微米中药功能衬材对家兔骨折愈合的影响
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摘要
[背景]
骨折是临床上的常见病多发病,骨折愈合是一个极其复杂的骨组织再生修复过程,受到全身和局部许多因素的影响和调节。约5%～10%的骨折由于各种原因发生延迟愈合或不愈合,严重影响人们的生存质量。如何加速骨折愈合,提高愈合质量一直是医学界共同关注的重大课题之一
中药外治法是指运用非口服药物刺激经络、穴位、皮肤、粘膜、肌肉、筋骨等以达到防病治病目的的一种传统医学疗法。中药外治法治疗骨折,历史悠久,疗效显著。目前已被研究证实的作用机制主要有如下几方面：改善血液循环；促进血肿的吸收和机化；增加胶原的合成；促进钙盐沉积；增加细胞数量、促进细胞活性；调节内分泌系统；促进骨痂生成,提高骨痂质量；干预骨形成调节因子的作用；提高微量元素的含量等。
伴随着科技的发展,皮肤给药已成为第三大给药途径,中医在这个领域可谓独具特色,中药外治的优势在于：药物的作用力直接、集中,能直达病所,使其病变局部内的药物浓度显著高于血药浓度,故发挥作用充分,奏效迅捷。且所用药量和副反应远小于内服药。将传统中医外治与现代透皮技术结合,形成中药透皮治疗系统必将是今后中药外治研究的发展趋势。
目前中药外治骨折的剂型主要有中药加工制成的膏剂、酊剂和现代气压罐装的喷雾剂等,文献已有报道,但均各自有其不足和局限性。如何选用有效的方药和采用科学合理的剂型治疗骨折,是中医药现代化发展的趋势。本课题运用目前中药现代化最前沿的创新成果——微纳米中药技术,将中药验方微米化并与纺织工业重整技术结合,制作成一种新型医用材料——微米中药功能衬材。目的是把复位、固定和局部用药有机地结合起来,除了作为石膏绷带或小夹板固定时的内层衬垫使用外,还具有消炎镇痛,减轻创伤反应,加快组织的修复,加速骨折愈合及功能恢复的作用。
微米中药是指采用现代高科技与传统炮制技术和制剂技术相结合而研制的能保持传统中药固有药效学物质基础的粒度为微米级(平均直径≤15μm)的新型中药。它是中药微米化后的产物,不是一种新的药种。微米中药可大幅度提高药物的活性和生物利用度,降低用药量,减少毒副作用,与中药现代化的核心“安全、有效、可控、稳定”息息相关。利用超微粉碎技术制备的中药微米颗粒,既能保持中药的有效成分不被破坏,同时由于制得的颗粒比表面积增大,能有效增进药物有效成分的溶出,提高药物的生物利用度；其次它可以提高药物的靶向性,有助于控制药物在体内的分布等；本研究产品——微米中药功能衬材,既便于给药剂量的标准化、规范化,同时又便于制剂生产工艺、质量控制的标准化、规范化。这种新型的医用功能复合材料可作为石膏绷带或小夹板固定时的内层衬材使用,使骨折在外固定的基础上,与局部用药有机结合,将有效地促进骨折愈合及功能的恢复。本实验将从血清生化指标、血液流变学指标、X线及组织形态学四个方面探讨微米中药功能衬材对家兔骨折愈合的影响。
[目的]
研究微米中药功能衬材对家兔骨折愈合的影响,为其临床应用和成果转化提供依据。
[方法]
1.实验分组和模型制作：24只普通级雄性新西兰兔随机分为4组：微米组、中药对照组、衬材对照组和模型对照组,每组6只。参照柴本甫造模方法,将全部家兔造成右侧桡骨中段骨缺损骨折模型。
2.衬材制作：利用高能球磨法制备微米三七粉,加入冰片等其它药物,结合纺织工业的重整技术制作出微米中药功能衬材。
3.处理方式：所有家兔均造模成功,微米组给予外敷微米中药功能衬材后加石膏外固定,中药对照组给予外敷普通中药功能衬材后加石膏外固定,衬材对照组给予外敷普通衬材(无中药)后加石膏外固定,模型对照组不予任何治疗。每周换1次衬材,连续观察6周。
4.指标检测方式：三七粉微米化前后在扫描电镜下行粒径对比；于造模前和造模后第2,4,6周行血清碱性磷酸酶活性,血钙、血磷浓度及血液流变学指标(全血高切、中切、低切粘度及血浆粘度)的检测；造模后第2,4,6周各组X射线下观察骨折愈合情况,并行外骨痂和骨痂线模糊程度评分；造模后第6周末将所有家兔处死,以骨折断端为中心0.5cm为取样部位,行脱钙固定、脱水及石蜡包埋后,连续切片,行苏木精-伊红染色(HE染色),光学显微镜下观察骨折区修复情况。
[统计学方法]
实验数据计量资料以均数±标准差((?)±s)表示。采用SPSS13.0软件进行统计分析。多组间计量资料比较使用单因素方差分析(one-way ANOVA方法),组内不同时间点的血清碱性磷酸酶活性、血钙、血磷浓度、血液流变学指标(全血高切、中切、低切粘度及血浆粘度)及外骨痂和骨痂线模糊程度评分比较采用重复测量的方差分析。检验标准α定为0.05。
[结果]
1.血清生化指标检测：结果显示组间家兔的血清碱性磷酸酶活性、血钙含量、血磷含量在骨折愈合过程的不同时间点中均存在显著差异(F值分别为3.147,3.427和6.452,P值分别为0.048,0.037和0.003),造模后第2周微米组血清碱性磷酸酶活性值达到高峰,和模型对照组、中药对照组比较均存在显著差异(P值分别为0.001,0.000)；微米组血钙含量和模型对照组、中药对照组比较均存在显著差异(P值分别为0.001,0.002)；4组动物血磷值均有所降低,微米组血磷含量和模型对照组比较存在显著差异(P=0.042)。造模后第4周各组血钙含量开始下降,其中微米组下降较多,和模型对照组、中药对照组比较均存在显著差异(P值均为0.000)；血磷值开始升高,其中微米组尤为明显,微米组和模型对照组、中药对照组比较均存在显著差异(P值分别为0.002,0.048)。造模后第6周微米组血清碱性磷酸酶活性及血钙含量均降至正常值附近,微米组血清碱性磷酸酶活性及血钙含量和模型对照组、中药对照组比较均存在显著差异(P<0.05)；4组动物血磷均持续在较高水平,微米组和模型对照组、中药对照组比较存在显著差异(P值分别为0.000,0.014)。同一时间点衬材对照组所有血清生化指标和模型对照组比较均无显著差异(P>0.05)。
2.血液流变学指标检测：微米组几乎所有指标均低于对照组,第2周时微米组全血低切粘度、全血高切粘度及血浆粘度和模型对照组相比存在显著差异(P值分别为0.000,0.017,0.000)；第4周时微米组四个指标和模型对照组相比存在显著差异(P=0.000),微米组全血低切粘度、血浆粘度与中药对照组比较存在显著差异(P值分别为0.000,0.001)；第6周时微米组四个指标和模型对照组相比存在显著差异(P=0.000),微米组全血低切粘度、血浆粘度与中药对照组比较存在显著差异(P值分别为0.001,0.036)。
3.X射线检查及外骨痂和骨折线模糊程度评分：造模后第6周显示微米组外骨痂接近完全吸收,骨折处皮质骨密度接近正常皮质骨,骨髓腔完全再通；中药对照组骨折线消失,外骨痂开始吸收,骨髓腔仍未通；模型对照组中有出现断端硬化不愈合现象。外骨痂和骨折线模糊程度评分结果显示造模后第2周时衬材对照组和模型对照组比较无显著差异(P=0.819),微米组和模型对照组比较存在显著差异(P=0.001)；造模后第4周时衬材对照组和模型对照组比较无显著差异(P=0.778),微米组和模型对照组比较存在显著差异(P=0.000),微米组与中药对照组比较存在显著差异(P=0.010)；造模后第6周时衬材对照组和模型对照组比较无显著差异(P=0.293),微米组和模型对照组、中药对照组比较均存在显著差异(P值分别为0.000,0.027)。
4.组织形态学：造模后第6周时,微米组骨折端小梁间血管扩张,数目较多,软骨内成骨完成,可见大量成骨细胞,编织骨开始塑形,骨髓腔再通；中药对照组可见编织骨、软骨细胞和软骨内成骨共存,断端内、外骨痂相连,骨髓腔未再通；而同期衬材对照组和模型对照组骨小梁较细,小梁间血管较少,断端内、外骨痂大多尚未相连,骨髓腔未再通。
[结论]
微米中药功能衬材能改善组织微循环,增加碱性磷酸酶的活性,降低血钙水平,升高血磷和钙磷乘积,加速成骨细胞增生,促进骨痂形成,具有促进骨折愈合的作用且强于普通中药功能衬材。
Background
Fracture is a kind of clinically frequently-occurring disease. Fracture healing is an extremely complex process of bone regeneration and repair, under the influence and regulation of a number of systemic and local factors. About 5% to 10% of fractures occur delayed union or disunion for various reasons, as seriously affects people's quality of life. How to accelerate fracture healing and how to improve the healing quality have been one of major topics that medical community are working together to focus on.
External treatment with traditional Chinese medicine is a traditional medicine therapy that could use non-oral drugs stimulating meridians, acupoints, skin, mucosa, muscle, bones, etc. to achieve the purpose of preventing and curing diseases. External treatment with traditional Chinese medicine on fractures has a long history and significant effect. At present the main mechanism confirmed by researches includes the following aspects:improving blood circulation; promoting hematoma absorption and organization; increasing collagen synthesis; promoting calcium deposition; increasing the number of cells and promoting cell activity; regulating endocrine system; promoting callus formation and improving the quality of callus; interventing the effect of regulatory factors of bone formation; improving the content of trace elements, etc.
Along with technology development, transdermal drug delivery has become the third largest route of administration. Traditional Chinese Medicine can be described as unique in this field. Advantages of external treatment with traditional Chinese medicine are that the effect of drug is direct and focused and able to direct the disease to make drug concentration within the local lesion significantly higher than blood drug level so that drugs play a full role and have a rapid effect and the dosage and side effects are much less than those of oral medicine. Transdermal therapeutic system of traditional Chinese medicine that conbines traditional external treatment with traditional Chinese medicine with modern transdermal technology will be the development trend of research on external treatment with traditional Chinese medicine in the future.
At present dosage forms of external treatment with traditional Chinese medicine on fracture are mainly slurry, tincture and air pressure-canned nebula that are made by traditional Chinese medicine, as is reported in literatures. But they all have shortcomings and limitations. How to choose a valid prescription and use scientific and rational dosage forms for fracture hailing is the development trend of modernization of traditional Chinese medicine. The subject will conbine the most cutting-edge innovational outcome of modernization of traditional Chinese medicine——micron and nano medicine technology with restructuring technology of textile industry and create a new type of medical material——micron traditional Chinese medicine functional lining material, in order to conbine reposition, fixation with local application organically. Micron traditional Chinese medicine functional lining material is not only used as plaster bandage or inner liner when splintage, but also has the role of eliminating inflammation and analgesia and reducing trauma response and accelerating tissue repair and fracture healing and functional recovery.
Micron-medicine is a new medicine whose particle size is micron level (average diameter≤15μm)combining modern technology with traditional processing techniques and preparation techniques, maintaining inherent material basis of pharmacodynamics of traditional Chinese medicine. It is the product of traditional Chinese medicine with micron-technology, not a new drug species. Micron-medicine can greatly improve drug activity and bioavailability and reduce dosage and side effects, which is closely related with the core of modernization of traditional Chinese medicine"safe, effective, controllable, and stability". First of all, not only micron-medicine particles made with ultrafine grinding technology can keep the active ingredients of traditional Chinese medicine not to be damaged, but aslo enhance the dissolution active ingredients of drug effectively and increase drug bioavailability because of increasing surface area of particles. Secondly, it can improve drug targeting to help control the distribution of drugs in vivo; This research product——micron traditional Chinese medicine functional lining material, is convenient for not only dose standardization but aslo standardization of manufacturing processes and quality control of the standard. This new type of medical function materials can be used as plaster bandage or inner liner when splintage and effectively promote fracture healing and functional recovery by conbining external fixation with local application organically. This experiment is to study the effects of micron traditional Chinese medicine functional lining material on the healing of fracture in rabbits from serum biochemical parameters, hemorheology, X ray and histomorphology aspects.
Objective
To study the effect of micron traditional Chinese medicine functional lining material on the healing of fracture in rabbits, so as to offer theoretical references for clinical applications and transformation.
Methods
1. Groups dividing and models establishment:24 male New Zealand white rabbits were randomly divided into 4 groups:micron group, traditional Chinese medicine control group,lining material control group and model control group. Each group consisted of 6 rabbits. With Chai Benfu's modeling method for the reference, all rabbits were made into fracture models with bone defect in the middle of the right radial
2. Lining materials production:Micron traditional Chinese medicine functional lining material was made with micron-panax notoginseng powder made with high-energy ball milling with borneol and other medicines with reforming technology of textile industry.
3.Treatment methods:All rabbits were modelled successfully. Rabbits in the micron group were treated with micron traditional Chinese medicine functional lining material plus plaster external fixation; those in the traditional Chinese medicine control group were treated with ordinary Chinese medicine functional material plus plaster external fixation; those in the lining material control group were treated with ordinary lining material added plaster external fixation; those in the natural healing control group had no special treatments. Lining materials were replaced weekly. All rabbits had been observed continuously for six weeks.
4.Outcome measures:The size of notoginseng powder was compared before and after micron process in SEM. The serum concentration of AKP, Ca, Pand hemorheology indexes(ηB.200,ηB.30,ηB.1 andηp) were detected respectively before modeling settlement and after modeling for 2,4 and 6 weeks. All the animals of the fracture healing were observed under the X-ray and Scores of fracture callus and the fuzzy degree of the fracture line were assessed after modeling for 2,4 and 6 weeks. All the rabbits were killed 6 weeks after modeling. Those samples taken from the position to 0.5cm fracture fragments as the center were made HE staining after decalcified fixation, dehydration, paraffin imbedding and serial sections. The repair of fracture zone was observed under optical microscope.
Statistical methods
The statistical analysis was performed with SPSS 13.0 software package and the data were presented with the mean±standard deviation (x±s). Multiple comparison between the measured data were performed by using one-way ANOVA. Multiple comparisons of the serum concentration of AKP, Ca, P, hemorheology indexes(ηB.200,ηB.30,ηB.1 andηp) and scores of fracture callus and the fuzzy degree of the fracture line among different time spots in each group were analyzed by using repeated measures analysis of variance. Statistical significance was set at P <0.05.
Result
1. Detection of serum biochemical parameters:The results showed that the serum concentration of AKP, Ca and P at different time points of the fracture healing process were significantly different (F values were 3.147,3.427 and 6.452, P values were 0.048,0.037and 0.003). AKP in micron group reached a peak after 2 weeks of modeling, AKP in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.001,0.000); Ca in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.001,0.002); P in 4 groups were both dropped, P in micron group and the model group were significantly different (P= 0.042<0.05). After 4 weeks of modeling, Ca in every group began to decrease, which in micron group fell more. Ca in micron group, model control group and traditional Chinese medicine control group were significantly different (P= 0.000<0.05); P began to increase, which in micron group was apparent. P in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.002,0.048). After 6 weeks of modeling, AKP and P in micron group had been reduced to near normal. AKP and P in micron group, model control group and traditional Chinese medicine control group were significantly different (P<0.05).P in 4 groups remained at a high level. P in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.000,0.014). All serum biochemical indixes in lining material control group and model control group at the same time showed no significant difference (P> 0.05).
2. Detection of hemorheology:All indixes of micron group were almost lower than other control groups. After 2 weeks of modeling,ηB.1,ηB.200 andηp in micron group and model control group were signific, antly different (P values were 0.000,0.017,0.000); After 4 weeks of modeling, four indixes in micron Group and model control group were significantly different (P= 0.000).ηB.1 andηp in micron group and traditional Chinese medicine control group were significantly different (P values were 0.000,0.001); After 6 weeks of modeling, four indixes in micron group and model control group were significantly different (P= 0.000).ηB.1 andηp in micron group and traditional Chinese medicine control group were significantly different (P values were 0.001,0.036).
3. X-ray examination and external callus and fracture line blurring score:It showed that external callus were almost completely absorbed, cortical bone mineral density in the location of fracture closed to normal cortical bone and bone marrow cavity was completely recanalized after 6 weeks of modeling; It showed that fracture line disappeared, external callus began to absorb and bone marrow cavity remained unconnected in traditional Chinese medicine control group;There was a hardening and nonunion phenomenon of broken ends in model control group. External callus and fracture line blurring score showed that scores of lining material control group and model control group showed no significant difference (P= 0.819), scores of micron group and model control group were significantly different (P= 0.001) after 2 weeks of modeling; 4 weeks after modeling, scores of lining material control group and model control group showed no significant difference (P= 0.778). Scores of micron group and model control group were significantly different (P= 0.000). Scores of micron group and traditional Chinese medicine control group were significantly different (P= 0.010).6 weeks after modeling, scores of lining material control group and model control group showed no significant difference (P= 0.293), scores of micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.000,0.027).
4. Histomorphology:The first 6 weeks after modeling, vasculars between trabeculars in the location of fracture expanded and the number was larger; Endochondral bone was completed; A large number of osteoblasts were saw; Woven bone began shaping; The bone marrow cavity was recanalized in micron groups. Weaving bone, cartilage cells and endochondral bone were coexisted; In callus and external callus of stump were connected; Bone marrow cavity was not recanalized in traditional Chinese medicine group. While trabecular bone was smaller and blood vessels between trabeculars were less and in callus and external callus of stump were mainly unconnected and the bone marrow cavity was not recanalized.
Conclusion
Micron traditional Chinese medicine functional lining material can improve microcirculation, increase the activity of AKP, decrease the level of Ca, elevate the product of serum P and Ca, accelerate the proliferation of osteoblasts and promote callus formation and fracture healing.lt was stronger than ordinary traditional Chinese medicine functional lining material.
骨折是临床上的常见病多发病,骨折愈合是一个极其复杂的骨组织再生修复过程,受到全身和局部许多因素的影响和调节。约5%～10%的骨折由于各种原因发生延迟愈合或不愈合,严重影响人们的生存质量。如何加速骨折愈合,提高愈合质量一直是医学界共同关注的重大课题之一
中药外治法是指运用非口服药物刺激经络、穴位、皮肤、粘膜、肌肉、筋骨等以达到防病治病目的的一种传统医学疗法。中药外治法治疗骨折,历史悠久,疗效显著。目前已被研究证实的作用机制主要有如下几方面：改善血液循环；促进血肿的吸收和机化；增加胶原的合成；促进钙盐沉积；增加细胞数量、促进细胞活性；调节内分泌系统；促进骨痂生成,提高骨痂质量；干预骨形成调节因子的作用；提高微量元素的含量等。
伴随着科技的发展,皮肤给药已成为第三大给药途径,中医在这个领域可谓独具特色,中药外治的优势在于：药物的作用力直接、集中,能直达病所,使其病变局部内的药物浓度显著高于血药浓度,故发挥作用充分,奏效迅捷。且所用药量和副反应远小于内服药。将传统中医外治与现代透皮技术结合,形成中药透皮治疗系统必将是今后中药外治研究的发展趋势。
目前中药外治骨折的剂型主要有中药加工制成的膏剂、酊剂和现代气压罐装的喷雾剂等,文献已有报道,但均各自有其不足和局限性。如何选用有效的方药和采用科学合理的剂型治疗骨折,是中医药现代化发展的趋势。本课题运用目前中药现代化最前沿的创新成果——微纳米中药技术,将中药验方微米化并与纺织工业重整技术结合,制作成一种新型医用材料——微米中药功能衬材。目的是把复位、固定和局部用药有机地结合起来,除了作为石膏绷带或小夹板固定时的内层衬垫使用外,还具有消炎镇痛,减轻创伤反应,加快组织的修复,加速骨折愈合及功能恢复的作用。
微米中药是指采用现代高科技与传统炮制技术和制剂技术相结合而研制的能保持传统中药固有药效学物质基础的粒度为微米级(平均直径≤15μm)的新型中药。它是中药微米化后的产物,不是一种新的药种。微米中药可大幅度提高药物的活性和生物利用度,降低用药量,减少毒副作用,与中药现代化的核心“安全、有效、可控、稳定”息息相关。利用超微粉碎技术制备的中药微米颗粒,既能保持中药的有效成分不被破坏,同时由于制得的颗粒比表面积增大,能有效增进药物有效成分的溶出,提高药物的生物利用度；其次它可以提高药物的靶向性,有助于控制药物在体内的分布等；本研究产品——微米中药功能衬材,既便于给药剂量的标准化、规范化,同时又便于制剂生产工艺、质量控制的标准化、规范化。这种新型的医用功能复合材料可作为石膏绷带或小夹板固定时的内层衬材使用,使骨折在外固定的基础上,与局部用药有机结合,将有效地促进骨折愈合及功能的恢复。本实验将从血清生化指标、血液流变学指标、X线及组织形态学四个方面探讨微米中药功能衬材对家兔骨折愈合的影响。
[目的]
研究微米中药功能衬材对家兔骨折愈合的影响,为其临床应用和成果转化提供依据。
[方法]
1.实验分组和模型制作：24只普通级雄性新西兰兔随机分为4组：微米组、中药对照组、衬材对照组和模型对照组,每组6只。参照柴本甫造模方法,将全部家兔造成右侧桡骨中段骨缺损骨折模型。
2.衬材制作：利用高能球磨法制备微米三七粉,加入冰片等其它药物,结合纺织工业的重整技术制作出微米中药功能衬材。
3.处理方式：所有家兔均造模成功,微米组给予外敷微米中药功能衬材后加石膏外固定,中药对照组给予外敷普通中药功能衬材后加石膏外固定,衬材对照组给予外敷普通衬材(无中药)后加石膏外固定,模型对照组不予任何治疗。每周换1次衬材,连续观察6周。
4.指标检测方式：三七粉微米化前后在扫描电镜下行粒径对比；于造模前和造模后第2,4,6周行血清碱性磷酸酶活性,血钙、血磷浓度及血液流变学指标(全血高切、中切、低切粘度及血浆粘度)的检测；造模后第2,4,6周各组X射线下观察骨折愈合情况,并行外骨痂和骨痂线模糊程度评分；造模后第6周末将所有家兔处死,以骨折断端为中心0.5cm为取样部位,行脱钙固定、脱水及石蜡包埋后,连续切片,行苏木精-伊红染色(HE染色),光学显微镜下观察骨折区修复情况。
[统计学方法]
实验数据计量资料以均数±标准差((?)±s)表示。采用SPSS13.0软件进行统计分析。多组间计量资料比较使用单因素方差分析(one-way ANOVA方法),组内不同时间点的血清碱性磷酸酶活性、血钙、血磷浓度、血液流变学指标(全血高切、中切、低切粘度及血浆粘度)及外骨痂和骨痂线模糊程度评分比较采用重复测量的方差分析。检验标准α定为0.05。
[结果]
1.血清生化指标检测：结果显示组间家兔的血清碱性磷酸酶活性、血钙含量、血磷含量在骨折愈合过程的不同时间点中均存在显著差异(F值分别为3.147,3.427和6.452,P值分别为0.048,0.037和0.003),造模后第2周微米组血清碱性磷酸酶活性值达到高峰,和模型对照组、中药对照组比较均存在显著差异(P值分别为0.001,0.000)；微米组血钙含量和模型对照组、中药对照组比较均存在显著差异(P值分别为0.001,0.002)；4组动物血磷值均有所降低,微米组血磷含量和模型对照组比较存在显著差异(P=0.042)。造模后第4周各组血钙含量开始下降,其中微米组下降较多,和模型对照组、中药对照组比较均存在显著差异(P值均为0.000)；血磷值开始升高,其中微米组尤为明显,微米组和模型对照组、中药对照组比较均存在显著差异(P值分别为0.002,0.048)。造模后第6周微米组血清碱性磷酸酶活性及血钙含量均降至正常值附近,微米组血清碱性磷酸酶活性及血钙含量和模型对照组、中药对照组比较均存在显著差异(P<0.05)；4组动物血磷均持续在较高水平,微米组和模型对照组、中药对照组比较存在显著差异(P值分别为0.000,0.014)。同一时间点衬材对照组所有血清生化指标和模型对照组比较均无显著差异(P>0.05)。
2.血液流变学指标检测：微米组几乎所有指标均低于对照组,第2周时微米组全血低切粘度、全血高切粘度及血浆粘度和模型对照组相比存在显著差异(P值分别为0.000,0.017,0.000)；第4周时微米组四个指标和模型对照组相比存在显著差异(P=0.000),微米组全血低切粘度、血浆粘度与中药对照组比较存在显著差异(P值分别为0.000,0.001)；第6周时微米组四个指标和模型对照组相比存在显著差异(P=0.000),微米组全血低切粘度、血浆粘度与中药对照组比较存在显著差异(P值分别为0.001,0.036)。
3.X射线检查及外骨痂和骨折线模糊程度评分：造模后第6周显示微米组外骨痂接近完全吸收,骨折处皮质骨密度接近正常皮质骨,骨髓腔完全再通；中药对照组骨折线消失,外骨痂开始吸收,骨髓腔仍未通；模型对照组中有出现断端硬化不愈合现象。外骨痂和骨折线模糊程度评分结果显示造模后第2周时衬材对照组和模型对照组比较无显著差异(P=0.819),微米组和模型对照组比较存在显著差异(P=0.001)；造模后第4周时衬材对照组和模型对照组比较无显著差异(P=0.778),微米组和模型对照组比较存在显著差异(P=0.000),微米组与中药对照组比较存在显著差异(P=0.010)；造模后第6周时衬材对照组和模型对照组比较无显著差异(P=0.293),微米组和模型对照组、中药对照组比较均存在显著差异(P值分别为0.000,0.027)。
4.组织形态学：造模后第6周时,微米组骨折端小梁间血管扩张,数目较多,软骨内成骨完成,可见大量成骨细胞,编织骨开始塑形,骨髓腔再通；中药对照组可见编织骨、软骨细胞和软骨内成骨共存,断端内、外骨痂相连,骨髓腔未再通；而同期衬材对照组和模型对照组骨小梁较细,小梁间血管较少,断端内、外骨痂大多尚未相连,骨髓腔未再通。
[结论]
微米中药功能衬材能改善组织微循环,增加碱性磷酸酶的活性,降低血钙水平,升高血磷和钙磷乘积,加速成骨细胞增生,促进骨痂形成,具有促进骨折愈合的作用且强于普通中药功能衬材。
Background
Fracture is a kind of clinically frequently-occurring disease. Fracture healing is an extremely complex process of bone regeneration and repair, under the influence and regulation of a number of systemic and local factors. About 5% to 10% of fractures occur delayed union or disunion for various reasons, as seriously affects people's quality of life. How to accelerate fracture healing and how to improve the healing quality have been one of major topics that medical community are working together to focus on.
External treatment with traditional Chinese medicine is a traditional medicine therapy that could use non-oral drugs stimulating meridians, acupoints, skin, mucosa, muscle, bones, etc. to achieve the purpose of preventing and curing diseases. External treatment with traditional Chinese medicine on fractures has a long history and significant effect. At present the main mechanism confirmed by researches includes the following aspects:improving blood circulation; promoting hematoma absorption and organization; increasing collagen synthesis; promoting calcium deposition; increasing the number of cells and promoting cell activity; regulating endocrine system; promoting callus formation and improving the quality of callus; interventing the effect of regulatory factors of bone formation; improving the content of trace elements, etc.
Along with technology development, transdermal drug delivery has become the third largest route of administration. Traditional Chinese Medicine can be described as unique in this field. Advantages of external treatment with traditional Chinese medicine are that the effect of drug is direct and focused and able to direct the disease to make drug concentration within the local lesion significantly higher than blood drug level so that drugs play a full role and have a rapid effect and the dosage and side effects are much less than those of oral medicine. Transdermal therapeutic system of traditional Chinese medicine that conbines traditional external treatment with traditional Chinese medicine with modern transdermal technology will be the development trend of research on external treatment with traditional Chinese medicine in the future.
At present dosage forms of external treatment with traditional Chinese medicine on fracture are mainly slurry, tincture and air pressure-canned nebula that are made by traditional Chinese medicine, as is reported in literatures. But they all have shortcomings and limitations. How to choose a valid prescription and use scientific and rational dosage forms for fracture hailing is the development trend of modernization of traditional Chinese medicine. The subject will conbine the most cutting-edge innovational outcome of modernization of traditional Chinese medicine——micron and nano medicine technology with restructuring technology of textile industry and create a new type of medical material——micron traditional Chinese medicine functional lining material, in order to conbine reposition, fixation with local application organically. Micron traditional Chinese medicine functional lining material is not only used as plaster bandage or inner liner when splintage, but also has the role of eliminating inflammation and analgesia and reducing trauma response and accelerating tissue repair and fracture healing and functional recovery.
Micron-medicine is a new medicine whose particle size is micron level (average diameter≤15μm)combining modern technology with traditional processing techniques and preparation techniques, maintaining inherent material basis of pharmacodynamics of traditional Chinese medicine. It is the product of traditional Chinese medicine with micron-technology, not a new drug species. Micron-medicine can greatly improve drug activity and bioavailability and reduce dosage and side effects, which is closely related with the core of modernization of traditional Chinese medicine"safe, effective, controllable, and stability". First of all, not only micron-medicine particles made with ultrafine grinding technology can keep the active ingredients of traditional Chinese medicine not to be damaged, but aslo enhance the dissolution active ingredients of drug effectively and increase drug bioavailability because of increasing surface area of particles. Secondly, it can improve drug targeting to help control the distribution of drugs in vivo; This research product——micron traditional Chinese medicine functional lining material, is convenient for not only dose standardization but aslo standardization of manufacturing processes and quality control of the standard. This new type of medical function materials can be used as plaster bandage or inner liner when splintage and effectively promote fracture healing and functional recovery by conbining external fixation with local application organically. This experiment is to study the effects of micron traditional Chinese medicine functional lining material on the healing of fracture in rabbits from serum biochemical parameters, hemorheology, X ray and histomorphology aspects.
Objective
To study the effect of micron traditional Chinese medicine functional lining material on the healing of fracture in rabbits, so as to offer theoretical references for clinical applications and transformation.
Methods
1. Groups dividing and models establishment:24 male New Zealand white rabbits were randomly divided into 4 groups:micron group, traditional Chinese medicine control group,lining material control group and model control group. Each group consisted of 6 rabbits. With Chai Benfu's modeling method for the reference, all rabbits were made into fracture models with bone defect in the middle of the right radial
2. Lining materials production:Micron traditional Chinese medicine functional lining material was made with micron-panax notoginseng powder made with high-energy ball milling with borneol and other medicines with reforming technology of textile industry.
3.Treatment methods:All rabbits were modelled successfully. Rabbits in the micron group were treated with micron traditional Chinese medicine functional lining material plus plaster external fixation; those in the traditional Chinese medicine control group were treated with ordinary Chinese medicine functional material plus plaster external fixation; those in the lining material control group were treated with ordinary lining material added plaster external fixation; those in the natural healing control group had no special treatments. Lining materials were replaced weekly. All rabbits had been observed continuously for six weeks.
4.Outcome measures:The size of notoginseng powder was compared before and after micron process in SEM. The serum concentration of AKP, Ca, Pand hemorheology indexes(ηB.200,ηB.30,ηB.1 andηp) were detected respectively before modeling settlement and after modeling for 2,4 and 6 weeks. All the animals of the fracture healing were observed under the X-ray and Scores of fracture callus and the fuzzy degree of the fracture line were assessed after modeling for 2,4 and 6 weeks. All the rabbits were killed 6 weeks after modeling. Those samples taken from the position to 0.5cm fracture fragments as the center were made HE staining after decalcified fixation, dehydration, paraffin imbedding and serial sections. The repair of fracture zone was observed under optical microscope.
Statistical methods
The statistical analysis was performed with SPSS 13.0 software package and the data were presented with the mean±standard deviation (x±s). Multiple comparison between the measured data were performed by using one-way ANOVA. Multiple comparisons of the serum concentration of AKP, Ca, P, hemorheology indexes(ηB.200,ηB.30,ηB.1 andηp) and scores of fracture callus and the fuzzy degree of the fracture line among different time spots in each group were analyzed by using repeated measures analysis of variance. Statistical significance was set at P <0.05.
Result
1. Detection of serum biochemical parameters:The results showed that the serum concentration of AKP, Ca and P at different time points of the fracture healing process were significantly different (F values were 3.147,3.427 and 6.452, P values were 0.048,0.037and 0.003). AKP in micron group reached a peak after 2 weeks of modeling, AKP in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.001,0.000); Ca in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.001,0.002); P in 4 groups were both dropped, P in micron group and the model group were significantly different (P= 0.042<0.05). After 4 weeks of modeling, Ca in every group began to decrease, which in micron group fell more. Ca in micron group, model control group and traditional Chinese medicine control group were significantly different (P= 0.000<0.05); P began to increase, which in micron group was apparent. P in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.002,0.048). After 6 weeks of modeling, AKP and P in micron group had been reduced to near normal. AKP and P in micron group, model control group and traditional Chinese medicine control group were significantly different (P<0.05).P in 4 groups remained at a high level. P in micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.000,0.014). All serum biochemical indixes in lining material control group and model control group at the same time showed no significant difference (P> 0.05).
2. Detection of hemorheology:All indixes of micron group were almost lower than other control groups. After 2 weeks of modeling,ηB.1,ηB.200 andηp in micron group and model control group were signific, antly different (P values were 0.000,0.017,0.000); After 4 weeks of modeling, four indixes in micron Group and model control group were significantly different (P= 0.000).ηB.1 andηp in micron group and traditional Chinese medicine control group were significantly different (P values were 0.000,0.001); After 6 weeks of modeling, four indixes in micron group and model control group were significantly different (P= 0.000).ηB.1 andηp in micron group and traditional Chinese medicine control group were significantly different (P values were 0.001,0.036).
3. X-ray examination and external callus and fracture line blurring score:It showed that external callus were almost completely absorbed, cortical bone mineral density in the location of fracture closed to normal cortical bone and bone marrow cavity was completely recanalized after 6 weeks of modeling; It showed that fracture line disappeared, external callus began to absorb and bone marrow cavity remained unconnected in traditional Chinese medicine control group;There was a hardening and nonunion phenomenon of broken ends in model control group. External callus and fracture line blurring score showed that scores of lining material control group and model control group showed no significant difference (P= 0.819), scores of micron group and model control group were significantly different (P= 0.001) after 2 weeks of modeling; 4 weeks after modeling, scores of lining material control group and model control group showed no significant difference (P= 0.778). Scores of micron group and model control group were significantly different (P= 0.000). Scores of micron group and traditional Chinese medicine control group were significantly different (P= 0.010).6 weeks after modeling, scores of lining material control group and model control group showed no significant difference (P= 0.293), scores of micron group, model control group and traditional Chinese medicine control group were significantly different (P values were 0.000,0.027).
4. Histomorphology:The first 6 weeks after modeling, vasculars between trabeculars in the location of fracture expanded and the number was larger; Endochondral bone was completed; A large number of osteoblasts were saw; Woven bone began shaping; The bone marrow cavity was recanalized in micron groups. Weaving bone, cartilage cells and endochondral bone were coexisted; In callus and external callus of stump were connected; Bone marrow cavity was not recanalized in traditional Chinese medicine group. While trabecular bone was smaller and blood vessels between trabeculars were less and in callus and external callus of stump were mainly unconnected and the bone marrow cavity was not recanalized.
Conclusion
Micron traditional Chinese medicine functional lining material can improve microcirculation, increase the activity of AKP, decrease the level of Ca, elevate the product of serum P and Ca, accelerate the proliferation of osteoblasts and promote callus formation and fracture healing.lt was stronger than ordinary traditional Chinese medicine functional lining material.
引文
[1]Rutten S, Nolte PA, Korstjens CM, etal. Low-intensity pulsed ultrasound affects RUNX2 immunopositive osteogenic cells in delayed clinical fracture healing[J]. Bone,2009,45(5):862-869.
[2]Schnoke M, Midura RJ. Pulsed electromagnetic fields rapidly modulate intracellular signaling events in osteoblastic cells:Comparison to parathyroid hormone and insulin[J]. J Orthop Res,2007,25(7):933-940.
[3]Jingushi S, Mizuno K, Matsushita T, etal. Low-intensity pulsed ultrasound treatment for postoperative delayed union or nonunion of long bone fractures[J]. J Orthop Sci,2007,12(1):35-41.
[4]张俐,张安祯,移平.针灸促进骨折愈合的红外热像图观察[J].中国骨伤,2001,14(8)：468-469.
[5]周晶亚.中草药接骨膏促进骨折愈合作用的实验研究[J].时珍国医国药,2008,19(08)：2014-2015.
[6]陈履平,倪正,韩祖斌,等.复方中药促骨生酊剂对骨折愈合影响的实验研究[J].中国骨伤,1998,11(04)：14-16.
[7]宋锁行.伤科灵喷雾剂在临床骨科中的应用[J].临床医药实践杂志,2005,14(12)：929.
[8]刘志宇,周学平.中医药治疗骨关节炎疗效机理研究进展[J].辽宁中医药大学学报,2008,10(11)：62-63.
[9]张建国,陈良金,蒋文跃.外敷中药对骨折愈合微血管重建的影响[J].中国骨伤,2000,13(02)：86-87.
[10]赵明杰,周丰慧,刘斌.超声波中药离子透入促进骨折愈合的实验研究及临床观察[J].广东医学,2006,27(06)：788-789.
[11]熊辉.中药干预骨折愈合过程的实验研究概况[J].湖南中医学院学报,2002,22(01)：69-70.
[12]陈海松,黄光才,刘维俊,等.骨肤康擦剂接骨作用的实验研究[J].中草药,1996,27(09)：129-130.
[13]张永斌,黄永,邹移海,等.壮筋补骨丸促进家兔人工骨折愈合的组织学观察[J].动物医学进展,2007,28(11)：46-49.
[14]顾坚毅,汤荣光,罗建中,等.新伤续断汤对骨折愈合中胶原影响的实验研究[J].中国中医骨伤科杂志,2001,9(04)：28-31-
[15]沈海琦,马勇,李运蔓.”接骨灵”对家兔骨折愈合相关血生化学指标的影响[J].江苏中医,1999,20(11)：47-48.
[16]刘钟华,杨世荣,回春.中药促进骨折愈合的实验研究概况[J].长春中医学院学报,2004,20(02)：63-65.
[17]洪定钢,时光达.淫羊藿影响骨折愈合的骨组织学及骨组织形态计量学研究[J].中医正骨,1999,11(02)：67-70.
[18]林燕萍,王和鸣,陈奕权.煅狗骨对实验性骨折愈合作用的组织学和组织化学研究[J].中国中医骨伤科杂志,1991,17(6)：4.
[19]朱建伟,李贵海,任宁,等.骨愈速对大鼠实验性骨折的影响[J].中药药理与临床,2004,20(01)：43-45.
[20]孙玉鹏,张皖清.生长因子与骨愈合[J].中华骨科杂志,1997,7(4)：265.
[21]吴官保,杨军辉,谢心军,等.中药促进骨折愈合研究进展[J].湖南中医学院学报,1997,17(2)：72.
[22]刘献祥,余希杰,许书亮.无名异冲剂在骨折愈合中DNA和BMP含量影响的实验研究[J].中国骨伤,1999,12(3)：21.
[23]汤耿民,沈霖,涂意辉.补肾活血方对成骨细胞生长因子TGF-pmRNA表达的影响[J].中国中医骨伤科杂志,1999,7(5)：5.
[24]汤耿民,沈霖,夏志道,等.补肾活血方对实验性骨折愈合过程中IGF-Ⅰ的影响[J].中国中医药骨伤科杂志,2000,8(3)：3.
[25]秦军志,王贤淑.丹参对大鼠胫骨骨折早期愈合过程中血清、骨痂及骨组织中钙、锌、铜的影响[J].中国中西医结合杂志,1992,12(6)：354.
[26]刘献详,许书亮,王和鸣,等.麦饭石促进骨折愈合的实验研究[J].中国中医骨伤科杂志,1995,3(2)：5.
[27]梁东辉.中药透皮治疗骨关节疼痛性疾病[J].临床医学工程,2008,15(9)：7-11.
[28]袁鹏程,袁万里.大毛救急粉外敷治疗骨折100例临床观察[J].湖南中医药导报,1998,4(5)：24.
[29]范和平,杨晓恒,范花平,等.黄龙接骨膏治疗四肢闭合性骨50例总结[J].湖南中药杂志,2002,18(5)：13-14.
[30]郭维玉.三七散油膏治疗骨折延迟愈合的疗效观察[J].医学文选,2002,21(5)：624-626.
[31]董发明.七叶红花药酒治疗620例骨折的临床分析[J].中国民族民间医药杂志,1998,(5)：21-22.
[32]陈玉丽,郑文兰.中药薰洗促进骨折愈合的临床观察[J].齐齐哈尔医学院学报,2004,25(11)：1251.
[33]刘晓梅.中药薰洗在骨折后期的治疗体会[J].医学信息手术学分册,2007,(4)：70-71.
[34]殴伦,米琨,俸志斌.骨髁间粉碎骨折术后30例[J].陕西中医.2007,28(12)：1621-1623.
[35]周丰慧,赵明杰,赵海燕.中药离子导入促进骨折愈合62例疗效观察[J].新中医,2004,36(6)：19-20.
[36]秦建军,刘立杰.中药电离子导入治疗胫骨疲劳骨折52例[J].河北中医,2001,23(6)：433.
[37]吴海忠.中药热熨加推拿治疗四肢骨折后期关节功能障碍[J].广西中医学院学报,2000,17(4)：33-34.
[38]吴峰,张兆华,何仁荣,等.电磁场配合骨宝液离子导入治疗股骨干骨折[J].广东医学,2005,26(4)：558-559.
[39]魏晓艳,韩洪舜,韩靖,等.中草药超声透入促进骨折愈合的临床研究 [J].现代中西医结合杂志,2003,12(2)：139-140.
[40]沈平姨,何筑生.制药粉体工程技术与中药现代化[J].世界科学技术——中药现代化,1999,1(3)：26.
[41]董洪涛.纳米与中医药学[J].辽宁中医杂志,2001,28(12)：715.
[42]吕文海等.炮制与超微粉碎对水蛭药效影响的初步实验研究[J].中国中药杂志,2001。26(4)：241.
[43]杜晓敏,刘璐.原生药材超细微粉制剂的药效学研究[J].中草药1999,30(90)：680—684.
[44]朱莉.超微粉碎技术及其在中药加工中的应用[J].云南大学学报(自然科学版增刊).2004,(26)：128—131.
[45]徐辉碧,谢长生.纳米技术在中药研究中的应用[J].中国药科大学学报,2001,32(8)：161-165.
[46]李秀凤,殷辉安,唐明,等.纳米中药研究进展[J].中医药通报,2004,3(5)： 53-56.
[47]杨祥良,徐辉碧,吴继洲,等.基于纳米技术的中药基础问题研究[J].华中科技大学学报,2000,(12)：104-105.
[48]Ying D, Huibi Xu, Kaixun Huang, etal. Size effects of realgar particles on apoptosis in a human umbilical vein endothelial cell line: ECV-304. Pharmacological Research,2001,44(6):513.
[49]方琴.纳米技术在医药领域中的应用[J].贵州医学,2002,26(11)：1040.
[50]杨惠森,张拴,宁显唯.纳米技术在生物医药研究中的应用[J].陕西中医学院学报,2003,26(3)：67-70.
[51]Wei H, Li Y G. Nanotechnology apply on biomedicine engineering-research actuality and development current[J]. Foreign Med Sci:Sect Biomed Engin(国外医学：生物医学工程分册),1999,22(6)：340.
[52]Wang Y,Wu W.Recessive nanograin inner target[J].Chin Tradit Herb Drugs(中草药),2004,39(1)：7-11.
[53]吴焕林,王薇,罗文杰,等.纳米中药的研究进展[J].中国现代医学杂志,2007,(21)：2625-2628.
[54]肖延龄,李伯.载药纳米微粒与中药现代化[J].中草药,2002,(05)：385-388.
[55]Schroder U, Sabel BA. Nanoparticles, a drug carrier system to pass the blood-brain barrier, permit centralanalgesic effects of i.v. dalargin injections[J]. Brain Res,1996,710(1-2):121-124.
[56]胥沙订,葛宝丰,徐印坎.实用骨科学[M].北京：人民军医出版社,20032.
[57]Ricky W.K. Wong, A. Bakr M. Rabie.Traditional Chinese Medicines and Bone Formation—A Review,Journal of Oral and Maxillofacial Surgery, 2006,64(05):828-837.
[58]徐东艳,应健跃.中药促进骨折愈合的研究进展[J].浙江中医杂志,2005,40(3)： 136-137.
[59]龚志贤,卢敏,谢心军,等.中医药治疗骨折的研究进展[J].中国中医骨伤科杂志,2006,14(3)：60-63.
[60]柴本甫,过邦辅.理气药物对骨折愈合影响的初步研究[J].中华外科杂志,1962,14(5)：298-299.
[61]孙保勇,陈文直.低强度脉冲超声促进骨折愈合的实验研究[J].中国矫形外科杂志,2005,3(6)：443-446.
[62]段伟利,杨海韵.骨折愈合的中西医治疗进展[J].中医正骨,2008,20(03)：66～68.
[63]刘彩兵,盛勇,涂铭旌.三七的超细化及纳米化研究[J].食品科技,2004,(11)：21～24.
[64]张莉,于燕莉,潘菡清.超微粉碎技术及设备国内中药领域应用概述[J].实用医药杂志,2006,23(06)：752-753.
[65]楼丹飞,周丹.中药超微研究进展[J].河南中医,2008,28(08)：102-104.
[66]杨连威,赵晓燕,李婷,等.中药超微粉碎后对其性能的影响研究[J].世界科学技术-中医药现代化,2008,(06)：77-81.
[67]陈力,吴懿平.微米中药及其制备技术[J].中草药,2002,(10)：865-868.
[68]王爱武,吕文海,耿晖.超微粉碎在中药生产中应用概况及展望[J].时珍国医国药,2000,(07)：669.
[69]陈旭.三七超微粉碎对生物体内吸收影响的初步试验[J].西北药学杂志,2002,17(05)：203-204.
[70]董华,胡乃武,董清平.骨痛仙胶囊对家兔骨折血清碱性磷酸酶、钙、磷影响的实验研究[J].中医药学报,2003,31(2)：51.
[71]SimankHG, ManggoldJ, SebaldW, etal. Bone morphogenetic protein-2 and growth and differentiation factor-5 enhance the healing of necrotic bone in a sheep mode.Growth Factors,2001,19(4):247-257.
[72]金惠铭,王建枝.病理生理学[M].北京：人民卫生出版社,2004：44.
[73]王怡,翁维良.止痛活血化瘀药物对血液流变学影响的研究[J].中国血液流变学杂志,1997,7(3)：1.
[74]张建国,陈良金,蒋文跃.外敷中药对骨折愈合微血管重建的影响[J].中国骨伤,2000,13(2)：86.
[75]卢敏,谢进, 戴致波.小针刀对兔骨折愈合影响的实验研究[J].湖南中医药大学学报,2007,27(04)：70-72.
[76]林燕萍,王和鸣,周瑞祥.补骨素对实验性骨折愈合的影响[J].中国骨伤,1993,6(1)：8.
[77]孙炳烈,郭建刚,赵然,等.中药电热夹板促进骨折愈合的实验研究[J].中国中医骨伤科杂志,1991,7(4)：15.
[2]Schnoke M, Midura RJ. Pulsed electromagnetic fields rapidly modulate intracellular signaling events in osteoblastic cells:Comparison to parathyroid hormone and insulin[J]. J Orthop Res,2007,25(7):933-940.
[3]Jingushi S, Mizuno K, Matsushita T, etal. Low-intensity pulsed ultrasound treatment for postoperative delayed union or nonunion of long bone fractures[J]. J Orthop Sci,2007,12(1):35-41.
[4]张俐,张安祯,移平.针灸促进骨折愈合的红外热像图观察[J].中国骨伤,2001,14(8)：468-469.
[5]周晶亚.中草药接骨膏促进骨折愈合作用的实验研究[J].时珍国医国药,2008,19(08)：2014-2015.
[6]陈履平,倪正,韩祖斌,等.复方中药促骨生酊剂对骨折愈合影响的实验研究[J].中国骨伤,1998,11(04)：14-16.
[7]宋锁行.伤科灵喷雾剂在临床骨科中的应用[J].临床医药实践杂志,2005,14(12)：929.
[8]刘志宇,周学平.中医药治疗骨关节炎疗效机理研究进展[J].辽宁中医药大学学报,2008,10(11)：62-63.
[9]张建国,陈良金,蒋文跃.外敷中药对骨折愈合微血管重建的影响[J].中国骨伤,2000,13(02)：86-87.
[10]赵明杰,周丰慧,刘斌.超声波中药离子透入促进骨折愈合的实验研究及临床观察[J].广东医学,2006,27(06)：788-789.
[11]熊辉.中药干预骨折愈合过程的实验研究概况[J].湖南中医学院学报,2002,22(01)：69-70.
[12]陈海松,黄光才,刘维俊,等.骨肤康擦剂接骨作用的实验研究[J].中草药,1996,27(09)：129-130.
[13]张永斌,黄永,邹移海,等.壮筋补骨丸促进家兔人工骨折愈合的组织学观察[J].动物医学进展,2007,28(11)：46-49.
[14]顾坚毅,汤荣光,罗建中,等.新伤续断汤对骨折愈合中胶原影响的实验研究[J].中国中医骨伤科杂志,2001,9(04)：28-31-
[15]沈海琦,马勇,李运蔓.”接骨灵”对家兔骨折愈合相关血生化学指标的影响[J].江苏中医,1999,20(11)：47-48.
[16]刘钟华,杨世荣,回春.中药促进骨折愈合的实验研究概况[J].长春中医学院学报,2004,20(02)：63-65.
[17]洪定钢,时光达.淫羊藿影响骨折愈合的骨组织学及骨组织形态计量学研究[J].中医正骨,1999,11(02)：67-70.
[18]林燕萍,王和鸣,陈奕权.煅狗骨对实验性骨折愈合作用的组织学和组织化学研究[J].中国中医骨伤科杂志,1991,17(6)：4.
[19]朱建伟,李贵海,任宁,等.骨愈速对大鼠实验性骨折的影响[J].中药药理与临床,2004,20(01)：43-45.
[20]孙玉鹏,张皖清.生长因子与骨愈合[J].中华骨科杂志,1997,7(4)：265.
[21]吴官保,杨军辉,谢心军,等.中药促进骨折愈合研究进展[J].湖南中医学院学报,1997,17(2)：72.
[22]刘献祥,余希杰,许书亮.无名异冲剂在骨折愈合中DNA和BMP含量影响的实验研究[J].中国骨伤,1999,12(3)：21.
[23]汤耿民,沈霖,涂意辉.补肾活血方对成骨细胞生长因子TGF-pmRNA表达的影响[J].中国中医骨伤科杂志,1999,7(5)：5.
[24]汤耿民,沈霖,夏志道,等.补肾活血方对实验性骨折愈合过程中IGF-Ⅰ的影响[J].中国中医药骨伤科杂志,2000,8(3)：3.
[25]秦军志,王贤淑.丹参对大鼠胫骨骨折早期愈合过程中血清、骨痂及骨组织中钙、锌、铜的影响[J].中国中西医结合杂志,1992,12(6)：354.
[26]刘献详,许书亮,王和鸣,等.麦饭石促进骨折愈合的实验研究[J].中国中医骨伤科杂志,1995,3(2)：5.
[27]梁东辉.中药透皮治疗骨关节疼痛性疾病[J].临床医学工程,2008,15(9)：7-11.
[28]袁鹏程,袁万里.大毛救急粉外敷治疗骨折100例临床观察[J].湖南中医药导报,1998,4(5)：24.
[29]范和平,杨晓恒,范花平,等.黄龙接骨膏治疗四肢闭合性骨50例总结[J].湖南中药杂志,2002,18(5)：13-14.
[30]郭维玉.三七散油膏治疗骨折延迟愈合的疗效观察[J].医学文选,2002,21(5)：624-626.
[31]董发明.七叶红花药酒治疗620例骨折的临床分析[J].中国民族民间医药杂志,1998,(5)：21-22.
[32]陈玉丽,郑文兰.中药薰洗促进骨折愈合的临床观察[J].齐齐哈尔医学院学报,2004,25(11)：1251.
[33]刘晓梅.中药薰洗在骨折后期的治疗体会[J].医学信息手术学分册,2007,(4)：70-71.
[34]殴伦,米琨,俸志斌.骨髁间粉碎骨折术后30例[J].陕西中医.2007,28(12)：1621-1623.
[35]周丰慧,赵明杰,赵海燕.中药离子导入促进骨折愈合62例疗效观察[J].新中医,2004,36(6)：19-20.
[36]秦建军,刘立杰.中药电离子导入治疗胫骨疲劳骨折52例[J].河北中医,2001,23(6)：433.
[37]吴海忠.中药热熨加推拿治疗四肢骨折后期关节功能障碍[J].广西中医学院学报,2000,17(4)：33-34.
[38]吴峰,张兆华,何仁荣,等.电磁场配合骨宝液离子导入治疗股骨干骨折[J].广东医学,2005,26(4)：558-559.
[39]魏晓艳,韩洪舜,韩靖,等.中草药超声透入促进骨折愈合的临床研究 [J].现代中西医结合杂志,2003,12(2)：139-140.
[40]沈平姨,何筑生.制药粉体工程技术与中药现代化[J].世界科学技术——中药现代化,1999,1(3)：26.
[41]董洪涛.纳米与中医药学[J].辽宁中医杂志,2001,28(12)：715.
[42]吕文海等.炮制与超微粉碎对水蛭药效影响的初步实验研究[J].中国中药杂志,2001。26(4)：241.
[43]杜晓敏,刘璐.原生药材超细微粉制剂的药效学研究[J].中草药1999,30(90)：680—684.
[44]朱莉.超微粉碎技术及其在中药加工中的应用[J].云南大学学报(自然科学版增刊).2004,(26)：128—131.
[45]徐辉碧,谢长生.纳米技术在中药研究中的应用[J].中国药科大学学报,2001,32(8)：161-165.
[46]李秀凤,殷辉安,唐明,等.纳米中药研究进展[J].中医药通报,2004,3(5)： 53-56.
[47]杨祥良,徐辉碧,吴继洲,等.基于纳米技术的中药基础问题研究[J].华中科技大学学报,2000,(12)：104-105.
[48]Ying D, Huibi Xu, Kaixun Huang, etal. Size effects of realgar particles on apoptosis in a human umbilical vein endothelial cell line: ECV-304. Pharmacological Research,2001,44(6):513.
[49]方琴.纳米技术在医药领域中的应用[J].贵州医学,2002,26(11)：1040.
[50]杨惠森,张拴,宁显唯.纳米技术在生物医药研究中的应用[J].陕西中医学院学报,2003,26(3)：67-70.
[51]Wei H, Li Y G. Nanotechnology apply on biomedicine engineering-research actuality and development current[J]. Foreign Med Sci:Sect Biomed Engin(国外医学：生物医学工程分册),1999,22(6)：340.
[52]Wang Y,Wu W.Recessive nanograin inner target[J].Chin Tradit Herb Drugs(中草药),2004,39(1)：7-11.
[53]吴焕林,王薇,罗文杰,等.纳米中药的研究进展[J].中国现代医学杂志,2007,(21)：2625-2628.
[54]肖延龄,李伯.载药纳米微粒与中药现代化[J].中草药,2002,(05)：385-388.
[55]Schroder U, Sabel BA. Nanoparticles, a drug carrier system to pass the blood-brain barrier, permit centralanalgesic effects of i.v. dalargin injections[J]. Brain Res,1996,710(1-2):121-124.
[56]胥沙订,葛宝丰,徐印坎.实用骨科学[M].北京：人民军医出版社,20032.
[57]Ricky W.K. Wong, A. Bakr M. Rabie.Traditional Chinese Medicines and Bone Formation—A Review,Journal of Oral and Maxillofacial Surgery, 2006,64(05):828-837.
[58]徐东艳,应健跃.中药促进骨折愈合的研究进展[J].浙江中医杂志,2005,40(3)： 136-137.
[59]龚志贤,卢敏,谢心军,等.中医药治疗骨折的研究进展[J].中国中医骨伤科杂志,2006,14(3)：60-63.
[60]柴本甫,过邦辅.理气药物对骨折愈合影响的初步研究[J].中华外科杂志,1962,14(5)：298-299.
[61]孙保勇,陈文直.低强度脉冲超声促进骨折愈合的实验研究[J].中国矫形外科杂志,2005,3(6)：443-446.
[62]段伟利,杨海韵.骨折愈合的中西医治疗进展[J].中医正骨,2008,20(03)：66～68.
[63]刘彩兵,盛勇,涂铭旌.三七的超细化及纳米化研究[J].食品科技,2004,(11)：21～24.
[64]张莉,于燕莉,潘菡清.超微粉碎技术及设备国内中药领域应用概述[J].实用医药杂志,2006,23(06)：752-753.
[65]楼丹飞,周丹.中药超微研究进展[J].河南中医,2008,28(08)：102-104.
[66]杨连威,赵晓燕,李婷,等.中药超微粉碎后对其性能的影响研究[J].世界科学技术-中医药现代化,2008,(06)：77-81.
[67]陈力,吴懿平.微米中药及其制备技术[J].中草药,2002,(10)：865-868.
[68]王爱武,吕文海,耿晖.超微粉碎在中药生产中应用概况及展望[J].时珍国医国药,2000,(07)：669.
[69]陈旭.三七超微粉碎对生物体内吸收影响的初步试验[J].西北药学杂志,2002,17(05)：203-204.
[70]董华,胡乃武,董清平.骨痛仙胶囊对家兔骨折血清碱性磷酸酶、钙、磷影响的实验研究[J].中医药学报,2003,31(2)：51.
[71]SimankHG, ManggoldJ, SebaldW, etal. Bone morphogenetic protein-2 and growth and differentiation factor-5 enhance the healing of necrotic bone in a sheep mode.Growth Factors,2001,19(4):247-257.
[72]金惠铭,王建枝.病理生理学[M].北京：人民卫生出版社,2004：44.
[73]王怡,翁维良.止痛活血化瘀药物对血液流变学影响的研究[J].中国血液流变学杂志,1997,7(3)：1.
[74]张建国,陈良金,蒋文跃.外敷中药对骨折愈合微血管重建的影响[J].中国骨伤,2000,13(2)：86.
[75]卢敏,谢进, 戴致波.小针刀对兔骨折愈合影响的实验研究[J].湖南中医药大学学报,2007,27(04)：70-72.
[76]林燕萍,王和鸣,周瑞祥.补骨素对实验性骨折愈合的影响[J].中国骨伤,1993,6(1)：8.
[77]孙炳烈,郭建刚,赵然,等.中药电热夹板促进骨折愈合的实验研究[J].中国中医骨伤科杂志,1991,7(4)：15.