Abstract
BACKGROUND: Animal models of peripheral partial epiphyseal plate closure are commonly used in related trials.
OBJECTIVE: To modify the original animal models of distal femoral partial epiphyseal plate closure.
DESIGN, TIME AND SETTING: A self-controlled trial was performed at the Experimental Animal Center of General Hospital of Chinese PLA from March to October 2007.
MATERIALS: Twenty healthy New Zealand rabbits of 4-6 weeks old weighing 1.0-1.5 kg were selected.
METHODS: A straight incision about 2 cm was made in lateral knee of 20 rabbits. The subcutaneous tissues and knee capsule were cut open to expose distal femoral condyle. The condylar plate line was clear. Two holes were drilled in the right lateral condyle, about 3.0-4.0 mm deep, and partial normal epiphyseal plate (about 33%-44% of the total growth plate) was removed. An incision deep to periosteum was created in the left side and regarded as self-control.
MAIN OUTCOME MEASURES: Femoral length, valgus angle of the distal femur, and femoral deformity were detected by X-ray photograph, and gross observation. Bone bridge formation of bilateral femora was observed by histological examination.
RESULTS: One rabbit died of diarrhea 31 days after surgery, and 19 were included in final analysis. ①Radiographs showed that the mean length of left femur (control side) was significantly longer but valgus angle was significantly smaller than the right side (experimental side) 4 months after surgery (P < 0.01). The left distal femur averagely grew for 2.27 cm, accounted for 57.6% of the overall femoral growth, and the right side averagely grew for 0.45 cm, accounted for 21.4% of the overall femoral growth. ②Four months after surgery, femoral appearance of the control side was normal, with smooth and complete condylar and facies articularis patellae. While, the experimental side femur was significantly shortened, and severe valgus deformity was observed in the distal femur. ③Five weeks after surgery, layer cells of the epiphyseal plate arranged regularly in the control side. Bone trabecula with many fibers was found in bone defect cavity of the experimental side, and the cell layer of the remnant epiphyseal plate was decreased. The epiphyseal plate was closed in the control side, and bone trabecula of the experimental side was thickened 10 weeks postoperatively.
CONCLUSION: This modeling method is simple, efficient, and easy to manipulate; in addition, the bone bridge area is controllable. The models can meet the study demands for peripheral partial epiphyseal plate closure, and are significant for filling material selection during treatment.

INTRODUCTION

Partial epiphyseal plate closure is a disease caused by bony union between long bone epiphyseal center and metaphysis in childhood, which impedes epiphyseal plate normal growth, leading to articular angulation deformity or limb shortening even permanent disability. Although many studies have reported various animal models, there are many drawbacks. In this study, we established animal models of partial epiphyseal plate closure by distal femoral epiphyseal plate resection. It is a simple method with controlled experiment error, high repeatability, and adjustable bone bridge area compared with original modeling methods.

MATERIALS AND METHODS

Materials
The experiment was performed at the Experimental Animal Center of General Hospital of Chinese PLA from March to October 2007. Twenty healthy New Zealand rabbits of 4-6 weeks weighing 1.0-1.5 kg, irrespective of gender, were provided by the Experimental Animal Center of General Hospital of Chinese PLA [No. SCXK (Jing) 2005-0013]. Bilateral femoral lateral condyles served as experimental sites.

Methods
Modeling
The rabbits were anesthetized with intramuscular injection of 20 mg/kg compound ketamine solution (Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences). The rabbits were fixed at prone position to depilate and spread out aseptic towel. A straight incision about 2 cm was made in the lateral knee. The subcutaneous tissues and knee capsule were cut open to expose distal femoral lateral condyle. The condylar plate line was clear. At 1 cm above epiphyseal plate, one puncture needle was used to puncture femoral cortical bone till medullary canal, and one L-shaped stainless steel needle was inserted as marker needle. The capsule of knee of the left side was sutured, and the incision was injected with 200 000 U penicillin and sutured. The incision covered with membrane served as control. Two holes were drilled in the epiphyseal plate of right lateral condyle using biopsy needle of 4 mm diameter, about 3.0-4.0 mm deep, and partial normal growth plate was removed. Then the right side was treated using the previous procedure as the left side.

Postoperative treatment
Radiographs was taken for both lower extremities immediately after surgery, and then the rabbits were freely placed in metal cage and fed with particle forage, with no external fixation. Penicillin (800 000 U per day) was injected into the rabbits for 3 days.

Observation
Radiographs: Radiographs of both lower extremities were taken every week after the rabbits were anesthetized. Bilateral femoral length and valgus angle of the distal femur were measured on the basis of radiographs. The line from femoral greater trochanter top to the midpoint of distal femoral epiphyseal plate was regarded as femoral Y-axis; the angle α between tangent line of distal femoral two condyles and the straight line vertical to femoral Y-axis was regarded as the valgus angle of the distal femur; femoral length was the distance “a” from the top of greater trochanter to distal medial condyle of femur; distal femoral growth length was the distance “b” from the highest point of marker to distal medial condyle of femur (Figure 1). Gross samples: Sixteen animals were executed to harvest bilateral femoral samples. Femoral length and the valgus angle of the distal femur were measured; femoral deformity was observed. The differences were compared after statistical analysis. Histology: Fresh femoral samples were fixed in 0.1 volume fraction formalin solution for 72 hours above, and washed with water for 24 hours, following by decalcification with 50% formic acid for 72 hours. The samples were made into 4 mm-thick bone blocks, dehydrated by alcohol, and embedded with paraffin. Serial sections on sagittal plane were prepared in 5 μm thick, and stained with hematine-eosin (HE).

Statistical analysis
The data were analyzed using SPSS 13.0 assisted by Department of Statistics, General Hospital of Chinese PLA. Measurement data were presented by Mean±SD, and paired t-test was performed.

RESULTS

Quantitative analysis of experimental animals
Of the 20 selected New Zealand rabbits, 1 rabbit died of diarrhea 31 days after surgery, and 19 were included in final analysis with no incision infection or other complications.

Femoral deformity
The left femur grew normally with no shortening or angular deformity, and the epiphyseal plate line was clear. The epiphyseal plate began to close about 15 weeks postoperatively. While since the second week after surgery in the right femur, the density of sites after epiphyseal plate resection began to increase, and the range was enlarged; valgus deformity of the distal femur occurred at 3 weeks postoperatively; femoral length was significantly shortened at 4 weeks after surgery, and femoral deformity was progressively aggravated; the remnant epiphyseal plate was thinned compared with the left side at the same stage, and the whole epiphyseal plate closed at 9 weeks after surgery. The closure time was earlier than the left side.

Radiograph measurement
Immediate radiographs showed that there were no significant differences in femoral length and the valgus angle of distal femur. At 16 weeks after surgery, the femur of the control side (left side) grew normally for 3.94 cm in average, and valgus angle only increased 1.33°; femoral shortening or angular deformity did not occur, and the epiphyseal plate developed normally with no bone bridge. The femur of the experimental side (right side) grew 2.05 cm in average, and the valgus angle increased 43°. Compared with the control side, the femoral length of the experimental side was significantly shortened, and valgus angle was significantly increased since the first week (P < 0.05). The distal femur of the control side averagely grew for 2.27 cm, accounted for 57.6% of overall femoral growth, while the experimental side averagely grew for 0.45 cm, accounted for 21.4% of overall femoral growth.

Gross observation (Table 1)

 

Four months after surgery, femoral appearance of the control side was normal, with smooth and complete condylar and facies articularis patellae. While in the experimental side, the femur was significantly shortened, and severe valgus deformity was observed in distal femur. Partial femur developed twisting deformity, i.e. the distal femur was backward and laterally twisted along femoral Y-axis. Cartilage surface defects were found in the distal femur; the epiphyseal plate line disappeared completely, and was replaced by conglobate new-bone formation. In addition, patellar dislocation was found in 7 cases of the experimental group.

Histological examination
Five weeks after surgery, layer cells of the epiphyseal plate in the control side arranged regularly and blue stained (Figure 2). Bone trabecula with many fibers was found in bone defect cavity of the experimental side, filling by scattering red blood cells. The cell layer of the remnant epiphyseal plate was decreased, and cells arranged irregularly (Figure 3). At 10 weeks postoperatively, the cell layer of the epiphyseal plate in the control side was significantly decreased with no lamellar arrangement (Figure 4), while the bone trabecula of the experimental side was enlarged, and conglobate red blood cells and remnant epiphyseal plate cell structure disappeared with only some blue-stained region (Figure 5).

 

DISCUSSION

Partial epiphyseal plate closure is commonly caused by traumatic epiphyseal injury[1-2], operative complications[3], bone tumor and tumor treatment-associated procedure [4-6], novel technique such as arthroscope[7], vascular embolism[8], even intravenous transfusion extravasation[9]. Although the causes are different, the partial normal epiphyseal plate between osteoepiphysis and metaphysis also disappears, but is replaced by osseous union, also named bone bridge, which leads to skeletal growth retardation, limb shortening or angular deformity. Partial epiphyseal plate closure is classified into central type, peripheral type and linear type, and peripheral bone bridge is most frequent. Currently, partial epiphyseal plate closure is mainly treated with bone bridge resection arthroscopically or surgically following by filling into bone defect cavity [7, 10-11]. Accordingly, filling selection becomes study focus [12-13].
Since 1950’s, the primary studies have been conducted to explore the remnant epiphyseal plate after partial resection[14]. In 1972, Osterman[15] firstly reported the animal models of peripheral epiphyseal plate closure made by distal femora of New Zealand rabbits. Subsequent experimental studies were conducted about partial epiphyseal plate closure [16-19] by making models in distal femora or proximal tibias of rabbit, murine, swine, dog and sheet. Cady [20] and Cottalorda [21] also established models of central epiphyseal plate closure in the distal femur of rabbits, but the procedure was too complex and excavated central partial epiphyseal plate through distal femoral intercondylar fossa, which damaged the integrity of knee joint, and led to knee exudation, even joint motion limitation. In addition, the diameter of perforator was limited within 4.5 mm to avoid bone fracture, but the epiphyseal injury area would be less than 25% of overall epiphyseal plate, and accordingly cannot meet the demands of experimental studies on large area epiphyseal plate closure. By epiphyseodesis method, Sudmann [17] created animal models of peripheral epiphyseal plate closure in rabbits. A 2 mm × 6 mm × 2 mm bone flap was resected from epiphyseal plate of femoral lateral condyle, including 2 mm in the distal epiphyseal plate and 4 mm in the proximal. Then the bone flap was rotated by 180°, and reimplanted into the rabbits. Peripheral bone bridge was found 3 weeks later. However, the success rate was only about 75%, and the area of bone bridge was limited.
In this study, we modified the modeling method on the basis of Osterman[15] to establish peripheral epiphyseal plate closure in the distal femora of New Zealand rabbits. New Zealand rabbits are inbred strain animals; they are gentle and easy to manage. Six to eight-week-old rabbits have good tolerance to surgery, and it will take 4-6 months for the physiologic healing of epiphyseal plate. The epiphyseal plate in distal femur with big area and irregular appearance has a high incidence rate of partial epiphyseal plate closure in children. Therefore, the epiphyseal plate in distal femur of rabbits is ideal material to made models. First, the epiphyseal plate of lateral condyle was exposed, and two adjacent holes, slightly overlapped (1 mm) and 3.0-4.0 mm deep, were drilled through the epiphyseal plate using a round hollowed biopsy needle (diameter, 4 mm). Due to the irregular appearance of the epiphyseal plate in distal femur, the superior border of the biopsy needle should be placed on the surface of the epiphyseal plate to ensure that the excavated tissues contained epiphyseal plate tissues. The overall area of the epiphyseal plate in distal femur of 6-week-old New Zealand rabbits measured by CT was about 63.2 mm2 [21], in our models, the area of the excavated epiphyseal plate was 21 -28 mm2 (7 mm×3 mm -7 mm×4 mm), about 33%-44% of the overall area according to the calculation.
In this study, bone bridge occurs in all experimental animals 16 weeks after surgery, resulting in severe femoral shortening and knee joint valgus deformity, which significantly differs from the control group. There are several differences in modeling between our study and Osterman: ①In the study of Osterman, the epiphyseal plate was excavated with no immediate filling until the secondary resection of bone bridge. While, by our method, we immediately filled the wound after partial resection of epiphyseal plate, which can better control the shape and size of bone defect region. This is helpful to study the treatment effects of different fillings, decreases the experimental errors caused by the size of bone defect, and improves the precision of the trials. ②If subsequent procedure is conducted after bone bridge formation, limb shortening or angular deformity has occurred before bone bridge resection and filling, and will affect the identification to the fillings. ③Bone bridge formation takes several weeks, so if we conduct the trials after bone bridge formation, there is little time for normal epiphyseal plate closure. In addition, it is not vigorous proliferative stage of epiphyseal plate, so the identification for the fillings will be limited. In our study, peripheral bone bridge occurs in the distal femur of the experimental side. The epiphyseal plate of distal femur in the experimental side has grown for 0.45 cm at 16 weeks, significantly smaller than the control side, accompanied by femoral growth disturbance and severe valgus deformity. The subsequent trials of filling can be conducted directly.
To sum up, this modeling method is reliable and simple, which produces controllable area of bone bridge and practical animal models of partial epiphyseal plate closure.

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部分骺板挖除后立即充填构建股骨远端周围型骺板部分早闭实验兔模型*☆

朱志宏1，许瑞江2
解放军总医院，1急诊科，2小儿外科，北京市 100853
朱志宏☆，男，1968年生，北京市人，汉族，2006年解放军军医进修学院毕业，博士，主治医师，主要从事创伤急救及骺板部分早闭的研究。
通讯作者：许瑞江，主任医师，教授，解放军总医院小儿外科，北京市 100853
国家自然科学基金资助项目（30772276）*
摘要
背景：用于骺板部分早闭的实验动物模型多为周围型骺板部分早闭。
目的：在周围型骺板部分早闭模型的基础上，改进建立股骨远端周围型骺板部分早闭动物模型。
设计、时间及地点：自身对照设计实验，于2007-03/10在解放军总医院实验动物中心完成。
材料：健康普通级新西兰幼兔20只，兔龄4~6周，体质量1.0~1.5 kg。
方法：将20只新西兰白兔取膝外侧直切口，长约2 cm，切开皮肤、皮下组织，并于外侧髌旁切开膝关节囊后显露股骨远端外髁，此时股骨远端的骺板线清晰可见。将右侧股骨外髁的骺板挖两个相邻的孔，深度约3.0~4.0 mm，以去除部分正常骺板，面积为股骨远端骺板总面积的33%~44%。左侧仅切开至骨膜作为自身对照。
主要观察指标：术后X射线片测量同时大体观察双侧股骨的长度、股骨远端外翻角度及股骨的畸形情况；组织学检查双侧股骨远端的骨桥形成情况。
结果：1只实验兔术后31 d 因腹泻死亡，19只术后生长良好，进入结果分析。①术后4个月，X射线观察显示左侧股骨平均长度>右侧，外翻角度 < 右侧，两组之间相比，差异显著（P < 0.01）。股骨远端左侧平均生长2.27 cm，占股骨生长的57.6%，右侧平均生长0.45 cm，占股骨生长的21.4%。②术后4个月，对照组股骨形态正常，两髁及髌骨关节面光滑完整。实验侧股骨明显短缩，远端出现严重的外翻畸形。③术后5周，对照组骺板各层细胞排列整齐。实验组骨缺损腔内可见大量纤细的骨小梁，周围剩余骺板细胞层数减少。术后10周，对照组骺板已基本闭合；实验组骨小梁变得更为粗大。
结论：本方法建立的模型操作简单，成功率高，应用灵活，造成的骨桥面积大小可以控制，适用骺板部分早闭疾病动物模型的要求，对治疗过程中充填物的选择亦有一定意义。
关键词：生长面；疾病动物，模型；组织构建
中图分类号: R332 文献标识码: A 文章编号: 1673-8225(2008)15-02993-04
朱志宏，许瑞江. 部分骺板挖除后立即充填构建股骨远端周围型骺板部分早闭实验兔模型[J].中国组织工程研究与临床康复,2008,12(15):2993-2996
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2993(ps).pdf]
(Edited by Lu W/Su LL/Wang L)