可注射骨修复材料结合骨碎补总黄酮修复鼠骨缺损
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摘要
研究背景
骨折不愈合、骨缺损发生率为5%～10%,对大范围骨缺损的修复是世界性难题。
自体骨移植具有骨传导和骨诱导的双重作用,被认为是修复骨缺损的金标准,但来源有限。异种骨或同种异体骨移植有众所周知的免疫排斥限制了其使用。清华大学材料科学与工程系按照仿生思路设计的纳米复合多孔材料,以胶原分子为模板,调制钙磷盐沉积到有序排列的胶原纤维上,自组成装具有天然骨分级结构和特性的纳米晶磷酸钙／胶原复合材料(nano-hydroxyapatite/collagen composite,简称nHAC),有良好的生物相容性和可吸收降解性,以nHAC为基础制备微创原位成型可注射骨修复材料(injectable bone regeneration composite, IBRC),既可以保持仿天然骨结构的特征,又赋予了材料可注射性能,具有广阔的应用前景。
中药骨碎补(Drynaria fortunei (Kunze) J. S,简称DR)具有强筋壮骨、益肾固精、散瘀止痛的作用,现代药理研究证实骨碎补总黄酮(assemble flavone of rhizome drynaria,简称AFDR)对骨折的修复有促进作用,改善微循环障碍,治疗骨质疏松症,并有一定抗炎、镇痛等作用。骨形态发生蛋白2 (Bone morphogenetic protein,BMP2)是在骨折愈合和骨改建中发挥重要作用的生长因子,已成功用于骨折不愈合及骨缺损的治疗,
骨缺损应用IBRC材料修复,结合使用AFDR或BMP2,可能减轻创伤反应,提高修复质量,是中西医结合治疗骨缺损一种模式的探索。
研究目的
观察和对比研究IBRC结合AFDR或BMP2对骨缺损的修复效果,初步探讨中西医结合治疗骨缺损的一种模式。
研究方法
1)随机对照实验,以SD大鼠80只,均行双侧直径5mm的颅骨缺损模型,左侧空置做对照。分为3组处理, A组(IBRC+AFDR, n=30):右侧颅骨缺损以IBRC材料修复,AFDR灌胃2-8周；B组[IBRC+去离子水(deionized water,DW), n=30)]:右侧颅骨缺损以IBRC修复,DW灌胃2-8周；C组[IBRC-HA (Hydroxyapatite, HA)+DW,n=20]：右侧颅骨缺损以IBRC-HA材料修复,DW灌胃灌胃2-8周。分2、4、8周三个时间点处死大鼠,抽取腹主动脉血化验血清钙、磷、碱性磷酸酶、肌酐、谷丙转氨酶；颅骨标本蜡块包埋并切片,进行苏木精-伊红(Hematoxylin-Eosin,HE)染色、Masson染色观察组织学变化,碱性磷酸酶和Ⅰ型胶原免疫组化染色,以及BMP2原位杂交,进行显微镜图像采集和分析光密度值,指标进行统计学处理。
2)随机对照实验,以SD大鼠40只,颅骨行8mm直径的极量骨缺损,随机分为4组,A组(IBRC+AFDR,n=12)植入IBRC材料,AFDR灌胃2-8周；B组(IBRC+DW,n=12)：植入可注射IBRC材料,DW灌胃2-8周；C组(IBRC+rhBMP2, n=13)IBRC材料复合rhBMP2(15ug/ml);D组(n=3)：空白不植骨。分时间点2、4、6、8、12周处死大鼠,分别进行Micro-CT检查和HE及Masson染色组织学检查,观察修复效果。
研究结果
IBRC结合AFDR修复颅骨缺损实验中,血清钙、磷值及钙磷乘积在AFDR组2周时无变化,4周时均较对照组明显升高(P<0.05),8周时两组无明显差异；碱性磷酸酶在AFDR组2周时明显升高(P<0.05),4周及8周时无差异；肌酐在8周时AFDR组与DW组对比显著降低(P<0.05)。
组织学观察骨缺损修复效果：IBRC+AFDR组优于IBRC+DW灌胃组,而HA组修复效果较前两者均差；碱性磷酸酶和Ⅰ型胶原免疫组化染色,以及BMP2原位杂交的图像分析表明,IBRC+AFDR组较后两组阳性表达明显。
颅骨极量骨缺损的实验中,IBRC+rhBMP2组修复效果最为理想,IBRC+AFDR组优于IBRC+DW组。
结论
1)AFDR在IBRC材料修复骨缺损的过程中,2周时提高血清碱性磷酸酶的水平,4周时可以提高血清钙、磷及钙磷乘积水平,从而对骨修复有促进作用。
2)IBRC材料结合AFDR修复骨缺损,能提高修复质量,缩短修复时间。
3)IBRC材料结合AFDR修复骨缺损,可以提高BMP2的阳性表达,促进修复材料的诱导成骨作用。
4)IBRC械料结合AFDR修复骨缺损,能够提高Coll-Ⅰ和ALP的阳性表达,有利于骨修复的骨基质形成和矿化。
5)本实验观察IBRC材料修复骨缺损主要是软骨内成骨模式。
6)IBRC材料可用于修复大鼠极量骨缺损,结合AFDR能明显提高修复速度及质量,但不及IBRC材料复合BMP2的修复速度及质量。
7)IBRC材料结合AFDR修复骨缺损,其效果不及IBRC材料结合BMP2的修复效果,验证了rhBMP2的有效,同时对中药促进骨修复提出挑战,传统中医治疗骨折以复方为主,深入开展复方的研究成为必要。
8)AFDR按人鼠换算剂量连续灌胃8周大鼠未见肝肾毒性,并可能有增加肾小球滤过功能的作用。
Introduction
Based on the fact that the accidents rate of bone nonunions, bone defects are approximately 5-10%, the repair of critical-sized bone defects is a challeging problem around the world.The autogenous bone graft, which is with limited resource, has been deemed as the golden criterion in bone repair for its osteoconductivity and osteoinductivity. And it is known to all that the xenogeneic bone or allogenic bone would cause immunological rejections which limite their application in bone repair. The Department of Materials Science and Engineering of Tsinghua University has designed a nanoporous material according to biomimetic method. Based on the template of tropocollagen molecule, a bone-like nano-hydroxyapatite/collagen (nHAC) composite was synthesized biomimetically by the orderly deposition of hydroxyapatite mineral with low crystallinity, hierarchical structure. It has a good biocompatibility and biodegradability. Recentaly, we have developed an injectable bone regeneration composite (IBRC) by using a calcium alginate hydrogel as matirx vehicle. It has good potential application in future for it not only maintained the natural bone-like characterics of nHAC but also a good injectability.
The Chinese medicine drynaria fortunei (DR) has many therapeutic functions, such as strenthening tendons and bones, invigorating the kindey and spermium, as well as pain and bruises relieving. It has been vertified by the contemporary pharmacological studies that assemble flavone of rhizome drynaria (AFDR) can promote fracture repair, and improve microcirulation, treat the osteoporosis, as well as certain anti-inflamatory and analgesic effects. The bone growth factor BMP2, which plays a crucial role in fracture healing and bone remodling, has been used for fracture healing and bone defect treatment successfully.
Combine the IBRC with AFDR or BMP2 in the application of bone defect repair may reduce the trauma response and improve the repair quality. It has been an exploration mode of combination of Chinese and western medicine for bone defects treatment.
Objective
This article discusses the mode of combing Chinese and western medicine in bone defects repair, which is based on observasion and comparative studies of the effect by the application of IBRC in combination with AFDR or BMP2.
Methods
1) andomized controlled trials. Arranged 80 SD rats into three groups, and made a control test with a 5mm diameter wide calvarial defects on the right side and the left skull defects as comparison. Group A (IBRC+AFDR, n=30):Implanted IBRC to the bone skull defect and fed the rats with AFDR for 2-8 weeks. Groud B(IBRC+DW,n=30):Implanted IBRC to the skull defect on the right, fed the rats with DW for 2-8 weeks. Group C (IBRC-HA+DW, n=20):Implanted IBRC-HA to the skull defect on the right, fed the rats with DW for 2-8 weeks. Executed the rats on week 2,4 and 8, and drawn the blood in the abdominal arteriae aorta for the test of serum calcium, phosphorus, alkaline phosphatase, creatinine, and alanine aminotransferase. Inbedded the skull specimens with paraffin and sliced, and observed the changes of the tissues after HE and Masson stained. After alkaline phosphatase and type I collagen immunohistochemical staining, as well as BMP2 in situ hybridization, we observed the microsope image and analyzed the OD value, and the index was analyzed by statistic treatment.
2) Randomized controlled trials:40 rats were randomly arranged into 4 groups and made critical-sized calvairal defects with 8mm diameter wide. Group A (IBRC+AFDR, n=12): Implanted IBRC on the defects, and fed the rats with AFDR for 2-8 weeks; Group B (IBRC+DW, n=12):Implanted IBRC and fed the rats for 2-8 weeks; Group C (IBRC+rhBMP2.n=13):Implanted the combination of IBRC and rhBMP2 (15ug/ml); Group D (n=3); No implant. Removed calvairal bones from the rats at week 2,4,6,8,12 and examined them with Micro-CT and HE stainning for repair effect evaluation
Results
In the experiment of skull bone defects repair by the combination of IBRC and AFDR, there wasn't any change of the value of serum calcium, phosphorus or calcium phoshate on week 2. But a significant raise (P< 0.05) was found in the control group on week 4. The result remained the same on week 4 and 8, but the creatinine has siginficantly decreased (P<0.05) in the AFDR group in the comparision with DW group on week 8. The repair effects in histological observation:Repair effect of the IBRC+AFDR group is better than the IBRC+DW group's, HA group is the worst one of the three. It is showed in the type I collagen immunohistochemical stained and the analysis of the BMP2 ISH graphics that the positive expression of the IBRC+AFDR group is much obvious than the other two. In the experiment of critical-sized calvarial defects, the repair effect of IBRC+rhBMP2 was the best, and the IBRC+AFDR group is better than the IBRC+DW group. Conclusion
1) In the experiment of combining AFDR and IBRC for calvarial defect repair, alkaline phosphatase has significantly improved in two weeks, and the amount of calcium in blood, phosphorus and calcium phosphate product level is higher in four weeks. All of these have proved that AFDR has the effect on promoting bone repair.
2) The repair time can be shorten and the quality can be improved when defects are repaired by IBRC in the combination with AFDR.
3) The positive expression of BMP2 can be improved and sequentially boost the osteoinductivity of the material when defects are repaired by the combination of IBRC and AFDR.
4) It can improve the positive expression of Coll-I and ALP, facilitating the formation and mineralization of bone matrix when the bone defects are repaired by the combination of IBRC and AFDR.
5) The observation method of this experiment of IBRC bone defects repair is based on the endochondral bone formation.
6) IBRC can be used in the critical-sized bone defect on rats, and with the combination of AFDR can distinctly raise the speed and quality of repairment, however, its effect is not as good as the combination of IBRC and BMP2.
7) The effect of IBRC and AFDR combination for bone defects are not as good as the combination of IBRC and BMP2, which also verify the effetivity of rhBMP2. At the same time, it also challenges the effectiveness of Chinese medicine for promoting bone repair. Since the traditional Chinese medicine treatment has been focused on compoud medicine, it is necessary to carry out an in-depth research on compound medicine.
8) There is no hepatorenal toxicity symdrome after feeding the rats with conversed dose of AFDR for 8 weeks. And it may increase the function of glomerular filtration.
骨折不愈合、骨缺损发生率为5%～10%,对大范围骨缺损的修复是世界性难题。
自体骨移植具有骨传导和骨诱导的双重作用,被认为是修复骨缺损的金标准,但来源有限。异种骨或同种异体骨移植有众所周知的免疫排斥限制了其使用。清华大学材料科学与工程系按照仿生思路设计的纳米复合多孔材料,以胶原分子为模板,调制钙磷盐沉积到有序排列的胶原纤维上,自组成装具有天然骨分级结构和特性的纳米晶磷酸钙／胶原复合材料(nano-hydroxyapatite/collagen composite,简称nHAC),有良好的生物相容性和可吸收降解性,以nHAC为基础制备微创原位成型可注射骨修复材料(injectable bone regeneration composite, IBRC),既可以保持仿天然骨结构的特征,又赋予了材料可注射性能,具有广阔的应用前景。
中药骨碎补(Drynaria fortunei (Kunze) J. S,简称DR)具有强筋壮骨、益肾固精、散瘀止痛的作用,现代药理研究证实骨碎补总黄酮(assemble flavone of rhizome drynaria,简称AFDR)对骨折的修复有促进作用,改善微循环障碍,治疗骨质疏松症,并有一定抗炎、镇痛等作用。骨形态发生蛋白2 (Bone morphogenetic protein,BMP2)是在骨折愈合和骨改建中发挥重要作用的生长因子,已成功用于骨折不愈合及骨缺损的治疗,
骨缺损应用IBRC材料修复,结合使用AFDR或BMP2,可能减轻创伤反应,提高修复质量,是中西医结合治疗骨缺损一种模式的探索。
研究目的
观察和对比研究IBRC结合AFDR或BMP2对骨缺损的修复效果,初步探讨中西医结合治疗骨缺损的一种模式。
研究方法
1)随机对照实验,以SD大鼠80只,均行双侧直径5mm的颅骨缺损模型,左侧空置做对照。分为3组处理, A组(IBRC+AFDR, n=30):右侧颅骨缺损以IBRC材料修复,AFDR灌胃2-8周；B组[IBRC+去离子水(deionized water,DW), n=30)]:右侧颅骨缺损以IBRC修复,DW灌胃2-8周；C组[IBRC-HA (Hydroxyapatite, HA)+DW,n=20]：右侧颅骨缺损以IBRC-HA材料修复,DW灌胃灌胃2-8周。分2、4、8周三个时间点处死大鼠,抽取腹主动脉血化验血清钙、磷、碱性磷酸酶、肌酐、谷丙转氨酶；颅骨标本蜡块包埋并切片,进行苏木精-伊红(Hematoxylin-Eosin,HE)染色、Masson染色观察组织学变化,碱性磷酸酶和Ⅰ型胶原免疫组化染色,以及BMP2原位杂交,进行显微镜图像采集和分析光密度值,指标进行统计学处理。
2)随机对照实验,以SD大鼠40只,颅骨行8mm直径的极量骨缺损,随机分为4组,A组(IBRC+AFDR,n=12)植入IBRC材料,AFDR灌胃2-8周；B组(IBRC+DW,n=12)：植入可注射IBRC材料,DW灌胃2-8周；C组(IBRC+rhBMP2, n=13)IBRC材料复合rhBMP2(15ug/ml);D组(n=3)：空白不植骨。分时间点2、4、6、8、12周处死大鼠,分别进行Micro-CT检查和HE及Masson染色组织学检查,观察修复效果。
研究结果
IBRC结合AFDR修复颅骨缺损实验中,血清钙、磷值及钙磷乘积在AFDR组2周时无变化,4周时均较对照组明显升高(P<0.05),8周时两组无明显差异；碱性磷酸酶在AFDR组2周时明显升高(P<0.05),4周及8周时无差异；肌酐在8周时AFDR组与DW组对比显著降低(P<0.05)。
组织学观察骨缺损修复效果：IBRC+AFDR组优于IBRC+DW灌胃组,而HA组修复效果较前两者均差；碱性磷酸酶和Ⅰ型胶原免疫组化染色,以及BMP2原位杂交的图像分析表明,IBRC+AFDR组较后两组阳性表达明显。
颅骨极量骨缺损的实验中,IBRC+rhBMP2组修复效果最为理想,IBRC+AFDR组优于IBRC+DW组。
结论
1)AFDR在IBRC材料修复骨缺损的过程中,2周时提高血清碱性磷酸酶的水平,4周时可以提高血清钙、磷及钙磷乘积水平,从而对骨修复有促进作用。
2)IBRC材料结合AFDR修复骨缺损,能提高修复质量,缩短修复时间。
3)IBRC材料结合AFDR修复骨缺损,可以提高BMP2的阳性表达,促进修复材料的诱导成骨作用。
4)IBRC械料结合AFDR修复骨缺损,能够提高Coll-Ⅰ和ALP的阳性表达,有利于骨修复的骨基质形成和矿化。
5)本实验观察IBRC材料修复骨缺损主要是软骨内成骨模式。
6)IBRC材料可用于修复大鼠极量骨缺损,结合AFDR能明显提高修复速度及质量,但不及IBRC材料复合BMP2的修复速度及质量。
7)IBRC材料结合AFDR修复骨缺损,其效果不及IBRC材料结合BMP2的修复效果,验证了rhBMP2的有效,同时对中药促进骨修复提出挑战,传统中医治疗骨折以复方为主,深入开展复方的研究成为必要。
8)AFDR按人鼠换算剂量连续灌胃8周大鼠未见肝肾毒性,并可能有增加肾小球滤过功能的作用。
Introduction
Based on the fact that the accidents rate of bone nonunions, bone defects are approximately 5-10%, the repair of critical-sized bone defects is a challeging problem around the world.The autogenous bone graft, which is with limited resource, has been deemed as the golden criterion in bone repair for its osteoconductivity and osteoinductivity. And it is known to all that the xenogeneic bone or allogenic bone would cause immunological rejections which limite their application in bone repair. The Department of Materials Science and Engineering of Tsinghua University has designed a nanoporous material according to biomimetic method. Based on the template of tropocollagen molecule, a bone-like nano-hydroxyapatite/collagen (nHAC) composite was synthesized biomimetically by the orderly deposition of hydroxyapatite mineral with low crystallinity, hierarchical structure. It has a good biocompatibility and biodegradability. Recentaly, we have developed an injectable bone regeneration composite (IBRC) by using a calcium alginate hydrogel as matirx vehicle. It has good potential application in future for it not only maintained the natural bone-like characterics of nHAC but also a good injectability.
The Chinese medicine drynaria fortunei (DR) has many therapeutic functions, such as strenthening tendons and bones, invigorating the kindey and spermium, as well as pain and bruises relieving. It has been vertified by the contemporary pharmacological studies that assemble flavone of rhizome drynaria (AFDR) can promote fracture repair, and improve microcirulation, treat the osteoporosis, as well as certain anti-inflamatory and analgesic effects. The bone growth factor BMP2, which plays a crucial role in fracture healing and bone remodling, has been used for fracture healing and bone defect treatment successfully.
Combine the IBRC with AFDR or BMP2 in the application of bone defect repair may reduce the trauma response and improve the repair quality. It has been an exploration mode of combination of Chinese and western medicine for bone defects treatment.
Objective
This article discusses the mode of combing Chinese and western medicine in bone defects repair, which is based on observasion and comparative studies of the effect by the application of IBRC in combination with AFDR or BMP2.
Methods
1) andomized controlled trials. Arranged 80 SD rats into three groups, and made a control test with a 5mm diameter wide calvarial defects on the right side and the left skull defects as comparison. Group A (IBRC+AFDR, n=30):Implanted IBRC to the bone skull defect and fed the rats with AFDR for 2-8 weeks. Groud B(IBRC+DW,n=30):Implanted IBRC to the skull defect on the right, fed the rats with DW for 2-8 weeks. Group C (IBRC-HA+DW, n=20):Implanted IBRC-HA to the skull defect on the right, fed the rats with DW for 2-8 weeks. Executed the rats on week 2,4 and 8, and drawn the blood in the abdominal arteriae aorta for the test of serum calcium, phosphorus, alkaline phosphatase, creatinine, and alanine aminotransferase. Inbedded the skull specimens with paraffin and sliced, and observed the changes of the tissues after HE and Masson stained. After alkaline phosphatase and type I collagen immunohistochemical staining, as well as BMP2 in situ hybridization, we observed the microsope image and analyzed the OD value, and the index was analyzed by statistic treatment.
2) Randomized controlled trials:40 rats were randomly arranged into 4 groups and made critical-sized calvairal defects with 8mm diameter wide. Group A (IBRC+AFDR, n=12): Implanted IBRC on the defects, and fed the rats with AFDR for 2-8 weeks; Group B (IBRC+DW, n=12):Implanted IBRC and fed the rats for 2-8 weeks; Group C (IBRC+rhBMP2.n=13):Implanted the combination of IBRC and rhBMP2 (15ug/ml); Group D (n=3); No implant. Removed calvairal bones from the rats at week 2,4,6,8,12 and examined them with Micro-CT and HE stainning for repair effect evaluation
Results
In the experiment of skull bone defects repair by the combination of IBRC and AFDR, there wasn't any change of the value of serum calcium, phosphorus or calcium phoshate on week 2. But a significant raise (P< 0.05) was found in the control group on week 4. The result remained the same on week 4 and 8, but the creatinine has siginficantly decreased (P<0.05) in the AFDR group in the comparision with DW group on week 8. The repair effects in histological observation:Repair effect of the IBRC+AFDR group is better than the IBRC+DW group's, HA group is the worst one of the three. It is showed in the type I collagen immunohistochemical stained and the analysis of the BMP2 ISH graphics that the positive expression of the IBRC+AFDR group is much obvious than the other two. In the experiment of critical-sized calvarial defects, the repair effect of IBRC+rhBMP2 was the best, and the IBRC+AFDR group is better than the IBRC+DW group. Conclusion
1) In the experiment of combining AFDR and IBRC for calvarial defect repair, alkaline phosphatase has significantly improved in two weeks, and the amount of calcium in blood, phosphorus and calcium phosphate product level is higher in four weeks. All of these have proved that AFDR has the effect on promoting bone repair.
2) The repair time can be shorten and the quality can be improved when defects are repaired by IBRC in the combination with AFDR.
3) The positive expression of BMP2 can be improved and sequentially boost the osteoinductivity of the material when defects are repaired by the combination of IBRC and AFDR.
4) It can improve the positive expression of Coll-I and ALP, facilitating the formation and mineralization of bone matrix when the bone defects are repaired by the combination of IBRC and AFDR.
5) The observation method of this experiment of IBRC bone defects repair is based on the endochondral bone formation.
6) IBRC can be used in the critical-sized bone defect on rats, and with the combination of AFDR can distinctly raise the speed and quality of repairment, however, its effect is not as good as the combination of IBRC and BMP2.
7) The effect of IBRC and AFDR combination for bone defects are not as good as the combination of IBRC and BMP2, which also verify the effetivity of rhBMP2. At the same time, it also challenges the effectiveness of Chinese medicine for promoting bone repair. Since the traditional Chinese medicine treatment has been focused on compoud medicine, it is necessary to carry out an in-depth research on compound medicine.
8) There is no hepatorenal toxicity symdrome after feeding the rats with conversed dose of AFDR for 8 weeks. And it may increase the function of glomerular filtration.
引文
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