摘要
目的:由于感染，外伤，肿瘤，先后天畸形，内分泌系统疾病造成的骨缺损，畸形，功能障碍，给患者的生活质量带来很大的影响，尤其是对于口腔颌面部。因此寻找一种简便、快捷、有效的方法促进骨创的修复，成为外科领域研究的热点和难点。
目前对如何促进骨愈合的研究很多。一些高分子生物材料植入，人工替代物植入，自体骨移植，组织工程化骨移植，低强度脉冲电磁场治疗等均有一定疗效，由于都存在或多或少的缺点，未能在临床上广泛应用。
多年以来，国内外学者借助组织学、组织化学，组织形态计量学，生物力学等手段对骨创愈合进行了大量的实验观察，对骨创愈合机制也进行了广泛的探讨。近年来，由于基因技术的发展应用，人们对骨创愈合的认识已从细胞生物学向分子生物学水平深化，并随着分子生物学的不断发展，己经认识到骨创愈合与多种生长因子有关，除内分泌、代谢因子等全身因子外，骨创局部尚有大量的生长因子，对骨创愈合起着重要的调节作用，如：TGF-β(转化生长因子)，VEGF(血管内皮细胞生长因子)，FGF(成纤维细胞生长因子)，BMP(骨形态发生蛋白)，神经生长因子(nerve growthfactor, NGF)等。
中药对加速骨折愈合机理的实验研究始自上世纪60年代，大致有如下几个方面的成果：中药改善骨折部的血液供应，促进血肿的吸收与机化；促进矿物质的沉积；增加成骨细胞的数量和活性，提高微量元素等。淫羊藿(Herba epimedii)是一种传统的补益中药，临床上用来治疗骨折及与骨代谢有关的许多疾病，并取得了良好的效果。研究结果表明，淫羊藿具有增强机体免疫功能，抗肿瘤，影响内分泌加强性腺功能、延缓衰老和影响心血管系统等多种重要的药理活性。淫羊藿的有效活性成分淫羊藿苷(icariin，C33H40O15，分子量：676.67)，属黄酮类化合物。李芳芳等将淫羊藿苷与大鼠卵泡颗粒细胞和肾上腺皮质细胞共同培养，显示一定浓度的淫羊藿苷可促使卵泡颗粒细胞分泌雌二醇，高浓度时还可促进肾上腺皮质细胞分泌皮质类固醇，提示淫羊藿补肾壮阳功效：作用下丘脑垂体-性腺轴及肾上腺轴，可有效调节体内激素的水平,改善骨代谢，促进蛋白质合成。由于淫羊藿苷在骨创愈合，修复中起到积极的作用，淫羊藿在促进骨愈合方面的研究成为研究热点，淫羊藿提取物：淫羊藿苷对成骨细胞增殖代谢的影响，体外实验董福生课题组已经完成。
本研究通过制作新西兰大白兔颅骨模型，术后采用淫羊藿苷灌胃，应用组织学和骨计量学方法，观察在骨缺损不同的愈合阶段，颅骨缺损区新生骨量，骨愈合速度和成骨质量。
同时提取颅骨缺损修复区新生骨组织mRNA，采用RT-PCR(realtime-polymerase chain reaction)方法，检测成骨细胞促进骨生成的基因变化情况，进一步研究在新骨形成的过程中和应用淫羊藿苷治疗后，相关基因的变化，相互联系，寻找成骨的分子信号传导通路。
在以上研究的基础上，通免疫组化方法，观察新骨形成区骨钙素和骨桥素的表达，初步探讨淫羊藿苷促进骨形成的机制。为淫羊藿苷在促进骨愈合的临床应用提供理论依据，奠定基础。
方法：
1淫羊藿苷对兔颅骨缺损修复愈合质量的影响
选用12周龄雌性新西兰大白兔36只，随机分为用药组和对照组，制作颅骨缺损动物模型，用药组术后给予淫羊藿苷灌胃，分别于术后4周、8周、12周处死动物，留取标本，拍射X线片观察；制作组织切片，HE染色，观察颅骨缺损区愈合情况；骨计量学观察新骨形成区骨小梁平均宽度(Traberculae Width，Tb·Wi μm)，骨小梁面积比(Trabercular AreaTb.Ar%)，成骨细胞个数(Osteoblast Number, OB·N)，破骨细胞个数(Osteoclast Number, OC·N)，观察新骨形成区骨量的变化，研究淫羊藿苷对兔颅骨缺损愈合的影响。
2淫羊藿苷促进兔颅骨缺损修复RANKL基因、RUNX2基因表达的影响
选用12周龄雌性新西兰大白兔36只，动物分组，动物模型制作，动物给药方法和剂量同第一部分，分别于术后4周、8周、12周留取标本后，提取颅骨缺损修复区新生骨组织mRNA，采用RT-PCR(realtime-polymerase chain reaction)方法，检测成骨细胞促进骨生成的基因变化情况，本部分实验所研究差异表达基因的重点：是关于成骨细胞增殖分化调节、成骨细胞功能调节和骨代谢调节等方面。
3淫羊藿苷促进兔颅骨缺损修复区成骨相关蛋白骨钙素、骨桥素的表达
选用12周龄雌性新西兰大白兔36只，动物分组，动物模型制作，动物给药方法和剂量同第一部分，分别于术后4周、8周、12周留取标本后，通过免疫组化方法，观察新骨形成区成骨细胞骨基质蛋白的表达情况：骨钙蛋白和骨桥蛋白的表达。
结果：
1淫羊藿苷对兔颅骨缺损修复愈合质量的影响
1.1一般观察
术后4周，对照组骨缺损区微凹陷，为纤维组织覆盖，边界清晰可辩，周边未见明显新骨形成。实验组颅骨缺损区中央局部凹陷，边界模糊不清，周围可见少量薄、半透明新骨生长。
术后8周，对照组骨缺损区表面及周边部骨痂形成，中央部略凹陷，较边缘为薄。实验组骨缺损区被均匀的硬组织覆盖，与周边骨质有明显骨融合。
术后12周，对照组骨缺损区表面及周边部骨痂形成，形态基本接近正常颅骨外形。实验组骨缺损区可见新形成的骨组织覆盖，与边缘无明显界限。
1.2X线观察
术后4周，对照组骨缺损处为低密度影，骨缺损边清晰。实验组缺损处为低密度影，骨缺损边缘模糊。
术后8周，对照组骨缺损区域密度低于缺损周围骨质，与正常组织间界限明显。实验组骨缺损区域内密度略高于对照组，界限模糊，缺损范围明显缩小。
术后12周，对照骨组缺损区域呈骨性低密度区，与正常组织间界限明显。实验组缺损区域与周围骨质无明显界限，密度基本一致。
1.3组织学观察
术后4周，对照组可见缺损边缘有少量类骨质和新骨形成，中央为纤维组织，大量的炎性细胞广泛浸润。实验组缺损区中央为纤维结缔组织充填，可见粗大的胶原纤维，中间散在少量炎性细胞，部分区域出现纤维骨样结构，大量新生骨质，成骨细胞较多成栅栏状排列在骨小梁周围。
术后8周，对照组缺损区存在大量结构疏松的纤维结缔组织，且炎症细胞仍广泛存在，缺损区边界有部分骨样和纤维骨样组织生成，其内有一定量的成骨细胞存在。实验组缺损区内有大量骨质形成，骨小梁连续，增厚，形成板层结构，部分成网织状，为较成熟骨，部分区域内骨质致密，形成板层状骨，中央为骨髓腔。
术后12周，对照组新生骨向中央逐渐充填骨缺损，缺损内可见较多新生的编织骨及板层骨。骨小梁连续，细胞成分较少，纤维成分较实验组多。实验组骨缺损处为成熟骨质，骨细胞丰富，骨小梁粗大，排列整齐，较明显的骨髓腔出现并连续。
1.4骨计量学观察
术后4周，用药组与对照组比较：用药组的松质区骨量(Tb·Ar%)，骨小梁宽度(Tb·Wi μm)，成骨细胞个数（OB·N）均明显大于对照组（P<0.01）；破骨细胞个数（CB·N）明显低于对照组（P<0.01）。术后8周，用药组与对照组比较：用药组的松质区骨量(Tb·Ar%)，骨小梁宽度(Tb·Wi μm)，成骨细胞个数（OB·N）均明显大于对照。组（P<0.01）；破骨细胞个数（CB·N）明显低于对照组（P<0.01）。术后12周，用药组与对照组比较：用药组的松质区骨量(Tb·Ar%)，骨小梁宽度(Tb·Wi μm)，成骨细胞个数（OB·N）均明显大于对照组（P<0.01）；破骨细胞个数（CB·N）两组之间无显著性差异（P>0.05）。
2淫羊藿苷促进兔颅骨缺损修复RANKL基因、RUNX2基因表达的影响
2.1mRNA纯度及完整性鉴定
分光光度法测定结果显示：对照组及用药组总RNA的OD260/OD280比值在1.8~2.0之间，纯度及浓度符合实验要求。
琼脂糖凝胶电泳结果显示：对照组及用药组总RNA经变性凝胶电泳后可见明显的28S及18S条带出现，28S条带亮度约为18S条带的两倍，各组提取的RNA完整性良好，符合实验要求。
2.2Real-time PCR定量分析结果
在术后4周，8周，12周时用药组和对照组Rankl、Runx2基因的mRNA表达结果：在术后4周，8周，12周时Rankl基因表达水平用药组均明显低于对照组，Runx2基因的表达水平用药组均明显高于对照组。
3淫羊藿苷促进兔颅骨缺损修区成骨相关蛋白骨钙素，骨桥素的表达
3.1骨钙素OC在新骨形成区的表达
用药后4周，实验组骨钙素OC的表达呈强阳性；对照组，OC在成骨细胞的胞浆和骨基质中阴性表达。8周时实验组OC的表达呈强阳性，对照组呈弱阳性表达。12时周实验组OC的表达呈强阳性，对照组呈弱阳性表达。
3.2骨桥素OPN在新骨形成区的表达
用药后4周实验组骨桥素OPN在成骨细胞的胞浆中呈强阳性表达，在骨基质中呈强阳性表达，对照组呈阴性表达。8周时实验组OPN的表达为强阳性；对照组呈弱阳性表达。12周时实验组OPN在呈强阳性表达；对照组呈弱阳性表达。
结论：
1淫羊藿苷能够增加兔颅骨缺损区成骨骨量，提高愈合质量。
2淫羊藿苷能够促进兔颅骨缺损愈合，加快颅骨缺损的愈合速度。
3淫羊藿苷在成骨过程中，发挥雌激素样作用，通过下调Rankl基因，减少破骨细胞的活化，抑制破骨细胞的功能，增加成骨。
4淫羊藿苷能上调成骨基因Runx2，通过经典Wnt/β-catenin途径，有效地促进骨愈合。
5淫羊藿苷促进颅骨缺损区成骨细胞成骨蛋白骨钙素的分泌，增加新骨形成的骨量，促进骨组织的愈合。
6淫羊藿苷促进颅骨缺损区成骨细胞成骨蛋白骨桥素的分泌，抑制破骨细胞的活性，增加缺损区骨量，加速骨组织愈合。
Objectives：Bone defects and bone deformity may occur by reason ofinflammation, trauma, tumor, endocrine diseases. The abnormal formations inparticular oral and maxillofacial region influence the patient’s quality of life. Itbecomes a research focus in the plastic surgery field to find a simple andeffective method to improve the repairmen of bone defects.
Nowadays there are a lot of techniques of promoting bone healing,including polymer biomaterial implants, artificial implants, autogenous bonegraft, tissue engineered bone graft, low intensity pulsed electromagnetictherapy. These techniques have not been widely applied in the clinical fielddue to some disadvantages.
Over the years, some scholars have done a large number of experimentson bone healing by means of histology, histochemistry, histomorphometry andhave discussed the mechanism of bone repair. In recent years, bone woundhealing has been probed from cell biology to molecular biology level due tothe development of application of gene technology. With the development ofmolecular biology, it has been recognized that bone wound healing is relatedwith various growth factors. A lot of bone growth factors play an importantrole in bone wound healing besides the endocrine and systemic factors. Thesegrowth factors include TGF-β (TGF), VEGF (vascular endothelial growthfactor), FGF (fibroblast growth factor), BMP (bone morphogenetic protein),and nerve growth factor (nerve growth factor, NGF), and so on.
Since the60's of the last century, Experimental research of TraditionalChinese medicine on accelerating fracture healing has been reported. Thereare several achievements as follows: the traditional Chinese medicine toimprove the blood supply of fracture area; to promote absorption of hematomaand deposits of mineral; to increase osteoblasts number and enhance theirs activity. Epimedium (Herba Epimedii) is a kind of traditional Chinesemedicines and is used clinically for the treatment of fracture and manydiseases of bone metabolism. Some research results have shown thatEpimedium can improve the immune function, resist to tumor, strengthengonadal, delay aging, and influence cardiovascular system. Icariin, the activecomponent of Herba Epimedii(C33H40O15, molecular weight:676.67), is aflavonoid compound. Li Fangfang has co-cultured rat granulosa cells andadrenocortical cells with icariin and found that icariin can increase estradiolsecreted by follicular granulosa cells, that high concentration can promote thesecretion of corticosterone and adrenal cortical cells. So conclusion aboveresults suggested that Epimedium kidney play a role on hypothalamicpituitary-gonadal axis and adrenal axis, which can effectively regulate bodyhormone level, improve the bone metabolism, promote protein synthesis.Icariin play an important role in the repair of bone wound healing. Theexperiment of Icariin on osteoblastic proliferation and metabolism in vitro hasbeen completed by group of Dong Fusheng.
This study set up New Zealand white rabbit skull defect models andobserves new bone mass, bone healing and bone quality of the defect area ofthe skull in different healing stages by gavage with icariin, by means ofhistology and histomorphometry method. In addition, mRNA of the bone ofskull defect areas was extracted and RT-PCR (real time-polymerase chainreaction) method was used to detect the gene change of bone formation and tofind the molecular signaling pathway of the bone.
On the basis of the above research, immunohistochemical method wasused to study the expression of osteocalcin and Osteopontin in new bone area.The objective is to probe the mechanism of icariin in promoting formation ofthe bone and provide the theory basis for the clinical application of icariin inpromoting bone healing.
Methods：
1The effects of Icariin on healing quality of the repair of rabbit skull defectThirty-six female New Zealand white rabbits (12weeks age) were randomly divided into two groups, treatment group and control group.Animals model of skull defect were established. One day after the operation,icariin was given orally at the dose of100mg/1Kg/day in the treatment groupand normal saline was given to the rabbits of the control group. After the4weeks,8weeks,12weeks postoperation the rabbits were killed respectively.The tissue samples of the rabbits were taken X-ray photograph. The sectionsof skull tissue were stained to observe bone trabecula width (Tb-Wi μ m),trabecula area ratio (Tb.Ar%), osteoblast number (OB·N), and osteoclastnumber (OC·N) of new bone by histomorphometry.
2The expression of the gene RANKL, RUNX2of icariin on repair of skulldefect of rabbits
Thirty-six female New Zealand white rabbits (12weeks age) wererandomly divided into two groups, treatment group and control group.Animals model of skull defect were established. One day after the operation,icariin was given orally at the dose of100mg/1Kg/day in the treatment groupand normal saline was given to the rabbits of the control group. After the4weeks,8weeks,12weeks postoperation the rabbits were killed respectively.The total mRNA of two groups was extracted respectively and purified afteridentification of the purity and integrity. RT-PCR method was used to detectthe osteoblast proliferation, differentiation, osteoblasts function and bonemetabolism.
3The expression of osteocalcin and osteopontin related to osteogenic ofIcariin on promoting repair of rabbit skull defect
Thirty-six female New Zealand white rabbits (12weeks age) wererandomly divided into two groups, treatment group and control group.Animals model of skull defect were established. One day after the operation,icariin was given orally at the dose of100mg/1Kg/day in the treatment groupand normal saline was given to the rabbits of the control group. After the4weeks,8weeks,12weeks postoperation the rabbits were killed respectively.The tissue samples were embedded in paraffin and were sectioned with athickness of4μm and were stained by using immunohistochemical method to observe the new bone, observed the expression of osteocalcin and osteopontin.
Results：
1The effects of Icariin on healing of the repair of rabbit skull defect
1.1General observations
At the4th week after surgery, the defect areas of the control group were alittle hollow, covered with fibrous tissue. The boundary was clear and therewas no obvious new bone formation around. The defect areas of theexperimental group were local depressions. The boundary was blurred andaround the boundary there were a lots of new bone.
At the8th week after surgery, the defect areas of the control group weresurrounded by calluses and the bone of the center was thinner than that of theedge. The bone defects in the experimental group were evenly covered andthere were fusion with the surrounding.
At the12th week after surgery, the surface of the defect of the controlgroup were surrounding by callus. The defect of the experimental group wascovered by mature bone tissue and boundary edge was not observed.
1.2X-ray observation
At the4th week after surgery, the density of the defect in the controlgroup was low and the defect edges are clear. The density of the defect in theexperimental group was low and the defect edge was blurred.
At the8th week after surgery, the density of the defect in the controlgroup was lower than that of the surrounding, and the boundaries between thedefect and normal tissues were distinct. The density of the defect in theexperimental group was slightly higher than that of control group.
At the12th week after surgery, the density of the defect in the controlgroup was lower than that of the experimental group. The density of the defectin the experimental group was similar to normal tissues.
1.3Histological observation
At the4th week after surgery, the control group showed that the defectedge had a small amount of osteoid formation and the central was fibroustissue and a lot of inflammatory cell. The defect of the experimental group was filling with thick collagen fiber and a few inflammatory cells. The trabeculaswere concentrated and there were a lot of osteoblasts with larger cell nucleolus.The osteoclasts were scarce.
At the8th week after surgery, the control group showed trabeculas werethin and was not contacted. There were a lot of osteoblasts (OB) andosteoclasts (OC). Fibrous tissue connective and inflammatory cells stillexisted in the center part of the defect. In the boundary part, osteoid, fibrousbone tissue and a certain amount of osteoblasts could be seen. In theexperimental group, bone trabeculas were continuous, thickening, formedlamellar, and reticular, marrow cavity was obviously.
At the12th week after surgery, the defects were filled with new bone inthe control group and woven bone and lamellar bone could be seen in themany newborn. Trabecular cells were less than those in the experimentalgroup. In experimental group, the newborn of the defect was mature andtrabeculae were thick and arranged in row. The marrow cavity was obviouslyand continuously.
1.4Bone histomorphometry
At the4th week after surgery, Traberculae Area (Tb·Ar%), TraberculaeWidth (Tb·Wi), Osteoblast Number (OB·N) of experimental group wassignificantly higher than that of control group (P<0.01). Osteoclast Number(OC·N) of experimental group was significantly lower than that of controlgroup (P<0.01).
At the8th week after surgery, Traberculae Area (Tb·Ar%), TraberculaeWidth (Tb·Wi), Osteoblast Number (OB·N) of experimental group wassignificantly higher than that of control group (P<0.01). Osteoclast Number(OC·N) of experimental group was significantly lower than that of controlgroup (P<0.01).
At the12th week after surgery, Traberculae Area (Tb·Ar%), TraberculaeWidth (Tb·Wi), Osteoblast Number (OB·N) of experimental group weresignificantly higher than that of control group (P<0.01), Osteoclast Number(OC·N) were no significant in experimental group compared with control group (P>0.05).
2The gene RANKL, RUNX2expression of icariin on repair of skull defect ofrabbits
2.1Purity and integrity of the total mRNA
The ratio of OD260/OD280of all the total RNA was between1.9and2.0.The28S and18S strip could be obviously appeared, and the brightness of28Sstrip was approximately twice compared with18S strip.
2.2quantitative analysis of Real-time PCR
The expression of Rankl of experimental group was significantly lowerthan that of control group. The expression of Runx2gene in experimentalgroup was obviously higher than that of control group at different times.
3Expression of osteocalcin and osteopontin related to osteogenic of icariin onpromoting repair of rabbit skull defect
3.1The expression of osteocalcin
At the4th week after treatment, the expression of osteocalcin (OC) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix in control group showed negative.
At the8th week after treatment, the expression of osteocalcin (OC) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed weakpositive.
At the12th week after treatment, the expression of osteocalcin (OC) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed weakpositive.
3.2The expression of osteopontin
At the4th week after treatment, the expression of osteopontin (OPN) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed negative.
At the8th week after treatment, the expression of osteopontin (OPN) inosteoblasts and matrix of experimental group showed strong positive while expression of OC in osteoblasts and matrix of control group showed weakpositive.
At the12th week after treatment, the expression of osteopontin (OPN) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed weakpositive.
Conclusions：
1Icariin can increase the quantity of the repair area about skull defects onrabbit and improve the quality of bone healing.
2Icariin can speed up the rate of repair about skull defects on rabbit.
3Icariin can provide estrogen-like function and promote osteogenesis byreducing expression of gene Rankl, lessening the number of osteoclasts,inhibiting the differentiation, activation and maturity of osteoclast.
4Icariin can up-regulate gene Runx2and promote the repair of defectthrough the Wnt/β-catenin pathway.
5Icariin can increase the quality of osteogenesis by augment secretion ofosteocalcin in osteoblasts.
6Icariin can increase the quality of osteogenesis by augment secretion ofosteopontin in osteoblasts.
引文
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