin is present throughout the matrix of the granulation tissue but it is not detectable in the scar after wound contraction is complete2,14. Fibronectin is present in foetal tissue, but not normally found in adult tissue. It is expressed during wound healing, rejection and tumour stroma13,5,26. In this study, highest amounts of Tn and Fn were found at week 3. From week 7, the immunoreactivity of Fn was clearly downregulated. Tenascin immunoreactivity as restricted layer was found up to week 24. High Tn and Fn immunoreactivities were detected in connective tissue capsules around PLA implants during the 36-week follow-up time, and it was concluded that high Fn and Tn contents found close to implants may hence be a sign of prolonged inflammation11. From that point of view, biocompatibility of POE might be better than that of PLA. Degradation by surface erosion rather than bulk hydrolysing might also explain minor foreign body reactions. More studies are needed to find out the suitability of solid POE as an implant material for fracture fixation devices;

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Address: Marja Ekholm Tuurinniitynkuja 4-6 02200 Espoo Finland Tel: +358 9 591 5620 (O)/022251 (R) Fax: +358 9 52594185. E-mail: [email protected]