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Absorbable membranes for bone repair: An experimental study on rabbits
Clinical Materials 17 (1994) 113-I 18 0 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0267-6605/94/$7Xil
0267-6605(95)00006-2
ELSEVIER
Absorbable Membranes for Bone Repair: An Experimental Study on Rabbits Nureddin Ashammakhi,” Antero M%kel&” Kimmo Vihtonen,’ Pentti Rokkanen” dz Pertti TiirmS$’ ‘Department of Orthopaedics and Traumatology, Helsinki University Central Hospital, Helsinki, Finland bBiomaterials Laboratory, University of Technology, Tampere, Finland (Received 19 August 1994; accepted 23 February
1995)
Abstract: To study the use of absorbable self-reinforced polyglycolide (SR-PGA) membranes for bone repair, distal femoral metaphyseal osteotomies were created in 10 rabbits and diaphyseal osteotomies in five. Osteotomies were fixed with intramedullary PLLA rods and PGA membranes were applied over the osteotomy. They were followed up for 6, 12 and 24 weeks. Radiography, histology, microradiography and oxytetracycline fluorescence labelling studies were used to evaluate the outcome. Healing occurred without complications in the metaphyseal series while failure was recorded in the diaphyseal series. In metaphyseal osteotomies, new bone formation was seen medially (on the side of PGA membrane), more proximal and medial to the membrane than at the inlet of osteotomy.
MATERIALS
INTRODUCTION
AND METHODS
Experimental animals
Polyglycolic acid (PGA) was first synthesized by Bischoff and Walden.’ The first absorbable synthetic sutures have gained a wide acceptance and clinical use.2 Self-reinforced polyglycolic acid (SR-PGA) devices, with adequate mechanical properties, have been applied for fixation purposes since 1984.3-9 Biocompatibility of PGA devices in animal and human tissues has been well documented.‘0-‘2 The SR-PGA membranes have been studied earlier by the authors.13”4 They appeared to be biocompatible with osseous tissu:sand to have a good effect on The application of these new bone formation. membranes was considered and a series of animal experiments was carried out.16 In this study membranes were used for the repair of osteotomies in rabbits. The results were encouraging in association with the repair of metaphyseal osteotomies but not with diaphyseal osteotomies.
Fourteen male and seven female New Zealand rabbits, 16-48 (average 33) weeks old, 3000-5050 (average 3900) g weight were used in this study. Implants Self-reinforced polyglycolic acid (SR-PGA) membranes of size 10 by 20 mm, 10 by 50 mm or 30 by 40 mm and 0.15 mm thick were used. The membranes were sterilized by ethylene oxide, thoroughly degassed, supplied in packages, each containing one piece (Biocon Ltd., Finland). The membrane packages were opened just before use in the operation theatre, according to the instruction of the manufacturer. Membranes were used as supplied or divided during operations into 5 by 10 mm and 10 by 10 mm. Self-reinforced 113
114
N. Ashammakhi
Table 1. Plan of experiments Operated bone
Follow up in weeks 6
12
24
Total
(1) Diaphysis (2) Metaphysis
_ 3
_ 3
5 4
5 10
Total
3
3
9
15
poly-L-lactide (SR-PLLA) rods of 4.5 mm diameter and 50 or 60mm long, cut during operations 410mm shorter, according to the length of femur, were used to fix the osteotomies. Surgical technique The rabbits were given 0.5 mg/kg atropine (AtropinR, Orion, Espoo, Finland) subcutaneously (s.c.) as premeditation and they were anaesthetized with S.C. medetomidine Laakefarmos, (Domitor@, Turku, Finland) 0.3 mg/kg, ketamine (Ketalar@, Parke-Davis, Barcelona, Spain) 0.4 mg/kg, and diazepam (Diapam@) 0.2 mg/g.17 Procaine penicillin (Procapen@, Orion, Espoo, Finland) 150 000 IU S.C.injections were also given. Softening eye drops (Oftan @ MC, Leiras, Turku, Finland) were applied to the rabbits’ eyes before operations to protect their corneas. The right hindthigh was shaved and scrubbed with antimicrobial solution (Neo-Amisept@). The rabbits were operated on in an experimental animal operation theatre, under aseptic conditions. Through a medial parapatellar approach the right knee was opened, the patella was dislocated laterally and the distal diaphysis of the femur exposed. A drill channel of 4*5mm in diameter was made through the intercondylar space into the femur. A transverse osteotomy was created using a circular saw (blade thickness of 0.15 mm), across the channel, not involving the posterior cortex (in three cases it did). Another oblique osteotomy joining proximally the transverse osteotomy was created, to form a wedge-shaped bone fragment on the ventral aspect of the femur. The osteotomy was fixed using an intramedullary SR-PLLA rod driven into the drill channel. PGA membrane was applied over the osteotomy and kept in place using a 3-O or 4-O USP PGA (Dexon @, Davis + Geck) wire (series 1). In series 2, the metaphysis was exposed, the same steps were followed, as in series 1, except that the
et al.
small fragment was not created. In addition, 1-O PGA thread was applied through transverse holes made proximal and distal to the osteotomy, and tied around to ensure fixation. Wounds were closed by Dexon@ sutures in both series. During drilling or applying the rod fractures resulted in six rabbits. These were stabilized by multiple circular Dexon@ cerclage. Postoperatively, the rabbits were returned to their cages and were given normal laboratory animal diet and care. They were left to use their limbs freely without any external support. Rabbits were followed up for 6, 12 and 24 weeks (Table 1). During the follow-up time attention was directed towards the rabbits’ general condition, wound healing and their use of the operated limb. Two days before killing the rabbits were given oxytetracycline (50 mg/kg body weight) for fluorescence studies. The rabbits were anaesthetized with Domitor@ and killed by intravenous air embolism. In the operation theatre, both femora with implants were excised en bloc and inspected for gross abnormalities.
Radiography After killing, radiographs were taken in anteroposterior and lateral projections. The technical values were 40 kV, 4mAs for 0.08 s, and 90 cm film focus distance. The aim was to evaluate the healing of osteotomies at the membrane side and on the opposite side, as well as any other radiographical changes.
Histology The distal parts of femurs were taken as specimens and were dehydrated by bathing successively in a graded series of 50% to absolute alcohol” which was replaced by xylene and later embedded successively in methylmethacrylate I-III.19 For histological analysis longitudinal sections of 5 pm were made using a Reichert-Jung microtome (Polycut S, 1983, Nussloch, West Germany) and stained by a modified Masson-Goldner method.*’ A (Leitz) light microscope was used for examination of the slides. Polarized light lenses were used to visualize the PGA fibres. Union was checked to see whether it occurred and whether it was fibrous, osteochondral or bony union. The effect of the implant on healing of the osteotomies was also examined.
Absorbable membranes for bone repair
Radiography
Table 2. Radiological evaluation of the osteotomy healing Union
Follow up in weeks 6
12
24
Total
1. Diaphysis Radiographic union Possible union Dislocation Nonunion External callusa
_ _ _ _
_ _ _
0 1 2 4 4
0 1 2 4 4
2. Metaphysis Radiographic union Possible union Dislocation Nonunion External callusb
1 1 1 0 2
3
4
0 0 3
0 0 2
7 1 1 0 7
Total a Mostly as collars but not a complete callus. b Callus seen more abundantly on the medial side (membrane side).
Microradiography fluorescence
and
oxytetracycline
115
(OTC)
Sections of 80 pm were cut with a Leitz saw microtome 1600 (Ernst Leitz GMBH Wetzlar, West Germany). Kodak Spectroscopic Plate Type 649-O (Eastman Kodak, Rochester, New York) films were used. Contact microradiographs were taken from these sections. Technical values were 21 kV for 15min. Using OTC (Terramycin@), as a labelling fluorochrome, the rabbits were injected subcutaneously 2 days before their killing (50mg/kg body weight). Longitudinal sections of 8Opm thick were examined microscopically under ultraviolet light to confirm the new bone observation.*l
RESULTS Diaphyseal fixation failed and bones dislocated in two rabbits on the second postoperative day, on the fifth postoperative day in one rabbit, after 5 weeks in one rabbit and after 9 weeks in another. Therefore, the rabbits were killed. Three of those rabbits already had intraoperative fractures. One week after the operation wound dehiscence occurred in two rabbits from the metaphyseal series, wounds were debrided, restitched and 150 000 IU s.c. penicillin given. At necropsy, in one rabbit (metaphyseal series), the right knee was found to be swollen and the patella dislocated laterally.
Radiographs taken from the diaphyseal osteotomies after 1 week showed fracture of the proximal or distal fragments but not complete dislocation. Radiographs taken from the same rabbits 2 months postoperatively showed nonunion that was clinically detectable. After 24 weeks, all but one diaphyseal osteotomies were dislocated and not united. External callus appeared as advancing collars which were not joined to each other and appeared sometimes to be funnel-like (Table 2). Radiographically rarefraction could be observed in the medulla, corresponding to the fixation PLLA rod, with no periosteal callus opposite to the membrane, while callus was seen abundantly proximal and distal to the area, corresponding to the membrane, with no evidence of internal callus as well. On palpation the callus could be felt. At 6 weeks, one metaphyseal osteotomy had dislocated laterally and posteriorly. Other cases united and showed external callus, more abundant on the medial side, proximal and distal to the membrane. After 12 and 24 weeks, all osteotomies had united. Histology Diaphyseal osteotomies showed fibrous union at 24 weeks. In one case, there was no sign of union at all and in another case malunion occurred. All metaphyseal osteotomies showed histological union (Table 2). Periosteal bone formation at the osteotomy site was seen more on the lateral side while new bone formation proximal and distal to the membrane was seen on the medial side. Membranes could be seen at 6 weeks and their remnants at 12 weeks (Fig. 1) and, in some cases, at 24 weeks; PLLA rods could be seen throughout the follow-up period with signs of degradation at 24 weeks. PGA threads used to ensure the fixation of the metaphyseal osteotomies were clearly seen at 6 weeks traversing the bone. The membrane did not always stay in intimate contact with the metaphyseal bone and where it did, periosteal osteoblastic proliferation could be seen, especially at 12 weeks. Microradiography and OTC fluorescence Results have confirmed the histological observations of new bone formation. OTC uptake was greater at areas showing new bone formation.
N. Ashammakhi
116
et al.
Table 3. Histological evaluation of osteotomy healing Union
Follow up in weeks 6
12
24
Total
(1) Diaphysis No sign of union Fibrous union Osteochondral union Bony union
_ _
_ _
1 3 0 0
1 3 0 0
(2) Metaphysis No sign of union Fibrous union Osteochondral union Bony union Peristeal new bone’
0 0 0 3 1
0 0 0 3 2
0 0 0 4 3
0 0 0 10 6
Total ’ New bone formation that was seen clearly on the medial side of osteotomy, opposite to the membrane, more than on the lateral side.
Fig. 1. A micrograph from a longitudinal section of femoral metaphyseal bone at 12 weeks after osteotomy fixed with PLLA rod and implantation of SR-PGA membrane over the bone. Osteotomy has healed. Note external callus (EC) and remnants of PGA membrane (arrows).
DISCUSSION
Rods made of PLA are superior to those made of PGA in their strength retention.22 They were successfully used in the fixation of nonweight bearing cancellous bone fractures and osteotomies;23 less satisfactory results being associated with their use in the fixation of weight bearing shaft fractures of long bones.24 Rotational deformities occurred frequently25 and absorbable threads have been used as an adjunct fixation device.26 In this study, in one case, the rod had passed the osteotomy, the PGA thread served as the only fixation device and the bone united without complications. Failures again occurred with the fixation of the diaphyseal but not with the metaphyseal osteotomies, due to biomechanical procedural reasons (threads
were not used in the fixation of diaphysis and lixation was less stable). The creation of bone fragment by a second osteotomy in the diaphyseal series, probably lessened the approximation of bone fragments as well as the fixation rigidity and stability, resulting in failures. The nonunions of diaphyseal osteotomies could also be a result of the interference with an adequate blood supply to the bone and hence with the blood supply of the fracture and the callus that forms consequently in normal situations. Medullary blood supply to the diaphyseal cortex is expected to be damaged by reaming.27 Periosteum remains, in such situations, as the most important source for revascularization of the diaphyseal cortex.28-30 The periosteum depends greatly on its connection to the muscles for blood supply, through the musculoperiosteal system of vessels.31232The PGA membrane was applied between the muscle and the periosteum after their separation, a procedure which could be criticized for affecting the periosteal bloc$s;pply33 and adversely affecting bone healThe importance of a viable periosteum ing. lies also in its function of sealing the fracture gap and preventing fibrous infiltration from the surrounding soft tissues.37 It forms advancing collars of callus that ultimately bridge the gap. But, as the membrane may also have interfered mechanically, and prevented the collars from joining up to each other, the gap was not sealed and fibrous tissue was able to infiltrate faster. Osteoblasts proliferate vigorously following fracture,38 and the callus forms. Vasodilatation and increased blood flow
Absorbable
membranes
precede the osteogenic process,39 so, adequate oxygenation and nutrient supply are ensured.“,41 But, since the revascularization was probably poor, osteogenesis was somehow not successful. The metaphyseal bone has a better vessel anastomoses, and it has been proved that combined medullary reaming and periosteal separation does not seriously affect the blood flow in the metaphysis as it does in the diaphysis.34 This can explain, in part, the observation of nonunions in this study seen in diaphysis but not in metaphysis.
CONCLUSIONS
The SR-PGA membranes can be successfully used over metaphyseal osteotomies in the rabbit distal femur, fixed with intramedullary PLLA rods. This can be of benefit for bone repair when there is an associated bone fragment along with the osteotomy (fracture).
ACKNOWLEDGEMENTS
The research grants of The Academy of Finland and Finnish Orthopedic Foundation to support this research are greatly appreciated.
REFERENCES 1. B&off, C. A. & Walden, P., cited by Higgins, N. A., US Patent No. 2,676,945, 1954. 2. Frazza, E. J. & Schmitt, E. E., A new absorbable suture. J. Biomed, Mater. Res. Symp. 1 (1971) 43-58 3. Rokkanen, P., Vainionpal, S., T&mall, P., Kilpikari, J., Biistman, O., Vihtonen, K., Laiho, I. & Tammimaki, M., Biodegradable implants in fracture fixation: Early results of treatment of fractures of the ankle. The Lancet, 22 June 1985, 1422-4. 4. Biistman, O., Vainionpaa, S., Hirvensalo, E., Make& A., Vihtonen K., Tiirmala, P. & Rokkanen, P., Biodegradable intenal fixation for malleolar fractures. A prospective randomized trial. J. Bone Jt Surg., 69-B (1987) 615-9. 5. Biistman, O., Makehi, E. A., Tiirmlla, P. & Rokkanen, P., Transphyseal fracture fixation using biodegradable pins. J. Bone Jt Surg., 71-B (1989) 706-7. 6. Hirvensalo, E., Fracture fixation with biodegradable rods. Forty-one cases of severe ankle fractures. Acta Orthop. Stand., 60 (1989) 601-6. 7. Hirvensalo, E., Bostman, 0. & Rokkanen, P., Absorbable
polyglycolide pins in fixation of displaced fractures of the radial head. Arch. Orthop. Trauma Surg., 109 (1990) 25861. 8. Hirvensalo,
E., Biistman, O., VainiopSiP, S., Tiirmila, P. & Rokkanen, P., Biodegradable fixation in chevron osteotomy
117
for bone repair
for hallwr valgus. Acta Orthop. Stand. (Suppl. 227), 59 (1988) 71. 9. Rokkanen, P., Biistman, O., Make@ E. A., Hirvensalo, E., Partio, E., P&till& H., Vihtonen, K., Vainionpaa, S. & Tiirmlla, P., Eight-year experience of the use of absorbable fracture fixation devices. XXVZZZCongress of the European Society for Surgical Research (ESSR), Turku, Finland, 23-26 May 1993, Abstract 198. 10. VainionpM, S., Biodegradation of polyglycolic acid in bone tissue: an experimental study on rabbits. Arch. Orthop. Trauma Surg., 104 (1986) 333-8. 11. Makela, E. A., Fixation properties and biodegradation of absorbable implants in growing bone. An experimental and clinical study. Academic dissertation, Helsinki University, Finland, 1989. 12. Rokkanen, P., Bostman, O., Hirvensalo, E., Vainionpla, S. & Tiirmala, P., Three years’ audit of biodegradable osteofixation in orthopedic surgery. Acta Orthop. Stand., (Suppl. 227), 59 (1988) 18-19. 13. Ashammakhi, N. A., Makehi, E. A., Vihtonen, K., Rokkanen, P. & T6rmHlH, P., The behaviour of SR-PGA membranes implanted around cortical bone. An experimental study on rats. XXVIII Congress of the European Society for Surgical Research (ESSR), Turku, Finland, 23-26 May 1993. 14. Ashammakhi, N. A., MlikelH, E. A., Vihtonen, K., Rokkanen, P., Kuisma, H. & Tormala, P., Strength retention of self-reinforced polyglycolide (SR-PGA) membrane. An exnerimental studv on rats. XXIX Congress of the European Society for Surgical Research (ESSR) , M”ontpellier, France, 16-19 May 1994. Abstract No 182. 15. Ashammakhi, N., Make& E. A., Vihtonen, K., Rokkanen, P. & T&mall, P., The effect of self reinforced polyglycolide (SR-PGA) membrane on cancellous bone. An experimental study on rats. Combined Meeting of the European Orthopaedie Research Society (EORS) and British Research Society (BORS), London, UK, 11-12 April, 1994.
16. Ashammakhi, N. A., Make@ E. A., Vihtonen, K., Rokkanen, P. & Tormala, P., Absorbable polyglycolide membranes. Behaviour and effect on bone. Acta Orthop. Stand., (Suppl. 260) 65 (1994) 20. 17. Mero, M., Vainionpaa, S., Vasenius, J., Vihtonen, K. & Rokkanen, P., Meditomidine-letamine-diazepam anaesthesia in the rabbit. Acta Vet. Stand., 85 (1989) 135-7. 18. Junqueira, L. C. & Carneiro, J., Methods of study. In Basic Histology, 3rd Edn. Lange Medical Publications, Los Altos, California, 1980, pp. 1-17. 19. Schenk, R., cited by Makela, E. A., VainionpHB, S., Vihtonen, K., Mero, M., Helevirta, P., Tormala, P. & Rokkanen, P., The effect of a diagonally placed penetrating biodegradable implant on the epiphyseal plate. An experimental study on growing rabbits with special regard to polydioxanone. Clin. Mater., 3 (1988) 223333. 20. Goldner, J., A modification of the Masson trichrome technique for routine laboratory purposes. Am. J. Pathol., 14 (1938) 237-43.
21. Milch, R. A., Rall, D. P. & Tobie, J. E., Fluorescence of tetracycline antibiotics in bone. J. Bone Jt Surg., 40-A (1958) 897-910.
22. Vainionpai, applications
S., Rokkanen, P. & Tormala, P. Surgical of biodegradable polymers in human tissues.
Prog. Polym. Sci., 14 (1989) 679-716.
23. Majola, A., Vainionpaa, S., Vihtonen, K., Mero, M., Vasenius, J., Rokkanen, P. & T&mail& P. Absorption, biocompatibility, and fixation properties of polylactic acid in bone tissue: An experimental study in rats. Clin. Orthop., 268 (1991) 266-9.
24. Eitenmuller, J., cited by Mannen, M., Intramedullary
poly-
118
25.
26.
21.
28.
29.
30.
N. Ashammakhi lactide rods in fixation of cortical bone osteotomies. Acta Orthop. Stand. (Suppl. 248), 63 (1992) 80. Miettinen, H., Make& E. A., Rokkanen, P. & TiirmHla, P., Fixation of femoral shaft osteotomy with intramedullary metallic or absorbable rod: an experimental study on growing dogs. J. Biomater. Sci. Polym. Ed., 4 (1992) 135-43. Vasenius, J., Vainionplii, S., Vihtonen, K., Mero, M., Make@ A., Tiirmala, P. & Rokkanen, P., A histomorphological study on self-reinforced polyglycolide (SR-PGA) osteosynthesis implants coated with slowly absorbable polymers. J. Biomed. Mater. Res., 24 (1990) 1615-35. McKibbin, B., Repair of fractures. In Watson-Jones Fractures and Joint Injuries, Vol. I, 6th Edn, ed. J. N. Wilson. Churchil Livingstone, Edinburgh, 1982, pp. 14-28. Gothman, L., The normal arterial pattern of the rabbit’s tibia. A microangiographic study. Acta Chir. Stand., 120 (1960) 201-10. Giithman, L., The arterial pattern of the rabbit’s tibia after the application of an intramedullary nail. A microradiographic study. Acta Chir. Stand., 120 (1960) 21 l-9. Reichert, I. L. H., McCarthy, I. D. & Hughes, S. P. F., The acute vascular response to intramedullary reaming of the tibia. Combined Meeting of the European Orthopaedic Research Society (EORS) and British Orthopaedic Research Society (BORS), London, 11-12 April, 1994,
Abstract 41. 31. Zucman, J., Studies on the vascular connections between periosteum, bone and muscle. Part one. Br. J. Surg., 48, (1960) 324-8. 32. Whiteside, L., Circulation in the bone. In Surgery of the
et al. Musculoskeletal
33. 34.
35.
36. 37.
38.
39.
40. 41.
System. Evarts, Churchil Livingstone Inc., USA, 1983, pp. 1:51-1:63. Whiteside, L. A. & Lesker, P. A., The effects of extraperiosteal and subperiosteal dissection. II. On fracture healing. J. Bone Jt Surg., 60-A (1978) 26-30. Rhinelander, F. W., Tibia1 blood supply in relation to fracture healing. Clin. Orthop., 105, (1974) 34-81. Whiteside, L. A., Ogata, K., Lesker, P. & Reynolds, F. C., The acute effects of periosteal stripping and medullary reaming on regional bone blood flow. Clin. Orthop., 131 (1978) 266-72. Eyre-Brook, L., The periosteum: Its function reassessed. Clin. Orthop., 189 (1984) 300-7. Macnab, I. & De Haas, W.G., The role of periosteal blood supply in the healing of fractures of the tibia. Clin. Orthop., 105 (1974) 27-33. Tonna, E. A. & Cronkite, E. P., Autoradiographic studies of cell proliferation in the periosteum of intact and fractured femora of mice utilizing DNA labelling with H3-thymidine. Proc. Sot. Exp. Biol. Med., 107 (1961) 719-21. Wray, J. B., Periosteal vessel changes in the immediate postfracture period. Surg. Gynaecol. Obestet., 117 (1963) 311-4. Ketenjian, A. Y. & Arsenis, C., Morphological and biochemical studies during differentiation and calcification of fracture callus cartilage. Clin Orthop., 107 (1975) 266-73. Kuhlman, R. E. & Bakowski, M. J., The biochemical activity of fracture callus in relation to bone production. Clin. Orthop., 107 (1975) 258-65.
0267-6605(95)00006-2
ELSEVIER
Absorbable Membranes for Bone Repair: An Experimental Study on Rabbits Nureddin Ashammakhi,” Antero M%kel&” Kimmo Vihtonen,’ Pentti Rokkanen” dz Pertti TiirmS$’ ‘Department of Orthopaedics and Traumatology, Helsinki University Central Hospital, Helsinki, Finland bBiomaterials Laboratory, University of Technology, Tampere, Finland (Received 19 August 1994; accepted 23 February
1995)
Abstract: To study the use of absorbable self-reinforced polyglycolide (SR-PGA) membranes for bone repair, distal femoral metaphyseal osteotomies were created in 10 rabbits and diaphyseal osteotomies in five. Osteotomies were fixed with intramedullary PLLA rods and PGA membranes were applied over the osteotomy. They were followed up for 6, 12 and 24 weeks. Radiography, histology, microradiography and oxytetracycline fluorescence labelling studies were used to evaluate the outcome. Healing occurred without complications in the metaphyseal series while failure was recorded in the diaphyseal series. In metaphyseal osteotomies, new bone formation was seen medially (on the side of PGA membrane), more proximal and medial to the membrane than at the inlet of osteotomy.
MATERIALS
INTRODUCTION
AND METHODS
Experimental animals
Polyglycolic acid (PGA) was first synthesized by Bischoff and Walden.’ The first absorbable synthetic sutures have gained a wide acceptance and clinical use.2 Self-reinforced polyglycolic acid (SR-PGA) devices, with adequate mechanical properties, have been applied for fixation purposes since 1984.3-9 Biocompatibility of PGA devices in animal and human tissues has been well documented.‘0-‘2 The SR-PGA membranes have been studied earlier by the authors.13”4 They appeared to be biocompatible with osseous tissu:sand to have a good effect on The application of these new bone formation. membranes was considered and a series of animal experiments was carried out.16 In this study membranes were used for the repair of osteotomies in rabbits. The results were encouraging in association with the repair of metaphyseal osteotomies but not with diaphyseal osteotomies.
Fourteen male and seven female New Zealand rabbits, 16-48 (average 33) weeks old, 3000-5050 (average 3900) g weight were used in this study. Implants Self-reinforced polyglycolic acid (SR-PGA) membranes of size 10 by 20 mm, 10 by 50 mm or 30 by 40 mm and 0.15 mm thick were used. The membranes were sterilized by ethylene oxide, thoroughly degassed, supplied in packages, each containing one piece (Biocon Ltd., Finland). The membrane packages were opened just before use in the operation theatre, according to the instruction of the manufacturer. Membranes were used as supplied or divided during operations into 5 by 10 mm and 10 by 10 mm. Self-reinforced 113
114
N. Ashammakhi
Table 1. Plan of experiments Operated bone
Follow up in weeks 6
12
24
Total
(1) Diaphysis (2) Metaphysis
_ 3
_ 3
5 4
5 10
Total
3
3
9
15
poly-L-lactide (SR-PLLA) rods of 4.5 mm diameter and 50 or 60mm long, cut during operations 410mm shorter, according to the length of femur, were used to fix the osteotomies. Surgical technique The rabbits were given 0.5 mg/kg atropine (AtropinR, Orion, Espoo, Finland) subcutaneously (s.c.) as premeditation and they were anaesthetized with S.C. medetomidine Laakefarmos, (Domitor@, Turku, Finland) 0.3 mg/kg, ketamine (Ketalar@, Parke-Davis, Barcelona, Spain) 0.4 mg/kg, and diazepam (Diapam@) 0.2 mg/g.17 Procaine penicillin (Procapen@, Orion, Espoo, Finland) 150 000 IU S.C.injections were also given. Softening eye drops (Oftan @ MC, Leiras, Turku, Finland) were applied to the rabbits’ eyes before operations to protect their corneas. The right hindthigh was shaved and scrubbed with antimicrobial solution (Neo-Amisept@). The rabbits were operated on in an experimental animal operation theatre, under aseptic conditions. Through a medial parapatellar approach the right knee was opened, the patella was dislocated laterally and the distal diaphysis of the femur exposed. A drill channel of 4*5mm in diameter was made through the intercondylar space into the femur. A transverse osteotomy was created using a circular saw (blade thickness of 0.15 mm), across the channel, not involving the posterior cortex (in three cases it did). Another oblique osteotomy joining proximally the transverse osteotomy was created, to form a wedge-shaped bone fragment on the ventral aspect of the femur. The osteotomy was fixed using an intramedullary SR-PLLA rod driven into the drill channel. PGA membrane was applied over the osteotomy and kept in place using a 3-O or 4-O USP PGA (Dexon @, Davis + Geck) wire (series 1). In series 2, the metaphysis was exposed, the same steps were followed, as in series 1, except that the
et al.
small fragment was not created. In addition, 1-O PGA thread was applied through transverse holes made proximal and distal to the osteotomy, and tied around to ensure fixation. Wounds were closed by Dexon@ sutures in both series. During drilling or applying the rod fractures resulted in six rabbits. These were stabilized by multiple circular Dexon@ cerclage. Postoperatively, the rabbits were returned to their cages and were given normal laboratory animal diet and care. They were left to use their limbs freely without any external support. Rabbits were followed up for 6, 12 and 24 weeks (Table 1). During the follow-up time attention was directed towards the rabbits’ general condition, wound healing and their use of the operated limb. Two days before killing the rabbits were given oxytetracycline (50 mg/kg body weight) for fluorescence studies. The rabbits were anaesthetized with Domitor@ and killed by intravenous air embolism. In the operation theatre, both femora with implants were excised en bloc and inspected for gross abnormalities.
Radiography After killing, radiographs were taken in anteroposterior and lateral projections. The technical values were 40 kV, 4mAs for 0.08 s, and 90 cm film focus distance. The aim was to evaluate the healing of osteotomies at the membrane side and on the opposite side, as well as any other radiographical changes.
Histology The distal parts of femurs were taken as specimens and were dehydrated by bathing successively in a graded series of 50% to absolute alcohol” which was replaced by xylene and later embedded successively in methylmethacrylate I-III.19 For histological analysis longitudinal sections of 5 pm were made using a Reichert-Jung microtome (Polycut S, 1983, Nussloch, West Germany) and stained by a modified Masson-Goldner method.*’ A (Leitz) light microscope was used for examination of the slides. Polarized light lenses were used to visualize the PGA fibres. Union was checked to see whether it occurred and whether it was fibrous, osteochondral or bony union. The effect of the implant on healing of the osteotomies was also examined.
Absorbable membranes for bone repair
Radiography
Table 2. Radiological evaluation of the osteotomy healing Union
Follow up in weeks 6
12
24
Total
1. Diaphysis Radiographic union Possible union Dislocation Nonunion External callusa
_ _ _ _
_ _ _
0 1 2 4 4
0 1 2 4 4
2. Metaphysis Radiographic union Possible union Dislocation Nonunion External callusb
1 1 1 0 2
3
4
0 0 3
0 0 2
7 1 1 0 7
Total a Mostly as collars but not a complete callus. b Callus seen more abundantly on the medial side (membrane side).
Microradiography fluorescence
and
oxytetracycline
115
(OTC)
Sections of 80 pm were cut with a Leitz saw microtome 1600 (Ernst Leitz GMBH Wetzlar, West Germany). Kodak Spectroscopic Plate Type 649-O (Eastman Kodak, Rochester, New York) films were used. Contact microradiographs were taken from these sections. Technical values were 21 kV for 15min. Using OTC (Terramycin@), as a labelling fluorochrome, the rabbits were injected subcutaneously 2 days before their killing (50mg/kg body weight). Longitudinal sections of 8Opm thick were examined microscopically under ultraviolet light to confirm the new bone observation.*l
RESULTS Diaphyseal fixation failed and bones dislocated in two rabbits on the second postoperative day, on the fifth postoperative day in one rabbit, after 5 weeks in one rabbit and after 9 weeks in another. Therefore, the rabbits were killed. Three of those rabbits already had intraoperative fractures. One week after the operation wound dehiscence occurred in two rabbits from the metaphyseal series, wounds were debrided, restitched and 150 000 IU s.c. penicillin given. At necropsy, in one rabbit (metaphyseal series), the right knee was found to be swollen and the patella dislocated laterally.
Radiographs taken from the diaphyseal osteotomies after 1 week showed fracture of the proximal or distal fragments but not complete dislocation. Radiographs taken from the same rabbits 2 months postoperatively showed nonunion that was clinically detectable. After 24 weeks, all but one diaphyseal osteotomies were dislocated and not united. External callus appeared as advancing collars which were not joined to each other and appeared sometimes to be funnel-like (Table 2). Radiographically rarefraction could be observed in the medulla, corresponding to the fixation PLLA rod, with no periosteal callus opposite to the membrane, while callus was seen abundantly proximal and distal to the area, corresponding to the membrane, with no evidence of internal callus as well. On palpation the callus could be felt. At 6 weeks, one metaphyseal osteotomy had dislocated laterally and posteriorly. Other cases united and showed external callus, more abundant on the medial side, proximal and distal to the membrane. After 12 and 24 weeks, all osteotomies had united. Histology Diaphyseal osteotomies showed fibrous union at 24 weeks. In one case, there was no sign of union at all and in another case malunion occurred. All metaphyseal osteotomies showed histological union (Table 2). Periosteal bone formation at the osteotomy site was seen more on the lateral side while new bone formation proximal and distal to the membrane was seen on the medial side. Membranes could be seen at 6 weeks and their remnants at 12 weeks (Fig. 1) and, in some cases, at 24 weeks; PLLA rods could be seen throughout the follow-up period with signs of degradation at 24 weeks. PGA threads used to ensure the fixation of the metaphyseal osteotomies were clearly seen at 6 weeks traversing the bone. The membrane did not always stay in intimate contact with the metaphyseal bone and where it did, periosteal osteoblastic proliferation could be seen, especially at 12 weeks. Microradiography and OTC fluorescence Results have confirmed the histological observations of new bone formation. OTC uptake was greater at areas showing new bone formation.
N. Ashammakhi
116
et al.
Table 3. Histological evaluation of osteotomy healing Union
Follow up in weeks 6
12
24
Total
(1) Diaphysis No sign of union Fibrous union Osteochondral union Bony union
_ _
_ _
1 3 0 0
1 3 0 0
(2) Metaphysis No sign of union Fibrous union Osteochondral union Bony union Peristeal new bone’
0 0 0 3 1
0 0 0 3 2
0 0 0 4 3
0 0 0 10 6
Total ’ New bone formation that was seen clearly on the medial side of osteotomy, opposite to the membrane, more than on the lateral side.
Fig. 1. A micrograph from a longitudinal section of femoral metaphyseal bone at 12 weeks after osteotomy fixed with PLLA rod and implantation of SR-PGA membrane over the bone. Osteotomy has healed. Note external callus (EC) and remnants of PGA membrane (arrows).
DISCUSSION
Rods made of PLA are superior to those made of PGA in their strength retention.22 They were successfully used in the fixation of nonweight bearing cancellous bone fractures and osteotomies;23 less satisfactory results being associated with their use in the fixation of weight bearing shaft fractures of long bones.24 Rotational deformities occurred frequently25 and absorbable threads have been used as an adjunct fixation device.26 In this study, in one case, the rod had passed the osteotomy, the PGA thread served as the only fixation device and the bone united without complications. Failures again occurred with the fixation of the diaphyseal but not with the metaphyseal osteotomies, due to biomechanical procedural reasons (threads
were not used in the fixation of diaphysis and lixation was less stable). The creation of bone fragment by a second osteotomy in the diaphyseal series, probably lessened the approximation of bone fragments as well as the fixation rigidity and stability, resulting in failures. The nonunions of diaphyseal osteotomies could also be a result of the interference with an adequate blood supply to the bone and hence with the blood supply of the fracture and the callus that forms consequently in normal situations. Medullary blood supply to the diaphyseal cortex is expected to be damaged by reaming.27 Periosteum remains, in such situations, as the most important source for revascularization of the diaphyseal cortex.28-30 The periosteum depends greatly on its connection to the muscles for blood supply, through the musculoperiosteal system of vessels.31232The PGA membrane was applied between the muscle and the periosteum after their separation, a procedure which could be criticized for affecting the periosteal bloc$s;pply33 and adversely affecting bone healThe importance of a viable periosteum ing. lies also in its function of sealing the fracture gap and preventing fibrous infiltration from the surrounding soft tissues.37 It forms advancing collars of callus that ultimately bridge the gap. But, as the membrane may also have interfered mechanically, and prevented the collars from joining up to each other, the gap was not sealed and fibrous tissue was able to infiltrate faster. Osteoblasts proliferate vigorously following fracture,38 and the callus forms. Vasodilatation and increased blood flow
Absorbable
membranes
precede the osteogenic process,39 so, adequate oxygenation and nutrient supply are ensured.“,41 But, since the revascularization was probably poor, osteogenesis was somehow not successful. The metaphyseal bone has a better vessel anastomoses, and it has been proved that combined medullary reaming and periosteal separation does not seriously affect the blood flow in the metaphysis as it does in the diaphysis.34 This can explain, in part, the observation of nonunions in this study seen in diaphysis but not in metaphysis.
CONCLUSIONS
The SR-PGA membranes can be successfully used over metaphyseal osteotomies in the rabbit distal femur, fixed with intramedullary PLLA rods. This can be of benefit for bone repair when there is an associated bone fragment along with the osteotomy (fracture).
ACKNOWLEDGEMENTS
The research grants of The Academy of Finland and Finnish Orthopedic Foundation to support this research are greatly appreciated.
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