THE FEATURES OF FRACTURE HEALING IN CATS AFTER IMMEDIATE AND DELAYED OPEN REDUCTION

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1 THE FEATURES OF FRACTURE HEALING IN CATS AFTER IMMEDIATE AND DELAYED OPEN REDUCTION M. A. EMERY and H. MURAKAMI, EDt1oN1 on, CANADA From the Surgical-Medical Research Institute, University of Alberta, Edmonton The problem of delayed union and non-union after fractures, particularly if open reduction has been necessary, continues to be a problem. Various methods of overcoming this problem have been suggested. That most widely investigated concerns the method of fixation. In 1959 Smith reported the effect on fracture healing of delay in open reduction for one to three weeks. He claimed that delay in operation markedly improved the incidence of fracture union after open reduction. Other papers have also noted this difference (Table I) (Charnley and Guindy 1961, Lam 1964, Smith 1964, Emery 1965). This paper reports investigations to determine the difference in the mechanism of fracture healing in the fractured femurs of cats after a deliberate delay of two weeks before internal fixation. TABLE CLINICAL EVALUATION or IMMEDIATE AND DELAYED OPERATION IN ADULT HUMANS I Incidence of delayed union A th Fracture Number of and non-union (per cent) U ON site cases - Early Delayed operation operation Smith (1959)... Forearm CharnleyandGuindy(l96l) Femur Lam (1964)... Femur Smith(1964)... Femur Emery(1965)... Forearm III MATERIALS AND METHODS In fifty-one female cats the left femur was fractured at midshaft by external manual force. This produced a transverse fracture, often with a butterfly fragment. Twenty-seven cats were operated on immediately after fracture. The fracture was reduced and fixed rigidly by an intramedullary Steinmann s pin. These cats were classified as the immediate operation group. In the other twenty-four cats the fractured leg was strapped to the abdomen by adhesive tapes. This immobilised the fracture satisfactorily and, although it resulted in some shortening, it simulated a splint or traction fixation as used for human fractures. Two weeks later internal fixation was done in the same way as in the immediate operation group. These cats were classified as the delayed operation group. From one to seven weeks after operation two to six cats were killed at weekly intervals. Radiographs were taken of the fracture; then the femur was removed and examined macroscopically. Twenty-seven of the fractured femurs were tested on the Baldwin tensiometer to determine the maximum longitudinal tension necessary to break down the callus around the fracture site. All specimens were then examined histologically, specimens being taken VOL. 49 B, NO. 3, AUGUST

2 572 M. A. EMERY AND H. MURAKAMI through the centre of the callus and also from each end. Sections were stained with haematoxylin and eosin and with toluidine-blue after decalcification (Table II). RESULTS The results have been analysed under four headings: clinical union, radiological union, tensile strength and histological examination. Clinical union-this was determined by the disappearance of movement at the fracture site (Table Ill). In the immediate operation group, bone union was seen after the sixth week after operation in most specimens. In the delayed operation group, most of the specimens were united by the fourth week after operation, and all by the fifth week. TABLE NUMBER OF CATS EXAMINED II Weeksafteroperation Total Immediate operation Delayed operation. 3 2 S TABLE CLINICAL UNION IN THE FEMORA OF CATS III Weeksafteroperation Immediateoperation Delayed operation. Numberexamined Numberwithunion I I 5 2. Numberexamined : Numberwithunion TABLE RADIOLOGICAL UNION IN THE FEMORA OF CATS IV Weeksafteroperation Immediate operation. No sign e fracture site ofc1u Radiological Dela ed 0 r t n Callus at Bridging Bridging Radiological Radiological y PC a 10. fracture site of callus of callus union union Radiological union-a significant difference in the amount and the maturity of the callus was noted between the immediate and delayed operation groups (Table IV). Radiological union was seen at the end of five weeks after operation in the delayed operation group, but not until the seventh week in the immediate operation group. It was not until three weeks after fracture in the immediate operation group that any evidence ofhealing was present. This was represented by a moderate degree of periosteal thickening. At two weeks after the delayed operation, or four weeks after the fracture, a large amount of callus was observed at the fracture site. By four weeks the callus had bridged the fracture site, and by six weeks radiological union was evident. At four weeks periosteal new bone formation was noted in the immediate operation group, and at seven weeks bridging had occurred, but the shadow of the cortical bone was still sharp and the callus immature. THE JOURNAL OF BONE AND JOINT SURGERY

3 FRACTURE HEALING IN CATS AFTER IMMEDIATE AND DELAYED OPEN REDUCTION 573 Tension testing-after the testing of a number of early specimens it became evident that the soft tissues alone have a testing tension strength of about twenty pounds. Compared histologically, when a significant degree of cartilage was present about forty pounds of tension strength was found. The tension strength then increased rapidly as bone formed at the fracture site (Fig. 1). The immediate operation group showed about twenty pounds of tension strength from the second to the fourth weeks. This strength then gradually increased. The delayed operation group increased its tension strength from the second week after operation, and at the end of the third week showed almost the same tension strength as at the fifth week after immediate operation. Histological examination. Immediate operaiion-ln the immediate operation group the periosteum, which was extremely thin, was disrupted extensively by the fracture and this was noted at the time of the operation. At the end of the second week after operation a large amount offibrous proliferation was seen all around the fractured ends (Fig. 2). The periosteum was seen only well away from the fracture site. Beneath this a small amount of cancellous new bone had formed. Occasional small islands of cartilage were also noted. After the third week fibrous 100 tissue proliferation had increased, as o #{176} 7 had the amount of cartilage (Fig. 3) 80 1 but there was no indirect new bone formation. Beneath the remaining MAXIMUM.. TENSION 60 periosteum cancellous bone had in- OF CALLUS creased in amount (Fig. 4). After POUNDS the fourth week cartilage and fibrous tissue were still the predominant 30 features. Endochondral new bone 20.x formation could be seen for the. S. first time (Fig. 5). Ossification.. centres were few, and appeared to be.... WEEKS AFTER OPERATION originating from regions of the F 1 old cortical bone, and from the new To show the strength of;he callus. The white dot bone formed by the periosteum. represents the tension necessary to pull apart the After the fifth week the ro res- callus in femur after a delayed operation. The. F b black dot represents the same, but after an imsion of endochondral bone formation mediate operation. had been occurring slowly, and was still relatively small in amount (Fig. 6). Cartilage was still the dominant tissue. It was noted that the number of ossification centres were still few in number (Fig. 7). The progress of the endochondral bone formation continued slowly through the seventh week. Maturation occurred in its deeper portions. At the end of the seventh week the new bone had become the dominant structure of the callus. Some reorganisation had occurred, but the trabeculae were still immature and showed the presence of cartilage components within the bone matrix (Fig. 8). In summary, after immediate open reduction the periosteum was usually severely damaged. Fibrous tissue and cartilage formation were the dominant feature of the healing process. Replacement of cartilage by endochondral bone formation occurred slowly from a small number of ossification centres. Delayedoperation-At the time ofoperation two observations were made. The most important was the condition of the periosteum, which had become much thicker and tougher and was therefore much more easily preserved. The blood supply to the area had greatly increased and there was relatively more bleeding during the operation. VOL. 49 B, NO. 3, AUGUST 1967

4 574 M. A. EMERY AND H. MURAKAMI Histologically, at the first week after operation, a layer of haematoma was seen between the fractured bone surface and the already formed cartilage. A thin layer of cancellous bone had already formed on the fractured bone surface (Fig. 9). A short distance from the fracture 4. 4 : #{149} -: S #{149} q.d S.#{234}#{149}4#{149}#{149} #{149}5%. #{149},.. K. #{149} _,.#{149} I #{149}. S,#{149}#{149},., I #{149} _bl, l,&% #{149} #{149}. 4. ::.-,z,.. -:r.: #{149} : : Ti FIG. 2 FIG. 3 Figure 2-Three weeks after an immediate operation there is extensive fibrous proliferation around the fractured bone. The periosteum cannot be seen. (Haematoxylin and eosin, x 125.) Figure 3-Three weeks after an immediate operation there is extensive cartilage formation on the fractured bone surface. In the middle of the section the cartilage matrix around the blood vessels has calcified. No new bone formation is seen as yet. (Haematoxylin and eosin, x 75.) FIG. 4 FIG. 5 Figure 4-Three weeks after an immediate operation an increased amount of cancellous bone formation is seen where the periosteum has remained intact. Fibrous proliferation outside the periosteum is slight and there is no cartilage. (Haematoxylin and eosin, x 50.) Figure 5-Four weeks after an immediate operation a thin layer of cancellous bone has formed on the fractured bone surface. A large amount of cartilage is seen adjacent to the new bone. Endochondral bone formation has started. (Haematoxylin and eosin, x 75.) site a thick layer of mature cancellous bone had already formed under the intact periosteum (Fig. 10). After the second week two layers ofcancellous bone were seen beneath the periosteum, the result of stripping of the periosteum at operation. Adjacent to the fractured bone surface THE JOURNAL OF BONE AND JOINT SURGERY

5 FRACTURE HEALING IN CATS AFTER IMMEDIATE AND DELAYED OPEN REDUCTION 575. \1 I #{231} Z: S. #{149} #{149}:-. [#{231}.,#{149}4 1 h -,. -t-- ±... - FIG. 6 FIG. 7 Figure 6-Five weeks after an immediate operation cartilage is still the dominant structure of the callus. Endochondral bone formation is progressing slowly and still shows the presence oi cartilage components within the bone matrix ofthe new trabeculae. (Haematoxylin and eosin, x 50.) Figure 7-Six weeks after an immediate operation endochondral bone formation is proceeding from the new bone formed on the fractured bone surface. A large amount of cartilage is still present. (Haematoxylin and eosin, x 30.) a relatively mature layer of bone was noted. Next a layer of fibrous tissue and cartilage was seen, then a second layer of less mature cancellous bone formed beneath the intact periosteum. Endochondral bone formation was seen proceeding into the intermediate cartilage layer from both layers of cancellous bone (Fig. 1 1). The histological sections suggest that the periosteum was preserved during the operative procedure, and that subperiosteal new bone formation continued after the operation from the stripped periosteum. After the third week subperiosteal new bone formation continued to occur rapidly. Mature looking endochondral bone formation had started adjacent to the old cortical bone and was already quite abundant (Fig. 12). By the end of the fourth week subperiosteal new bone had become abundant and quite mature. In some locations, close to the fractured bone ends, the split made in the cancellous bone beneath the periosteum at operation was still filled with cartilage (Fig. 13). Elsewhere the cartilage had nearly been replaced by endochondral new bone (Fig. 14). All the delayed operation group showed the presence of numerous endochondral ossification centres from which the replacement of cartilage by new bone rapidly occurred. Rapid replacement of cartilage by bone continued through the fifth week, and by the end of the sixth week the callus was almost completely composed of maturing new bone trabeculae (Fig. IS). Essentially, the histological differences in the development of new bone formation between the two groups were as follows. In the immediate operation group the periosteum was extensively destroyed. Subperiosteal new bone formation was slight and a large amount of fibrous tissue and cartilage was formed. This was replaced slowly by bone from a limited number of ossification centres. In the delayed operation group the thickened periosteum was preserved. Subperiosteal new bone formation was the primary source of new bone. Fibrous tissue and cartilage formed, but was very rapidly replaced by new bone from multiple ossification centres. In both groups, because of the intramedullary nailing, endosteal bone formation was slight and did not appear to take a significant part in the healing process. VOL. 49 B, NO. 3, AUGUST 1967

6 576 M. A. EMERY AND H. MURAKAMI :.? FIG. 8 FIG. 9 Figure 8-Seven weeks after an immediate operation cancellous bone has almost completely replaced cartilage and has become the dominant structure ofthe callus. New immature trabeculae still contain cartilage components within bone matrix. Well reorganised fractured bone is seen at the bottom. (Haematoxylin and eosin, x 26.) Figure 9-One week after a delayed operation a layer of haematoma is seen between the fractured bone surface and the preformed cartilage. A small amount of cancellous bone has already formed on the fractured bone surface. (Haematoxylin and eosin, < 40.) ; %r I j y S.. fi. -. FIG. 10 FIG. 11 Figure 10-One week after a delayed operation, a short distance from the fracture site, a thick layer of mature cancellous bone had already formed under the intact periosteum. (l-iaematoxylin and eosin, x 30.) Figure 11- Two weeks after a delayed operation two layers of cancellous bone are seen beneath the periosteum. The deeper layer is attached to the fractured bone surface. Between the two layers fibrous tissue and cartilage has formed. Endochondral bone formation is proceeding from both layers of bone. The periosteal surface is at the top, the fractured bone is at the bottom. (Haematoxylin and eosin, x 26.) THE JOURNAL OF BONE AND JOINT SURGERY

7 FRACTURE HEALING IN CATS AFTER IMMEDIATE AND DELAYED OPEN REDUCTION 577 f.:.d. ;, :,. ( - 0..i_ FIG. 12 FIG. 13 Figure 12-Three weeks after a delayed operation a layer of well matured cancellous bone is seen on the fractured bone surface. Immature new bone is forming adjacent to the mature cancellous bone. Cartilage is seen at the periphery. (Haematoxylin and eosin, x 50.) Figure 13-Four weeks after a delayed operation two layers of cancellous bone are seen in the callus. Between them a layer of cartilage has formed, suggesting the separation of preformed young osteoid tissue by the operation. The split made in the cancellous bone at operation can still be seen to be filled with cartilage The endochondral bone formation has increased and matured. (Haematoxylin and eosin, x 30.) FIG. 14 FIG. 15 Figure 14-Four weeks after a delayed operation a thick layer of well matured cancellous bone is seen on the fractured bone surface. (Haematoxylin and eosin, x 30.) Figure 15-Six weeks after a delayed operation further maturation of the ness bone is seen on the fractured bone surface. Immature new bone is also seen at the periphery of the callus. Between them a thin layer of fibrous tissue is still present. (Haematoxylin and eosin, x30.) VOL. 49 B, NO. 3, AUGUST

8 578 M. A. EMERY AND H. MURAKAMI DISCUSSION In cats delayed union or non-union is very uncommon after any form of internal fixation. The one case of delayed union was noted in the immediate operation group at four weeks. Because of the difficulty in producing non-union in cats by ordinary operative treatment our main consideration was to demonstrate the different mechanisms of healing of fractures after immediate and delayed open reduction, in an attempt to explain the significant clinical differences between the two methods. Histologicalfindings-Two types of bone formation are known to occur in the healing fracture. Direct bone formation occurs under the intact periosteum and from the endosteum. Indirect bone formation occurs where the periosteum has been disrupted and destroyed and is usually seen most prominently around and between the fracture ends. It is characterised initially by the presence of extensive fibrous tissue proliferation and cartilage formation. The cartilage is then replaced with endochondrally formed bone. The initiation of the endochondrally formed bone is usually from the already directly formed bone and takes place slowly by a creeping substitution. Fibrous proliferation and cartilage formation are important stepping stones to endochondral bone formation necessary for the union of fractures. Several recent histological (Ham 1930, Urist and McLean 1941, Koekenberg 1963) and autoradiographical studies (Tonna and Cronkite 1961) on fracture healing have indicated that the main site of osteogenic cell proliferation and bone production after fracture is the osteogenic cell layer of the periosteum. The osteogenic cells are considered to be primitive or slightly differentiated mesenchymal cells (Kember 1960, Young 1962a, b), and their differentiation into definitive cells is largely caused by the environment under which they must reproduce (Young l962b, Murakami and Emery 1966). The condition of the blood supply is most important. It is Ham s (1930, 1952) contention that osteogenic cells differentiate into osteoblasts when the blood supply is sufficient, but when the local blood supply is disturbed they differentiate into chondroblasts. This was confirmed by Koekenberg (1963) in his detailed angiographical and histological studies on tibial fractures in rabbits; after intramedullary nailing the blood vessels inside the marrow space were nearly completely destroyed, and the blood supply to the fracture site consisted of periosteal blood vessels only. He believed that preservation of the periosteum with its soft-tissue connections was most important in the maintenance of an adequate blood supply to the fracture site. Our results confirm the importance of preserving the periosteum. When an operation is done within the first week of fracture the already damaged periosteum, still thin and easily torn, may be destroyed. The blood supply is greatly diminished, giving an inadequate environment for the differentiation of osteoblasts. As a result, a large amount of fibrous tissue and cartilage form. Only when the blood supply is re-established can new bone formation occur ; if differentiation into osteoblasts is delayed or fails delayed union or non-union can result. The delay of two weeks before open operation, providing the fracture is immobilised, keeps the periosteum from further immediate injury. The blood supply improves, and osteogenic cells can grow within the periosteum and can start differentiation into osteoblasts; new bone is formed before the two weeks waiting time has ended. At operation the thickened periosteum is much more resistant to damage and, in spite of the interference at operation, retains its ability to continue to produce new bone, thus providing more advantageous conditions for fracture healing. To elucidate further the difference in vascular supply after immediate and delayed open reduction, micro-angiographical studies are being done. CONCLUSIONS The mechanism of fracture repair in the cat after immediate and delayed open reduction shows distinct differences. Delayed open reduction shows definite advantages: first, the THE JOURNAL OF BONE AND JOINT SURGERY

9 FRACTURE HEALING IN CATS AFTER IMMEDIATE AND DELAYED OPEN REDUCTION 579 periosteum is thick, easily preserved and rapidly develops a lot of periosteal new bone. Second, endochondral bone formation occurs rapidly from multiple ossification centres. These observations support the clinical findings that a delay in open reduction decreases the incidence of non-union. SUMMARY 1. Clinical studies in humans have indicated that a delay of one to three weeks in the open reduction of a fracture decreases the incidence of delayed union and non-union. 2. Studies in cats indicate that a delay of two weeks before open reduction causes a different repair mechanism from that following immediate operation. 3. Repair after delayed operation is characterised by increased periosteal new bone formation and more rapid endochondral bone formation. After immediate operation periosteal new bone is slow to develop; much more fibrous tissue and cartilage develop, followed by slow endochondral bone formation. I wish to thank the Canadian Legion, Edmonton Branch, for their financial support. I also wish to thank Mr E. Burton and Dr A. Axsel of the Surgical-Medical Research Laboratory, University of Alberta, for the photomicrography and histological slides. REFERENCES CHARNLEY, J., and GUINDY, A. (1961): Delayed Operation in the Open Reduction of Fractures of Long Bones. Journal of Bone and Joi,zt Surgery, 43-B, 664. EMERY, M. A. (1965): The Incidence of Delayed Union and Non-Union Following Fractures of Both Bones of the Forearm in Adults. Canadian Journal of Surgery, 8, 285. HAM, A. W. (1930): A Histological Study of the Early Phases of Bone Repair. Journal ofbone andjoint Surgery, 12, 827. HAM, A. W. (1952): Some Histophysiological Problems Peculiar to Calcified Tissues. Journal ofbone and Joint Surgery, 34-A, 701. KEMBER, N. F. (1960): Cell Division in Endochondral Ossification: A Study of Cell Proliferation in Rat Bones by the Method of Tritiated Thymidine Autoradiography. Journal of Bone and Joint Surgery, 42-B, 824. KOEKENBERG, L. J. L. (1963): Vascularisation in the HealingofFractures. Springfield, Illinois: Charles C. Thomas. LAM, S. J. (1964): The Place of Delayed Internal Fixation in the Treatment of Fractures of the Long Bones. Journal of Bone and Joint Surgery, 46-B, 393. MURAKAMI, H., and EMERY, M. A. (1966): Personal conununication. SMITH, J. E. M. (1959): Internal Fixation in the Treatment of Fractures of the Shafts of the Radius and Ulna in Adults. Journal of Bone and Joint Surgery, 41-B, 122. SMITH, J. E. M. (1964): The Results of Early and Delayed Internal Fixation of Fractures of the Shaft of the Femur. Journal of Bone and Joint Surgery, 46-B, 28. TONNA, E. A., and CRONKITE, E. P. (1961): Cellular Response to Fracture Studied with Tritiated Thymidine. Journal of Bone and Joint Surgery, 43-A, 352. URIST, M. R., and MCLEAN, F. C. (1941): Calcification and Ossification: I. Calcification in the Callus in Healing Fractures in Normal Rats. Journal of Bone and Joint Surgery, 23, 1. YOUNG, R. W. (1962a): Regional Differences in Cell Generation Time in Growing Rat Tibiae. Experimental Cell Research, 26, 562. YOUNG, R. W. (l962b): Cell Proliferation and Specialization During Endochondral Osteogenesis in Young Rats. Journal of Cell Biology, 14, 357. VOL. 49 B, NO. 3, AUGUST 1967