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Osteogenic ability of free perichondreal autografts in canine tibial defects: An experimental study
Experimental Animal Science
Osteogenic ability of free perichondreal autografts in canine tibial defects: An experimental study* M. AR1CAN2, M. ORTATATL1l, K. YI~ITARSLAN 2 and C. CEYLAN2 1Department of Pathology, Faculty of Veterinary Science, The University of Selguk, 42031 Campus/Konya Turkey 2Department of Surgery, Faculty of Veterinary Science, The University of Selquk, 42031 Campus/Konya Turkey
Summary This study set out to establish the effect of transplanting perichondreum on bone healing at sites of ribial bone defects in an experimental dog model. Transplantation of free, autologous, non-vascularised, perichondreal grafts to the distal of right anteromedial plane side of the tibia was compared with non-transplantation on the proximal side of the same bone. In experimental dogs (n = 7), a 5 cm piece segment of perichondreum, that has been excised from the thirteenth rib of the same animal, was transplanted to the midddle defect fracture site of bone, but not to the control proximal defect fracture site. The dogs were allowed to recover from the operation and were kept 21 days in cages, with freerange. On days 30 (Group I) and 45 (Group II) after operations, the dogs were euthanatized. Histopathologically, defects in 30 days treated perichondreum group were filled by new ossified tissue while control defects in the same period were not fully resurface& The new ossified tissue consisted of a thin and inadequate trabeculae. In 45 days treated groups, defects with transplanting perichondreum were filled by thick trabeculae converting into a compact bone tissue. The control defects of this group, however, were filled by an extreme callus overflowing to medulla and bone surface. This study has provided evidence to show that autologous, non-vascularized perichondreum retains an osteogenic ability when transplanted to tibial bone defect sites. It appears that callus formarion occurred within the perichondreum grafting which resembles that of enchondral and intramembranous ossification.
Key words: Canine, tibia, perichondreal grafts *All procedures have been performed in accordance with national or local animal welfare legislation or respectively is based on the European Council Directive. J. Exp. Anim. Sci. 2003; 42:203-217 Urban & Fischer Verlag http://www.urbanfischer.de/joumals/jeansc 0939-8600/03/42/04-203 $15.00/0
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Introduction Healing of fractures not simply is a process via scar tissue healing but actually also depends on a process of bone regeneration (OLuSOLAand GREGG, 1991). For instance, in many reports on the bone fracture healing process, it has been suggested that the surrounding soft tissues contribute a significant number of osteogenic cells to the fracture callus (FRosT, 1989). It has long been recognised that the healing of bone fractures is related greater decrease the rate of bone healing to the severity of associated bone and soft tissue damage (DoI and SAKAI,1994). Severe soft-tissue loss with periosteal detachment and compromised vascularity, rather than fracture configuration, are major determinants of the outcome of untreated fractures. All the conventional methods of fracture repair may be applied to fractures of the tibia. Simple fractures may be effectively treated with an intramedullary pin, with or without a combination of cerclage or meni- cerclage wire, plates or Kirschner splints (ENGKVIST,1979; DE YOUNGand PROBST, 1993) In this study, our aim was to improve the osteogenic ability of bone defects above that of these conventional techniques. Periosteal and osteoperiosteal grafts have been used experimentally in the treatment of fractures, loss of bone substance such as osteoporosis, pseudoarthrosis and in the reconstruction of articular defects and femoral neck fractures (KING, 1976; VAN DEN WILDENBERG,1984; CAMILLIand PANTEADO, 1994; REYNDERS,et al. 1998). It has been shown that in fracture healing, the periostenm is responsible for bridging callus formation through both an external woven bone formation and enchondral bone formation process (RANTAet al., 1981; LI et al., 1989a,b). When the periosteum is detached from the bone it carries osteogenic cells with it. Furthermore, stripping of the periosteum undoubtedly affects the extra-osseous blood supply to the bone (ONI and GREGG, 1990; SAKAI and DoI, 1994; UTVAG 1996). Little is known about the effect of perichondreal stripping on the partial fracture, which is the type of bone defect we are concentrating on in this experimental study. Especially, the role of vascularity for healing procedure has not been well described. Some authors claim that healing is accomplished almost exclusively by the activities of the periosteum and endosteum (OLuSOLAet al. 1989; HULTn,1990; ONI and GREGG, 1990; OLUSOLAet al. 1991). Cells of the periosteum (which develops from the embryonic perichondrium) have mesenchymal-like properties to develop cartilage or bone, depending on the conditions of the environment (SKOOG et al., 1972). Rib perichondrium has been employed successfully to resurface the rabbit femoral condyle and canine patella (ENGKVIST, 1979; ENGKVIST,and WILANDER, 1979; AMIELet al., 1985; KWANet al., 1989; COUTTS et al., 1992). Johansson and Engkvist (1981) have reported of the use of rib perichondrium to resurface human finger joints but with various levels of success (COUTTS et al., 1992). Fibrocartilage may form at a site of bone fracture rather than bone. This fibrocartilage was thought to originate from the periosteum ie. source of fibroblasts. Movement of the fracture was thought to cause fibrocartilage forming rather than osteoblast-osteocyte-bone formation. Engkvist and Ohlsen (1979) used rabbit ear perichondrium to heal lesions of the rabbit glenoid, but found that rib perichondrium has a higher potential to compare to ear perichondrium because it has a higher proportion of
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elastin compared to glenoids. Cartilage grown from ear perichondrium contained an increased level of elastin and furthermore transplanting a more pluripotent perichondrium tissue gives it the opportunity to form the correct cartilage at the transplanted area. Our aim was to discover whether perichondreal detachment impairs healing of the bone defect. Therefore, the present study is to evaluate the osteogenic capability of perichondreal grafts taken from rib perichondrium and transplanted to sites of transverse/partial bone defects in a dog trauma model. Thus, the role of vascularity for healing procedure also was described in the study.
Materials and M e t h o d s Surgical Model: Seven, skeletally mature dogs of unselected breed, age and sex were used. Their weight ranged from 10-20 kg. The absence of skeletal diseases was confirmed by clinical examination and radiography. The operation procedures were carried out by quarantine and conventional health measures. All procedures have been performed in accordance with national or local animal welfare legislation or respectively is based on the European Council Directive. The animals were anesthesized with intravenous sodium thiopenthal (20 mg/kg). They were entubated with 4% halotane for afterwards. The surgical area was shaved and prepared with Betadine. A 10 cm-long incision was made on the anterior chest wall lateral to the sternum and a 15 cm portion of the first attached 5 centimetre cartilaginous rib was harvested from the thirteenth rib. The removed perichondrium was placed in saline solutions. The proximal to lower third of the right tibia of the dogs was exposed through an anteromedial incision. The skin edges and muscles were retracted from the shaft and a 1 cm transverse partial osteotomy was created until the medullar cavity of bone (5-6 ram) in the proximal and middle diaphyssis of tibia. In addition, a 5-cm length of periosteum on either side of the osteotomy was excised to exclude a position perichondreal contribution to healing. The graft was then transplanted transversely on the middle partial defect.
Table 1. Callus area in control and transplanted perichondreal groups. Number Experiof mental cases period
21 3 61 71 1 4 5
30 days 30 days 30 days 30 days 45 days 45 days 45 days
Control group
With perichondreum
Original defect gap (ram)
Callus area (mm)
The filling rate of callus area (%)
Original defect gap (ram)
Callus area (ram)
The filling rate of callus area (%)
5.2 6.0 5.2 5.9 5.0 6.0 5.4
11.2 2.6 10.7 9.8 7.5 8.4 8.0
215 43 205 166 150 140 148
5.1 6.0 5.0 5.6 5.2 5.8 5.1
4.3 4.8 4.0 5.1 5.0 6.1 4.9
84 80 80 91 96 105 96
The valuesrepresentthe thickness of callus or originaldefect gaps as milimeters(mm) 1The dogs with fracture (in controldefect site of tibia)
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The animals were divided into two groups, the healing of the bone defects was investigated. The animals were allowed to recover and were euthanatized for pathological examination after 30 (Group I) and 45 days (Group II) with each animal acting as its own control throughout the experiment. Histology: The tibial segments including defect and intact side were fixed in 10 % buffered formalin and than decalcified in 5 % nitric acid solution. The specimens were transferred into alcoholxylol-paraffin sections and embedded in paraffin blocks. The sections were cut 5-6 mm pieces and stained with hemotoxylin and eosin for histological evaluation. Radiography: X-rays were taken 5 times for group I and 7 times for group II until the dogs euthanatized. The first radiography was taken immediately after surgery. And than carried out for one week. Measures of callus area: The defect gaps produced experimentally and thickness of callus were measured with a calliger compass (composing stick), and the callus sizes were also measured using an ocular micrometer in a light microscope.
Results
Pathological findings • 30 days treated group (Group I) - Control f o r group I." The control site of three animals (dogs number 2, 6, 7) the tibia show that the callus was not absolutely filled (Fig. 1). Histopathologically it was seen that the defects sites did not fairly recover. This callus consisted of mostly fibrous tissue which was rich in collagen (Fig. 2). Because the callus loaded the medulla, the bone marrow could not be detected. Among these callus tissues, there was a spongious, regenerating ossified tissue including weak and worse arranged bone trabeculae. Some cases also show that findings of regeneration were more evident.
Fig. 1. Perichondreum treated defect was totally filled by callus (arrow), while control defect was not absolutely filled by callus yet (arrow head) in 30 days treated group.
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Fig. 2. A mixture of fibrous (f), cartilaginous (c) and new ossified tissue (o) with large gaps (g) in 30 days treated control group with fracture (H&E, x90).
New bone tissue consisted of disordered, weak and inadequate bone trabeculae which were made of hypercelulare and with large gaps (Fig. 3). These new bone trabeculae were covered by a newly produced and hypertrophic periosteum. • Autologous nonvascularized perichondreal group I: Thirty days postoperatively, the defects were filled nearly totally by new ossified tissue consisting of thick trabeculae. This tissue appeared to be converting into a compact bone by decreasing its cellularity (Fig. 4). Some trabeculae contained primitive haversian canals and lacunae, in which there were buried mature bone cells known as osteocytes. Among the trabeculae there was connective tissue rich in blood vessels,numerous osteoblasts some osteoclast (Fig. 5) or there was tissue resembling bone marrow. Osteoblasts were producing new bone tissue while osteoclasts were reducing the primitive and disordered bone. Fibrous tissue was more clear in the area near to periosteum. Intramembranous ossification was still seen under the periosteum which spread into the healing area. Some cases showed that thickened periosteum and healing areas carrying on bone formation. • 45 days treated group (Group II) - Control f o r group II: An enormous hypertrophic callus was seen (Fig. 6) and the defects were filled by fibro-cartilaginous and ossified tissue which spread in the direction of both the medullae and bone surface. This tissue con-
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Fig. 3. Disordered, weak and inadequate bone trabeculae with large gaps under the regenerated and hypercellulare periosteum (p) in 30 days treated control group (H&E, x75).
tained usually thick or sometimes thin trabeculae which were not distinct and very irregular. Decrease of cellular responses was seen in the trabeculae near to medullae while hypercellular responses were more active in periosteum. There was new bone and cartilage formation in this side. Osteoblasts and osteoclasts were found in islands of fibrous
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Fig. 4. New ossified tissue consisted of thick trabeculae in which there are primitive Haversian canals (arrows) in 30 days treated with perichondreal group (H&E, x75).
tissue among the trabeculae (Fig. 7). The callus was longer, apparently as a result of additional bone and cartilage, and extended onto intact bone surface (Fig. 8). Osteoblasts were producing new bone tissue. On the other hand, osteoclasts were reabsorbing former bone in order to remodel this regenerating bone tissue.
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Fig. 5. Primitive Haversian canals (H) and osteocytes (thin arrows) in the thick trabeculae, and osteoclasts (arrow heads) and numerous osteoblasts (thick arrow) in the intertrabeculare areas in 30 days treated with perichondreal group (H&E, x190).
Fig. 6. An enormous hypertrophic callus has been seen in the control site (arrow head), while the perichondreal defect has been smoothly resurfaced (arrow) in 45 days treated group.
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Fig. 7. Close up appearance of osteoblasts (arrows) and osteoclasts (arrow heads) in the intertrabeculare area in 45 days treated control defect (H&E, ×300).
• Autologous nonvascuIarized perichondreal group 11: From 45 days after operation it was detected that the defects had been absolutely filled with a regenerating bone tissue. New bone trabeculae were more thickened and distinct in 45 days treated group compared to 30 days the group with perichondreum. Compact trabeculae were solid and almost reached its greatest thickness, remaining smaller areas of fibrous tissue among them. The cellularity of trabeculae near to the medullary cavity was fairly decreased. The regenerating bone was converting into compact bone, forming the Haversian canals and concentric larnellae (remodelling and reconstruction) (Fig. 9). This regenerating bone tissue was covered by hypercellular perichondreum, under which osteoblasts were maintaining bone formation. Intertrabeculare gaps near to periosteum were filled by a fibrousossify tissue. The new bone tissue was firmly stuck to old and original bones (Fig. 10). • Radiography : Enormous thickening was seen in the control group compared to autologous periosteum group by x-ray (not data shown). The hypertrophic callus was revealed in nonperiosteal group compared to periosteal group. Periosteal bridging callus was external and was the only callus revealed significantly by standard radiological examination.
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Fig. 8. The exuberant new bone trabeculae (t) related to hypertrophic callus are overflowed from the original bone (ob) surface in 45 days treated control defect (H&E, ×75).
Fig. 10. New bone tissue (nb) which firmly stuck to original bone (ob). The border (between arrows) has been hardly detected in 45 days treated with perichondreal group (H&E, x90).
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Fig. 9. New forming Haversian canals (H) and concentric lamellae (arrows) within regenerating bone tissue which is converting into compact bone in 45 days treated with perichondreal group (H&E, x170).
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Discussion This study was undertaken to investigate the osteogenic potential of a free autologous pericbondreum in a poorly vascularized setting, a condition often found in all species after traumatic injury. The dog bone defect has been modified in this study. The osteogenic capability of longitudinal autologous nonvascularized periosteal grafts were investigated using tibia trauma model in rabbits (REYNDERSet al., 1998; REYNDERSet al., 1999). Similar size of bone defects were made in tibia for this study. Autologous perichondreal grafts were applied to distal site of tibia. The proximal side of tibia was used as a control. There is clear evidence that new bone formation by perichondreal grafts may be influenced by several factors, including the vascularity of the periosteal flap (RoMAYA and MASQU~LET, 1990) the donor site (PucKErr et al., 1979), contact with living cortical bone (FINLEYet al., 1978; CANALISand BURSTEIN,1985), stress from weight-beating (FINLEYet al., 1978; PUCV,ETT et al., 1979) and the vascularity of the recipient site (RtTSmA et al., 1994) It has also been shown that micro surgical revascularization of periosteal grafts promotes superior bone formation, (HAw and O'BRIEN, 1978). Microsurgery is a protracted and very demanding technique that simply is not practical for immediate bone coverage of open tibial fractures in the presence of severe soft tissue damage. Therefore, it was claim that the main reason choosing a practical and reproducible technique of periosteal transplantation that can be used effectively in the emergency setting to cover bone after trauma. The defects in control side of group 1 were not fully resurfaced yet, and new bone tissue consisted of disordered, weak and inadequate bone trabeculae which were made of hypercelulare and with large gaps. In perichondreum grafted defects were filled nearly totally by new ossified tissue, consisting of thick and solid trabeculae. The findings of this study show that periost grafted defects were completed in less time and with more solid trabeculae than in the control defects, suggesting that periost accelerates the healing of bone injury. Furthermore, in 45 days group, defects were resurfaced by more restricted and well arranged bone tissue, which appeared to be converting into a compact bone, unlike control defects in the same period. These findings have supported previous records that in transformation into compact bone, resorption cavities appear continuously, containing osteoblasts and osteoclasts, and are replaced by higher orders of haversian systems (FROST, 1989) It was shown that perichondreum autograft can be used for treatment of articular defects (JAROMAand RITSmA, 1987; COUTTSet al., 1992; RITSILA, 1994). Jaroma and Ritsila (1987) was concluded that free perichondreum grafts on the chondrectomised articular surface of patella differentiation into cartilage. Chondrocyte proliferation was somewhat slower in the series with the fibrous layer of the periosteum facing the subchondral bone than in the series with cambium layer facing the articular surface. In this study, perichondreal autografts were used for bone defects. Partial bone defect the dog tibia have been shown by our research group to heal at 4 weeks. The present study has also shown that, in the dogs with full weight-bearing a free perichondreal graft with viable periosteal contact produced bridging callus. Initially, bone healing occured
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mainly trough enchondral ossification, then was reconstructed and remodelled by intramembranous ossification, which was carried out by work of osteoblasts and osteoclasts. The findings in the study revealed that the defects were filled from medulla to bone surface and new bone has been maturated in the same way. Several researches have noted the influence of mechanical stress on the rapidity and quality of new bone formed by the periosteum (STAFFORDet al., 1992; REYNDERS et al., 1999). This finding confirms the view, previously expressed by others (RuBAK 1982; DE YOUNG and PROBST, 1993) that the perichondreum is most important for normal healing. It also raises the possibility that if the periosteum is not present as in open fractures healing can come about only through the activities of the remaining undamaged periosteum and the endosteum. The ability of perichondreum to provide a source of proliferative chondrogenic cells has been demonstrated by this study and confirms the results of other investigators (ENGKWSa', 1979; ENGKV~STand O~-mSEN, 1979; COUTTS et al., 1992). In this study, autologous perichondreum grafts were transplanted from costal perichondreum. It was easy to applicate and more than 5 cm length of perichondreum was excised. Engkvist and Ohlsen (1979) used rabbit ear perichondrium to heal lesions of the rabbit glenoid but found that rib perichondrium had greater potential because cartilage grown from ear perichondrium contained an increased level of elastin. Rib perichondrium has been employed successfully to resurface the rabbit femoral condyle and canine patella. Jaroma and Ritsila (1987) show that periosteal grafts transplanted on the costal cartilage of adolescent rabbits formed a cartilaginous zone on the costal cartilage side of the recipient bed. This and other experiments indicate that the first osseous union in the repair of longbone fractures and ostectomies is by medullar bridging callus. Perichondreum autografts may be important in our understanding delayed union and nonunion of fractures and bone defects which increase morbidity. It is hoped that studies on perichondreal autografts will also give further insight into the principles of fracture healing and may provide useful information for man.
References AMIEL, D., R. D. Courts, M. ABEL, W. STEWART, F. HARWOOD,and W. H. AKESON,1985. Rib perichondrial grafts for the repair of full-thickness articular cartilage defects: A morphological and biochemical study in rabbits. J. Bone Joint Surg. 67:911. CAMILLI,J. A. and C. V. PANTEADO,1994. Bone formation by vascularized periosteal and osteoperiosteal grafts. An experimental study in rats. Arch. Orthop. Trauma. Surg. 114:18-24. CANALISR. F. and D. BURSTEIN,1985. Osteogenesis in vascularized periosteum. Arch. Otolaryngol. 111:511-516. COUTTS,R. D., L.-Y. W. SAVIO, D. AMIEL, H. P. VON SCHROEDER, and M. K. KWAN,1992. Rib perichondrial autografts in full-thickness articular cartilage defects in rabbits. Clin. Orthop. 275: 263-273. DE YOUNG,D. J. and C. W. PROBST,1993. Methods of internal fracture fixation: General Principles. In SLATTER,D., ed., Textbook of small animal surgery. W. B. Saunders Company, Philadelphia. 1610-1631.
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DoI, K. and K. SAKAI,1994. Vasularized periosteal bone graft from the supracondylar region of the femur. Microsurgery 15: 305-315. ENGKVIST,O. 1979. Reconstruction of patellar articular cartilage with free autologous perichondrial grafts. An experimental study in dogs. Scan. J. Plast. Reconstr Surg. 13: 361-369. ENGKVIST,O. and L. OHLSEN, 1979. Reconstruction of articular cartilage with free autologous perichondrial grafts. An experimental study in rabbits. Scan. J. Plast. Reconstr Surg. 13: 269-274. ENGKVIST, O. and E. WmANDER, 1979. Formation of cartilage from rib pericondrium grafted to an articular defect in the femur condyle of the rabbit. Scand. J. Plast. Reconstr Surg. 13:371-276. FINLEY, J. M., R. D. ACLAND,and M. B. WOOD, 1978. Revascularized periosteal grafts. A new method to produce functional new bone without bone grafting. Plast Reconstr Surg. 61: 1-6. FROST, H. M. 1989. The biology of fracture healing. Clin. Orthop. 248: 283-309. HAW, C. S. B., O'BP,n~N, and T. KUP,ATA, 1978. The microsurgical revascularization of resected segments of tibia in the dog. J Bone Joint Surg. 60B: 266-269. HULTH, A. 1989. Current concepts of fracture healing. Clin Orthop. 249: 265-284. JAROMA, H. J. and V.A. RITSILA, 1987. Reconstruction of patellar cartilage defects with free periosteal grafts. An experimental study. Scand. J. Plast. Reconstr Surg. 21: 175-181. JOHANNSON,S. H. and O. ENGKVIST,1981. Small joint reconstruction by perichondrium artroplasty. Clin. Plast. Surg. 8: 107. KING, K. F. 1976. Periosteal pedicle grafting in dogs. J Bone Joint Surg. 58:117-121. KWAN,M. K., R. D. CourTs, S. L. Y. Woo, and F. P. FmLD, 1989. Morphological and biomechanic evaluations of neocartilage from the repair of full-thickness articular cartilage defects using rib perichondrium autografts: A long-term study. J. Biomech. 22: 921-930. LI, W. G., J. Q. TANG,Q. L. CuI, and R. Z. Znou, 1989. Experimental study of free periosteal autograft. II. Increasing osteogenesis of periosteum. Chin. Med. J. 102:411-415. LI, W. G., J. Q. TANG, Q. L. Cu1, and R. Z. ZNOU, 1989. Experimental study of free periosteal autograft. Animals age and periosteal osteogenesis. Chin. Med. J. 102: 361-364. OLUSOLA,O. A. O., H. STAFFORD,and P. J. GREGG, 1989. An experimental study of the patterns of periosteal and endosteal damage in tibial shaft fractures using a rabbit trauma model. J. Orthop. Trauma. 3: 142-147. OLUSOLA,O. A. O. and P. J. GP,EGG, 1991. An investigation of the contribution of the extraosseous tissues to the diaphyseal fracture callus using a rabbit tibial fracture model. J. Orthop. Trauma. 5: 480-484. OLUSOLA,O. A. O. and P. J. GREGG, 1990. The relative contribution of individual osseous circulations to diaphyseal cortical blood supply. J. Orthop. Trauma 4: 441-448. PERV,~N, S. M. 1993. Primary bone healing. In Bow,AB, M. J., D. D. Smeak, M. S. Bloomberg, eds., Disease mechanism in small animal surgery. Philadelphia 663-671. PUCKETT,C. L., J. S. HURVITZ,M. H. METZLER,and D. SILVER,1979. Bone formation by revascularized periosteal and bone grafts, compared with traditional bone grafts. Plast. Reconstr. Surg. 64: 361-365. RANTA, R., P. YLIPAAVALNIEMI1,M. ALTONEN, mad P. E. CALONIUS, 1981. Transplantation of free tibial periosteal graft on alveolar bone defect in adult rabbit. Int. J. Oral Surg. 10: 122-127. REYNDERS,P., J. H. R. BECKER, and P. BROOS, 1998. The osteogenic potential of free periosteal grafts. An experimental study in rats. Acta Orthop. Belg. 64: 184-192. REYNDERS, P., J. H. R. BECKER, and P. BROOS, 1999. Osteogenic Ability of free pefiosteal autografts in tibial fractures with severe soft tissue damage: An experimental study. J Orthop Trauma 13: 121-128. RITSILA,V. A., S. SANTAVIRTA,S. ALHOPURO,M. POUSSA,H. JAROMA,J. M. Rubak, A. ESKOLA,V. HOIKKA,O. SNELLMAN,and K. OSTERMAN,1994. Periosteal and perichondral grafting in reconstructive surgery. Clin Orthop 302: 259-265. ROMANA,M. C. and A. C. MASQUELET, 1990. Vascularized periosteum associated with cancellous bone graft. An experimental study. Plast Reconstr Surg. 85: 587-592.
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RUBAK J. M. 1982. Reconstruction of articular cartilage defects with free peIiosteal grafts. An experimental study. Acta Orthop Scand 53:175-180. SKOOG,T., L. OHLSEN,and S. A. SO~N, 1972. Perichondrial potential for cartilagenous regeneration Scand. J. Plast. Reconstr. Surg. 6: 123. STAFFORD,U., O. O. A. ONI, J. HAY, and P. J. GREGG, 1992. An investigation of the contribution of the extraosseous tissues to the diaphyseal fracture callus using a rabbit tibial fracture model and in situ immunocytochemical localisation of osteocalcin. J. Orthop. Trauma. 6: 190-194. UTVAG, S. E., O. GRUNDNESand O. REIKERAOS,1996. Effects of periosteal stripping on healing of segmental fractures in rats. J. Orthop. Trauma. 10: 279-284. VAN DEN WILDENBERG,F. A., R. J'. GORIS,and M. B. TUTErN NOLTHENIUS-PUYLAERT,1984. Free revascularised periosteum transplantation: an experimental study. Br. J. Plast. Surg. 37: 226-235.
Corresponding author: Dr. MUSTAFAARICAN,Department of Surgery, Faculty of Veterinary Science, The University of Sel~uk 42031 Campus/Konya Turkey Tel.: ++90-332-2410041-2748; Fax: ++90-332-2140063; e-mail: [email protected] or [email protected]
Osteogenic ability of free perichondreal autografts in canine tibial defects: An experimental study* M. AR1CAN2, M. ORTATATL1l, K. YI~ITARSLAN 2 and C. CEYLAN2 1Department of Pathology, Faculty of Veterinary Science, The University of Selguk, 42031 Campus/Konya Turkey 2Department of Surgery, Faculty of Veterinary Science, The University of Selquk, 42031 Campus/Konya Turkey
Summary This study set out to establish the effect of transplanting perichondreum on bone healing at sites of ribial bone defects in an experimental dog model. Transplantation of free, autologous, non-vascularised, perichondreal grafts to the distal of right anteromedial plane side of the tibia was compared with non-transplantation on the proximal side of the same bone. In experimental dogs (n = 7), a 5 cm piece segment of perichondreum, that has been excised from the thirteenth rib of the same animal, was transplanted to the midddle defect fracture site of bone, but not to the control proximal defect fracture site. The dogs were allowed to recover from the operation and were kept 21 days in cages, with freerange. On days 30 (Group I) and 45 (Group II) after operations, the dogs were euthanatized. Histopathologically, defects in 30 days treated perichondreum group were filled by new ossified tissue while control defects in the same period were not fully resurface& The new ossified tissue consisted of a thin and inadequate trabeculae. In 45 days treated groups, defects with transplanting perichondreum were filled by thick trabeculae converting into a compact bone tissue. The control defects of this group, however, were filled by an extreme callus overflowing to medulla and bone surface. This study has provided evidence to show that autologous, non-vascularized perichondreum retains an osteogenic ability when transplanted to tibial bone defect sites. It appears that callus formarion occurred within the perichondreum grafting which resembles that of enchondral and intramembranous ossification.
Key words: Canine, tibia, perichondreal grafts *All procedures have been performed in accordance with national or local animal welfare legislation or respectively is based on the European Council Directive. J. Exp. Anim. Sci. 2003; 42:203-217 Urban & Fischer Verlag http://www.urbanfischer.de/joumals/jeansc 0939-8600/03/42/04-203 $15.00/0
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Introduction Healing of fractures not simply is a process via scar tissue healing but actually also depends on a process of bone regeneration (OLuSOLAand GREGG, 1991). For instance, in many reports on the bone fracture healing process, it has been suggested that the surrounding soft tissues contribute a significant number of osteogenic cells to the fracture callus (FRosT, 1989). It has long been recognised that the healing of bone fractures is related greater decrease the rate of bone healing to the severity of associated bone and soft tissue damage (DoI and SAKAI,1994). Severe soft-tissue loss with periosteal detachment and compromised vascularity, rather than fracture configuration, are major determinants of the outcome of untreated fractures. All the conventional methods of fracture repair may be applied to fractures of the tibia. Simple fractures may be effectively treated with an intramedullary pin, with or without a combination of cerclage or meni- cerclage wire, plates or Kirschner splints (ENGKVIST,1979; DE YOUNGand PROBST, 1993) In this study, our aim was to improve the osteogenic ability of bone defects above that of these conventional techniques. Periosteal and osteoperiosteal grafts have been used experimentally in the treatment of fractures, loss of bone substance such as osteoporosis, pseudoarthrosis and in the reconstruction of articular defects and femoral neck fractures (KING, 1976; VAN DEN WILDENBERG,1984; CAMILLIand PANTEADO, 1994; REYNDERS,et al. 1998). It has been shown that in fracture healing, the periostenm is responsible for bridging callus formation through both an external woven bone formation and enchondral bone formation process (RANTAet al., 1981; LI et al., 1989a,b). When the periosteum is detached from the bone it carries osteogenic cells with it. Furthermore, stripping of the periosteum undoubtedly affects the extra-osseous blood supply to the bone (ONI and GREGG, 1990; SAKAI and DoI, 1994; UTVAG 1996). Little is known about the effect of perichondreal stripping on the partial fracture, which is the type of bone defect we are concentrating on in this experimental study. Especially, the role of vascularity for healing procedure has not been well described. Some authors claim that healing is accomplished almost exclusively by the activities of the periosteum and endosteum (OLuSOLAet al. 1989; HULTn,1990; ONI and GREGG, 1990; OLUSOLAet al. 1991). Cells of the periosteum (which develops from the embryonic perichondrium) have mesenchymal-like properties to develop cartilage or bone, depending on the conditions of the environment (SKOOG et al., 1972). Rib perichondrium has been employed successfully to resurface the rabbit femoral condyle and canine patella (ENGKVIST, 1979; ENGKVIST,and WILANDER, 1979; AMIELet al., 1985; KWANet al., 1989; COUTTS et al., 1992). Johansson and Engkvist (1981) have reported of the use of rib perichondrium to resurface human finger joints but with various levels of success (COUTTS et al., 1992). Fibrocartilage may form at a site of bone fracture rather than bone. This fibrocartilage was thought to originate from the periosteum ie. source of fibroblasts. Movement of the fracture was thought to cause fibrocartilage forming rather than osteoblast-osteocyte-bone formation. Engkvist and Ohlsen (1979) used rabbit ear perichondrium to heal lesions of the rabbit glenoid, but found that rib perichondrium has a higher potential to compare to ear perichondrium because it has a higher proportion of
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elastin compared to glenoids. Cartilage grown from ear perichondrium contained an increased level of elastin and furthermore transplanting a more pluripotent perichondrium tissue gives it the opportunity to form the correct cartilage at the transplanted area. Our aim was to discover whether perichondreal detachment impairs healing of the bone defect. Therefore, the present study is to evaluate the osteogenic capability of perichondreal grafts taken from rib perichondrium and transplanted to sites of transverse/partial bone defects in a dog trauma model. Thus, the role of vascularity for healing procedure also was described in the study.
Materials and M e t h o d s Surgical Model: Seven, skeletally mature dogs of unselected breed, age and sex were used. Their weight ranged from 10-20 kg. The absence of skeletal diseases was confirmed by clinical examination and radiography. The operation procedures were carried out by quarantine and conventional health measures. All procedures have been performed in accordance with national or local animal welfare legislation or respectively is based on the European Council Directive. The animals were anesthesized with intravenous sodium thiopenthal (20 mg/kg). They were entubated with 4% halotane for afterwards. The surgical area was shaved and prepared with Betadine. A 10 cm-long incision was made on the anterior chest wall lateral to the sternum and a 15 cm portion of the first attached 5 centimetre cartilaginous rib was harvested from the thirteenth rib. The removed perichondrium was placed in saline solutions. The proximal to lower third of the right tibia of the dogs was exposed through an anteromedial incision. The skin edges and muscles were retracted from the shaft and a 1 cm transverse partial osteotomy was created until the medullar cavity of bone (5-6 ram) in the proximal and middle diaphyssis of tibia. In addition, a 5-cm length of periosteum on either side of the osteotomy was excised to exclude a position perichondreal contribution to healing. The graft was then transplanted transversely on the middle partial defect.
Table 1. Callus area in control and transplanted perichondreal groups. Number Experiof mental cases period
21 3 61 71 1 4 5
30 days 30 days 30 days 30 days 45 days 45 days 45 days
Control group
With perichondreum
Original defect gap (ram)
Callus area (mm)
The filling rate of callus area (%)
Original defect gap (ram)
Callus area (ram)
The filling rate of callus area (%)
5.2 6.0 5.2 5.9 5.0 6.0 5.4
11.2 2.6 10.7 9.8 7.5 8.4 8.0
215 43 205 166 150 140 148
5.1 6.0 5.0 5.6 5.2 5.8 5.1
4.3 4.8 4.0 5.1 5.0 6.1 4.9
84 80 80 91 96 105 96
The valuesrepresentthe thickness of callus or originaldefect gaps as milimeters(mm) 1The dogs with fracture (in controldefect site of tibia)
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The animals were divided into two groups, the healing of the bone defects was investigated. The animals were allowed to recover and were euthanatized for pathological examination after 30 (Group I) and 45 days (Group II) with each animal acting as its own control throughout the experiment. Histology: The tibial segments including defect and intact side were fixed in 10 % buffered formalin and than decalcified in 5 % nitric acid solution. The specimens were transferred into alcoholxylol-paraffin sections and embedded in paraffin blocks. The sections were cut 5-6 mm pieces and stained with hemotoxylin and eosin for histological evaluation. Radiography: X-rays were taken 5 times for group I and 7 times for group II until the dogs euthanatized. The first radiography was taken immediately after surgery. And than carried out for one week. Measures of callus area: The defect gaps produced experimentally and thickness of callus were measured with a calliger compass (composing stick), and the callus sizes were also measured using an ocular micrometer in a light microscope.
Results
Pathological findings • 30 days treated group (Group I) - Control f o r group I." The control site of three animals (dogs number 2, 6, 7) the tibia show that the callus was not absolutely filled (Fig. 1). Histopathologically it was seen that the defects sites did not fairly recover. This callus consisted of mostly fibrous tissue which was rich in collagen (Fig. 2). Because the callus loaded the medulla, the bone marrow could not be detected. Among these callus tissues, there was a spongious, regenerating ossified tissue including weak and worse arranged bone trabeculae. Some cases also show that findings of regeneration were more evident.
Fig. 1. Perichondreum treated defect was totally filled by callus (arrow), while control defect was not absolutely filled by callus yet (arrow head) in 30 days treated group.
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Fig. 2. A mixture of fibrous (f), cartilaginous (c) and new ossified tissue (o) with large gaps (g) in 30 days treated control group with fracture (H&E, x90).
New bone tissue consisted of disordered, weak and inadequate bone trabeculae which were made of hypercelulare and with large gaps (Fig. 3). These new bone trabeculae were covered by a newly produced and hypertrophic periosteum. • Autologous nonvascularized perichondreal group I: Thirty days postoperatively, the defects were filled nearly totally by new ossified tissue consisting of thick trabeculae. This tissue appeared to be converting into a compact bone by decreasing its cellularity (Fig. 4). Some trabeculae contained primitive haversian canals and lacunae, in which there were buried mature bone cells known as osteocytes. Among the trabeculae there was connective tissue rich in blood vessels,numerous osteoblasts some osteoclast (Fig. 5) or there was tissue resembling bone marrow. Osteoblasts were producing new bone tissue while osteoclasts were reducing the primitive and disordered bone. Fibrous tissue was more clear in the area near to periosteum. Intramembranous ossification was still seen under the periosteum which spread into the healing area. Some cases showed that thickened periosteum and healing areas carrying on bone formation. • 45 days treated group (Group II) - Control f o r group II: An enormous hypertrophic callus was seen (Fig. 6) and the defects were filled by fibro-cartilaginous and ossified tissue which spread in the direction of both the medullae and bone surface. This tissue con-
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Fig. 3. Disordered, weak and inadequate bone trabeculae with large gaps under the regenerated and hypercellulare periosteum (p) in 30 days treated control group (H&E, x75).
tained usually thick or sometimes thin trabeculae which were not distinct and very irregular. Decrease of cellular responses was seen in the trabeculae near to medullae while hypercellular responses were more active in periosteum. There was new bone and cartilage formation in this side. Osteoblasts and osteoclasts were found in islands of fibrous
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Fig. 4. New ossified tissue consisted of thick trabeculae in which there are primitive Haversian canals (arrows) in 30 days treated with perichondreal group (H&E, x75).
tissue among the trabeculae (Fig. 7). The callus was longer, apparently as a result of additional bone and cartilage, and extended onto intact bone surface (Fig. 8). Osteoblasts were producing new bone tissue. On the other hand, osteoclasts were reabsorbing former bone in order to remodel this regenerating bone tissue.
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Fig. 5. Primitive Haversian canals (H) and osteocytes (thin arrows) in the thick trabeculae, and osteoclasts (arrow heads) and numerous osteoblasts (thick arrow) in the intertrabeculare areas in 30 days treated with perichondreal group (H&E, x190).
Fig. 6. An enormous hypertrophic callus has been seen in the control site (arrow head), while the perichondreal defect has been smoothly resurfaced (arrow) in 45 days treated group.
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Fig. 7. Close up appearance of osteoblasts (arrows) and osteoclasts (arrow heads) in the intertrabeculare area in 45 days treated control defect (H&E, ×300).
• Autologous nonvascuIarized perichondreal group 11: From 45 days after operation it was detected that the defects had been absolutely filled with a regenerating bone tissue. New bone trabeculae were more thickened and distinct in 45 days treated group compared to 30 days the group with perichondreum. Compact trabeculae were solid and almost reached its greatest thickness, remaining smaller areas of fibrous tissue among them. The cellularity of trabeculae near to the medullary cavity was fairly decreased. The regenerating bone was converting into compact bone, forming the Haversian canals and concentric larnellae (remodelling and reconstruction) (Fig. 9). This regenerating bone tissue was covered by hypercellular perichondreum, under which osteoblasts were maintaining bone formation. Intertrabeculare gaps near to periosteum were filled by a fibrousossify tissue. The new bone tissue was firmly stuck to old and original bones (Fig. 10). • Radiography : Enormous thickening was seen in the control group compared to autologous periosteum group by x-ray (not data shown). The hypertrophic callus was revealed in nonperiosteal group compared to periosteal group. Periosteal bridging callus was external and was the only callus revealed significantly by standard radiological examination.
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Fig. 8. The exuberant new bone trabeculae (t) related to hypertrophic callus are overflowed from the original bone (ob) surface in 45 days treated control defect (H&E, ×75).
Fig. 10. New bone tissue (nb) which firmly stuck to original bone (ob). The border (between arrows) has been hardly detected in 45 days treated with perichondreal group (H&E, x90).
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Fig. 9. New forming Haversian canals (H) and concentric lamellae (arrows) within regenerating bone tissue which is converting into compact bone in 45 days treated with perichondreal group (H&E, x170).
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Discussion This study was undertaken to investigate the osteogenic potential of a free autologous pericbondreum in a poorly vascularized setting, a condition often found in all species after traumatic injury. The dog bone defect has been modified in this study. The osteogenic capability of longitudinal autologous nonvascularized periosteal grafts were investigated using tibia trauma model in rabbits (REYNDERSet al., 1998; REYNDERSet al., 1999). Similar size of bone defects were made in tibia for this study. Autologous perichondreal grafts were applied to distal site of tibia. The proximal side of tibia was used as a control. There is clear evidence that new bone formation by perichondreal grafts may be influenced by several factors, including the vascularity of the periosteal flap (RoMAYA and MASQU~LET, 1990) the donor site (PucKErr et al., 1979), contact with living cortical bone (FINLEYet al., 1978; CANALISand BURSTEIN,1985), stress from weight-beating (FINLEYet al., 1978; PUCV,ETT et al., 1979) and the vascularity of the recipient site (RtTSmA et al., 1994) It has also been shown that micro surgical revascularization of periosteal grafts promotes superior bone formation, (HAw and O'BRIEN, 1978). Microsurgery is a protracted and very demanding technique that simply is not practical for immediate bone coverage of open tibial fractures in the presence of severe soft tissue damage. Therefore, it was claim that the main reason choosing a practical and reproducible technique of periosteal transplantation that can be used effectively in the emergency setting to cover bone after trauma. The defects in control side of group 1 were not fully resurfaced yet, and new bone tissue consisted of disordered, weak and inadequate bone trabeculae which were made of hypercelulare and with large gaps. In perichondreum grafted defects were filled nearly totally by new ossified tissue, consisting of thick and solid trabeculae. The findings of this study show that periost grafted defects were completed in less time and with more solid trabeculae than in the control defects, suggesting that periost accelerates the healing of bone injury. Furthermore, in 45 days group, defects were resurfaced by more restricted and well arranged bone tissue, which appeared to be converting into a compact bone, unlike control defects in the same period. These findings have supported previous records that in transformation into compact bone, resorption cavities appear continuously, containing osteoblasts and osteoclasts, and are replaced by higher orders of haversian systems (FROST, 1989) It was shown that perichondreum autograft can be used for treatment of articular defects (JAROMAand RITSmA, 1987; COUTTSet al., 1992; RITSILA, 1994). Jaroma and Ritsila (1987) was concluded that free perichondreum grafts on the chondrectomised articular surface of patella differentiation into cartilage. Chondrocyte proliferation was somewhat slower in the series with the fibrous layer of the periosteum facing the subchondral bone than in the series with cambium layer facing the articular surface. In this study, perichondreal autografts were used for bone defects. Partial bone defect the dog tibia have been shown by our research group to heal at 4 weeks. The present study has also shown that, in the dogs with full weight-bearing a free perichondreal graft with viable periosteal contact produced bridging callus. Initially, bone healing occured
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mainly trough enchondral ossification, then was reconstructed and remodelled by intramembranous ossification, which was carried out by work of osteoblasts and osteoclasts. The findings in the study revealed that the defects were filled from medulla to bone surface and new bone has been maturated in the same way. Several researches have noted the influence of mechanical stress on the rapidity and quality of new bone formed by the periosteum (STAFFORDet al., 1992; REYNDERS et al., 1999). This finding confirms the view, previously expressed by others (RuBAK 1982; DE YOUNG and PROBST, 1993) that the perichondreum is most important for normal healing. It also raises the possibility that if the periosteum is not present as in open fractures healing can come about only through the activities of the remaining undamaged periosteum and the endosteum. The ability of perichondreum to provide a source of proliferative chondrogenic cells has been demonstrated by this study and confirms the results of other investigators (ENGKWSa', 1979; ENGKV~STand O~-mSEN, 1979; COUTTS et al., 1992). In this study, autologous perichondreum grafts were transplanted from costal perichondreum. It was easy to applicate and more than 5 cm length of perichondreum was excised. Engkvist and Ohlsen (1979) used rabbit ear perichondrium to heal lesions of the rabbit glenoid but found that rib perichondrium had greater potential because cartilage grown from ear perichondrium contained an increased level of elastin. Rib perichondrium has been employed successfully to resurface the rabbit femoral condyle and canine patella. Jaroma and Ritsila (1987) show that periosteal grafts transplanted on the costal cartilage of adolescent rabbits formed a cartilaginous zone on the costal cartilage side of the recipient bed. This and other experiments indicate that the first osseous union in the repair of longbone fractures and ostectomies is by medullar bridging callus. Perichondreum autografts may be important in our understanding delayed union and nonunion of fractures and bone defects which increase morbidity. It is hoped that studies on perichondreal autografts will also give further insight into the principles of fracture healing and may provide useful information for man.
References AMIEL, D., R. D. Courts, M. ABEL, W. STEWART, F. HARWOOD,and W. H. AKESON,1985. Rib perichondrial grafts for the repair of full-thickness articular cartilage defects: A morphological and biochemical study in rabbits. J. Bone Joint Surg. 67:911. CAMILLI,J. A. and C. V. PANTEADO,1994. Bone formation by vascularized periosteal and osteoperiosteal grafts. An experimental study in rats. Arch. Orthop. Trauma. Surg. 114:18-24. CANALISR. F. and D. BURSTEIN,1985. Osteogenesis in vascularized periosteum. Arch. Otolaryngol. 111:511-516. COUTTS,R. D., L.-Y. W. SAVIO, D. AMIEL, H. P. VON SCHROEDER, and M. K. KWAN,1992. Rib perichondrial autografts in full-thickness articular cartilage defects in rabbits. Clin. Orthop. 275: 263-273. DE YOUNG,D. J. and C. W. PROBST,1993. Methods of internal fracture fixation: General Principles. In SLATTER,D., ed., Textbook of small animal surgery. W. B. Saunders Company, Philadelphia. 1610-1631.
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DoI, K. and K. SAKAI,1994. Vasularized periosteal bone graft from the supracondylar region of the femur. Microsurgery 15: 305-315. ENGKVIST,O. 1979. Reconstruction of patellar articular cartilage with free autologous perichondrial grafts. An experimental study in dogs. Scan. J. Plast. Reconstr Surg. 13: 361-369. ENGKVIST,O. and L. OHLSEN, 1979. Reconstruction of articular cartilage with free autologous perichondrial grafts. An experimental study in rabbits. Scan. J. Plast. Reconstr Surg. 13: 269-274. ENGKVIST, O. and E. WmANDER, 1979. Formation of cartilage from rib pericondrium grafted to an articular defect in the femur condyle of the rabbit. Scand. J. Plast. Reconstr Surg. 13:371-276. FINLEY, J. M., R. D. ACLAND,and M. B. WOOD, 1978. Revascularized periosteal grafts. A new method to produce functional new bone without bone grafting. Plast Reconstr Surg. 61: 1-6. FROST, H. M. 1989. The biology of fracture healing. Clin. Orthop. 248: 283-309. HAW, C. S. B., O'BP,n~N, and T. KUP,ATA, 1978. The microsurgical revascularization of resected segments of tibia in the dog. J Bone Joint Surg. 60B: 266-269. HULTH, A. 1989. Current concepts of fracture healing. Clin Orthop. 249: 265-284. JAROMA, H. J. and V.A. RITSILA, 1987. Reconstruction of patellar cartilage defects with free periosteal grafts. An experimental study. Scand. J. Plast. Reconstr Surg. 21: 175-181. JOHANNSON,S. H. and O. ENGKVIST,1981. Small joint reconstruction by perichondrium artroplasty. Clin. Plast. Surg. 8: 107. KING, K. F. 1976. Periosteal pedicle grafting in dogs. J Bone Joint Surg. 58:117-121. KWAN,M. K., R. D. CourTs, S. L. Y. Woo, and F. P. FmLD, 1989. Morphological and biomechanic evaluations of neocartilage from the repair of full-thickness articular cartilage defects using rib perichondrium autografts: A long-term study. J. Biomech. 22: 921-930. LI, W. G., J. Q. TANG,Q. L. CuI, and R. Z. Znou, 1989. Experimental study of free periosteal autograft. II. Increasing osteogenesis of periosteum. Chin. Med. J. 102:411-415. LI, W. G., J. Q. TANG, Q. L. Cu1, and R. Z. ZNOU, 1989. Experimental study of free periosteal autograft. Animals age and periosteal osteogenesis. Chin. Med. J. 102: 361-364. OLUSOLA,O. A. O., H. STAFFORD,and P. J. GREGG, 1989. An experimental study of the patterns of periosteal and endosteal damage in tibial shaft fractures using a rabbit trauma model. J. Orthop. Trauma. 3: 142-147. OLUSOLA,O. A. O. and P. J. GP,EGG, 1991. An investigation of the contribution of the extraosseous tissues to the diaphyseal fracture callus using a rabbit tibial fracture model. J. Orthop. Trauma. 5: 480-484. OLUSOLA,O. A. O. and P. J. GREGG, 1990. The relative contribution of individual osseous circulations to diaphyseal cortical blood supply. J. Orthop. Trauma 4: 441-448. PERV,~N, S. M. 1993. Primary bone healing. In Bow,AB, M. J., D. D. Smeak, M. S. Bloomberg, eds., Disease mechanism in small animal surgery. Philadelphia 663-671. PUCKETT,C. L., J. S. HURVITZ,M. H. METZLER,and D. SILVER,1979. Bone formation by revascularized periosteal and bone grafts, compared with traditional bone grafts. Plast. Reconstr. Surg. 64: 361-365. RANTA, R., P. YLIPAAVALNIEMI1,M. ALTONEN, mad P. E. CALONIUS, 1981. Transplantation of free tibial periosteal graft on alveolar bone defect in adult rabbit. Int. J. Oral Surg. 10: 122-127. REYNDERS,P., J. H. R. BECKER, and P. BROOS, 1998. The osteogenic potential of free periosteal grafts. An experimental study in rats. Acta Orthop. Belg. 64: 184-192. REYNDERS, P., J. H. R. BECKER, and P. BROOS, 1999. Osteogenic Ability of free pefiosteal autografts in tibial fractures with severe soft tissue damage: An experimental study. J Orthop Trauma 13: 121-128. RITSILA,V. A., S. SANTAVIRTA,S. ALHOPURO,M. POUSSA,H. JAROMA,J. M. Rubak, A. ESKOLA,V. HOIKKA,O. SNELLMAN,and K. OSTERMAN,1994. Periosteal and perichondral grafting in reconstructive surgery. Clin Orthop 302: 259-265. ROMANA,M. C. and A. C. MASQUELET, 1990. Vascularized periosteum associated with cancellous bone graft. An experimental study. Plast Reconstr Surg. 85: 587-592.
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RUBAK J. M. 1982. Reconstruction of articular cartilage defects with free peIiosteal grafts. An experimental study. Acta Orthop Scand 53:175-180. SKOOG,T., L. OHLSEN,and S. A. SO~N, 1972. Perichondrial potential for cartilagenous regeneration Scand. J. Plast. Reconstr. Surg. 6: 123. STAFFORD,U., O. O. A. ONI, J. HAY, and P. J. GREGG, 1992. An investigation of the contribution of the extraosseous tissues to the diaphyseal fracture callus using a rabbit tibial fracture model and in situ immunocytochemical localisation of osteocalcin. J. Orthop. Trauma. 6: 190-194. UTVAG, S. E., O. GRUNDNESand O. REIKERAOS,1996. Effects of periosteal stripping on healing of segmental fractures in rats. J. Orthop. Trauma. 10: 279-284. VAN DEN WILDENBERG,F. A., R. J'. GORIS,and M. B. TUTErN NOLTHENIUS-PUYLAERT,1984. Free revascularised periosteum transplantation: an experimental study. Br. J. Plast. Surg. 37: 226-235.
Corresponding author: Dr. MUSTAFAARICAN,Department of Surgery, Faculty of Veterinary Science, The University of Sel~uk 42031 Campus/Konya Turkey Tel.: ++90-332-2410041-2748; Fax: ++90-332-2140063; e-mail: [email protected] or [email protected]