中药调控种子细胞复合生物材料修复骨缺损的实验研究
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摘要
目的:各种原因引起的大段骨缺损很难愈合,随着组织工程学理论与技术的发展,将具有成骨能力的种子细胞复合可降解生物材料构建成具有生命活力的组织工程人工骨移植到骨缺损处,发挥骨诱导、骨传导甚至直接成骨作用,成为这一棘手问题的新的解决方案。
中医认为,骨折后如肾生养精髓不足,则无以养骨而难以愈合,中医治疗骨折促进骨折愈合,无不从补肾壮骨入手。如果能够证实补肾壮骨中药具有促进骨髓基质细胞(mesenchymai stem cells,MSC)增殖、分化或两者兼有的功能,就能从骨生长的细胞学层面上阐述这类药物的具体作用机制和靶点。通过本研究希望解答以下问题:1.中药骨碎补促进骨折愈合(接骨续筋)的细胞学机制,即是否是骨髓基质细胞—成骨细胞—骨生长—骨愈合的过程,为印证髓满骨充理论提供依据;2.中药参与培养的骨髓基质细胞特性维持的能力,即是否具有稳定增殖和传代的能力;3.中药参与培养的骨髓基质细胞能否分化为成骨细胞,即骨髓基质细胞的多潜能分化和定向分化能力的维持;4.骨碎补提取物在细胞培养过程中的量效关系;5.这种特定条件下培养的种子细胞与生物基质材料的复合是怎样的,即能否制成具有生命活性的组织工程人工骨;6.该人工骨植入骨缺损处后的成骨效能如何。
一、骨碎补提取液对兔骨髓基质细胞增殖的影响
方法:
1.分组:实验共分五组:①空白对照组(标准培养液);②诱导对照组(成骨条件诱导培养液);③中药Ⅰ组(含骨碎补20mg/ml的培养液);④中药Ⅱ组(含骨碎补2mg/ml的培养液);⑤中药Ⅲ组(含骨碎补0.2mg/ml的培养液)。将从2月龄纯种新西兰大白兔提取的骨髓悬液分离漂洗出骨髓基质细胞,加入以上培养液中培养。
结果:
1.细胞生长状况
中药Ⅰ组细胞贴壁数量少,生长缓慢,细胞形态细长,胞质较少,细胞萎缩脱落。空白组细胞基本呈长梭形,其他各组细胞逐渐增多,伸出伪足,呈梭形、星形、多角形等不同形状。胞浆内有较多小颗粒,核仁明显,一周左右细胞基本长满并呈集落存在。
2.细胞活力及生长曲线的测定结果
诱导组与空白组未见显著性差异,中低浓度提取液有促进骨髓基质细胞增殖作用,而低浓度较中浓度作用更强,高浓度提取液抑制骨髓基质细胞的增殖,还能导致其凋亡。
3.细胞分裂指数的测定结果
测量分裂指数记录并绘制成曲线,将贴片的细胞行HE染色,中药Ⅰ组明显抑制细胞生长,曲线低平。
结论中低浓度骨碎补提取液促进骨髓基质细胞增殖,而高浓度提取液抑制细胞增殖,甚至引起凋亡,条件诱导培养液对细胞增殖的影响不明显。
二、骨碎补提取液对兔骨髓基质细胞分化的影响
方法骨髓基质细胞培养同上。
结果
1.倒置相差显微镜观察
中药Ⅰ组细胞脱壁漂浮,数量明显减少,细胞边缘粗糙皱缩,核固缩,裂解。中药Ⅱ、Ⅲ组、诱导对照组细胞呈梭形、多角形,融合为单层后继续增殖成复层生长,形成多个岛状致密细胞群,中间细胞排列紧密,渐被含有高折射的空泡和深色颗粒的基质包埋形成白色钙化结节。空白对照组,融合为单层后细胞发生接触抑制,最终停止分裂增殖。
2.扫描电镜观察
中药Ⅱ、Ⅲ两组细胞多为单层细胞结构,细胞为梭形或多角形,带多个突起,数目不等,长短粗细各异,可见细长微丝形成细胞间的连接,细胞表面及细胞间可见钙盐结晶沉积。
3.碱性磷酸酶染色
中药Ⅱ、Ⅲ组和诱导组染色阳性率分别为35.6%、31.4%和72.4%,空白组仅为3.6%。诱导组与空白对照组及中药各组之间有极显著性差异(P＜0.001),中药Ⅱ、Ⅲ组与空白组之间也有极显著性差异(P＜0.001),中药两组之间无明显差异(P＞0.05)。
4.Ⅰ型胶原免疫组织化学染色
各组Ⅰ型胶原在细胞内和基质均有阳性染色,诱导组与空白组有极显著性差异(P＜0.001),中药Ⅱ、Ⅲ组虽明显低于诱导组(P＜0.05),但都明显或显著高于空白组(分别为P＜0.05,P＜0.01),而中药两组之间无明显差异(P＞0.05)。
5.钙结节染色
中药Ⅱ、Ⅲ组在每个标本上均可见到金黄色钙结节,低倍视野平均为1～2个,少于诱导组的平均3～4个,而空白组仅偶见钙结节。
结论高浓度骨碎补醇提液引起了BMSCs的死亡或凋亡,不利于BMSCs向成骨细胞分化。中、低浓度骨碎补醇提液具有促进BMSCs向成骨细胞分化的作用。
三、成骨细胞与多孔支架材料的生物相容性研究
方法传代增殖的兔成骨细胞制成5×10~4/ml浓度的细胞悬液,置入已有支架材料的培养板中,使细胞悬液充分渗入支架材料孔隙内联合培养,8小时后加入条件培养液,其后隔日更换培养液,连续培养。
结果
1.倒置相差显微镜和扫描电镜观察
培养7天,紧密贴附于支架表面的成骨细胞分化增殖连接成片,细胞多为梭形、三角形向周围伸出伪足,分泌的基质包绕于细胞周围,充填于支架材料周边孔隙中,可见钙结节。
2.碱性磷酸酶染色
培养2周钙钴法在成骨细胞的胞浆中呈现浅棕色至棕黑色颗粒。
3.成骨细胞在支架材料上的成活和增殖能力
测定值为0.221±0.017,对照组为0.140±0.015,(P＜0.01),表明支架有利于细胞的附着和增殖。
结论成骨细胞在生物支架上分布均匀,粘附力强,并能很好地分化增殖及分泌新的骨基质,生物活性玻璃能够为种子细胞提供良好的载体和生存环境,二者生物相容性好。
四、组织工程人工骨修复兔骨缺损的实验研究
方法
将20只新西兰大白兔分为4组,在其桡骨缺损处分别植入组织工程人工骨(Ⅰ组)、生物活性玻璃(Ⅱ组)、自体髂骨(Ⅲ组),每组6只,另2只保留骨缺损作为空白对照组(Ⅳ组)。缝合肌层皮肤包扎。术后每天肌注青霉素,连续三天,常规饲养。
结果
1.大体观察
12周Ⅰ组骨缺损区修复良好,与正常骨无差异;Ⅱ组骨缺损初步修复,有少量连续骨痂通过;Ⅲ组骨缺损完全修复,有连续性骨痂通过骨折端,质地坚硬;Ⅳ组假关节形成。
2.X线观察
12周ⅠⅢ组骨缺损完全修复,可见皮质骨及髓腔形成,皮质骨厚度接近正常骨;Ⅱ组有少量连续性骨痂通过断端;Ⅳ组骨端硬化分离,形成假关节。
3.组织学观察
12周Ⅰ组移植颗粒间骨桥和小梁状结构已建立,髓腔进一步开通,逐步恢复自然骨的特点。Ⅱ组颗粒周围形成少量新骨,颗粒间骨桥连接不紧密,骨小梁也不明显。
4.扫描电镜
12周Ⅲ组已形成隆起密集的结合,Ⅰ组分界模糊过渡自然,结合部密集。Ⅱ组也有明显的修复,但不紧密。Ⅳ组全部为纤维组织,中央有裂隙。
5.生物力学测试结果显示:
Ⅲ组桡骨扭转强度最高,与Ⅰ组无明显差异(P＞0.05)。Ⅱ组及Ⅳ组修复效果较差,有明显差异。
结论成骨细胞与生物活性玻璃复合的组织工程骨的成骨能力与自体髂骨的修复结果相似,明显优于其它组,该人工骨能很好地修复骨缺损。
Aim:A desirable clinical orthopedic requirement is the healing of segmental bone defect.with the development of technology and theory,a promising approach is the development of tissue engineering artificial bone that compound seed cell and absorbed biomaterial play the role of inducement,conduction even directly formatted bone.
According to TCM,bone union after fracture get delayed if the kidney is deficient at nourishing bone marrow thus the treatment of promoting the union of fracture always start with invigorating the kidney and strengthen the bone.If it could be verified that Chinese medicine of invigorating the kidney and strengthen the bone have the function of enhancing the marrow stoma cells(MSC) to proliferate or differentiate or both,so the specific mechanism and target could be explained from the point of cytology about bone growing.This research is expected to answer the following questions:1.if the cytological mechanism of drynaria(Chinese herb Gusuibu) on promoting the union of fracture is the procedure from MSC to osteoblast to bone growing then to the union of fracture.Thus some theoretical support could be found to confirm the theory of "if the bone marrow get well nourished then the bone get strengthened."2.If MSC interfered with Chinese medicine have the ability of stabilized proliferation and passage.3.If MSC interfered with Chinese medicine can differentiate to osteoblast.4.If there is dose-effect relationship during the cell culture interfered with extraction of drynaria.5.Is there a way to combine this specific cultured seed cells with biological matrix materials to make some organic tissue engineering artificial bone? 6.How is the efficacy of such artificial bone when it is implanted into bone defect area?
Part 1:the influence to proliferation of bone stromal cells with Gu suibu extract
Methods:
1.grouping:①blank controlled groups(standarding culture)②induce controlled groups(condition culture fluid)③herbⅠmedia(gu suibu 20mg/ml)④herbⅡ(contain gu suibu 2mg/ml)⑤herbⅢ(contain gu suibu 0.2mg/ml).Bone marrow stromal cell were established from adult new Zealand white rabbits and culture in above culture solution respectively.
Results:
1.Situation of cell growth
Adherent cell of Chinese medicine groupⅠhad a small number,Slow growths, morphology slender,less cytoplasm and cell shrinkage loss.Most cells of blank group were long spindle,other cells in each group gradually increased,extended pseudopodia,were fusiform,stellate,polygonal and etc.There were more small intracytoplasmic particles,nucleolus obviously,and cell basicly covered and there was colony a week or so.
2.Cell viability and growth curve of the measured results
There is no significant difference between induction groups with blank group. The low and middle concentration of extract can promote the proliferation of bone marrow stromal cell and the low concentration had a greater role than the middle concentration,while high concentration of extract can inhibit the proliferation of bone marrow stromal cells and lead to apoptosis.
3.Cell viability and growth curve of the measured results
By measurement mitotic index records,mapping it into a curve,and observing the HE staining of the cell line,we drew the conclusion that Chinese medicine group significantly inhibited cell growth,and had the low curve.
Conclusion:The low and middle concentration of extract can promote the proliferation of bone marrow stromal cell,while high concentration of extract can inhibited the proliferation of bone marrow stromal cells and lead to apoptosis.the influence of conditional media to cell proliferation was unobvious.
Part 2、the influence to differentiation of bone stromal cells with Gusuibu extract
Methods:bone marrow stromal cells culture same above.
Results:
1.Inverted phase contrast microscope and scanning electron microscopy
Cells of ChineseⅠgroup floated off the wall,the number of cells significantly reduced,cells edge became rough and shrinked,and nuclear condensation,cracking. Cells oftraditi0nal Chinese MedicineⅡ,Ⅲgroups and the induction control group were uniform,polygonal,and kept growing into complex layer after integration into a single layer and formatted several dense cells groups like islands,in which the closely middle cell were gradually embedded by empty vesicles and dark particles and turned into the matrix of white calcified nodule formation.Blank control group,contacted inhibition after the integration of the single layer,and ultimately to stop proliferation.
2.Scanning electron microscope
Most cells of Chinese MedicineⅡ,Ⅲgroups were single-cell structure, spindle or polygonal cells with multiple processes,and different in the number of ranges,the length and the thickness.We can see the formation of long filaments between connected cells,the cell surface and cell gap can be seen the deposition of calcium salt crystallization.
3.Alkaline phosphatase staining
The positive rate of staining of cells of traditional Chinese MedicineⅡ,Ⅲgroups and the induction control group was 35.6%,31.4%and 72.4%,however blank group was only 3.6%.Induced group,blank group and each Chinese medicine group had a very significant difference(P＜0.001).Chinese MedicineⅡ,Ⅲgroups and blank group had also a very significant difference(P＜0.001).There is no significant difference between the two Chinese medicine groups(P＞0.05).
4.TypeⅠcollagen immunohistochemistry
Each group of typeⅠcollagen both in the cells and matrix were positive staining,induced group and the blank group was significant difference(P＜0.001). Chinese MedicineⅡ,Ⅲgroup,although obviously not as good as induced group (P＜0.05),but significantly higher than the blank group(P＜0.05,P＜0.01),while there is no significant difference between the two Chinese medicine groups(P＞0.05).
5.Calcium nodules staining
Specimens of Chinese MedicineⅡ,Ⅲgroups can be seen on the golden calcium nodules,low-fold vision of an average of 1～2,tess than the average 3 to 4 of inducted group,and the blank group can be seen nodules of calcium only Occasional
Conclusion:high concentration of extract can inhibited the proliferation of bone marrow stromal cells and leads to apoptosis or death,went against to differentiate to osteoblast.The low and middle concentration of extract can promote the differentiation of bone marrow stromal cell,
Part 3 the biocompatibility study of osteoblast with multiple-hole scaffold
Method:The passaged rabbit osteoblasts cell suspension were adjusted to density of 5×10~4/ml,and put in Scaffold materials in the culture plate,so that the cell suspension can fully infiltrate into porous scaffolds with co-culture.Added conditioned medium after 8 hours,replaced the culture medium the next day,and continuously cultured.
Results:
1.Inverted phase contrast microscope and scanning electron microscopy
After 7 days culture,osteoblasts closely attached to the stent surface differentiated and proliferated,and connected into a film,most cells were long spindle and triangle, extending pseudopodia,matrix enveloping the cells.Cells filled the pores around the stent material in which calcium nodules can be seen.
2.ALP(alkaline phosphatase) staining:
Osteoblasts cells can be seen in the cytoplasm of light brown to brownish-black particles after Ca-Co-culture 2 weeks.
3.Osteoblast survival and proliferation ability on scaffolds
Test value 0.221±0.017,contrast is 0.140±0.015,(P＜0.01),Showed:scaffold was propitious to the adhesion and proliferation of osteobtast.
Conclusion:Osteoblast cell distribution in biomaterial scaffold was uniform,strong adhesion,differentiation and secrete new matrix,bioglass that provide survive space and carrier as bone tissue engineering scaffolds have good biocompatibility with osteoblast.
Part 4 Experimental study of repairing bone defect with tissue engineering artificial bone,
Methods:24 new Zealand white rabbits were randomly divided into 4 groups, making 1.5 cm bone defect models in middle segment of radius and implant different material,ⅠArtificial bone,Ⅱbioglass、Ⅲauto iliac graft,Ⅳblank contrast。Fixed with muscle and skin,Penicillin im everyday after operation.Routine feed.At 8,12 weeks after implantation,the effectiveness of bone formation was evaluated by means of gross,histological observation and scanning electronic microscope(SEM) examination and biomechanical analysis.
Results:
1.Gross observe:
12 weeks:bone defect region completely repaired was same to normal bone inⅠgroups,primary repairing little consecutive callus through inⅡgroups,a lot of consecutive callus through inⅢgroups,pseudoarthrosis formed inⅣgroups.
2.X-ray observes:
12weeks,completely renovated inⅠandⅢsubstantial compacted and marrow cavity formatted,thickness of substantial Compact closely to normal bone.little consecutive callus across breaking gapingⅡgroups,sclerosis and separated form pseudoarthrosis in defect region.
3.Histological observe:
12 weeks:bone bridge and girder structure has been established between grafts grains,marrow cavity dredged,restore characteristic of natural bone in groupⅠ. Little new bones formation,bone bridge link sparsely.Trabeculae inconspicuous. Between graft grains.
4.Scanning electron microscope:
12 weeks,dense link have shaped in groupsⅢ,obvious repairing has been seen, link wasn't thick in groupsⅡ,all of region is fiber tissue,and crack has been seen on middle in groupsⅣ.
5.Biomechanical test:
Torsion intensity of GroupⅢwas same as that groupⅠ,higher than any other two groups.
Results:repairing ability of tissue-engineering artificial bone of osteoblast and bioglass almost same that auto iliac-implant,excel that any other tow groups.And this artificial bone can achieve the repairing bone defect.
中医认为,骨折后如肾生养精髓不足,则无以养骨而难以愈合,中医治疗骨折促进骨折愈合,无不从补肾壮骨入手。如果能够证实补肾壮骨中药具有促进骨髓基质细胞(mesenchymai stem cells,MSC)增殖、分化或两者兼有的功能,就能从骨生长的细胞学层面上阐述这类药物的具体作用机制和靶点。通过本研究希望解答以下问题:1.中药骨碎补促进骨折愈合(接骨续筋)的细胞学机制,即是否是骨髓基质细胞—成骨细胞—骨生长—骨愈合的过程,为印证髓满骨充理论提供依据;2.中药参与培养的骨髓基质细胞特性维持的能力,即是否具有稳定增殖和传代的能力;3.中药参与培养的骨髓基质细胞能否分化为成骨细胞,即骨髓基质细胞的多潜能分化和定向分化能力的维持;4.骨碎补提取物在细胞培养过程中的量效关系;5.这种特定条件下培养的种子细胞与生物基质材料的复合是怎样的,即能否制成具有生命活性的组织工程人工骨;6.该人工骨植入骨缺损处后的成骨效能如何。
一、骨碎补提取液对兔骨髓基质细胞增殖的影响
方法:
1.分组:实验共分五组:①空白对照组(标准培养液);②诱导对照组(成骨条件诱导培养液);③中药Ⅰ组(含骨碎补20mg/ml的培养液);④中药Ⅱ组(含骨碎补2mg/ml的培养液);⑤中药Ⅲ组(含骨碎补0.2mg/ml的培养液)。将从2月龄纯种新西兰大白兔提取的骨髓悬液分离漂洗出骨髓基质细胞,加入以上培养液中培养。
结果:
1.细胞生长状况
中药Ⅰ组细胞贴壁数量少,生长缓慢,细胞形态细长,胞质较少,细胞萎缩脱落。空白组细胞基本呈长梭形,其他各组细胞逐渐增多,伸出伪足,呈梭形、星形、多角形等不同形状。胞浆内有较多小颗粒,核仁明显,一周左右细胞基本长满并呈集落存在。
2.细胞活力及生长曲线的测定结果
诱导组与空白组未见显著性差异,中低浓度提取液有促进骨髓基质细胞增殖作用,而低浓度较中浓度作用更强,高浓度提取液抑制骨髓基质细胞的增殖,还能导致其凋亡。
3.细胞分裂指数的测定结果
测量分裂指数记录并绘制成曲线,将贴片的细胞行HE染色,中药Ⅰ组明显抑制细胞生长,曲线低平。
结论中低浓度骨碎补提取液促进骨髓基质细胞增殖,而高浓度提取液抑制细胞增殖,甚至引起凋亡,条件诱导培养液对细胞增殖的影响不明显。
二、骨碎补提取液对兔骨髓基质细胞分化的影响
方法骨髓基质细胞培养同上。
结果
1.倒置相差显微镜观察
中药Ⅰ组细胞脱壁漂浮,数量明显减少,细胞边缘粗糙皱缩,核固缩,裂解。中药Ⅱ、Ⅲ组、诱导对照组细胞呈梭形、多角形,融合为单层后继续增殖成复层生长,形成多个岛状致密细胞群,中间细胞排列紧密,渐被含有高折射的空泡和深色颗粒的基质包埋形成白色钙化结节。空白对照组,融合为单层后细胞发生接触抑制,最终停止分裂增殖。
2.扫描电镜观察
中药Ⅱ、Ⅲ两组细胞多为单层细胞结构,细胞为梭形或多角形,带多个突起,数目不等,长短粗细各异,可见细长微丝形成细胞间的连接,细胞表面及细胞间可见钙盐结晶沉积。
3.碱性磷酸酶染色
中药Ⅱ、Ⅲ组和诱导组染色阳性率分别为35.6%、31.4%和72.4%,空白组仅为3.6%。诱导组与空白对照组及中药各组之间有极显著性差异(P＜0.001),中药Ⅱ、Ⅲ组与空白组之间也有极显著性差异(P＜0.001),中药两组之间无明显差异(P＞0.05)。
4.Ⅰ型胶原免疫组织化学染色
各组Ⅰ型胶原在细胞内和基质均有阳性染色,诱导组与空白组有极显著性差异(P＜0.001),中药Ⅱ、Ⅲ组虽明显低于诱导组(P＜0.05),但都明显或显著高于空白组(分别为P＜0.05,P＜0.01),而中药两组之间无明显差异(P＞0.05)。
5.钙结节染色
中药Ⅱ、Ⅲ组在每个标本上均可见到金黄色钙结节,低倍视野平均为1～2个,少于诱导组的平均3～4个,而空白组仅偶见钙结节。
结论高浓度骨碎补醇提液引起了BMSCs的死亡或凋亡,不利于BMSCs向成骨细胞分化。中、低浓度骨碎补醇提液具有促进BMSCs向成骨细胞分化的作用。
三、成骨细胞与多孔支架材料的生物相容性研究
方法传代增殖的兔成骨细胞制成5×10~4/ml浓度的细胞悬液,置入已有支架材料的培养板中,使细胞悬液充分渗入支架材料孔隙内联合培养,8小时后加入条件培养液,其后隔日更换培养液,连续培养。
结果
1.倒置相差显微镜和扫描电镜观察
培养7天,紧密贴附于支架表面的成骨细胞分化增殖连接成片,细胞多为梭形、三角形向周围伸出伪足,分泌的基质包绕于细胞周围,充填于支架材料周边孔隙中,可见钙结节。
2.碱性磷酸酶染色
培养2周钙钴法在成骨细胞的胞浆中呈现浅棕色至棕黑色颗粒。
3.成骨细胞在支架材料上的成活和增殖能力
测定值为0.221±0.017,对照组为0.140±0.015,(P＜0.01),表明支架有利于细胞的附着和增殖。
结论成骨细胞在生物支架上分布均匀,粘附力强,并能很好地分化增殖及分泌新的骨基质,生物活性玻璃能够为种子细胞提供良好的载体和生存环境,二者生物相容性好。
四、组织工程人工骨修复兔骨缺损的实验研究
方法
将20只新西兰大白兔分为4组,在其桡骨缺损处分别植入组织工程人工骨(Ⅰ组)、生物活性玻璃(Ⅱ组)、自体髂骨(Ⅲ组),每组6只,另2只保留骨缺损作为空白对照组(Ⅳ组)。缝合肌层皮肤包扎。术后每天肌注青霉素,连续三天,常规饲养。
结果
1.大体观察
12周Ⅰ组骨缺损区修复良好,与正常骨无差异;Ⅱ组骨缺损初步修复,有少量连续骨痂通过;Ⅲ组骨缺损完全修复,有连续性骨痂通过骨折端,质地坚硬;Ⅳ组假关节形成。
2.X线观察
12周ⅠⅢ组骨缺损完全修复,可见皮质骨及髓腔形成,皮质骨厚度接近正常骨;Ⅱ组有少量连续性骨痂通过断端;Ⅳ组骨端硬化分离,形成假关节。
3.组织学观察
12周Ⅰ组移植颗粒间骨桥和小梁状结构已建立,髓腔进一步开通,逐步恢复自然骨的特点。Ⅱ组颗粒周围形成少量新骨,颗粒间骨桥连接不紧密,骨小梁也不明显。
4.扫描电镜
12周Ⅲ组已形成隆起密集的结合,Ⅰ组分界模糊过渡自然,结合部密集。Ⅱ组也有明显的修复,但不紧密。Ⅳ组全部为纤维组织,中央有裂隙。
5.生物力学测试结果显示:
Ⅲ组桡骨扭转强度最高,与Ⅰ组无明显差异(P＞0.05)。Ⅱ组及Ⅳ组修复效果较差,有明显差异。
结论成骨细胞与生物活性玻璃复合的组织工程骨的成骨能力与自体髂骨的修复结果相似,明显优于其它组,该人工骨能很好地修复骨缺损。
Aim:A desirable clinical orthopedic requirement is the healing of segmental bone defect.with the development of technology and theory,a promising approach is the development of tissue engineering artificial bone that compound seed cell and absorbed biomaterial play the role of inducement,conduction even directly formatted bone.
According to TCM,bone union after fracture get delayed if the kidney is deficient at nourishing bone marrow thus the treatment of promoting the union of fracture always start with invigorating the kidney and strengthen the bone.If it could be verified that Chinese medicine of invigorating the kidney and strengthen the bone have the function of enhancing the marrow stoma cells(MSC) to proliferate or differentiate or both,so the specific mechanism and target could be explained from the point of cytology about bone growing.This research is expected to answer the following questions:1.if the cytological mechanism of drynaria(Chinese herb Gusuibu) on promoting the union of fracture is the procedure from MSC to osteoblast to bone growing then to the union of fracture.Thus some theoretical support could be found to confirm the theory of "if the bone marrow get well nourished then the bone get strengthened."2.If MSC interfered with Chinese medicine have the ability of stabilized proliferation and passage.3.If MSC interfered with Chinese medicine can differentiate to osteoblast.4.If there is dose-effect relationship during the cell culture interfered with extraction of drynaria.5.Is there a way to combine this specific cultured seed cells with biological matrix materials to make some organic tissue engineering artificial bone? 6.How is the efficacy of such artificial bone when it is implanted into bone defect area?
Part 1:the influence to proliferation of bone stromal cells with Gu suibu extract
Methods:
1.grouping:①blank controlled groups(standarding culture)②induce controlled groups(condition culture fluid)③herbⅠmedia(gu suibu 20mg/ml)④herbⅡ(contain gu suibu 2mg/ml)⑤herbⅢ(contain gu suibu 0.2mg/ml).Bone marrow stromal cell were established from adult new Zealand white rabbits and culture in above culture solution respectively.
Results:
1.Situation of cell growth
Adherent cell of Chinese medicine groupⅠhad a small number,Slow growths, morphology slender,less cytoplasm and cell shrinkage loss.Most cells of blank group were long spindle,other cells in each group gradually increased,extended pseudopodia,were fusiform,stellate,polygonal and etc.There were more small intracytoplasmic particles,nucleolus obviously,and cell basicly covered and there was colony a week or so.
2.Cell viability and growth curve of the measured results
There is no significant difference between induction groups with blank group. The low and middle concentration of extract can promote the proliferation of bone marrow stromal cell and the low concentration had a greater role than the middle concentration,while high concentration of extract can inhibit the proliferation of bone marrow stromal cells and lead to apoptosis.
3.Cell viability and growth curve of the measured results
By measurement mitotic index records,mapping it into a curve,and observing the HE staining of the cell line,we drew the conclusion that Chinese medicine group significantly inhibited cell growth,and had the low curve.
Conclusion:The low and middle concentration of extract can promote the proliferation of bone marrow stromal cell,while high concentration of extract can inhibited the proliferation of bone marrow stromal cells and lead to apoptosis.the influence of conditional media to cell proliferation was unobvious.
Part 2、the influence to differentiation of bone stromal cells with Gusuibu extract
Methods:bone marrow stromal cells culture same above.
Results:
1.Inverted phase contrast microscope and scanning electron microscopy
Cells of ChineseⅠgroup floated off the wall,the number of cells significantly reduced,cells edge became rough and shrinked,and nuclear condensation,cracking. Cells oftraditi0nal Chinese MedicineⅡ,Ⅲgroups and the induction control group were uniform,polygonal,and kept growing into complex layer after integration into a single layer and formatted several dense cells groups like islands,in which the closely middle cell were gradually embedded by empty vesicles and dark particles and turned into the matrix of white calcified nodule formation.Blank control group,contacted inhibition after the integration of the single layer,and ultimately to stop proliferation.
2.Scanning electron microscope
Most cells of Chinese MedicineⅡ,Ⅲgroups were single-cell structure, spindle or polygonal cells with multiple processes,and different in the number of ranges,the length and the thickness.We can see the formation of long filaments between connected cells,the cell surface and cell gap can be seen the deposition of calcium salt crystallization.
3.Alkaline phosphatase staining
The positive rate of staining of cells of traditional Chinese MedicineⅡ,Ⅲgroups and the induction control group was 35.6%,31.4%and 72.4%,however blank group was only 3.6%.Induced group,blank group and each Chinese medicine group had a very significant difference(P＜0.001).Chinese MedicineⅡ,Ⅲgroups and blank group had also a very significant difference(P＜0.001).There is no significant difference between the two Chinese medicine groups(P＞0.05).
4.TypeⅠcollagen immunohistochemistry
Each group of typeⅠcollagen both in the cells and matrix were positive staining,induced group and the blank group was significant difference(P＜0.001). Chinese MedicineⅡ,Ⅲgroup,although obviously not as good as induced group (P＜0.05),but significantly higher than the blank group(P＜0.05,P＜0.01),while there is no significant difference between the two Chinese medicine groups(P＞0.05).
5.Calcium nodules staining
Specimens of Chinese MedicineⅡ,Ⅲgroups can be seen on the golden calcium nodules,low-fold vision of an average of 1～2,tess than the average 3 to 4 of inducted group,and the blank group can be seen nodules of calcium only Occasional
Conclusion:high concentration of extract can inhibited the proliferation of bone marrow stromal cells and leads to apoptosis or death,went against to differentiate to osteoblast.The low and middle concentration of extract can promote the differentiation of bone marrow stromal cell,
Part 3 the biocompatibility study of osteoblast with multiple-hole scaffold
Method:The passaged rabbit osteoblasts cell suspension were adjusted to density of 5×10~4/ml,and put in Scaffold materials in the culture plate,so that the cell suspension can fully infiltrate into porous scaffolds with co-culture.Added conditioned medium after 8 hours,replaced the culture medium the next day,and continuously cultured.
Results:
1.Inverted phase contrast microscope and scanning electron microscopy
After 7 days culture,osteoblasts closely attached to the stent surface differentiated and proliferated,and connected into a film,most cells were long spindle and triangle, extending pseudopodia,matrix enveloping the cells.Cells filled the pores around the stent material in which calcium nodules can be seen.
2.ALP(alkaline phosphatase) staining:
Osteoblasts cells can be seen in the cytoplasm of light brown to brownish-black particles after Ca-Co-culture 2 weeks.
3.Osteoblast survival and proliferation ability on scaffolds
Test value 0.221±0.017,contrast is 0.140±0.015,(P＜0.01),Showed:scaffold was propitious to the adhesion and proliferation of osteobtast.
Conclusion:Osteoblast cell distribution in biomaterial scaffold was uniform,strong adhesion,differentiation and secrete new matrix,bioglass that provide survive space and carrier as bone tissue engineering scaffolds have good biocompatibility with osteoblast.
Part 4 Experimental study of repairing bone defect with tissue engineering artificial bone,
Methods:24 new Zealand white rabbits were randomly divided into 4 groups, making 1.5 cm bone defect models in middle segment of radius and implant different material,ⅠArtificial bone,Ⅱbioglass、Ⅲauto iliac graft,Ⅳblank contrast。Fixed with muscle and skin,Penicillin im everyday after operation.Routine feed.At 8,12 weeks after implantation,the effectiveness of bone formation was evaluated by means of gross,histological observation and scanning electronic microscope(SEM) examination and biomechanical analysis.
Results:
1.Gross observe:
12 weeks:bone defect region completely repaired was same to normal bone inⅠgroups,primary repairing little consecutive callus through inⅡgroups,a lot of consecutive callus through inⅢgroups,pseudoarthrosis formed inⅣgroups.
2.X-ray observes:
12weeks,completely renovated inⅠandⅢsubstantial compacted and marrow cavity formatted,thickness of substantial Compact closely to normal bone.little consecutive callus across breaking gapingⅡgroups,sclerosis and separated form pseudoarthrosis in defect region.
3.Histological observe:
12 weeks:bone bridge and girder structure has been established between grafts grains,marrow cavity dredged,restore characteristic of natural bone in groupⅠ. Little new bones formation,bone bridge link sparsely.Trabeculae inconspicuous. Between graft grains.
4.Scanning electron microscope:
12 weeks,dense link have shaped in groupsⅢ,obvious repairing has been seen, link wasn't thick in groupsⅡ,all of region is fiber tissue,and crack has been seen on middle in groupsⅣ.
5.Biomechanical test:
Torsion intensity of GroupⅢwas same as that groupⅠ,higher than any other two groups.
Results:repairing ability of tissue-engineering artificial bone of osteoblast and bioglass almost same that auto iliac-implant,excel that any other tow groups.And this artificial bone can achieve the repairing bone defect.
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