中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (16): 2493-2498.doi: 10.3969/j.issn.2095-4344.2014.16.006
• 纳米生物材料 nanobiomaterials • 上一篇 下一篇
多壁碳纳米管改善激素性股骨头坏死模型兔的股骨头形态
戚 超1，王效军1，王效强2，冯 鑫1，张笑生1，赵 夏1，于腾波1
- 1青岛大学附属医院关节外科，山东省青岛市 266003；2寿光市市立医院，山东省寿光市 262700
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修回日期:2014-03-05出版日期:2014-04-16发布日期:2014-04-16 -
通讯作者:于腾波，博士，主任医师，教授，青岛大学附属医院关节外科，山东省青岛市 266003 -
作者简介:戚超，博士，主治医师，主要从事关节外科方面的研究。 共同第一作者：王效军，青岛大学在读硕士，主要从事关节外科方面的研究。
Multiwalled carbon nanotubes improve the morphology of the femoral head of a rabbit model of steroid-induced necrosis of femoral head
Qi Chao1, Wang Xiao-jun1, Wang Xiao-qiang2, Feng Xin1, Zhang Xiao-sheng1, Zhao Xia1, Yu Teng-bo1
- 1Department of joint Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China; 2Shouguang Municipal Hospital, Shouguang 262700, Shandong Province, China
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Revised:2014-03-05Online:2014-04-16Published:2014-04-16 -
Contact:Yu Teng-bo, M.D., Chief physician, Professor, Department of joint Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China -
About author:Qi Chao, M.D., Attending physician, Department of Joint Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China Wang Xiao-jun, Studying for master’s degree, Department of Joint Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China Qi Chao and Wang Xiao-jun contributed equally to this work.
摘要：
背景：碳纳米管可以促进成骨细胞的增殖和分化，可能对激素性股骨头坏死发挥治疗作用。
目的：观察多壁碳纳米管在激素性股骨头坏死模型建立过程中的作用。
方法：36只新西兰大耳白兔随机选出32只等分为治疗组和模型组，剩余4只作为空白组。治疗组兔每天臀肌注射地塞米松2.5 mg/kg，每周向双侧股骨的骨髓腔内各注射0.1 g/L多壁碳纳米管悬浊液1 mL；模型组每天臀肌注射地塞米松2.5 mg/kg，每周向双侧股骨的骨髓腔内各注射生理盐水1 mL，空白组每天臀肌注射生理盐水2 mL，每周向双侧股骨的骨髓腔内各注射生理盐水1 mL。
结果与结论：模型组兔股骨头组织中骨小梁开始少量出现变细断裂，骨髓脂肪化十分明显，脂肪细胞肥大，微血管开始出现血栓；而经多壁碳纳米管悬浊液治疗的兔股骨头病理损伤出现明显改善。提示多壁碳纳米管能在一定程度上改善激素性股骨头坏死的病理损伤。
中图分类号:
引用本文
戚 超，王效军，王效强，冯 鑫，张笑生，赵 夏，于腾波. 多壁碳纳米管改善激素性股骨头坏死模型兔的股骨头形态[J]. 中国组织工程研究, 2014, 18(16): 2493-2498.
Qi Chao, Wang Xiao-jun, Wang Xiao-qiang, Feng Xin, Zhang Xiao-sheng, Zhao Xia, Yu Teng-bo. Multiwalled carbon nanotubes improve the morphology of the femoral head of a rabbit model of steroid-induced necrosis of femoral head[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(16): 2493-2498.
Quantitative analysis of experimental animals
Rabbits in the treatment group had no obvious side reactions and biocompatibility problems. All of the 36 rabbits in the treatment group survived until the expected dates.
Multiwalled carbon nanotubes improve the pathological pattern of the rabbit models of SNFH
No necrotic bone collapses were found through gross observation in the three groups when the femoral heads were cut along the coronal plane. Four weeks after hormone injection, the number of empty bone lacunae in subchondral area and trabeculae in the model group significantly increased than that in the control group, the trabeculae began to have a small amount of thinner fractures, extensive pimelosis in the bone marrow was detectable, fat cells in the bone marrow became bigger and microvascular thrombosis appeared, while there was no positive histopathological manifestation in the treatment group. At the 6th and 8th week, the model group appeared to have the following changes: necrotic bone appeared in the subchondral area, parts of the trabeculae were fractured, the number of osteoblasts and blood vessels decreased, the number of osteoclasts and empty bone lacunae increased and the empty bone lacunae distributed focally, a large number of bone marrow and blood vessels became necrotic. The treatment group appeared to have the following changes: the bone trabeculae became thinner, the number of osteoblasts and blood vessels decreased while the number of osteoclasts and empty bone lacunae increased compared with the control group, intraosseous vessels were slightly damaged. Twelve weeks after hormone injection, the necrosis situation of bone in the subchondral area became even worse and the number of empty bone lacunae increased further more in the model group, while in the treatment group, the number of empty bone lacunae increased further, parts of the trabeculae were fractured, small pieces of necrotic bone marrow appeared. The number of osteoblasts decreased while the number of osteoclasts and fat cells increased obviously in the model group (Table 1). Due to the use of multiwalled carbon nanotubes, the number of osteoblasts decreased while the number of osteoclasts and fat cells increased in the treatment group, but they were not as worse as the model group. As osteoblasts, osteoclasts and bone fat cells were all differentiated from BMSCs, the results proved that multiwalled carbon nanotubes can inhibit the differentiation of BMSCs to osteoclasts and bone fat cells and promote the differentiation of BMSCs to osteoblasts. And this may be the mechanism of SNFH (Figure 1).| [1] Bao SY, Zhao GQ, Bi LQ. Changes in apoptosis related gene expression following alendronate intervention in the early srage of steroid-induced necrosis of the femoral head. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2008;12(46):9095-9099
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As the most common type of non-traumatic osteonecrosis of femoral head, steroid-induced necrosis of femoral head (SNFH), also called immortal illness, has higher morbidity and disability rate than before[1-11]. It has been imminent for us to find an effective way to prevent and treat SNFH. However, the pathogenesis of SNFH has not been clarified yet. To explore its pathogenesis then becomes the most important thing we should do to prevent and treat SNFH. The known pathological characteristics include that the number of osteoblasts and osteocytes reduces, empty bone lacunae and tiny blood clots widely distribute, and hypertrophic fat cells function to extrude normal bone marrow cells. Many pathogenesis theories of SNFH have been proposed, such as cell apoptosis theory, fat embolism theory, intraosseous hypertension theory, intravascular coagulation theory, and microvascular damage theory and so on. As a very promising material of bone formation, scientists have found that carbon nanotubes have the function of promoting cell proliferation, differentiation and tissue regeneration. Zanello et al [12] firstly found that osteocytes could grow, proliferate and differentiate normally on the carbon nanotubes. Elias et al [13-17] found that carbon nanotubes could promote the proliferation and differentiation of osteoblasts. In a study performed by Li et al [18], researchers found the mechanism of promoting osteoblast function by carbon nanotubes. Narita et al [19] found that carbon nanotubes could inhibit the proliferation and differentiation of bone marrow stromal cells into osteoclasts and the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). These studies creatively utilized multiwalled carbon nanotubes’ characteristics of promoting BMSCs to differentiate to osteoblasts and vascular endothelial cells and the proliferation of
osteoblasts as well as the characteristics of inhibiting BMSCs to differentiate to fat cells and the proliferation of osteoclasts[18-19]. BMSCs transplantation has been shown to improve the symptoms of SNFH to a certain extent, which cannot only enhance the blood supply of the femoral head by vascular proliferation, but also get direct access to the bone lacunae, thereby differentiating into osteoblasts to increase bone density[20]. Nonetheless, our study was designed to observe the role of multiwalled carbon nanotubes in treatment of SNFH.
osteoblasts as well as the characteristics of inhibiting BMSCs to differentiate to fat cells and the proliferation of osteoclasts[18-19]. BMSCs transplantation has been shown to improve the symptoms of SNFH to a certain extent, which cannot only enhance the blood supply of the femoral head by vascular proliferation, but also get direct access to the bone lacunae, thereby differentiating into osteoblasts to increase bone density[20]. Nonetheless, our study was designed to observe the role of multiwalled carbon nanotubes in treatment of SNFH.
Design
Randomized and controlled animals experiment.
Time and setting
The experiment was conducted at the Animal Experimental Center of Affiliated Hospital of Medical College, Qingdao University, China between May and August 2013.
Materials
Animals
Thirty-six healthy adult New Zealand rabbits (18 males, 18 females), weighing (3.50±0.16) kg, were provided by Institute for Drug Control, Qingdao, China (Licenses: SCXK (Lu) 2003-0010), and housed separately at 24 ℃. All experimental disposals of animals were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[21].
Biomaterials
High purity carboxylated multiwalled carbon nanotubes provided by Beijing Boyu Hi-Tech New Material Co., Ltd. (China). The multiwalled carbon nanotube is cracked by natural gas under the effect of cobalt catalyst (OD: 20-40 nm, Length: 10-30 µm, purity: > 95%).
Methods
Establishment of a rabbit model of early SNFH and intervening of multiwalled carbon nanotubes
Totally 36 adult rabbits were randomly divided into three groups: control group (n=4), model group (n=16) and treatment group (n=16). In treatment group, the 16 rabbits were given intragluteally injection of dexamethasone (2.5 mg/kg) every day and injection of 1 mL liquor of multiwalled carbon nanotubes (0.1 g/L, disposal of sterilize and ultrasonic oscillate before use) into the bilateral femur medullary space every week for 12 weeks. In model group, the 16 rabbits were given intragluteally injection of dexamethasone (2.5 mg/kg per day) and injection of 1 mL normal saline into bilateral femur medullary space every week for 12 weeks. In control group, the four rabbits were given intragluteally injection of 2 mL normal saline per day and injection of 1 mL normal saline into the bilateral femur medullary space every week for 12 weeks. During the whole 12 weeks, each rabbit was given intragluteally injection of 4×105 units of penicillin (North China Pharmaceutical Co., Ltd., China) twice a week for prevention of infection.
Histopathological assay
Rabbits in each group were sacrificed at the 4th, 6th, 8th, 12th weeks under intravenous anesthesia with pentobarbital sodium (35 mg/kg). After the animal was killed, the bilateral femoral heads including the thighbones and metaphyses were all removed, observed by macrography, then fixed by formaldehyde solution and demineralized by ethylene diamine tetraacetie acid. The samples were washed by current water, dehydrated using graded ethanol, vitrified by dimethylbenzene, embedded in wax, and cut into thin layers. Then the layers were stained with hematoxylin-eosin and observed by light microscopy (LEICA DM2000B). And changes in the cartilage, trabeculae, and hematopoietic tissue were observed. Calculating the empty bone lacunae rate of the femoral head at random 10 fields (hematoxylin-eosin staining, ×100) and 50 bone lacunae were counted in each. The number of empty bone lacunae was counted and the proportion of empty bone lacunae was calculated. Then, the severity of bone tissue necrosis of the three groups in different periods was detected based on the empty bone lacunae rate. At the same time, 10 bone lacunae in the femoral head were randomly selected, and we compared the number of bone fat cells, osteoblasts and osteoclasts of the three groups in different periods to explore the mechanism of SNFH.
Main outcome measures
We observed the pathological changes in the femoral head.
Statistical analysis
The data were expressed as mean±SD, and analyzed by SPSS 19.0 software (SPSS, USA). Student’s t test was carried out for analysis and P value which less than 0.05 meant statistical significance.
As one of the most common types of osteonecrosis of the femoral head, SNFH keeps a high incidence level and its pathogenesis is still unclear although many theories have been proposed, such as theories of cell apoptosis, fat embolism, intraosseous hypertension, intravascular coagulation, and microvascular damage and so on[1-11, 22-41]. Along with further research on SNFH in recent years, the theory of adipogenic differentiation of BMSCs has been put forward in which the adipogenic differentiation of BMSCs is the initial factor resulting in SNFH, while cell apoptosis, fat embolism, intraosseous hypertension, intravascular coagulation and microvascular damage are only secondary factors[42-50]. This theory also thinks steroid hormone leads to the decrease of BMSCs both in the number and activity but increases the activity of osteoclasts which breaks the balance between bone absorption and reconstruction, thereby causing the collapse of femoral head under the action of external load force. The theory explains the pathogenesis of SNFH from a new perspective and elaborates the all pathological changes. So we think this may be the real pathogenesis of SNFH.
With the development of science and technology, people have a further understanding of the biological function of carbon nanotubes. Carbon nanotubes have been shown to have the function of promoting cell proliferation, differentiation and tissue regeneration. On one hand, carbon nanotubes improve the imbalance between bone absorption and reconstruction caused by SNFH through promoting the differentiation BMSCs into osteoblasts and inhibiting the adipogenic differentiation of BMSCs. On the other hand, the morphological structure of carbon nanotubes is very similar to the triple helical structure of bone collagen fibers which can simulate the collagen skeleton on morphology and promote the deposition of calcium, phosphorus and other inorganic mineral and then induce the nucleation and growth of hydroxyl calcium phosphate. Coupled with its high tensile strength, elastic modulus and mechanical fatigue resistance properties, carbon nanotubes enhance the bone strength more to prevent the occurrence of the collapse of the femoral head. Some scholars believe carbon nanotubes have cytotoxicity based on verification tests[12, 51-56]. Nagai et al [57] found that carbon nanotubes could increase the risk of mesothelium cell tumor. But subsequent experiment results showed that the cytotoxicity detected in the previous test was due to the purity of carbon nanotubes. The residual metal catalysts during the process of producing carbon nanotubes such as nickel and iron entered the cells and triggered oxidation-reduction reaction. Then the hydrogen peroxide and free radicals produced by oxidation-reduction reaction damaged the cells, while the cytotoxicity of carbon nanotubes after further purification reduced significantly[58-60]. All in all, the current research on the toxicity of carbon nanotubes only focuses on the visceral toxicity of rodents and in vitro cytotoxicity, while toxicology studies for large mammals are very limited.
Our experiment proved that carbon nanotubes can reduce the occurrent of SNFH to a certain extent, by promoting the differentiation BMSCs into vascular endothelial cells and osteoblasts, inhibiting the adipogenic differentiation of BMSCs, promoting the proliferation of osteoblasts and inhibiting the proliferation of osteoclasts, while carbon nanotubes cannot improve the situation of fat embolism, intraosseous hypertension, intravascular coagulation, and microvascular damage directly. So we think steroid hormone inhibits BMSCs differentiating into osteoblasts and promotes adipogenic differentiation of BMSCs, which is the initial factor resulting in SNFH, while fat embolism, intraosseous hypertension, intravascular coagulation and microvascular damage are only secondary factors. But the best dose of carbon nanotubes and its best way of chemical modification remain to be discussed. Taken together, BMSCs transplantation in combination with functionalized carbon nanotubes may be an effective method for the treatment of early SNFH.
早期：多数患者的临床症状不明显，仅见于大腿部肌肉无力和内收肌疼痛，个别患者有远离部位的肢体疼痛，部分患者在劳累后可出现髋关节间歇性疼痛，双髋交替性疼痛或伴轻度跛行。临床检查时，可见髋部及腹股沟区轻微压痛，4字实验和托马征均可阳性。由于早期无典型临床症状体征及X射线表现。
中晚期：激素性股骨头坏死多为间歇性，隐袭性发病，随着中晚期病情演变，以发展缓慢的髋关节疼痛为主要临床症状，活动时加重，休息时不明显。约有四分之一的患者呈间歇性发作，其发作时表现为突然剧痛，又突然消失，在髋关节疼痛发作期间，个别患者常规止痛剂无效。
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