Abstract
BACKGROUND: Fixation of bone fracture is one of the fundamental methods for bone fracture healing. The technique of AO has a lot of defects, such as negative effect induced by "stress dodging". Recently, the technique of CO is praised highly by national and international scholars.
OBJECTIVE: To observe the influence of external fixation of small splint on healing of long bone fracture of rabbit, and compare to the internal fixation of steel plate.
DESIGN: Randomized and controlled animal trial.
SETTING: Research Institute of Orthopedics, Hubei College of Traditional Chinese Medicine.
MATERIALS: The experiment was performed at the Laboratory of Orthopedics, Hubei College of Traditional Chinese Medicine from April 2006 to April 2007. Thirty rabbits were randomly divided into small splint fixation group and steel plate fixation group with 15 rabbits in each group. Small splint was self-made of fir-barks with good elasticity, and composed of exterior, interior, front and back splints. The upper part of the splint was wide and the lower part was narrow. We sting an eyelet in the small splint that is used in front and behind part. A hole was drilled in the front and back splints close to the tubercle of tibia. Steel plate was provided by Jiangsu Golden Deer Group (Type HA2.0).
METHODS: The standard models of transverse fracture of 3 mm in the meta-infer 1/3 of left tibia were established. In small splint fixation group (SSF group), the fracture was fixed by plaster stone, and 5 days later, replaced by external fixation of small splint. The steel fixation group (SF group) was fixed by steel plate with 4 holes. Animals were executed 14, 24, and 34 days after surgery, respectively. The growth condition of bony callus in fracture sites was observed, and the histomorphology of bony callus and bone cell production during fracture healing was observed.
MAIN OUTCOME MEASURES: Macroscopic observation of rabbit tibial bony callus, and histomorphology of bony callus and bone cell formation.
RESULTS: In SSF group, the bony callus formed early, and there were plentiful and active osteoblast. Thirty-four days after surgery, bony union was observed in fracture sites. In SF group, there was little fibrous bony callus in the fracture ends 14 days after surgery, accompanied by granulation tissue. Twenty-four days after surgery, sparing cartilage synostosis was observed. On day 34 days, bony callus span the fracture ends, but fracture ends did not connect completely yet. Compared with the SF group, the quantity of bony callus and the speed of fracture healing were superior in SSF group.
CONCLUSION: The external fixation of small splint can promote osteoblastic differentiation and proliferation, absorption of hematoma, calcification of the bony callus, and the growth and rebuilding of bone trabecula.

INTRODUCTION

The intension and style of fixation for fracture decide the mode of fracture union, and it has influence on histomorphology and ultramicrostructure of the union of fracture. Small splint fixation is a kind of elastic fixation, which makes external callus grow plentifully, and the fracture heal at the second stage. In this study, we established rabbit models of transverse fracture pattern in tibia that was fixed by small splint or steel plate to observe macroscopic condition and morphology of bony callus as well as osteocytes.

MATERIALS AND METHODS

Materials
The experiment was performed at the Laboratory of Orthopedics, Hubei College of Traditional Chinese Medicine from April 2006 to April 2007. Thirty healthy 5-month-old New Zealand rabbits weighing 2.0-2.5 kg were selected with a half amount of male and female. The animals were supplied by Animal Experimental Center of Disease Control Institute of Hubei Province. All the animals were fed with standard diet. The experimental procedure was accorded with the animal ethical standards.
The small splint was self-made of fir-barks that have good elasticity. It consisted of four splints: exterior, interior, front and back. The upper part of the splint was wide and the lower part was narrow. We drilled a hole in the front and back small splints close to the tubercle of tibia. Steel plate was provided by Jiangsu Golden Deer Group (Type HA2.0). Light microscope produced by Olympus, Japanese and H-500 transmission electron microscope by Hitachi, Japanese were provided by General Hospital of Wuhan Military Area Command of Chinese PLA. LKB-Ⅱ ultramicrotome, produced by LKB, Sweden, was provided by Wuhan University Medical College.

Methods
Model establishment and grouping
Thirty rabbits were randomly divided into small splint fixation group and steel plate fixation group with 15 rabbits in each group. The left hind leg of rabbit was cleaned and soaked in 1% glutaral (GA) solution for 5 minutes, (intramuscularly injected) i.m 0.1 g phenobarbital sodium (PBS), fixed in dorsal position on operational table. The left hind leg was sheared; the operating field was sterilized by
vigor iodine, then the rabbit was locally anesthetized by 1% dolicaine, and aseptic towel was spread out. With 1/3 meta-inferior segment of tibia as center, about a 2-cm incision was slit along anterolateral tibia, then skin, fascial superficialis, muscle, and periost were cut open layer by layer to expose tibia. The tibia was sew by fret saw to make a bone defect of 3 mm. The standard model of transverse fracture was established. In steel plate fixation group, the fracture was fixed by a 3-hole steel plate on the ante-lateral position; after the wound was washed by 1% benzalkonium bromide normal saline, the wound was sutured. In small splint fixation group, the wound was directly washed by 1% benzalkonium bromide normal saline, sutured layer by layer, then fixed by plaster cast of external use. After surgery, each rabbit was i.m penicillin 40 000 U/(kg·d) once a day for 3 days. Five days later, small splint fixation group was fixed by small splint: i.m 0.1 g PBS, the rabbit was fixed to lie on the back on the fixed frame, the plaster was removed, and a Kirschner wire of 1.5 mm was penetrated in tibial tubercle, and crossed the hole of small splint to prevent splint slippage. Finally, the four small splints were packed using three bands, and the upward to downward range of motion was 0.3 cm.

Observational index and method
Macroscopic observation: external, internal bony callus and bridge bony callus 14, 24, and 34 days after surgery. Histomorphological observation: The rabbits were executed (5 animals at each time point). The tibial samples were sawed at 1 cm above and below the fracture, fixed by 10% formaldehyde solution, decalcificated by 5% nitric acid solution, flushed by lotic water, dehydrated by gradient alcohol, and embedded by paraffin. Longitudinal sections were prepared with each piece of 5 cm thick. The sections were stained using hymatine-eosin (HE) in routine method, then observed under a light microscope. Ultrastructural observation: Sample of 1 mm3 was harvested at each time point, and fixed by 2.5% GA solution, decalcificated by 1% EDTA solution, washed by balanced solution (pH, 7.2-7.4) for three times, then fixed by 1% osmic acid for 2 hours, dehydrated by alcohol, embedded by epoxy resin 812 to obtain extra thin section. The sections were stained using lead uranium and observed by transmission electron microscope.

RESULTS

Quantitative analysis of animals
Thirty rabbits were all included in final analysis with no loss.

Macroscopic observation of bony callus during fracture healing
In the small splint fixation group, the fractured fragment union was primarily observed 14 days after surgery, and little spindle external callus was found; 24 days after surgery, the callus was in spindle shape, and callus bridged the two broken ends; on day 34 days, the two fractured ends were conjugated firmly by the callus, and there was no range of motion in the broken ends. In the steel plate fixation group, no dislocation was found in the fractured ends at each time point. Fibrous joint was seen 14 days after surgery; on day 24, dispersing external callus was found, aligning regularly, and joint of spindle form was not observed in the broken ends; 34 days after surgery, the fractured ends were united by external callus in spindle shape, but not very firmly.

Morphology of bony callus and osteoblast production during fracture healing
Fourteen days after surgery, light microscopic observation showed that in small splint fixation group, plenty of osteoblast proliferated in periosteum, with fibrous callus formation; many mesenchymal cells differentiated and proliferated, and chondrocytes were found, but granulation tissue was very little; in steel plate fixation group, blood tumor was absorbed well; there was small amount of osteoblast in periosteum, and a small quantity of chondrocytes were found. Electron microscopic observation suggested that in small splint fixation group, there was abundant rough endoplasmic reticulum (RER) in osteoblast, and cell prominence was sharp, and grew into collagen fiber deeply; there were a lot of chondrosomes, plenty of collagen fibrils concentrated around osteoblast, aligning irregularly, and a large amount of fibroblast arranged disorderly. In steel plate fixation group, fibroblast had fairly affluent RER, with little vacuole in kytoplasm, and lysosome (LYS) was found in cells. Twenty-four days after surgery, light microscopic observation showed that in small splint fixation group, a lot of osteoblast proliferated and formed new bone trabecula in the fractured ends; woven bone transformed to plate-like bone gradually, and there were still some cartilages; in steel plate fixation group, few fibrous tissue and cartilaginous tissue were found in the fractured fragment, and os endochondrale was observed. Electron microscopic observation suggested that in small splint fixation group, chondrocytes transformed into bone cells with irregular or egg-round cell nucleus, clear plasmosome; there was swollen chondrosome in cytoplasm, and a lot of ER. In steel plate fixation group, affluent RER was found in osteoblast, cristae in chondrosome disappeared; chondrosome became swelling, and had marrow-like change. Thirty-four days after surgery, light microscopic observation showed that in small splint fixation group, the fractured ends were united, and canals of de Candolle was of partial recanalization; a lot of lamellar bone was seen in the fractured end, but woven bone was hardly observed; exterior and interior callus was thinning, and chondrocytes disappeard, no fibroblast was found. In steel plate fixation group, the fractured ends were not united completely, and cartilage was partly calcificated; in addition, there was some bridge callus and little bony external callus. Electron microscopic observation showed that in splint fixation group, bone cell shape was irregular, and the cell nucleus was egg-round; intranuclear heterochromatin distributed in mass, and intracytoplasm ER expanded; cell prominence was stretching into compact bone.

DISCUSSION

Fixation is an essential procedure in fracture healing. In different history periods, various medical institutions select different fixation patterns for fracture. After a half of century of popularity of AO internal fixation, its defects and deficiencies are gradually realized by national and international researchers. They all think elastic fixation is more reasonable and beneficial technique for fracture healing [1]. Elastic fixation is a repeated stress from tiny to great, and from great to tiny supplied to the fractured ends in axial direction during fracture healing. In recent years, national and international researchers have made a lot of studies about fracture micromovement and fracture healing. It is demonstrated [2-4] that in the early period of fracture healing, controllable micromovement can promote the growth of fracture, and accelerate fracture healing. Cornell et al [5] demonstrated that micromovement in the fractured end can cause repeated damage to callus, resulting in repeatedly earlier reaction of fracture, and release of many biochemical mediators, and bone formation factors, which accordingly induced lobus intermedius cell multiplication and differentiate towards osteoblast. In addition, it is reported by Goodship et al [6] that under non-rigid fixation, modulation of the mechanical stress environment can influence healing speed and growth extent of external callus; the movement of fracture fragments plays an important role in fracture healing. The micromovement in the fracture ends can promote the formation and calcification of callus, which is benefit to the union of fracture. Local external fixation using small splint can control the activity not benefit to the union of fracture, and balance body gravity and muscle tonus that may cause redislocation again by constraint force of band, lever force of splint and efficacy of pressure pad. Qiao et al [7] has proved that by the experiment that can control the micromovement by external fixation frame, micromovement can increase blood flow of the fracture ends, and promote the expression of growth factor. By means of the fixation of small splint, the muscle around the fracture ends can produce tensile force by function practice, and form a longitudinal pressure. It is a physiological stimulation for the fracture ends to bear this pressure and stress in the condition that the fracture ends are always in the force environment of repeated micromovement in a considerable range. The results of this study suggest both speed and quantity of bone callus formation in small splint fixation group are superior to steel plate fixation group. Light microscopic observation shows that during fracture healing in small splint fixation group, osteoblast appears earlier, and the quantity is more than in steel plate fixation group; besides, its function is active. Thirty-four days after surgery, plenty of bone cells with active function are seen in the fracture ends. In steel plate fixation group, common steel plate is used for fracture fixation. The style of fracture union is like secondary healing. Because of the lack of micromovement in axial direction, the callus is not abundant, and the degree of maturation is low in fracture ends. This results in a slower healing compared with small splint fixation group. Fibroblasts observed in the initial stage of fracture healing means the beginning of repair of the fracture. Fibroblast cannot only form fibrous callus but also bony callus. Osteoblast plays an important role in the repair of fracture. On one hand, it can synthetize and produce collagen and protein, and when it is surrounded by organic matrix which is secreted by itself, it participates osteoid mineralization. Besides, osteoblast can secrete cell factor. If the blood supply in the fracture ends is patency, the stem cells in bone tissues can differentiate into osteoblast, which can become bone cell at last. If the blood supply is fairly bad, the stem cells then differentiate into chondroblast and chondrocyte. Chondrocyte proliferates plentifully and forms cartilaginous cell, which can differentiates into bone cell, and bony callus finally. Bony cell is translated by the osteoblast with no osteogenesis. The integrity and activity of bone cell is the sign of the vitality of bone; moreover, bone cell is an important component to keep the integrity of bone tissue.

REFERENCES

1 Jin HB. Tendency and prospect of Chinese osteosythesis. Zhongguo Gushang 2005;18(2):65-66
2 Kershaw CJ, Cunningham JL, Kenwright J. Tibial external fixation, weight bearing, and fracture movement. Clin Orthop Relat Res 1993;(293):28-36
3 Kassis B, Glorion C, Tabib W, et al. Callus response to micromovement after elongation in the rabbit. J Pediatr Orthop 1996;16(4):480-483
4 Sarmiento A, McKellop HA, Llinas A, et al.Effect of loading and fracture motions on diaphyseal tibial fractures. J Orthop Res 1996;14(1):80-84
5 Cornell CN, Lane JM. Newest factors in fracture healing. Clin Orthop Relat Res 1992;(277):297-311
6 Goodship AE, Cunningham JL, Kenwright J. Strain rate and timing of stimulation in mechanical modulation of fracture healing. Clin Orthop Relat Res 1998;(355 Suppl):S105-S115
7 Qiao L, Hou SX, Li WF, et al. Effects of micromovement on microcirculation at the fracture part and expression of vascular endothelial growth factor. Zhonghua Guke Zazhi 2005;7(1):54-55

小夹板外固定与钢板内固定材料置入对骨折断端成骨活性的影响☆

李 瑛，邹 季，熊 勇
湖北中医学院骨伤研究所，湖北省武汉市 430061
李 瑛☆，女，1964年生，湖北省天门市人，汉族，湖北中医学院在读博士，副教授，主要从事骨与关节损伤研究。
摘要
背景： AO技术存在许多的缺陷，如"应力遮挡"产生的负面效应等。近年来国内外学者认为弹性固定法最合理，是最有利于骨折愈合的治疗理念。
目的：观察小夹板外固定对兔长管状骨骨折断端成骨活性的影响，并与钢板内固定材料置入方法比较。
设计：随机对照动物实验。
单位：湖北中医学院骨伤科研究所。
材料：实验于2006-04/2007-04在湖北中医学院骨伤实验室完成。30只家兔随机分成小夹板固定组、钢板固定组，每组15只。自制小夹板，由具有较好弹性的杉树皮制成。分前后、内外侧四块夹板，夹板上宽下窄，在前后侧夹板靠近胫骨结节部刺一小孔。钢板由江苏金鹿集团医疗有限公司提供。
方法：在左胫骨中下1/3处造成3 mm骨缺损横行骨折模型，小夹板固定组用石膏固定5 d后换成小夹板外固定，钢板固定组用4孔钢板内固定。术后14，24，34 d时分批处死各组动物，通过肉眼观察骨折处骨痂生长情况，并观察骨折愈合过程中骨痂组织形态学及骨生成细胞情况。
主要观察指标：不同时期兔胫骨骨痂肉眼观察，骨痂组织形态学和骨生成细胞情况。
结果：小夹板固定组骨痂形成早，早期成骨细胞丰富且活跃，34 d时骨折全部骨性连接。钢板固定组：14 d时骨折端见少量的纤维骨痂，仍有肉芽组织，24 d时见少量的软骨连接，34 d时骨痂已跨过骨折端，但未完全连接。小夹板外固定组与钢板固定组比较，骨折各期形成的骨痂量多，骨折愈合快
结论：小夹板外固定能促进骨折处成骨细胞的分化增殖和血肿的吸收、骨痂的钙化、骨小梁的生长改建。
关键词：成骨活性；骨折愈合；小夹板；钢板；骨痂大体观；骨痂形态学；骨生成细胞
中图分类号: R318 文献标识码: A 文章编号: 1673-8225(2008)13-02576-03
李瑛，邹季，熊勇.小夹板外固定与钢板内固定材料置入对骨折断端成骨活性的影响[J].中国组织工程研究与临床康复，2008，12(13):2576-2578
[www.zglckf.com/zglckf/ejournal/upfiles/08-13/13k-2576(ps).pdf]
（Edited by Zhang HM/Su LL/Wang L）