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Histological study of tissue reactions to ε-caprolactone–lactide copolymer in paste form
Biomaterials 20 (1999) 1257}1262
Histological study of tissue reactions to e-caprolactone}lactide copolymer in paste form Marja Ekholm *, Jarkko Hietanen, Christian Lindqvist , Jorma Rautavuori, Seppo Santavirta, Riitta Suuronen Department of Oral and Maxillofacial Surgery, Department of Surgery, Helsinki University Central Hospital, Kasarmikatu 11-13, 00130 Helsinki, Finland Department of Oral Pathology, Institute of Dentistry, University of Helsinki, Helsinki, Finland Department of Oral and Maxillofacial Surgery, Institute of Dentistry, University of Helsinki, Finland Institute of Biomaterials, Tampere University of Technology, Tampere, Finland Department of Orthopaedics and Traumatology, Helsinki University Central Hospital, Helsinki, Finland Received 30 September 1996; accepted 7 May 1997
Abstract In previous studies, e-caprolactone}lactide copolymer in solid form has been used in experimental animals as suture material, and as a biodegradable nerve guide. The aim of the study reported here was to assess tissue reactions to e-caprolactone}lactide copolymer in paste form, histologically, and to compare bone healing at the sites of implantation versus that at control sites. The other purpose of the study was to evaluate the properties of the implanted material as a "lling material for bone defects. Resorption time and intensity of in#ammatory reaction were also evaluated. Material was implanted into the abdominal walls and femurs of 34 rats. Follow-up times were from 2 weeks to 1 year. The results showed that e-caprolactone}lactide copolymer in paste form induces a severe in#ammatory reaction when placed in muscle, and moderate in#ammation when implanted into bone. The resorption time was more than 1 year. Bone healing at sites of implantation was slower than at control sites. 1999 Elsevier Science Limited. All rights reserved. Keywords: e-Caprolactone}lactide copolymer; Bone; Muscle; In#ammation
1. Introduction The most important biodegradable polymers currently used in medical and surgical applications are a-hydroxyacid derivatives, aliphatic polyesters. They are employed for tissue replacement, tissue augmentation, tissue support and drug delivery. Bioresorbable surgical materials are better than biostable materials when the need is for temporary presence of biomaterial. Typical implants for this kind of purpose are absorbable sutures [1], bone fracture "xation devices [2] and urological stents [3]. In cranio-maxillofacial surgery, bone transplants are needed in reconstructing skeletal defects resulting from trauma, infection or congenital deformity. Currently, autografts, allografts or arti"cial biomaterials are used in these connections. To avoid the risks and problems asso-
* Correspondence address: Tuurinniitynkuja 4, 02200 Espoo, Finland.
ciated with use of allografts or autografts, a search is in progress for new arti"cial bonegraft materials. Poly(e-caprolactone) (PCL) and poly(lactic acid) (PLA) are aliphatic polyesters. PCL has been used for drug delivery systems [4, 5] and PLA for fracture "xation devices in orthopaedics [2, 6, 7] and in maxillofacial surgery [2, 8]. When PCL and PLA are polymerized they form e-caprolactone}lactide copolymer. This is available in various forms, for example as a solid, paste or wax. The properties of the copolymer can be adjusted and balanced by modi"cations of monomer composition and molecular weight [9]. Studies have been carried out in which e-caprolactone}lactide copolymer in solid form has been used in experimental animals. A two-ply nerve guide constructed from poly(Llactide) and PCL copolymer has been implanted in the sciatic nerve of the rat [10}13]. Nakamura et al. [14] made sutures from a copolymer of e-caprolactone and L-lactide, and used rabbits as experimental animals. Films made from e-caprolactone and D,L-lactide have been used in dogs to prevent postoperative pleural and
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pericardial adhesion [15]. Tissue reactions in these studies have been reported to be minimal. Only mild foreignbody reactions were noted. The purpose of our study was to assess tissue reactions to e-caprolactone}lactide copolymer in paste form, histologically, and to evaluate bone healing at sites of implantation as compared to that at control sites. The aim was also to evaluate the copolymer as a "lling material for bone defects. The material was implanted into bone and muscle. Resorption time of the material from the tissue is also reported.
2. Materials and methods 2.1. Experimental animals and implant material Thirty-four adult male rats (Wistar), weighing 300}500 g were used as experimental animals. There were "ve rats in a 2-week study group and in a control group, and four rats in 9-, 26- and 52-week study and control groups (Table 1). The implanted material was a 40 : 60 w/w copolymer of e-caprolactone and D,L-lactide (e-CL/DL-LA) prepared in the Helsinki University of Technology, Polymer Technology Laboratory (Fig. 1). Polymerization is explained in detail by Hiljanen-Vainio et al. [15]. The ratio of D-lactide to L-lactide was 50 : 50. The initial molecular weight (M ) of the copolymer, measured before sterilization was 17 900. It was in paste form. The copolymer was sterilized using gamma radiation (Kolmi-Set Oy, Ilomantsi, Finland). The minimal dose was 25 kGy. 2.2. Operative procedure Preoperatively, the animals received 20 ng kg\ procaine penicillin (Maxipen, 300 mg ml\, P"zer, Canada Inc., London, Canada) subcutaneously (s.c.) to prevent infection. Anaesthesia was induced using 0.1 ml per 100 g (s.c.) #uanisone-fentanyl (Hypnorm 10 mg ml\ and 0.2 mg ml\, Janssen Pharmaceutica, Beerse, Belgium) and maintained with intraperitoneal doses of 0.1 ml per 100 g of #uanisone-fentanyl if necessary.
2.2.1. Group A1 (n"17) A midline incision was made in the abdominal skin, and muscle pouches were formed by blunt dissections on both sides of the linea alba in abdominal walls. The material was inserted into the muscle pouches and the incisions were closed in layers, using absorbable sutures. 2.2.2. Group B1 (n"17) The material was implanted in the left femur. A parapatellar incision was made medially, and soft tissues and the patella were dislocated laterally. The distal part of the femur was exposed and a small defect was drilled between the condyles with a dental burrure to the cancellous bone. The material was inserted into the hole and the incision was closed in layers with absorbable sutures (Fig. 2). Similar procedures without insertion of material were carried out in the control groups (group A2, n"17; group B2, n"17). 2.3. Postoperative procedure All of the animals were fed ad libitum after the surgical procedure, and were free to move about their cages. For postoperative pain control they also received 0.02 mg per 100 g bubrenorphinum (Temgesic, 0.3 mg ml\, Reckitt & Colman, Hull, UK) after surgery and subsequently every 12 h for 2 days. 2.4. Follow-up times and specimens The follow-up times were 2, 9, 26 and 52 weeks (Table 1). After follow-up, the animals were killed, the left femur was exarticulated and the muscle pouches in the abdominal wall were resected.
Fig. 1. Chemical structure of e-caprolactone}lactide copolymer.
Table 1 Follow-up times and numbers of samples Group
Location of implant
2 weeks follow-up
9 weeks follow-up
26 weeks follow-up
52 weeks follow-up
A1 A2 B1 B2
Abdominal wall Control Left femur Control
5 3 5 3
3 4 3 4
4 4 4 4
3 4 3 4
e-CL/DL-LA was implanted into left femurs and into abdominal walls. The same procedures were carried out in control group animals except for insertion of material.
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graded as mild, moderate or severe. A mild in#ammatory reaction was de"ned as a reaction with only a few in#ammatory cells, a moderate in#ammatory reaction was de"ned as a reaction with a moderate number of in#ammatory cells and a severe in#ammatory reaction was de"ned as a reaction with many in#ammatory cells. All specimens were "rst evaluated using light microscopy, and then re-evaluated and placed into one or other of the categories mentioned above, according to the intensity of in#ammatory reaction. The cell types were determined based on their morphology and staining.
3. Results Of the 34 experimental animals, four died, three during anasthesia and one during the 1 year follow up. In both groups visual inspection revealed no complications in healing and there were no infections during follow-up. 3.1. Soft tissue
Fig. 2. Schematic drawing of the surgical technique in bone.
2.4.1. Hard tissue The bony specimens were "xed with 70% alcohol and embedded in methyl-methacrylate [17]. They were cut into 5-lm sections and stained using the Masson}Goldner method [18]. 2.4.2. Soft tissue The muscle pouches from the left sides were snapfrozen immediately after resection. Before cutting the specimens into 6-lm sections, the area of implantation was "rst located by visual inspection, and the specimens were then cut into 20-lm sections and stained with Toluidine Blue to make sure that the right place had been selected. In the histological studies, sections were stained with haematoxylin}eosin and evaluated using light microscopy. The muscle pouches from the right sides were prepared for possible later immunohistochemical studies. The intensity of the in#ammatory reaction was
3.1.1. Two weeks In the group in which material had been implanted (n"5), there was moderate in#ammatory reaction around the implanted material and absorbable sutures. The material itself was not visible, but there were empty spaces of di!erent sizes indicating the sites of implanted polymer (Fig. 3). The implantation sites were clearly visible during follow-up. Lymphocytes, plasma cells and macrophages were abundant. Some eosinophilic leucocytes were also present. Foreign-body giant cells were found in one sample. In#ammatory reactions at sites of implantation and around sutures were equally intense. In the control group (n"3), in#ammatory reactions were moderate around sutures. When sutures were not visible in the sample, the in#ammation was mild. Lymphocytes, plasma cells, macrophages and some foreign-body giant cells were present, but no eosinophilic leucocytes. 3.1.2. Two months In the group in which material had been implanted (n"3), sutures were still visible in the operation area, as well as empty material spaces. In#ammation around sutures was moderate and slightly greater than 6 weeks earlier. In#ammatory reactions around implant areas were severe in two samples and moderate in one sample. The number of eosinophilic leucocytes was higher than after 2 weeks of follow-up (Fig. 4). Lymphocytes plasma cells, macrophages and also some epithelioid cells were present. In the control group (n"4), in#ammatory reactions around sutures were moderate and slightly greater than after 2 weeks of follow-up. No eosinophilic leucocytes were found, but lymphocytes, macrophages and plasma
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Fig. 3. Round spaces of di!erent sizes indicating the implanted polymer (m) are visible in a 2-week soft tissue specimen. Foreign-body giant cells are also found. Haematoxylin}eosin stain. Original magni"cation ;250.
Fig. 5. Severe in#ammation in seen in a 1-year soft tissue specimen around the implanted material (m). Haematoxylin}eosin stain. Original magni"cation ;125.
and macrophages predominated (Fig. 5). No eosinophilic leucocytes were found. In the control group (n"4), neither sutures nor in#ammation were found. 3.2. Hard tissue
Fig. 4. Numerous eosinophilic leucocytes (arrow) were found in a 2month soft tissue specimen situated near the hollow apaces. Haematoxylin}eosin stain. Original magni"cation ;1240.
cells were numerous, and some foreign-body giant cells were seen around sutures. 3.1.3. Six months In the group in which material has been implanted (n"4), no sutures were found. Empty material spaces were still evident and in#ammatory reactions surrounding them were severe. Lymphocytes, macrophages, plasma cells and eosinophils, together with some foreignbody giant cells, were seen. In the control group (n"4), neither sutures nor in#ammation were found. 3.1.4. One year In the group in which material had been implanted (n"3), no sutures were found. Only a few empty material spaces were seen at the sites of implantation. There was severe in#ammatory reaction, in which lymphocytes
3.2.1. Two weeks In the group in which material had been implanted (n"5), there was moderate in#ammatory reaction, in which lymphocytes, plasma cells, macrophages and foreign-body giant cells were seen (Fig. 6). Sites of implantation with empty material spaces were clearly visible in all samples. Osteoid formation and some osteoclasts were also found. Collagen "bres were abundant. There was formation of cartilage on the surfaces of the condyles. In the control group (n"3), there was a mild in#ammatory reaction at the sites of defects. Macrophages, plasma cells and lymphocytes were fewer in number than in the group in which material had been implanted. The amount of bone and osteoid formation was similar to that in the group in which the material had been implanted. Cartilage formation was also seen on the surfaces of the defects. 3.2.2. Two months In the group in which material had been implanted (n"3), sites of implantation were still visible in all samples. Empty material spaces were smaller and fewer in number than 6 weeks previously. There were also more collagen "bres. The in#ammatory reaction was moderate, and as intense as after 2 weeks of follow-up. The number of foreign-body giant cells was greater. The formation of osteoid varied between samples. In one sample there was more osteoid than 6 weeks previously, in another there was less, and in the third there was no
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Fig. 6. Implanted material (arrow) is seen inside a large foreign-body giant cell in a 2-week hard tissue specimen. Masson}Goldner stain. Polarized light. Original magni"cation ;500.
Fig. 7. Six months of follow-up. The defect is smaller than at previous follow-up times and is surrounded by more bone (B). Material spaces are small, but the in#ammation remains moderate. Masson}Goldner stain. Original magni"cation ;125.
osteoid formation. Cartilage formation on surfaces of condyles was not evident. In the control group (n"4), the area of implantation could still be recognized. Cartilage on the surfaces of defects was thin. Mononuclear in#ammatory cell in"ltration was evident. There was more bone and blood-forming bone marrow in the corresponding areas than in the group in which material had been implanted.
In the control group (n"4), the defect areas were no longer visible. Cartilage had regenerated, and defects had "lled with bone. In all samples relating to the same follow-up times there was some individual variation in intensity of tissue response.
4. Discussion 3.2.3. Six months In the group in which material had been implanted (n"4), the areas of implantation were smaller than after two months of follow-up, and there was more bone surrounding them (Fig. 7). A cartilage layer was also found on surfaces of defects. The in#ammatory reaction was moderate, but less than after the previous follow-up period. The amount of collagen was the same as after 2 months of follow-up. In one sample there was a connective tissue capsule surrounding the material space. Osteoid formation was also evident. Clinically, there was a subsiantial enlargement of the left condyle in one rat, but microscopic examination revealed only normal bone and osteoid formation. No malignancy was found. In the control group (n"4), the defect areas were no longer visible. Cartilage had regenerated and defects had "lled with bone. 3.2.4. One year In the group in which material had been implanted (n"3), the site of implantation was visible in only one sample. The material spaces were very small. In two samples the areas could no longer be detected. There was a connective tissue capsule surrounding the material space and in#ammation was mild. The amount of collagen seemed similar to that observed after 6 months of follow-up.
The in#ammatory reaction when e-CL/DL-LA was implanted in muscle was moderate after 2 weeks of follow-up, but was subsequently severe. There have been no previous reports of severe in#ammation around sites of implantation [10}15]. Taylor et al. [19] compared the degradation of six absorbable polymers and the acute toxicity of the accumulated degradation products of the polymers under uniform conditions of exposure in vitro. Taylor et al. also measured the toxicity of known quantities of pure degradation products of "ve absorbable polymers. They noted di!erences in the relative toxicities of the degradation products and concluded that toxicity is a factor that should be considered in material selection, but must be combined with degradation rates and local tissue clearances to predict the concentrations present in the tissues, and the resulting responses. In our study, e-CL/DL-LA was placed into muscle pouches. After 2 weeks of follow-up time, the in#ammatory reactions around the sutures and the sites of implantation were equally intense. Subsequently, they were greatest around the implant areas. The severe in#ammation elicited by the material may, in part, be a result of high local concentrations of degradation products and the transport potential of muscle tissue. Amounts of implanted polymer may have been higher than in previous studies. Had the amounts of implanted polymer been lower, resorption times could have been shorter, with no severe in#ammatory
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reactions. Macrophages and foreign-body giant cells were found from the outset. In one sample after the "rst follow-up period, giant cells were observed phagocytosing a piece of material. This "nding is in agreement with "ndings in previous studies [10, 11, 14]. The high numbers of eosinophilic leucocytes observed after all followup periods except the last one are interesting. There are marked increases in the numbers of eosinophilic leucocytes in many types of allergic and hypersensitive states in humans. High numbers of eosinophils can be a sign of some kind of immune response. Nakamura et al. [14] found eosinophils surrounding sutures made from a copolymer of e-caprolactone and L-lactide. Eosinophils have also been found from the tissue surrounding carbon-coated subperiosteal implants, where clinical incompatibility was "rst noticed [20]. The in#ammatory reaction in bone was less pronounced than that in muscle. It was moderate after the "rst two follow-up periods (2 weeks, 2 months) and mild to moderate after 6 months of follow-up. It was mild after 1 year of follow-up. The reason for more pronounced reaction in muscle may partly be the better vascularization of muscle tissue, and the greater amounts of implanted material. Foreign-body reactions with macrophages and giant cells were found in all samples except those after the last follow up period. A connective tissue capsule surrounded the empty material spaces in two samples after 6 months and 1 year of follow-up. Dunnen et al. [10] also found capsule surrounding the material in their studies. Bone healing at the sites of implantation was slower than at the control sites throughout all periods of follow-up, except the "rst. We conclude that e-CL/DL-LA elicits moderate in#ammatory reactions when it is implanted into bone and severe in#ammatory reactions when it is implanted into muscle. The resorption time is more than 1 year. The material implanted was sticky and di$cult to handle, but after implantation into a bone defect it remained there without di$culty. Bleeding of the cancellous bone also stopped immediately after the material had been placed into the defects. Further studies are now being carried out to improve the consistency of the material, and to reduce the in#ammatory reaction in bone. Acknowledgements The author would like to thank Dr Pekka Ylinen of the Orthopaedic Hospital of the Invalid Foundation for devising the surgical technique. This work was supported by a grant from the Finnish Dental Society. References [1] Frazza EJ, Schmitt EE. A new absorbable suture. J Biomed Res Symp 1971;1:43}58.
[2] ToK rmaK laK P, Pohjonen T. Ultra-high strength bioabsorbable polymeric composites for surgical applications. In: Rokkanen P, ToK rmaK laK P, editors. Self-reinforced bioabsorbable polymeric composites in surgery. Tampere: Tampereen Pikakopio oy, 1995:1}23. [3] Talja M, Tammela T, Petas A et al. Biodegradable self-reinforced polyglycolic acid spiral stent in prevention of postoperative urinary retention after visual laser ablation of the prostate-laser prostatectomy. J Urol 1995;154:2089}92. [4] Pitt CG. Poly e-caprolactone and its copolymers. In: Langer R, Chasin M, editors. Biodegradable polymers as drug delivery systems. New York: Marcel Dekker, 1990:71}120. [5] Darney PD, Klaisle CM, Monroe SE, Phillips NR, Schindler A. Evaluation of a 1-year levonorgestrel-releasing contraceptive implant: side e!ects, release rates, and biodegradability. Fertil Steril 1992;58:137}43. [6] PihlajamaK ki H, BoK stman O, ToK rmaK laK P, Rokkanen P. Absorbable pins of self-reinforced poly-L-lactic acid for "xation of fractures and osteotomies. J Bone Joint Surg (Br) 1992;74 B:853}7. [7] Juutilainen T, PaK tiaK laK H, Rokkanen P, ToK rmaK laK P. Biodegradable wire "xation in oleocranon and patella fractures combined with biodegradable screws or plugs and compared with metallic "xation. Arch Orthop Trauma Surg 1995;14:319}23. [8] Cordewener FW, Bos RR, Rozema FR, Houtman WA. Poly(Llactide) implants for repair of human orbital #oor defects: clinical and magnetic resonance imaging evaluation of long-term results. J Oral Maxillofac Surg 1996;54:9}14. [9] Pitt CG, Je!coat AR, Zweidinger R, Schindler A. Sustained drug delivery systems. I. The permeability of poly(e-caprolactone), poly(D,L-lactic acid), and their copolymers. J Biomed Mater Res 1979;13:497}507. [10] Dunnen WFA, Schakenraad JM, Zondervan GJ, Pennings AJ, van der Lei B, Robinson PH. A new PLLA/PCL copolymer for nerve regeneration. J Mater Sci Mater Med 1993;4:521}5. [11] Dunnen WFA, van der Lei B, Schakenraad JM et al. Long term evaluation of nerve regeneration in a biodegradable nerve guide. Microsurgery 1993;14:508}15. [12] Dunnen WFA, van der Lei B, Robinson PHA, Holwerda A, Pennings AJ, Schakenraad JM. Biological performance of a degradable poly(lactic acid}e-caprolactone) nerve guide: in#uence of tube dimensions. J Biomed Mater Res 1995;29:757}66. [13] Hoppen J, Leenslag JW, Pennings AJ, Lei van der B, Robinson PH. Two-ply biodegradable nerve guide: basic aspects of design, construction and biological performance. Biomaterials 1990;11: 286}9. [14] Nakamura T, Shimizu Y, Matsui T, Okumura N, Hyon SH, Nishiya K. A novel bioabsorbable mono"lament surgical suture made from (e-caprolactone, L-lactide) copolymer. In: Planck H, Dauner M, Renardy M, editors. Degradation phenomena on polymeric biomaterials. Berlin: Springer, 1992:153}62. [15] Nakamura T, Hitomi S, Shimamoto T, Hyon S-H, Watanabe S, Shimizu Y. Surgical application of biodegradable "lms prepared from lactide-e-caprolactone copolymers. In: Pizzoferrato A, Machetti PG, Ravaglioli A, Lee AJC, editors. Biomaterials and clinical applications. Amsterdam: Elsevier, 1987:759}64. [16] Hiljanen-Vainio M, Orava P, SeppaK laK J. The properties of caprolactone/D,L-lactide copolymers with a minor caprolactone content. J Biomed Mater Res 1997;34:39}46. [17] Schenk RK. Zur histologischen Verarbeitung von untenkalkten Knochen. Acta Anat 1965;60:3}19. [18] Goldner J. A modi"cation of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 1938;144:237}43. [19] Taylor MS, Daniels AU, Andriano KP, Heller J. Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. J Appl Biomater 1994;5:151}7. [20] Hess JA, Molinari JA, Mentag PJ. Two cases of incompatibility of carbon-coated subperiosteal implants. Oral Surg Oral Med Oral Pathol 1982;54:499}505.
Histological study of tissue reactions to e-caprolactone}lactide copolymer in paste form Marja Ekholm *, Jarkko Hietanen, Christian Lindqvist , Jorma Rautavuori, Seppo Santavirta, Riitta Suuronen Department of Oral and Maxillofacial Surgery, Department of Surgery, Helsinki University Central Hospital, Kasarmikatu 11-13, 00130 Helsinki, Finland Department of Oral Pathology, Institute of Dentistry, University of Helsinki, Helsinki, Finland Department of Oral and Maxillofacial Surgery, Institute of Dentistry, University of Helsinki, Finland Institute of Biomaterials, Tampere University of Technology, Tampere, Finland Department of Orthopaedics and Traumatology, Helsinki University Central Hospital, Helsinki, Finland Received 30 September 1996; accepted 7 May 1997
Abstract In previous studies, e-caprolactone}lactide copolymer in solid form has been used in experimental animals as suture material, and as a biodegradable nerve guide. The aim of the study reported here was to assess tissue reactions to e-caprolactone}lactide copolymer in paste form, histologically, and to compare bone healing at the sites of implantation versus that at control sites. The other purpose of the study was to evaluate the properties of the implanted material as a "lling material for bone defects. Resorption time and intensity of in#ammatory reaction were also evaluated. Material was implanted into the abdominal walls and femurs of 34 rats. Follow-up times were from 2 weeks to 1 year. The results showed that e-caprolactone}lactide copolymer in paste form induces a severe in#ammatory reaction when placed in muscle, and moderate in#ammation when implanted into bone. The resorption time was more than 1 year. Bone healing at sites of implantation was slower than at control sites. 1999 Elsevier Science Limited. All rights reserved. Keywords: e-Caprolactone}lactide copolymer; Bone; Muscle; In#ammation
1. Introduction The most important biodegradable polymers currently used in medical and surgical applications are a-hydroxyacid derivatives, aliphatic polyesters. They are employed for tissue replacement, tissue augmentation, tissue support and drug delivery. Bioresorbable surgical materials are better than biostable materials when the need is for temporary presence of biomaterial. Typical implants for this kind of purpose are absorbable sutures [1], bone fracture "xation devices [2] and urological stents [3]. In cranio-maxillofacial surgery, bone transplants are needed in reconstructing skeletal defects resulting from trauma, infection or congenital deformity. Currently, autografts, allografts or arti"cial biomaterials are used in these connections. To avoid the risks and problems asso-
* Correspondence address: Tuurinniitynkuja 4, 02200 Espoo, Finland.
ciated with use of allografts or autografts, a search is in progress for new arti"cial bonegraft materials. Poly(e-caprolactone) (PCL) and poly(lactic acid) (PLA) are aliphatic polyesters. PCL has been used for drug delivery systems [4, 5] and PLA for fracture "xation devices in orthopaedics [2, 6, 7] and in maxillofacial surgery [2, 8]. When PCL and PLA are polymerized they form e-caprolactone}lactide copolymer. This is available in various forms, for example as a solid, paste or wax. The properties of the copolymer can be adjusted and balanced by modi"cations of monomer composition and molecular weight [9]. Studies have been carried out in which e-caprolactone}lactide copolymer in solid form has been used in experimental animals. A two-ply nerve guide constructed from poly(Llactide) and PCL copolymer has been implanted in the sciatic nerve of the rat [10}13]. Nakamura et al. [14] made sutures from a copolymer of e-caprolactone and L-lactide, and used rabbits as experimental animals. Films made from e-caprolactone and D,L-lactide have been used in dogs to prevent postoperative pleural and
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pericardial adhesion [15]. Tissue reactions in these studies have been reported to be minimal. Only mild foreignbody reactions were noted. The purpose of our study was to assess tissue reactions to e-caprolactone}lactide copolymer in paste form, histologically, and to evaluate bone healing at sites of implantation as compared to that at control sites. The aim was also to evaluate the copolymer as a "lling material for bone defects. The material was implanted into bone and muscle. Resorption time of the material from the tissue is also reported.
2. Materials and methods 2.1. Experimental animals and implant material Thirty-four adult male rats (Wistar), weighing 300}500 g were used as experimental animals. There were "ve rats in a 2-week study group and in a control group, and four rats in 9-, 26- and 52-week study and control groups (Table 1). The implanted material was a 40 : 60 w/w copolymer of e-caprolactone and D,L-lactide (e-CL/DL-LA) prepared in the Helsinki University of Technology, Polymer Technology Laboratory (Fig. 1). Polymerization is explained in detail by Hiljanen-Vainio et al. [15]. The ratio of D-lactide to L-lactide was 50 : 50. The initial molecular weight (M ) of the copolymer, measured before sterilization was 17 900. It was in paste form. The copolymer was sterilized using gamma radiation (Kolmi-Set Oy, Ilomantsi, Finland). The minimal dose was 25 kGy. 2.2. Operative procedure Preoperatively, the animals received 20 ng kg\ procaine penicillin (Maxipen, 300 mg ml\, P"zer, Canada Inc., London, Canada) subcutaneously (s.c.) to prevent infection. Anaesthesia was induced using 0.1 ml per 100 g (s.c.) #uanisone-fentanyl (Hypnorm 10 mg ml\ and 0.2 mg ml\, Janssen Pharmaceutica, Beerse, Belgium) and maintained with intraperitoneal doses of 0.1 ml per 100 g of #uanisone-fentanyl if necessary.
2.2.1. Group A1 (n"17) A midline incision was made in the abdominal skin, and muscle pouches were formed by blunt dissections on both sides of the linea alba in abdominal walls. The material was inserted into the muscle pouches and the incisions were closed in layers, using absorbable sutures. 2.2.2. Group B1 (n"17) The material was implanted in the left femur. A parapatellar incision was made medially, and soft tissues and the patella were dislocated laterally. The distal part of the femur was exposed and a small defect was drilled between the condyles with a dental burrure to the cancellous bone. The material was inserted into the hole and the incision was closed in layers with absorbable sutures (Fig. 2). Similar procedures without insertion of material were carried out in the control groups (group A2, n"17; group B2, n"17). 2.3. Postoperative procedure All of the animals were fed ad libitum after the surgical procedure, and were free to move about their cages. For postoperative pain control they also received 0.02 mg per 100 g bubrenorphinum (Temgesic, 0.3 mg ml\, Reckitt & Colman, Hull, UK) after surgery and subsequently every 12 h for 2 days. 2.4. Follow-up times and specimens The follow-up times were 2, 9, 26 and 52 weeks (Table 1). After follow-up, the animals were killed, the left femur was exarticulated and the muscle pouches in the abdominal wall were resected.
Fig. 1. Chemical structure of e-caprolactone}lactide copolymer.
Table 1 Follow-up times and numbers of samples Group
Location of implant
2 weeks follow-up
9 weeks follow-up
26 weeks follow-up
52 weeks follow-up
A1 A2 B1 B2
Abdominal wall Control Left femur Control
5 3 5 3
3 4 3 4
4 4 4 4
3 4 3 4
e-CL/DL-LA was implanted into left femurs and into abdominal walls. The same procedures were carried out in control group animals except for insertion of material.
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graded as mild, moderate or severe. A mild in#ammatory reaction was de"ned as a reaction with only a few in#ammatory cells, a moderate in#ammatory reaction was de"ned as a reaction with a moderate number of in#ammatory cells and a severe in#ammatory reaction was de"ned as a reaction with many in#ammatory cells. All specimens were "rst evaluated using light microscopy, and then re-evaluated and placed into one or other of the categories mentioned above, according to the intensity of in#ammatory reaction. The cell types were determined based on their morphology and staining.
3. Results Of the 34 experimental animals, four died, three during anasthesia and one during the 1 year follow up. In both groups visual inspection revealed no complications in healing and there were no infections during follow-up. 3.1. Soft tissue
Fig. 2. Schematic drawing of the surgical technique in bone.
2.4.1. Hard tissue The bony specimens were "xed with 70% alcohol and embedded in methyl-methacrylate [17]. They were cut into 5-lm sections and stained using the Masson}Goldner method [18]. 2.4.2. Soft tissue The muscle pouches from the left sides were snapfrozen immediately after resection. Before cutting the specimens into 6-lm sections, the area of implantation was "rst located by visual inspection, and the specimens were then cut into 20-lm sections and stained with Toluidine Blue to make sure that the right place had been selected. In the histological studies, sections were stained with haematoxylin}eosin and evaluated using light microscopy. The muscle pouches from the right sides were prepared for possible later immunohistochemical studies. The intensity of the in#ammatory reaction was
3.1.1. Two weeks In the group in which material had been implanted (n"5), there was moderate in#ammatory reaction around the implanted material and absorbable sutures. The material itself was not visible, but there were empty spaces of di!erent sizes indicating the sites of implanted polymer (Fig. 3). The implantation sites were clearly visible during follow-up. Lymphocytes, plasma cells and macrophages were abundant. Some eosinophilic leucocytes were also present. Foreign-body giant cells were found in one sample. In#ammatory reactions at sites of implantation and around sutures were equally intense. In the control group (n"3), in#ammatory reactions were moderate around sutures. When sutures were not visible in the sample, the in#ammation was mild. Lymphocytes, plasma cells, macrophages and some foreign-body giant cells were present, but no eosinophilic leucocytes. 3.1.2. Two months In the group in which material had been implanted (n"3), sutures were still visible in the operation area, as well as empty material spaces. In#ammation around sutures was moderate and slightly greater than 6 weeks earlier. In#ammatory reactions around implant areas were severe in two samples and moderate in one sample. The number of eosinophilic leucocytes was higher than after 2 weeks of follow-up (Fig. 4). Lymphocytes plasma cells, macrophages and also some epithelioid cells were present. In the control group (n"4), in#ammatory reactions around sutures were moderate and slightly greater than after 2 weeks of follow-up. No eosinophilic leucocytes were found, but lymphocytes, macrophages and plasma
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Fig. 3. Round spaces of di!erent sizes indicating the implanted polymer (m) are visible in a 2-week soft tissue specimen. Foreign-body giant cells are also found. Haematoxylin}eosin stain. Original magni"cation ;250.
Fig. 5. Severe in#ammation in seen in a 1-year soft tissue specimen around the implanted material (m). Haematoxylin}eosin stain. Original magni"cation ;125.
and macrophages predominated (Fig. 5). No eosinophilic leucocytes were found. In the control group (n"4), neither sutures nor in#ammation were found. 3.2. Hard tissue
Fig. 4. Numerous eosinophilic leucocytes (arrow) were found in a 2month soft tissue specimen situated near the hollow apaces. Haematoxylin}eosin stain. Original magni"cation ;1240.
cells were numerous, and some foreign-body giant cells were seen around sutures. 3.1.3. Six months In the group in which material has been implanted (n"4), no sutures were found. Empty material spaces were still evident and in#ammatory reactions surrounding them were severe. Lymphocytes, macrophages, plasma cells and eosinophils, together with some foreignbody giant cells, were seen. In the control group (n"4), neither sutures nor in#ammation were found. 3.1.4. One year In the group in which material had been implanted (n"3), no sutures were found. Only a few empty material spaces were seen at the sites of implantation. There was severe in#ammatory reaction, in which lymphocytes
3.2.1. Two weeks In the group in which material had been implanted (n"5), there was moderate in#ammatory reaction, in which lymphocytes, plasma cells, macrophages and foreign-body giant cells were seen (Fig. 6). Sites of implantation with empty material spaces were clearly visible in all samples. Osteoid formation and some osteoclasts were also found. Collagen "bres were abundant. There was formation of cartilage on the surfaces of the condyles. In the control group (n"3), there was a mild in#ammatory reaction at the sites of defects. Macrophages, plasma cells and lymphocytes were fewer in number than in the group in which material had been implanted. The amount of bone and osteoid formation was similar to that in the group in which the material had been implanted. Cartilage formation was also seen on the surfaces of the defects. 3.2.2. Two months In the group in which material had been implanted (n"3), sites of implantation were still visible in all samples. Empty material spaces were smaller and fewer in number than 6 weeks previously. There were also more collagen "bres. The in#ammatory reaction was moderate, and as intense as after 2 weeks of follow-up. The number of foreign-body giant cells was greater. The formation of osteoid varied between samples. In one sample there was more osteoid than 6 weeks previously, in another there was less, and in the third there was no
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Fig. 6. Implanted material (arrow) is seen inside a large foreign-body giant cell in a 2-week hard tissue specimen. Masson}Goldner stain. Polarized light. Original magni"cation ;500.
Fig. 7. Six months of follow-up. The defect is smaller than at previous follow-up times and is surrounded by more bone (B). Material spaces are small, but the in#ammation remains moderate. Masson}Goldner stain. Original magni"cation ;125.
osteoid formation. Cartilage formation on surfaces of condyles was not evident. In the control group (n"4), the area of implantation could still be recognized. Cartilage on the surfaces of defects was thin. Mononuclear in#ammatory cell in"ltration was evident. There was more bone and blood-forming bone marrow in the corresponding areas than in the group in which material had been implanted.
In the control group (n"4), the defect areas were no longer visible. Cartilage had regenerated, and defects had "lled with bone. In all samples relating to the same follow-up times there was some individual variation in intensity of tissue response.
4. Discussion 3.2.3. Six months In the group in which material had been implanted (n"4), the areas of implantation were smaller than after two months of follow-up, and there was more bone surrounding them (Fig. 7). A cartilage layer was also found on surfaces of defects. The in#ammatory reaction was moderate, but less than after the previous follow-up period. The amount of collagen was the same as after 2 months of follow-up. In one sample there was a connective tissue capsule surrounding the material space. Osteoid formation was also evident. Clinically, there was a subsiantial enlargement of the left condyle in one rat, but microscopic examination revealed only normal bone and osteoid formation. No malignancy was found. In the control group (n"4), the defect areas were no longer visible. Cartilage had regenerated and defects had "lled with bone. 3.2.4. One year In the group in which material had been implanted (n"3), the site of implantation was visible in only one sample. The material spaces were very small. In two samples the areas could no longer be detected. There was a connective tissue capsule surrounding the material space and in#ammation was mild. The amount of collagen seemed similar to that observed after 6 months of follow-up.
The in#ammatory reaction when e-CL/DL-LA was implanted in muscle was moderate after 2 weeks of follow-up, but was subsequently severe. There have been no previous reports of severe in#ammation around sites of implantation [10}15]. Taylor et al. [19] compared the degradation of six absorbable polymers and the acute toxicity of the accumulated degradation products of the polymers under uniform conditions of exposure in vitro. Taylor et al. also measured the toxicity of known quantities of pure degradation products of "ve absorbable polymers. They noted di!erences in the relative toxicities of the degradation products and concluded that toxicity is a factor that should be considered in material selection, but must be combined with degradation rates and local tissue clearances to predict the concentrations present in the tissues, and the resulting responses. In our study, e-CL/DL-LA was placed into muscle pouches. After 2 weeks of follow-up time, the in#ammatory reactions around the sutures and the sites of implantation were equally intense. Subsequently, they were greatest around the implant areas. The severe in#ammation elicited by the material may, in part, be a result of high local concentrations of degradation products and the transport potential of muscle tissue. Amounts of implanted polymer may have been higher than in previous studies. Had the amounts of implanted polymer been lower, resorption times could have been shorter, with no severe in#ammatory
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reactions. Macrophages and foreign-body giant cells were found from the outset. In one sample after the "rst follow-up period, giant cells were observed phagocytosing a piece of material. This "nding is in agreement with "ndings in previous studies [10, 11, 14]. The high numbers of eosinophilic leucocytes observed after all followup periods except the last one are interesting. There are marked increases in the numbers of eosinophilic leucocytes in many types of allergic and hypersensitive states in humans. High numbers of eosinophils can be a sign of some kind of immune response. Nakamura et al. [14] found eosinophils surrounding sutures made from a copolymer of e-caprolactone and L-lactide. Eosinophils have also been found from the tissue surrounding carbon-coated subperiosteal implants, where clinical incompatibility was "rst noticed [20]. The in#ammatory reaction in bone was less pronounced than that in muscle. It was moderate after the "rst two follow-up periods (2 weeks, 2 months) and mild to moderate after 6 months of follow-up. It was mild after 1 year of follow-up. The reason for more pronounced reaction in muscle may partly be the better vascularization of muscle tissue, and the greater amounts of implanted material. Foreign-body reactions with macrophages and giant cells were found in all samples except those after the last follow up period. A connective tissue capsule surrounded the empty material spaces in two samples after 6 months and 1 year of follow-up. Dunnen et al. [10] also found capsule surrounding the material in their studies. Bone healing at the sites of implantation was slower than at the control sites throughout all periods of follow-up, except the "rst. We conclude that e-CL/DL-LA elicits moderate in#ammatory reactions when it is implanted into bone and severe in#ammatory reactions when it is implanted into muscle. The resorption time is more than 1 year. The material implanted was sticky and di$cult to handle, but after implantation into a bone defect it remained there without di$culty. Bleeding of the cancellous bone also stopped immediately after the material had been placed into the defects. Further studies are now being carried out to improve the consistency of the material, and to reduce the in#ammatory reaction in bone. Acknowledgements The author would like to thank Dr Pekka Ylinen of the Orthopaedic Hospital of the Invalid Foundation for devising the surgical technique. This work was supported by a grant from the Finnish Dental Society. References [1] Frazza EJ, Schmitt EE. A new absorbable suture. J Biomed Res Symp 1971;1:43}58.
[2] ToK rmaK laK P, Pohjonen T. Ultra-high strength bioabsorbable polymeric composites for surgical applications. In: Rokkanen P, ToK rmaK laK P, editors. Self-reinforced bioabsorbable polymeric composites in surgery. Tampere: Tampereen Pikakopio oy, 1995:1}23. [3] Talja M, Tammela T, Petas A et al. Biodegradable self-reinforced polyglycolic acid spiral stent in prevention of postoperative urinary retention after visual laser ablation of the prostate-laser prostatectomy. J Urol 1995;154:2089}92. [4] Pitt CG. Poly e-caprolactone and its copolymers. In: Langer R, Chasin M, editors. Biodegradable polymers as drug delivery systems. New York: Marcel Dekker, 1990:71}120. [5] Darney PD, Klaisle CM, Monroe SE, Phillips NR, Schindler A. Evaluation of a 1-year levonorgestrel-releasing contraceptive implant: side e!ects, release rates, and biodegradability. Fertil Steril 1992;58:137}43. [6] PihlajamaK ki H, BoK stman O, ToK rmaK laK P, Rokkanen P. Absorbable pins of self-reinforced poly-L-lactic acid for "xation of fractures and osteotomies. J Bone Joint Surg (Br) 1992;74 B:853}7. [7] Juutilainen T, PaK tiaK laK H, Rokkanen P, ToK rmaK laK P. Biodegradable wire "xation in oleocranon and patella fractures combined with biodegradable screws or plugs and compared with metallic "xation. Arch Orthop Trauma Surg 1995;14:319}23. [8] Cordewener FW, Bos RR, Rozema FR, Houtman WA. Poly(Llactide) implants for repair of human orbital #oor defects: clinical and magnetic resonance imaging evaluation of long-term results. J Oral Maxillofac Surg 1996;54:9}14. [9] Pitt CG, Je!coat AR, Zweidinger R, Schindler A. Sustained drug delivery systems. I. The permeability of poly(e-caprolactone), poly(D,L-lactic acid), and their copolymers. J Biomed Mater Res 1979;13:497}507. [10] Dunnen WFA, Schakenraad JM, Zondervan GJ, Pennings AJ, van der Lei B, Robinson PH. A new PLLA/PCL copolymer for nerve regeneration. J Mater Sci Mater Med 1993;4:521}5. [11] Dunnen WFA, van der Lei B, Schakenraad JM et al. Long term evaluation of nerve regeneration in a biodegradable nerve guide. Microsurgery 1993;14:508}15. [12] Dunnen WFA, van der Lei B, Robinson PHA, Holwerda A, Pennings AJ, Schakenraad JM. Biological performance of a degradable poly(lactic acid}e-caprolactone) nerve guide: in#uence of tube dimensions. J Biomed Mater Res 1995;29:757}66. [13] Hoppen J, Leenslag JW, Pennings AJ, Lei van der B, Robinson PH. Two-ply biodegradable nerve guide: basic aspects of design, construction and biological performance. Biomaterials 1990;11: 286}9. [14] Nakamura T, Shimizu Y, Matsui T, Okumura N, Hyon SH, Nishiya K. A novel bioabsorbable mono"lament surgical suture made from (e-caprolactone, L-lactide) copolymer. In: Planck H, Dauner M, Renardy M, editors. Degradation phenomena on polymeric biomaterials. Berlin: Springer, 1992:153}62. [15] Nakamura T, Hitomi S, Shimamoto T, Hyon S-H, Watanabe S, Shimizu Y. Surgical application of biodegradable "lms prepared from lactide-e-caprolactone copolymers. In: Pizzoferrato A, Machetti PG, Ravaglioli A, Lee AJC, editors. Biomaterials and clinical applications. Amsterdam: Elsevier, 1987:759}64. [16] Hiljanen-Vainio M, Orava P, SeppaK laK J. The properties of caprolactone/D,L-lactide copolymers with a minor caprolactone content. J Biomed Mater Res 1997;34:39}46. [17] Schenk RK. Zur histologischen Verarbeitung von untenkalkten Knochen. Acta Anat 1965;60:3}19. [18] Goldner J. A modi"cation of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 1938;144:237}43. [19] Taylor MS, Daniels AU, Andriano KP, Heller J. Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. J Appl Biomater 1994;5:151}7. [20] Hess JA, Molinari JA, Mentag PJ. Two cases of incompatibility of carbon-coated subperiosteal implants. Oral Surg Oral Med Oral Pathol 1982;54:499}505.