Br. J. exp. Path. (1979) 60, 549
RESORPTION OF INSOLUBLE, HETEROLOGOUS, FLUORESCEINCOLLAGEN SPONGES IN SENSITIZED AND NON-SENSITIZED RATS D. J. ETHERINGTON, I. A. SILVER* AND D. J. RESTALL From the A.R.C. Meat Research Institute, Langford, Bristol BS18 7DY and the Department of Pathology*, Medical School, University of Bristol, Bristol BS8 ITD Received for publication May 1, 1979
Summary.-Sponges of insoluble bovine collagen were slowly resorbed over a 35-day period when implanted under the back skin of rats. The cellular picture was typical of a mild foreign-body reaction. The reaction to fluorescein-labelled collagen sponges was similar but there was evidence also of a weak immunological response. An acute inflammatory reaction with massive oedema was elicited when fluorescein -labelled collagen sponges were implanted in rats previously sensitized to either fluorescein-collagen or fluorescein-bovine serum albumin. The early invasion by PMN leucocytes subsided after 4 days and caused no observable breakdown of the sponge. The implanted material was rapidly encapsulated by fibrous tissue which was then resorbed along with the sponge between the 7th and 12th day. Macrophages were very active in the sponge at this time, sometimes forming giant cells. Fibroblasts were invading from the periphery with the development of the granulation tissue. The small residue which remained after this time was overrun by granulation tissue and was slowly resorbed up to the 35th day. Throughout the period of study there was only a weak local immunological response after the 28th day. The level of circulating antibodies against the fluorescein hapten was high, but the titre for the antibodies against bovine collagen remained low. The significance of these findings in the pathological destruction of connective tissue is discussed.
COLLAGEN DEGRADATION is an integral part of the progressive destruction of tissue in various chronic inflammatory diseases including the cartilage erosion characteristic of rheumatoid arthritis (Krane, 1975). Considerable interest exists in the enzymatic mechanisms for the resorption of these extracellular fibres and several recent reviews have been published (Harris and Krane, 1974; Etherington, 1977a; Harris and Cartwright, 1977; Burleigh, 1977). Collagen-anticollagen antibodies which have been identified at sites of rheumatoid inflammation may be an important factor in the aetiology and severity of this disease and may also influence the process of collagenolysis (Andriopoulos et al., 1976; Steffen et al., 1977; Menzel et al., 1978). Nevertheless, heterologous collagen pre37
parations have been used in surgical repairs and as dressings for large skin wounds, and collagen sponges and gels have also been employed to stimulate tissue repair in bone and cartilage defects (Benfer and Struck, 1973; Holmes, Volz and Chvapil, 1975; DelBalso and Adrian, 1976; Moskow, G-old and Gottsegen, 1976). The material is biodegradable and has been shown to elicit a low or negligible antigenic reaction, especially if the telopeptides have been removed by an enzymatic treatment. A variety of structures has been formed and tested and the degree of intermolecular cross-linking can be regulated at the tanning process (Stenzel, Miyata and Rubin, 1974). The ability of the host to resorb insoluble, cross-linked collagen, whether as dressings, sutures, arterial splints or other surgical prostheses,
550
D. J. ETHERINGTON, I. A. SILVER AND D. J. RESTALL
has been well documented and this is clearly related to the extent of the intermolecular cross-linking, so that the more highly tanned the preparation the more slowly is it resorbed (Stenzel et al., 1974; Shekhter et al., 1975; Berakha, Goulian and Watari, 1975; Bartone, Shervey and Gardner, 1976; Oliver et al., 1976; Chvapil, Owen and Clark, 1977). Histological examinations have been made of the cellular reaction in rats to the s.c. implantation of commercially prepared bovine collagen sponge (Bicol: Ethicon Inc; Cutright et al., 1973) and of intact dermal collagen allografts (Oliver et al., 1976). Polymorphonuclear neutrophil leucocytes (PMN) appeared in the Bicol sponges within 3 days and giant cell formation was observed from the 7th day. The sponges were completely resorbed within 40 days. The dermal allografts, cross-linked with glutaraldehyde, were also invaded by inflammatory cells initially and later evoked giant-cell formation. However, they were neither resorbed nor rejected and were shown subsequently to become repopulated with fibroblasts and new blood vessels. In the present paper we report on the tissue reaction to heterologous collagen sponges which have been labelled with fluorescein isothiocyanate. The fluorescein moiety is a powerful hapten to which the animals can be sensitized in order to elicit a strong antigenic reaction when the sponges are implanted. MATERIALS AND METHODS
Antigen preparation and sensitizing procedure. Soluble collagen was prepared from depilated and saline-washed calf skin pieces by extraction with 0-IM acetic acid for 48 h. The soluble collagen was collected by precipitation with 0-85M NaCl and then dissolved in and dialysed against 0-02M acetic acid. The collagen was complexed with fluorescein isothiocyanate (Steven, Torre-Blanco and Hunter, 1975) and the final preparation was dialysed exhaustively against 0-02M acetic acid. The fluoresceincollagen (F-collagen) antigen (1-62 mg/ml) contained 5-9 moles of fluorescein/mole of collagen as determined by spectral analysis (The and
Feltkamp, 1970). Fluorescein-labelled bovine serum albumin (F-BSA) was prepared in a similar manner. The final preparation was dialysed exhaustively against phosphate-buffered saline (PBS). The F-BSA antigen (1-4 mg/ml) contained 2-8 moles fluorescein/mole protein. The fluorescein-protein antigens were stored at -25°. PBS was used to dilute F-collagen (3 x ) and F-BSA (4 x ) before they were emulsified with Freund's complete adjuvant (Difco). Female Wistar rats (8 weeks old) were sensitized with 0 4 ml of antigen divided between 4 s.c. sites. A second divided injection of 0 4 ml was given s.c. 2 weeks after the first injection. The levels of circulating antibodies were determined by the passive haemagglutination assay (Herbert, 1967). Sera were collected from rats 2 weeks after the second sensitizing injection of either F-collagen or F-BSA. Soluble collagen and soluble F-collagen were adsorbed to separate portions of tanned sheep red blood cells as the test antigens. Preparation of collagen sponges.-Insoluble bovine Achilles-tendon collagen was prepared by exhaustive washing with 1M NaCl, pH 7-5 and 0-5M acetic acid and chemically analysed as previously described (Etherington, 1977b). The freeze-dried preparation contained 12-2% (w.w) hydroxyproline and was low in hexose (0-6% w/w) and uronic acid (0 22% w/w). The collagen was labelled with fluorescein isothiocyanate (Steven et al., 1975), washed finally with acetone and air-dried. Sponges were prepared from the labelled and unlabelled collagens by dispersion at 5 mg/ml in 0-2M acetic acid using a Polytron homogenizer (Kinematica GmbH). The dispersions were poured into flat-bottomed dishes to a depth of 5 mm and freeze-dried. The porous sheets that were obtained were cut into squares (10 x 10 mm). Sponges were sterilized by soaking for 30 min in 70%o (v/v) ethyl alcohol, blotted between sterile filter paper and then soaked in PBS contained 300 i.u./ml penicillin and 300 ,g/ ml streptomycin (Flow Laboratories). Surgical implantation of sponges. Sponges were implanted into sensitized rats 2 weeks after the second injection of antigen and into nonsensitized rats of the same age (12 weeks). The rats were anaesthetized with an i.m. injection of fluoranisone and fentanyl citrate (Hypnorm (Janssen) 50 ,l/100 g) and the dorsal area shaved. The skin was swabbed with Hibitane (I.C.I.) 0-5% (v/v) in 70%o (v/v) ethyl alcohol and a transverse incision made in the dorso-lumbar region. By the use of blunt-nosed scissors s.c. pockets were then extended longitudinally from this incision. Two sterile sponges were inserted, one cranially and one caudally, and the incision closed with polyamide sutures. The wounds were dusted with 2% Aureomycin
551
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RESORPTION OF INSOLUBLE, HETEROLOGOUS, FLUORESCEINCOLLAGEN SPONGES IN SENSITIZED AND NON-SENSITIZED RATS D. J. ETHERINGTON, I. A. SILVER* AND D. J. RESTALL From the A.R.C. Meat Research Institute, Langford, Bristol BS18 7DY and the Department of Pathology*, Medical School, University of Bristol, Bristol BS8 ITD Received for publication May 1, 1979
Summary.-Sponges of insoluble bovine collagen were slowly resorbed over a 35-day period when implanted under the back skin of rats. The cellular picture was typical of a mild foreign-body reaction. The reaction to fluorescein-labelled collagen sponges was similar but there was evidence also of a weak immunological response. An acute inflammatory reaction with massive oedema was elicited when fluorescein -labelled collagen sponges were implanted in rats previously sensitized to either fluorescein-collagen or fluorescein-bovine serum albumin. The early invasion by PMN leucocytes subsided after 4 days and caused no observable breakdown of the sponge. The implanted material was rapidly encapsulated by fibrous tissue which was then resorbed along with the sponge between the 7th and 12th day. Macrophages were very active in the sponge at this time, sometimes forming giant cells. Fibroblasts were invading from the periphery with the development of the granulation tissue. The small residue which remained after this time was overrun by granulation tissue and was slowly resorbed up to the 35th day. Throughout the period of study there was only a weak local immunological response after the 28th day. The level of circulating antibodies against the fluorescein hapten was high, but the titre for the antibodies against bovine collagen remained low. The significance of these findings in the pathological destruction of connective tissue is discussed.
COLLAGEN DEGRADATION is an integral part of the progressive destruction of tissue in various chronic inflammatory diseases including the cartilage erosion characteristic of rheumatoid arthritis (Krane, 1975). Considerable interest exists in the enzymatic mechanisms for the resorption of these extracellular fibres and several recent reviews have been published (Harris and Krane, 1974; Etherington, 1977a; Harris and Cartwright, 1977; Burleigh, 1977). Collagen-anticollagen antibodies which have been identified at sites of rheumatoid inflammation may be an important factor in the aetiology and severity of this disease and may also influence the process of collagenolysis (Andriopoulos et al., 1976; Steffen et al., 1977; Menzel et al., 1978). Nevertheless, heterologous collagen pre37
parations have been used in surgical repairs and as dressings for large skin wounds, and collagen sponges and gels have also been employed to stimulate tissue repair in bone and cartilage defects (Benfer and Struck, 1973; Holmes, Volz and Chvapil, 1975; DelBalso and Adrian, 1976; Moskow, G-old and Gottsegen, 1976). The material is biodegradable and has been shown to elicit a low or negligible antigenic reaction, especially if the telopeptides have been removed by an enzymatic treatment. A variety of structures has been formed and tested and the degree of intermolecular cross-linking can be regulated at the tanning process (Stenzel, Miyata and Rubin, 1974). The ability of the host to resorb insoluble, cross-linked collagen, whether as dressings, sutures, arterial splints or other surgical prostheses,
550
D. J. ETHERINGTON, I. A. SILVER AND D. J. RESTALL
has been well documented and this is clearly related to the extent of the intermolecular cross-linking, so that the more highly tanned the preparation the more slowly is it resorbed (Stenzel et al., 1974; Shekhter et al., 1975; Berakha, Goulian and Watari, 1975; Bartone, Shervey and Gardner, 1976; Oliver et al., 1976; Chvapil, Owen and Clark, 1977). Histological examinations have been made of the cellular reaction in rats to the s.c. implantation of commercially prepared bovine collagen sponge (Bicol: Ethicon Inc; Cutright et al., 1973) and of intact dermal collagen allografts (Oliver et al., 1976). Polymorphonuclear neutrophil leucocytes (PMN) appeared in the Bicol sponges within 3 days and giant cell formation was observed from the 7th day. The sponges were completely resorbed within 40 days. The dermal allografts, cross-linked with glutaraldehyde, were also invaded by inflammatory cells initially and later evoked giant-cell formation. However, they were neither resorbed nor rejected and were shown subsequently to become repopulated with fibroblasts and new blood vessels. In the present paper we report on the tissue reaction to heterologous collagen sponges which have been labelled with fluorescein isothiocyanate. The fluorescein moiety is a powerful hapten to which the animals can be sensitized in order to elicit a strong antigenic reaction when the sponges are implanted. MATERIALS AND METHODS
Antigen preparation and sensitizing procedure. Soluble collagen was prepared from depilated and saline-washed calf skin pieces by extraction with 0-IM acetic acid for 48 h. The soluble collagen was collected by precipitation with 0-85M NaCl and then dissolved in and dialysed against 0-02M acetic acid. The collagen was complexed with fluorescein isothiocyanate (Steven, Torre-Blanco and Hunter, 1975) and the final preparation was dialysed exhaustively against 0-02M acetic acid. The fluoresceincollagen (F-collagen) antigen (1-62 mg/ml) contained 5-9 moles of fluorescein/mole of collagen as determined by spectral analysis (The and
Feltkamp, 1970). Fluorescein-labelled bovine serum albumin (F-BSA) was prepared in a similar manner. The final preparation was dialysed exhaustively against phosphate-buffered saline (PBS). The F-BSA antigen (1-4 mg/ml) contained 2-8 moles fluorescein/mole protein. The fluorescein-protein antigens were stored at -25°. PBS was used to dilute F-collagen (3 x ) and F-BSA (4 x ) before they were emulsified with Freund's complete adjuvant (Difco). Female Wistar rats (8 weeks old) were sensitized with 0 4 ml of antigen divided between 4 s.c. sites. A second divided injection of 0 4 ml was given s.c. 2 weeks after the first injection. The levels of circulating antibodies were determined by the passive haemagglutination assay (Herbert, 1967). Sera were collected from rats 2 weeks after the second sensitizing injection of either F-collagen or F-BSA. Soluble collagen and soluble F-collagen were adsorbed to separate portions of tanned sheep red blood cells as the test antigens. Preparation of collagen sponges.-Insoluble bovine Achilles-tendon collagen was prepared by exhaustive washing with 1M NaCl, pH 7-5 and 0-5M acetic acid and chemically analysed as previously described (Etherington, 1977b). The freeze-dried preparation contained 12-2% (w.w) hydroxyproline and was low in hexose (0-6% w/w) and uronic acid (0 22% w/w). The collagen was labelled with fluorescein isothiocyanate (Steven et al., 1975), washed finally with acetone and air-dried. Sponges were prepared from the labelled and unlabelled collagens by dispersion at 5 mg/ml in 0-2M acetic acid using a Polytron homogenizer (Kinematica GmbH). The dispersions were poured into flat-bottomed dishes to a depth of 5 mm and freeze-dried. The porous sheets that were obtained were cut into squares (10 x 10 mm). Sponges were sterilized by soaking for 30 min in 70%o (v/v) ethyl alcohol, blotted between sterile filter paper and then soaked in PBS contained 300 i.u./ml penicillin and 300 ,g/ ml streptomycin (Flow Laboratories). Surgical implantation of sponges. Sponges were implanted into sensitized rats 2 weeks after the second injection of antigen and into nonsensitized rats of the same age (12 weeks). The rats were anaesthetized with an i.m. injection of fluoranisone and fentanyl citrate (Hypnorm (Janssen) 50 ,l/100 g) and the dorsal area shaved. The skin was swabbed with Hibitane (I.C.I.) 0-5% (v/v) in 70%o (v/v) ethyl alcohol and a transverse incision made in the dorso-lumbar region. By the use of blunt-nosed scissors s.c. pockets were then extended longitudinally from this incision. Two sterile sponges were inserted, one cranially and one caudally, and the incision closed with polyamide sutures. The wounds were dusted with 2% Aureomycin
551
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