J. Anat. (1975), 119, 2, pp. 359-367
359
With 11 figures Printed in Great Britain
Bone induction in implants of decalcified bone and dentine G. J. LINDEN Anatomy Department, Medical Biology Centre, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
(Accepted 1 November 1974) INTRODUCTION
In recent years Urist and his colleagues have shown that decalcified bone and dentine regularly induce new bone formation when implanted in muscles and other non-skeletal tissues in rats, rabbits and guinea-pigs (Urist et al. 1967; Urist, Iwata & Strates, 1972). The histology of the process, however, is still rather obscure and the
recalcification of the decalcified implants has not received adequate attention. Therefore samples of Sprague-Dawley rat bone kindly supplied by Dr Urist were implanted beneath the kidney capsule and between the muscle layers of the lateral abdominal wall of young Ash-Wistar rats. In addition, samples of decalcified AshWistar rat bone and dentine prepared locally by the author were implanted into rats of the same strain.The fate of the implants was investigated by conventional histological techniques, supplemented in some cases by the use of lead acetate as a vital stain for calcium (Scheiman-Tagger & Brodie, 1963). MATERIALS AND METHODS
In the course of these experiments 68 Ash-Wistar rats between 3 and 10 weeks old were used. Implants, prepared as described below, were placed either beneath the kidney capsule or between the muscle layers of the lateral abdominal wall. Although the local Ash-Wistar rats from which implants were prepared were not strictly speaking isogeneic with the rats into which they were placed, they were obviously genetically closer to one another than to the allogeneic Sprague-Dawley rats. Usually two, but sometimes three or four implants were placed in each rat (See Table 1). Preparation of the implants Decalcified allogeneic bone. The samples consisted of diaphyseal segments of the long bones of young Sprague-Dawley rats which had been treated as follows. After excision of the long bone and removal of the epiphyses and non-osseous tissues the marrow was scraped out. The diaphyseal cortical bone was defatted for 2 hours in 1: 1 chloroform: methanol, decalcified for 24 hours in 1 mm DMSO (dimethyl sulphoxide) in M-H3P04 at 2 °C and then rinsed in distilled water and frozen-dried. The material was supplied in sealed plastic envelopes. The implants were prepared by cutting this material into rectangular blocks, 3 mm x 2 mm x 0-4 mm, with the aid of a dissection
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361 Bone induction in decalcified implants microscope. The volume of these blocks always exceeded 1 mm3. The blocks were stored in sterilized glass phials prior to implantation. All the material placed beneath the kidney capsule was of this shape and size. However, some complete crosssectional cylindrical implants 2 mm long were prepared for intramuscular grafting. Control material was prepared by heating implants at 100 'C. for 60 minutes. This temperature has been shown to destroy all bone inducing activity (Urist et al. 1967). Decalcified isogeneic bone. The diaphyses of cleaned long bones from the local colony of Ash-Wistar rats were placed unfixed in a solution of 0 6 N-HCl at 4 'C for 48 hours. They were then rinsed in several changes of 70 % alcohol and cut into small rectangular pieces while still immersed in the alcohol. They were then rinsed in distilled water and implanted immediately. Decalcified 'isogeneic' dentine. Portions of the mandible containing the incisors were excised using bone shears. The teeth were removed and scraped free of cementum. These were decalcified in 0-6 N-HCl for 72 hours at 4 'C. They were then rinsed in several changes of 70 % alcohol and the enamel removed as far as possible with a scalpel. The remainder of each tooth, consisting mainly of decalcified dentine, served as the implant. It was rinsed in distilled water and implanted immediately. Undecalcified 'alcohol fixed bone'. Excised long bones were cleaned and left in 70 % alcohol for 24 hours. Pieces of suitable size were cut with bone shears, rinsed in distilled water and implanted immediately. Histological procedures with chloroform at planned intervals after operation. In the The rats were killed case of the muscle implants, first of all the whole of the right lateral abdominal wall was removed and immersed in 10 % formol saline, and then, after preliminary fixation, the implants were excised with some supporting muscle tissue. Similarly in the case of the subcapsular implants, the whole kidney was first excised and im** mersed in 10 % formol saline, and then later the implants were removed with some of the supporting renal tissue, care being taken not to disturb the relationship of an implant to the underlying parenchyma. The majority of the implants after fixation were decalcified in 5 % trichloroacetic acid and double-embedded. However, several implants were sectioned undecalcified. The sections were mostly stained with haemotoxylin and eosin, but some were stained with Weigert and van Gieson, and some with Alcian blue, either in conjunction with the stains mentioned or with neutral red. Lead acetate method The procedure described by Scheiman-Tagger & Brodie (1963) was followed. Seven Ash-Wistar rats between 5 and 7 weeks old were used. Intramuscular implants of Fig. 1. Subcapsular implant of decalc ified 'allogeneic' bone at 5 days. d, decalcified implant; i, inflammatory cells; f, fibroblasts. H. & E. x 108. Fig. 2. Subcapsular implant of decalcified 'allogeneic' bone at 14 days. m, multinucleate giant cells. H. & E. x 170. Fig. 3. Subcapsular implant of decalcified 'allogeneic' bone at 60 days. H. & E. x 108. Fig. 4. Subcapsular implant of decalcified 'allogeneic' bone at 25 days. v, vascularization; m, multinucleate giant cells. H. & E. x 108.
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359
With 11 figures Printed in Great Britain
Bone induction in implants of decalcified bone and dentine G. J. LINDEN Anatomy Department, Medical Biology Centre, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
(Accepted 1 November 1974) INTRODUCTION
In recent years Urist and his colleagues have shown that decalcified bone and dentine regularly induce new bone formation when implanted in muscles and other non-skeletal tissues in rats, rabbits and guinea-pigs (Urist et al. 1967; Urist, Iwata & Strates, 1972). The histology of the process, however, is still rather obscure and the
recalcification of the decalcified implants has not received adequate attention. Therefore samples of Sprague-Dawley rat bone kindly supplied by Dr Urist were implanted beneath the kidney capsule and between the muscle layers of the lateral abdominal wall of young Ash-Wistar rats. In addition, samples of decalcified AshWistar rat bone and dentine prepared locally by the author were implanted into rats of the same strain.The fate of the implants was investigated by conventional histological techniques, supplemented in some cases by the use of lead acetate as a vital stain for calcium (Scheiman-Tagger & Brodie, 1963). MATERIALS AND METHODS
In the course of these experiments 68 Ash-Wistar rats between 3 and 10 weeks old were used. Implants, prepared as described below, were placed either beneath the kidney capsule or between the muscle layers of the lateral abdominal wall. Although the local Ash-Wistar rats from which implants were prepared were not strictly speaking isogeneic with the rats into which they were placed, they were obviously genetically closer to one another than to the allogeneic Sprague-Dawley rats. Usually two, but sometimes three or four implants were placed in each rat (See Table 1). Preparation of the implants Decalcified allogeneic bone. The samples consisted of diaphyseal segments of the long bones of young Sprague-Dawley rats which had been treated as follows. After excision of the long bone and removal of the epiphyses and non-osseous tissues the marrow was scraped out. The diaphyseal cortical bone was defatted for 2 hours in 1: 1 chloroform: methanol, decalcified for 24 hours in 1 mm DMSO (dimethyl sulphoxide) in M-H3P04 at 2 °C and then rinsed in distilled water and frozen-dried. The material was supplied in sealed plastic envelopes. The implants were prepared by cutting this material into rectangular blocks, 3 mm x 2 mm x 0-4 mm, with the aid of a dissection
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361 Bone induction in decalcified implants microscope. The volume of these blocks always exceeded 1 mm3. The blocks were stored in sterilized glass phials prior to implantation. All the material placed beneath the kidney capsule was of this shape and size. However, some complete crosssectional cylindrical implants 2 mm long were prepared for intramuscular grafting. Control material was prepared by heating implants at 100 'C. for 60 minutes. This temperature has been shown to destroy all bone inducing activity (Urist et al. 1967). Decalcified isogeneic bone. The diaphyses of cleaned long bones from the local colony of Ash-Wistar rats were placed unfixed in a solution of 0 6 N-HCl at 4 'C for 48 hours. They were then rinsed in several changes of 70 % alcohol and cut into small rectangular pieces while still immersed in the alcohol. They were then rinsed in distilled water and implanted immediately. Decalcified 'isogeneic' dentine. Portions of the mandible containing the incisors were excised using bone shears. The teeth were removed and scraped free of cementum. These were decalcified in 0-6 N-HCl for 72 hours at 4 'C. They were then rinsed in several changes of 70 % alcohol and the enamel removed as far as possible with a scalpel. The remainder of each tooth, consisting mainly of decalcified dentine, served as the implant. It was rinsed in distilled water and implanted immediately. Undecalcified 'alcohol fixed bone'. Excised long bones were cleaned and left in 70 % alcohol for 24 hours. Pieces of suitable size were cut with bone shears, rinsed in distilled water and implanted immediately. Histological procedures with chloroform at planned intervals after operation. In the The rats were killed case of the muscle implants, first of all the whole of the right lateral abdominal wall was removed and immersed in 10 % formol saline, and then, after preliminary fixation, the implants were excised with some supporting muscle tissue. Similarly in the case of the subcapsular implants, the whole kidney was first excised and im** mersed in 10 % formol saline, and then later the implants were removed with some of the supporting renal tissue, care being taken not to disturb the relationship of an implant to the underlying parenchyma. The majority of the implants after fixation were decalcified in 5 % trichloroacetic acid and double-embedded. However, several implants were sectioned undecalcified. The sections were mostly stained with haemotoxylin and eosin, but some were stained with Weigert and van Gieson, and some with Alcian blue, either in conjunction with the stains mentioned or with neutral red. Lead acetate method The procedure described by Scheiman-Tagger & Brodie (1963) was followed. Seven Ash-Wistar rats between 5 and 7 weeks old were used. Intramuscular implants of Fig. 1. Subcapsular implant of decalc ified 'allogeneic' bone at 5 days. d, decalcified implant; i, inflammatory cells; f, fibroblasts. H. & E. x 108. Fig. 2. Subcapsular implant of decalcified 'allogeneic' bone at 14 days. m, multinucleate giant cells. H. & E. x 170. Fig. 3. Subcapsular implant of decalcified 'allogeneic' bone at 60 days. H. & E. x 108. Fig. 4. Subcapsular implant of decalcified 'allogeneic' bone at 25 days. v, vascularization; m, multinucleate giant cells. H. & E. x 108.
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