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Collagen types present at human fracture sites—a preliminary report
78 Injury (1986) 17,78-80
Printedin Great Britain
Collagen types present at human fracture sites preliminary report
a
A. M. A n d e r s o n , G. W. H a s t i n g s , T. R. Fisher and E. R. S. Ross Biomedical Engineering Unit, North Staffordshire Polytechnic, Stoke-on-Trent A. S h u t t l e w o r t h Department of Biochemistry, University of Manchester
Summary Collagen has been examined from normally and abnormally healing fractures. A higher proportion of type III collagen than was expected was found in abnormally healing fractures. Very little is known about collagen synthesis in healing fractures and how it is altered in abnormal healing states such as delayed and non-union. INTRODUCTION THE ossification process in healing bone may be influenced by the collagen type in the new bone. Type I collagen is the most prevalent kind in ossified tissues, but there is evidence to suggest that type II collagen is also capable of ossification (Von Der Mark and Von Der Mark, 1977). However, the evolution of collagen in normal and abnormal healing has received little attention and Sevitt (1981) refers to the scanty knowledge available. Since abnormalities of collagen type have been noted in other conditions affecting bone (Muller et al., 1975; Pope et al., 1975; Bateman et al., 1984) it is of interest to determine whether a change in type was evident in cases of non-union. This study was undertaken to examine the collagen types of the fibrous tissues biopsied from fracture sites in human femurs which had either united normally or had been diagnosed as showing non-union. This is part of a wider investigation of less rigid internal fixation (Tayton et al., 1982) and magnetic stimulation (Anderson, 1984).
MATERIALS A N D METHODS Samples were obtained by open biopsy. Since the fractures were to be treated surgically for various reasons (Table I), no ethical problem was posed. After the femur had been exposed, the fracture site was identified and tissue samples of approximately 1 0 x 5 x 5 m m were taken traversing the fracture line. An osteotome was used in preference to a saw, in order to minimize necrosis. When possible, a piece of the fibrous tissue within the defect was removed, complete with bony attachment at each end. Since the collection of samples took place over a long period of time, they were stored at - 7 0 ° C until enough had been obtained to make analysis worth while. The small specimen size permitted as near uniform freezing as possible. The sample was initially plunged into liquid nitrogen, then removed and placed on a glass microscope slide. The orientation of the sample with respect to the fracture site was recorded by labelling the proximal, medial and anterior aspects of the specimen. The whole slide was wrapped in aluminium foil and stored at - 70 ° C.
Samples were demineralized using solutions of ethylenediamine tetra-acetic acid. Then the pepsin soluble fraction of the collagen was extracted. The estimation of the quantity of types I and III collagen in the pepsin-extracted fracture was achieved on 5 per
Table L Details of samples from fracture sites Time
Patient no.
Male/ female Diagnosis
Site
since fracture (months)
1
M
Normal
Femur
4
2
M
Femur
3
3
F
Delayed union Non-union
Femur
12
4
F
Non-union
Neck of femur
13
No. of samples
Reason for operation Osteotomy for angulation Corrective osteotomy Non-union after functional brace Total hip replacement
79
Anderson et al. : Collagen types in fractures
cent acrylamide gels after interrupted electrophoresis (Neville, 1971).
RESULTS Type III collagen was present in all the samples of fractures but was absent in normal uninjured bone. The amount of type III collagen present in each sample is given in Table H as a percentage of the total pepsin-soluble collagen present. No quantitative assessment was made of the total amount of collagen present.
DISCUSSION Two processes of healing are recognized in fractures of long bones (McKibbin, 1978; Sevitt, 1981). External callus is always formed, except after intervention by plating. Sevitt (1981) describes direct and indirect processes of bony healing, the latter involving temporary fibrous bridging of the bone ends, later to be replaced by bone. McKibbin (1978), on the other hand, believes that this fibrous response is abnormal and that fibrous invasion of the fracture gap always halts osteogenic repair until fibrosis is removed. Both authors, however, agree that the removal of the fibrous mass is essential to final bony union but the nature of the collagen in the fibrous tissue is not known. Discussion of the sequence of formation of the collagen type is therefore confined to that in external callus, which may contain hyaline cartilage, especially when the degree of vascularity and therefore the oxygen level is low. This is believed to be the case in non-union (Brighton et al., 1977) and is borne out by histological examination of the samples used for this study. Ossification of this callus cartilage takes place by a process akin to endochondral ossification of the epiphyseal plate (Sevitt, 1981). Von Der Mark and Von Der Mark (1977) have shown the relationships of collagen type within this process. It is therefore possible to use this information to predict the collagen types present in a healing fracture or a non-union. The primary collagenous response to fracture is likely to be the production of type II, as in the formation of hyaline cartilage within the callus. This would be followed by type III production, the precursor of endochondral ossification, and finally by type I production in the completion of bone union. A little type III production would also be expected in the early stages, due to the proliferation of blood vessels. The collagen type of any interposed fibrous tissue is not known. The observation of 15 per cent of type III collagen in
a normally healing fracture is therefore readily explicable. It is likely that the sample was taken before endochondral ossification was complete, so that type III collagen had not been fully removed. The raised levels of type III collagen in non-unions (around 30 per cent) may have several explanations. First, the collagen type of the fibrous tissue between the bone ends is not known, and it could be predominantly type III. Second, it is possible that the levels found are the normal maximums achieved in fracture sites and that substitution by type I has not taken place. Third, there may have been over-stimulation of type III production. The observation of a raised type III collagen level in the sample diagnosed as delayed union (patient 2) compared with a lower level of type III collagen in the older sample of normal healing (patient 1) suggests high type III levels are not merely a time-dependent phenomenon. It is impossible in this small study to determine whether the observed change in proportions of the different types of collagen is the cause of non-union or a result of iL or even to conclude that this change would be seen in all sites of non-union. However, the results indicate that a greater study would be worth while, especially if immunohistochemical techniques were also employed to determine the distribution of collagen types throughout the specimens. It is interesting to note that although the highest level of type III collagen was found in the sample with the grossest change towards true synovial pseudarthrosis (patient 3), other samples with raised type III levels did not exhibit such marked histological changes. In the case of the clinically delayed union (patient 2) there was little histological difference from a normally healing fracture. It appears from this study that the increased levels of type III collagen show a good correlation with the clinical diagnoses.
CONCLUSIONS Different types of collagen are found in the callus of healing fractures and the proportion of type III appears to be greater in non-unions and delayed union of fractures than with normal healing.
Acknowledgement One of the authors (A.M.A.) wishes to acknowledge the support of an SERC 'CASE' award for. the duration of this study.
Table IL Amounts of type III collagen present at fracture sites
Patient no.
1 2 3 4
Diagnosis
Total pepsinsoluble collagen-% original sample
Amount of type Ill collagen (%)
4 3
0.5 15-4 63.4
Undetectable) 15.28 32-0
12 13
56.4 8.5
42.74 29.5
Time since fracture (months)
(Normal uninjured femur Normal Delayed union Non-union Non-union
80 REFERENCES Anderson A. M. (1984) The effect of pulsing electromagnetic fields on polymerising systems. PhD Thesis, North Staffordshire Polytechnic. Bateman J. F., Mascara T., Chan D. et al. (1984) Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem. J. 217, 103-115. Brighton C. T., Friedenberg Z. B., Mitchell E. I. et al. (1977) Treatment of nonunion with constant direct current. Clin. Orthop. 124, 106. McKibbin B. (1978) Biology of fracture healing in long bones. J. Bone Joint Surg. 60B, 150. Muller P. K., Lemmen C., Gay G. et al. (1975) Disturbance in the regulation of the type of collagen synthesised in a form of osteogenesis imperfecta. Eur. J. Biochem. 59, 97.
Injury: the British Journal of Accident Surgery (1986) Vol. 17/No. 2
Neville D. (1971) Molecular weight determination of protein dodecyl sulfate complexes by gel electrophoresis in discontinuous buffer system. J. Biol. Chem. 246, 6328. Pope F. M., Martin G. R., Lichtenstein J. R. et al. (1975) Patients with Ehlers Danlos syndrome type IV lack type III collagen. Proc. Natl Acad. Sci. USA 72, 1314. Sevitt S. (1981) Bone Repair and Fracture Healing in Man. Edinburgh: Churchill Livingstone. Tayton K., Johnson-Nurse C., McKibbin B. et al. (1982) The use of semi-rigid carbon-fibre reinforced plastic plates for fixation of human fractures. J. Bone Joint Surg. 64B, 105. Von Der Mark K. and Von Der Mark H. (1977) The role of three genetically distinct collagen types in endochondral ossification. J. Bone Joint Surg. 59B, 458. Paper accepted 7 May 1985.
Requests for reprints should be addressed to: Dr G. W. Hastings, Biomedical Engineering Unit, Medical Institute, Stoke-on-Trent ST4 7NY.
Printedin Great Britain
Collagen types present at human fracture sites preliminary report
a
A. M. A n d e r s o n , G. W. H a s t i n g s , T. R. Fisher and E. R. S. Ross Biomedical Engineering Unit, North Staffordshire Polytechnic, Stoke-on-Trent A. S h u t t l e w o r t h Department of Biochemistry, University of Manchester
Summary Collagen has been examined from normally and abnormally healing fractures. A higher proportion of type III collagen than was expected was found in abnormally healing fractures. Very little is known about collagen synthesis in healing fractures and how it is altered in abnormal healing states such as delayed and non-union. INTRODUCTION THE ossification process in healing bone may be influenced by the collagen type in the new bone. Type I collagen is the most prevalent kind in ossified tissues, but there is evidence to suggest that type II collagen is also capable of ossification (Von Der Mark and Von Der Mark, 1977). However, the evolution of collagen in normal and abnormal healing has received little attention and Sevitt (1981) refers to the scanty knowledge available. Since abnormalities of collagen type have been noted in other conditions affecting bone (Muller et al., 1975; Pope et al., 1975; Bateman et al., 1984) it is of interest to determine whether a change in type was evident in cases of non-union. This study was undertaken to examine the collagen types of the fibrous tissues biopsied from fracture sites in human femurs which had either united normally or had been diagnosed as showing non-union. This is part of a wider investigation of less rigid internal fixation (Tayton et al., 1982) and magnetic stimulation (Anderson, 1984).
MATERIALS A N D METHODS Samples were obtained by open biopsy. Since the fractures were to be treated surgically for various reasons (Table I), no ethical problem was posed. After the femur had been exposed, the fracture site was identified and tissue samples of approximately 1 0 x 5 x 5 m m were taken traversing the fracture line. An osteotome was used in preference to a saw, in order to minimize necrosis. When possible, a piece of the fibrous tissue within the defect was removed, complete with bony attachment at each end. Since the collection of samples took place over a long period of time, they were stored at - 7 0 ° C until enough had been obtained to make analysis worth while. The small specimen size permitted as near uniform freezing as possible. The sample was initially plunged into liquid nitrogen, then removed and placed on a glass microscope slide. The orientation of the sample with respect to the fracture site was recorded by labelling the proximal, medial and anterior aspects of the specimen. The whole slide was wrapped in aluminium foil and stored at - 70 ° C.
Samples were demineralized using solutions of ethylenediamine tetra-acetic acid. Then the pepsin soluble fraction of the collagen was extracted. The estimation of the quantity of types I and III collagen in the pepsin-extracted fracture was achieved on 5 per
Table L Details of samples from fracture sites Time
Patient no.
Male/ female Diagnosis
Site
since fracture (months)
1
M
Normal
Femur
4
2
M
Femur
3
3
F
Delayed union Non-union
Femur
12
4
F
Non-union
Neck of femur
13
No. of samples
Reason for operation Osteotomy for angulation Corrective osteotomy Non-union after functional brace Total hip replacement
79
Anderson et al. : Collagen types in fractures
cent acrylamide gels after interrupted electrophoresis (Neville, 1971).
RESULTS Type III collagen was present in all the samples of fractures but was absent in normal uninjured bone. The amount of type III collagen present in each sample is given in Table H as a percentage of the total pepsin-soluble collagen present. No quantitative assessment was made of the total amount of collagen present.
DISCUSSION Two processes of healing are recognized in fractures of long bones (McKibbin, 1978; Sevitt, 1981). External callus is always formed, except after intervention by plating. Sevitt (1981) describes direct and indirect processes of bony healing, the latter involving temporary fibrous bridging of the bone ends, later to be replaced by bone. McKibbin (1978), on the other hand, believes that this fibrous response is abnormal and that fibrous invasion of the fracture gap always halts osteogenic repair until fibrosis is removed. Both authors, however, agree that the removal of the fibrous mass is essential to final bony union but the nature of the collagen in the fibrous tissue is not known. Discussion of the sequence of formation of the collagen type is therefore confined to that in external callus, which may contain hyaline cartilage, especially when the degree of vascularity and therefore the oxygen level is low. This is believed to be the case in non-union (Brighton et al., 1977) and is borne out by histological examination of the samples used for this study. Ossification of this callus cartilage takes place by a process akin to endochondral ossification of the epiphyseal plate (Sevitt, 1981). Von Der Mark and Von Der Mark (1977) have shown the relationships of collagen type within this process. It is therefore possible to use this information to predict the collagen types present in a healing fracture or a non-union. The primary collagenous response to fracture is likely to be the production of type II, as in the formation of hyaline cartilage within the callus. This would be followed by type III production, the precursor of endochondral ossification, and finally by type I production in the completion of bone union. A little type III production would also be expected in the early stages, due to the proliferation of blood vessels. The collagen type of any interposed fibrous tissue is not known. The observation of 15 per cent of type III collagen in
a normally healing fracture is therefore readily explicable. It is likely that the sample was taken before endochondral ossification was complete, so that type III collagen had not been fully removed. The raised levels of type III collagen in non-unions (around 30 per cent) may have several explanations. First, the collagen type of the fibrous tissue between the bone ends is not known, and it could be predominantly type III. Second, it is possible that the levels found are the normal maximums achieved in fracture sites and that substitution by type I has not taken place. Third, there may have been over-stimulation of type III production. The observation of a raised type III collagen level in the sample diagnosed as delayed union (patient 2) compared with a lower level of type III collagen in the older sample of normal healing (patient 1) suggests high type III levels are not merely a time-dependent phenomenon. It is impossible in this small study to determine whether the observed change in proportions of the different types of collagen is the cause of non-union or a result of iL or even to conclude that this change would be seen in all sites of non-union. However, the results indicate that a greater study would be worth while, especially if immunohistochemical techniques were also employed to determine the distribution of collagen types throughout the specimens. It is interesting to note that although the highest level of type III collagen was found in the sample with the grossest change towards true synovial pseudarthrosis (patient 3), other samples with raised type III levels did not exhibit such marked histological changes. In the case of the clinically delayed union (patient 2) there was little histological difference from a normally healing fracture. It appears from this study that the increased levels of type III collagen show a good correlation with the clinical diagnoses.
CONCLUSIONS Different types of collagen are found in the callus of healing fractures and the proportion of type III appears to be greater in non-unions and delayed union of fractures than with normal healing.
Acknowledgement One of the authors (A.M.A.) wishes to acknowledge the support of an SERC 'CASE' award for. the duration of this study.
Table IL Amounts of type III collagen present at fracture sites
Patient no.
1 2 3 4
Diagnosis
Total pepsinsoluble collagen-% original sample
Amount of type Ill collagen (%)
4 3
0.5 15-4 63.4
Undetectable) 15.28 32-0
12 13
56.4 8.5
42.74 29.5
Time since fracture (months)
(Normal uninjured femur Normal Delayed union Non-union Non-union
80 REFERENCES Anderson A. M. (1984) The effect of pulsing electromagnetic fields on polymerising systems. PhD Thesis, North Staffordshire Polytechnic. Bateman J. F., Mascara T., Chan D. et al. (1984) Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem. J. 217, 103-115. Brighton C. T., Friedenberg Z. B., Mitchell E. I. et al. (1977) Treatment of nonunion with constant direct current. Clin. Orthop. 124, 106. McKibbin B. (1978) Biology of fracture healing in long bones. J. Bone Joint Surg. 60B, 150. Muller P. K., Lemmen C., Gay G. et al. (1975) Disturbance in the regulation of the type of collagen synthesised in a form of osteogenesis imperfecta. Eur. J. Biochem. 59, 97.
Injury: the British Journal of Accident Surgery (1986) Vol. 17/No. 2
Neville D. (1971) Molecular weight determination of protein dodecyl sulfate complexes by gel electrophoresis in discontinuous buffer system. J. Biol. Chem. 246, 6328. Pope F. M., Martin G. R., Lichtenstein J. R. et al. (1975) Patients with Ehlers Danlos syndrome type IV lack type III collagen. Proc. Natl Acad. Sci. USA 72, 1314. Sevitt S. (1981) Bone Repair and Fracture Healing in Man. Edinburgh: Churchill Livingstone. Tayton K., Johnson-Nurse C., McKibbin B. et al. (1982) The use of semi-rigid carbon-fibre reinforced plastic plates for fixation of human fractures. J. Bone Joint Surg. 64B, 105. Von Der Mark K. and Von Der Mark H. (1977) The role of three genetically distinct collagen types in endochondral ossification. J. Bone Joint Surg. 59B, 458. Paper accepted 7 May 1985.
Requests for reprints should be addressed to: Dr G. W. Hastings, Biomedical Engineering Unit, Medical Institute, Stoke-on-Trent ST4 7NY.