组织工程化骨修复人工关节周围骨缺损的实验研究
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摘要
研究背景:
Branemark于20世纪60年代提出了骨整合的概念,即在光镜水平下正常的改建骨和假体之间无软组织,假体与骨组织直接接触,其承受的负荷能通过直接接触持续不断地传递,并分散到骨组织中。骨整合被认为是人工关节置换术后的理想状态。
生物固定性假体要获得满意的远期效果,依赖于假体骨组织间的紧密接触、良好的骨长入和一定的骨生长时间。然而许多人工关节置换术或翻修术患者常合并有假体周围骨缺损,为恢复骨性结构并重建骨的稳定性,骨移植已成为修复骨缺损的一个必要手段。临床上根据骨缺损的大小、性质,可应用自体骨移植、异体骨移植治疗。但由于自体骨移植可提供的骨量有限,移植骨在形态、大小和数量上难以满足缺损处的要求,特别是巨大的或特殊部位骨缺损的修复,故其应用常常受到限制。1984年,Sloof等首先报告应用异体颗粒骨代替自体骨,打压植骨修复髋臼骨缺损。1993年,Ling等用同样的技术行股骨骨缺损的修复重建,从此该项技术被广泛的应用于人工关节置换术及翻修术中。但由于冻干的异体骨无成骨细胞,骨诱导活性低,新骨爬行替代及塑性的时间长,有移植骨吸收、塌陷、假体松动等并发症的可能。
上世纪八十年代兴起的骨组织工程学为骨缺损的修复提供了一个全新的思路和方法。组织工程涉及三个要素:即种子细胞、生物活性因子、支架材料。组织工程技术通过获取具有成骨活性的组织,经分离、培养、扩增,诱导为成骨细胞,并在体外与可吸收的支架材料复合回植于体内,依靠种子细胞的成骨作用及生物材料的降解吸收,最终形成骨组织。应用组织工程化骨修复长骨节段性骨缺损、颅骨缺损已有较多的报告,而应用组织工程化骨修复人工关节周围骨缺损,并观察其对假体骨界面骨整合的研究目前国内外尚未见文献报告。
目的:
1.建立骨髓间充质干细胞(MSCs)的分离、培养、骨向诱导方法;
2.体外骨髓MSCs与CHA(CHA)复合构建组织工程化骨,观察其修复植入体周围松质骨骨缺损的的效果。
3.观察组织工程化骨修复植入体周围骨缺损对植入体-骨界面骨整合的影响。
4.研究BMP促进组织工程化骨修复人工关节周围骨缺损,提高假体-骨界面骨接触的能力。
方法:
1.采用Ficoll(1.073g/ml)密度梯度离心加贴壁培养法分离兔骨髓MSCs,并培养、扩增;将第4代骨髓MSCs应用骨诱导培养液(10-8mol/L地塞米松+10mmol/L甘油磷酸钠+ 50mg/L维生素C)培养,骨向诱导。
2.诱导后的成骨细胞应用细胞形态学、细胞活力检测、细胞生长及增殖曲线、ALP活性检测、矿化结节茜素红染色、I型胶原免疫组化与ALP鉴定。
3.于兔左侧股骨髁制作一0.6×1.2cm的穿透性松质骨骨缺损,植入钛合金(Ti-6Al-4V)植入体于骨缺损的中央,在体外将同种异体骨髓MSCs与CHA复合构建组织工程化骨,修复植入体周围骨缺损,同法右侧骨缺损处仅植入CHA为对照组,术后4、8、12周行X射线检查、生物力学测试、放射性核素骨扫描、脱钙骨组织学HE染色,观察其骨缺损修复效果。
4.并于术后4、8、12周行X射线检查、剖面观察、植入体剪切强度测定、植入体表面扫描电镜、植入体骨界面能谱分析、不脱钙骨组织学Van Gieson苦味酸-品红染色及图像分析系统计算植入体骨接触率,观察组织工程化骨对植入体-骨界面骨整合的影响。
5.行犬人工全髋关节置换术,制作股骨骨缺损模型,随机分为三组,分别用rhBMP-2+自体骨髓MSCs +CHA、CHA+自体骨髓MSCs、CHA修复骨缺损,术后3个月处死动物,行X射线检查、生物力学测试、不脱钙骨组织学Van Gieson苦味酸-品红染色及图像分析系统分析计算假体骨接触率。
结果:
1.应用密度梯度离心结合贴壁培养法成功的分离、培养、扩增兔骨髓MSCs,培养3天后细胞的形态由圆形转为梭形;MSCs生长曲线分为生长潜伏期、对数生长期和平台期;MSCs表面标志物SH3呈阳性。
2.骨诱导后细胞培养5-8天,细胞形态逐渐变为多角形,呈现聚集生长。诱导后细胞ALP活性较未诱导组显著性增高。骨向诱导后第三代细胞ALP染色、I型胶原免疫组化染色均呈阳性,茜素红染色可见典型的矿化结节。
3.体外构建组织工程化骨修复植入体周围松质骨缺损结果:术后4、8、12周大体观察与剖面观察,实验组随时间的延长可见骨修复。X线表现为CHA颗粒吸收,被新生骨取代。生物力学结果示术后12周股骨髁最大压力载荷、载荷/应变比值均较对照组高,最大应变位移较对照组低(P<0.01)。放射性核素骨扫描实验组不同时间点ROI平均计数均较对照组高,有显著性差异(P<0.01)。实验组术后8周内ROI平均计数为快速增长期,术后8周开始进入缓慢增长期,12周达到峰值。组织学观察,实验组术后4周植入区内CHA颗粒之间有岛状新生骨组织;术后8周植入区内可见支架材料部分吸收,遗留空间被新生骨组织取代,骨缺损基本得到修复;术后12周可见明显编织骨、板层骨及骨小梁形成,CHA大部分吸收,但仍可见到未吸收的HA颗粒。对照组术后4、8周无新生骨,术后12周有少量的新生骨。
4.组织工程化骨对植入体-骨界面骨整合的影响:术后4、8、12周大体观察与剖面观察,见实验组随时间的延长骨缺损得到修复,植入体周围无间隙。X线检查见CHA颗粒吸收,被新生骨取代,植入体周围为新生骨,植入体位置良好,而对照组植入体周围主要为高密度的CHA颗粒。生物力学结果示不同时间点植入体骨界面剪切强度较对照组增高(P<0.01)。扫描电镜观察,可见实验组植入体表面孔隙内无定形物质随时间的变化而逐渐增加。能谱分析术后4周实验组Ca、P元素百分含量较对照组增高,8、12周Ca元素百分含量较对照组高(P<0.05),P元素百分含量与对照组比较无显著性差异;Ca/P比值随时间的延长逐渐增高,于术后12周达2.02(P<0.01)。不脱钙骨组织学观察,术后4周对照组植入体-骨界面为厚而疏松的软组织,实验组界面为结缔组织纤维膜,新生骨偶见;术后8周对照组植入体-骨界面为结缔组织纤维膜,厚薄不一,界面较松,周围可见少量新生骨组织,实验组界面有新骨沉积,植入体表面有部分点状骨接触;术后12周对照组植入体-骨界面为结缔组织纤维膜,界面有少量点状骨接触,实验组界面主要为骨组织,部分界面可见连续性骨接触,甚至呈环形包绕。术后4周对照组与实验组植入体骨接触率为0,术后8周、12周植入体骨接触率均高于对照组(P<0.01)。
5. rhBMP-2促进组织工程化骨修复假体周围骨缺损及假体-骨界面骨整合:术后3个月X线表现BMP组新骨生成明显,CHA颗粒完全吸收,假体稳定;细胞组有大量的新骨形成,但仍有CHA颗粒影,假体稳定;对照组新骨生成不明显,假体周围有透亮带。对照组、细胞组、BMP组假体骨界面剪切强度无显著性差异。不脱钙骨组织学观察,BMP组假体骨界面为骨组织充填,部分界面可见连续性骨接触,仅见少量纤维组织,新生骨以成熟的板层骨为主,与假体结合紧密。细胞组假体骨界面主要为骨组织充填,可见较多的骨接触,有部分纤维结缔组织。对照组假体骨界面为纤维结缔组织,边缘有少量编织骨,骨接触较少。图形分析计算对照组、细胞组、BMP组假体界面骨接触率(%)分别为15.47±8.05、38.78±19.40、67.23±18.36(P<0.01)。
结论:
1.本研究成功的分离、培养了兔骨髓MSCs,经骨向诱导后表达高水平的ALP活性与I型胶原,并能矿化细胞外基质,证实已分化为成熟的成骨细胞。
2.骨髓MSCs骨向诱导后与CHA复合可修复植入体周围松质骨骨缺损,移植物血管化、骨代谢活性前8周为快速发展阶段,8-12周进入缓慢发展渐趋成熟阶段。
3.骨髓MSCs骨向诱导后与CHA复合修复植入体周围骨缺损,促进新骨生成,改善植入体-骨界面骨整合的作用。
4.植入体骨界面能谱分析示随着时间的延长Ca、P元素百分比含量有增高趋势,Ca/P比值随着时间的延长逐渐接近正常骨组织,表明早期为新生骨形成阶段,逐渐转为骨改建与成熟阶段。
5. BMP促进组织工程化骨修复人工关节周围骨缺损,改善早期假体骨界面骨整合率。
Background:
In the 1960s, Branemark proposed the conception of osseointegration that there is no soft tissue between rebuilding bone and prosthesis under microscope, that bone contacts tightly with prosthesis, and that the loading taken on by prosthesis can pass and disperse constantly into bone by the bone-prosthesis contact. Osseointegration is regarded as the ideal state after arthroplasty.
Long-time satisfactory result of biological prosthesis replacement depends on the tight bone- prosthesis contact, favorable bone growth and bone growing time. Many patients who undergo an operation for prosthesis replacement or revision often suffer from bone defect around prosthesis. Bone grafts become a necessary method to recover the construction and stability of bone. According to the size and property of the bone defect, we usually adopt auto graft or allograft to treat bone defects in clinic. Due to the shape, size and quantity of bone, auto graft can not achieve the request of bone defect, especially of gigantic or special bone defect. The use of auto graft often is limited. Since Sloof etc firstly reported that bone defect of acetabular can be repaired by packing bone graft with variant granulation bone in 1984 and Ling etc used the same technique to treat bone defect of femur in 1993, the technique has been widely used in the operation for prosthesis replacement or revision. Because variant bone has no osteoblasts and much lower activity of bone induction, to achieve real creeping, substiution and remolding of new bone needs much more time. Allograft usually has many complications, such as absorption, collapse of grafting bone, and asceptic loosening of prosthesis.
In the 1980s, the emergence of tissue engineering provides us a brand-new thought and method in repairing bone defect. Tissue engineering has three important elements, namely seed cell, bioactivity factor, and scaffold material. Although there are many reports on repair of segmental defect of long bone and skull bone defect by use of tissue engineering bone, untill now any reports about repairing periprosthetic bone defect with tissue engineering bone and the effects of tissue engineering bone on osseointegration of the bone-impant interface have not been found in articles at home and abroad.
Aim:
1. To establish method of abstraction, cultivation and bone induction of mesenchymal stem cell (MSCs).
2. To construct the tissue engineering bone with MSCs and coral hydroxyapatite (CHA) in vitro, and observe the effects on repair of bone defect around implant.
3. To observe the effects of tissue engineering bone on osseointegration of the bone-implant interface.
4. To study the capability of BMP to boost tissue engineering bone to repair periprosthetic bone defect, promote bone growth around prosthesis and the osseointegration of the bone-prosthesis interface.
Method:
1. BMSCs were segregated by Ficol(l1.073g/ml)density gradient centrifugation and the adherent culture method, then cultivated and multiplied. The 4th generation MSCs were induced by bone induction culture fluid (10-8mol/L DEX+10mmol/L Sodium Glycerophosphate + 50mg/L Vitamin C).
2. Osteoblasts derived from BMSCs were verified by cell morphology, cell vigor detection, cell growth curve, ALP activity examination, alizarin Bordeaux dyeing of mineralization nodes , immunohistochemistry of collagen I and Gomori dyeing of ALP.
3. A penetrating transverse bone defect (0.6cm×1.2cm) was created in bilateral femoral condyles respectively in rabbits. The titanium alloy implants were implanted in centre of bone defects. Tissue engineering bone constructed with osteblasts induced from allogeneic induced BMSCs and CHA in vitro was embedded into defect around the impant in left as experimental group and only CHA was embedded into the defect in right as control group. The effects on repairing bone defect around implant were studied by X-ray examination, biomechanical examination, radionuclide bone imaging by ECT, decalcificated bone histology with HE dyeing.
4. The osseointegration of the bone-implant interface was observed by X-ray examination, section observation, push-out test of implant, scanning electron microscope, spectrum analysis, undecalcified bone histology with Van Gieson carbazotic acid–solferino dyeing, bone-to-implant contact calculated by datum from image analysis.
5. Total hip replacement with femoral bone defects was operated in right hip of dog. Animals were divided to three groups, called BMP group, cell group and CHA group The bone defects were imlpanted at random by the compound of rhBMP-2 and tissue engineering bone, tissue engineering bone and CHA respectively. Animals sacrificed at three months after operation. The effects of BMP on osseointegration of the bone-prosthesis interface and repairing bone defect around prosthesis were studied by X-ray examination, biomechanical examination, undecalcified bone histology with Van Gieson carbazotic acid–solferino dyeing as well as bone-to- prosthesis contact calculated by datum from image analysis.
Results:
1. BMSCs were segregated, cultivated and multiplied successfully by way of density gradient centrifugation and adherent culture. The round shape of cells turned to fusiform after culture for three days. Growth curve of BMSCs was divided into detention phase, logarithmic growth phase and platform phase. The surface market SH3 of BMSCs took on positive.
2. The shape of cells cultivated in bone induction culture fluid turned to polygon gradually. Cells began to growt in cluster after culture for 5-8 days. ALP activity was significantly higher in experimental group than that in control group(P<0.01). Both collegan I immunohistochemistry and ALP Gomori dyeing presented positive and alizarin Bordeaux dyeing of the 3th generation BMSCs after bone induction showed typical mineral nodus .
3. Cancelous bone defect was repaired by tissue engineering bone constructed in vitro. As time going by, bone repair was observed in experimental group by the way of general observation and section observation at 4th, 8th and 12th week postoperatively. Biomechanical results showed that both the maximum pressure load and ratio of load to straining in experimental group were significantly higher than that in control group; The maximum strain- displacement in experimental group was lower than that in control group(P<0.01). Radionuclide bone imaging showed that mean of ROI in experimental group increased significantly higher than that in control group at different time points. Mean of ROI increased at quicker epacme within 8 weeks and began to increase at slower epacme at 8th week and achieved peak amplitude at 12th week postoperately. Histology study demonstrated that little new bone around CHA granulation in experimental group was found at 4th week; Scaffold materials were absorbed partially and replaced by new bone tissue, and bone defects were repaired basically by some new bone which rebuild maturely in partial domain in experimental group at 8th week; The obvious woven bone, lamellar bone and bone trabecula with blood sinus were found, but unabsorbed CHA granulation still existed in experimental group at 12th week. Little new bone around CHA granulation in controll group was found at 8th and12th week.
4. The effects of tissue engneering bone on osseointegration of the bone-implant interface were observed. As time going by, the implants were embedded well by new bone in experimental group in general observation at 8th and 12th week postoperatively. X-ray showed that CHA granulations were absorbed and replaced by new bone and that the position of implant surrounded by new bone was good in experimental group; Gaps around implants in control group were found. Biomechanical examination showed that the shear-strength of bone-implant intreface in experimental group was significantly higher than that in control group at different time points. Scanning electron microscope notified that amount of amorphous material on pores of the implant surface increased gradually with time going by in experimental group. The percent content of calcium and phosphate was significantly higher in experimental group than that in control group at 4th week postoperatively; The percent content of calcium was significantely higher in experimental group than that in control group, but the percent content of phosphate increased unsignificantely at 8th, 12th week; The ratio calcium of and phosphate increased gradually with time going by and reached to 2.02 at 12th week postoperatively(P<0.01). Histology study: (1) At 4th week postoperatively, the thick and lax soft tissue on the implant surface and CHA granulation without new bone were found in control group; In experimental group the connective tissue membrane with little new bone on the implant surface was presented. (2) At 8th week postoperatively, uneven loosening connective tissue membrane with little new bone on the implant surface was found in control group; New bone formation with point bone-implant contact at interface existed in experimental group. (3) At 12th week postoperatively, the connective tissue fibrous membrane with little point bone-implant contact was presented in control group; In experimental group bone tissue with continuous and encircled bone-implant contact was found at the interface. The bone-to-implant contact (BIC) was zero both in experimental group and control group at 4th week postoperatively. The bone-to-implant contact in experimental group and control group was 15.36±3.57%, 6.09±1.46% at 8th week, and 46.54±6.89%, 17.70±6.32% at 12th week respectively. The difference between two groups had statistical sense.
5. Repair of bone defect around prosthesis and the effects of rhBMP-2 and tissue engineering bone on osseointegration of the bone-prosthesis interface were observed. X-ray study: Lots of new bone with stable femoral prosthesis was found and CHA granulation absorbed completely in BMP group. Although massive new bone formed with stable prosthesis, the shape of CHA granulation still was presented in cell group; New bone formation was not obvious with radiolucent zone around prosthesis in CHA group. Biomechanical study: The shear-strength of bone-implant interface was 1.868±0.351, 1.269±0.432, 0.588±0.226 respectively in BMP group, cell group and CHA group; The difference had no statistical significance. Histology study: New bone tissue mainly with mature lamellar bone and little fibrous tissue and continuous bone-prosthesis contact at the interface were found in BMP group; New bone tissue partially with fibrous connective tissue and point bone-prosthesis contact at the interface were presented in cell group; Fibrous connective tissue with little lamellar bone and verge and slight bone- prosthesis contact were presented in HA group. Image analysis: The bone-to-implant contact(BIC) was 67.23±18.36、38.78±19.40、15.47±8.05 respectively in BMP group, cell group and control group(P<0.01).
Conclusion:
1. BMSCs can be segregated and cultivated successfully. Cells express high level of ALP activity and collagen I and capability of mineralizing intracellular matrix after bone induction. This demonstrates that BMSCs have differentiated to mature osteoblasts.
2. Cancellous bone defect around the implant can be repaired by tissue engineering bone constructed with BMSCs and CHA.Vascularization and bone metabolic activity of bone graft is at stage of quick development within 8 weeks and enter into stage of ripeness and slow development at period of 8th to 12th week. CHA granulation still chould be found at 12th week in histology.
3. Tissue engineering bone constructed with BMSCs andCHA can repair the bone defect around implant and promote the osseointegration on the bone-implant interface.
4. Microanalysis of implant surface shows that percent contents of calcium and phosphate have the trend to increase and the ratio of calcium to phosphate gradually approaches the data of normal bone tissue with time going by in experimental period. This demonstrates that earlier period is the stage of new bone formation and gradually turns into stage of bone rebuilding and ripeness.
5. BMP improves the efects of tissue engineering bone on the repair of bone defect around prosthesis and the osseointegration on the bone-prosthesis interface.
Branemark于20世纪60年代提出了骨整合的概念,即在光镜水平下正常的改建骨和假体之间无软组织,假体与骨组织直接接触,其承受的负荷能通过直接接触持续不断地传递,并分散到骨组织中。骨整合被认为是人工关节置换术后的理想状态。
生物固定性假体要获得满意的远期效果,依赖于假体骨组织间的紧密接触、良好的骨长入和一定的骨生长时间。然而许多人工关节置换术或翻修术患者常合并有假体周围骨缺损,为恢复骨性结构并重建骨的稳定性,骨移植已成为修复骨缺损的一个必要手段。临床上根据骨缺损的大小、性质,可应用自体骨移植、异体骨移植治疗。但由于自体骨移植可提供的骨量有限,移植骨在形态、大小和数量上难以满足缺损处的要求,特别是巨大的或特殊部位骨缺损的修复,故其应用常常受到限制。1984年,Sloof等首先报告应用异体颗粒骨代替自体骨,打压植骨修复髋臼骨缺损。1993年,Ling等用同样的技术行股骨骨缺损的修复重建,从此该项技术被广泛的应用于人工关节置换术及翻修术中。但由于冻干的异体骨无成骨细胞,骨诱导活性低,新骨爬行替代及塑性的时间长,有移植骨吸收、塌陷、假体松动等并发症的可能。
上世纪八十年代兴起的骨组织工程学为骨缺损的修复提供了一个全新的思路和方法。组织工程涉及三个要素:即种子细胞、生物活性因子、支架材料。组织工程技术通过获取具有成骨活性的组织,经分离、培养、扩增,诱导为成骨细胞,并在体外与可吸收的支架材料复合回植于体内,依靠种子细胞的成骨作用及生物材料的降解吸收,最终形成骨组织。应用组织工程化骨修复长骨节段性骨缺损、颅骨缺损已有较多的报告,而应用组织工程化骨修复人工关节周围骨缺损,并观察其对假体骨界面骨整合的研究目前国内外尚未见文献报告。
目的:
1.建立骨髓间充质干细胞(MSCs)的分离、培养、骨向诱导方法;
2.体外骨髓MSCs与CHA(CHA)复合构建组织工程化骨,观察其修复植入体周围松质骨骨缺损的的效果。
3.观察组织工程化骨修复植入体周围骨缺损对植入体-骨界面骨整合的影响。
4.研究BMP促进组织工程化骨修复人工关节周围骨缺损,提高假体-骨界面骨接触的能力。
方法:
1.采用Ficoll(1.073g/ml)密度梯度离心加贴壁培养法分离兔骨髓MSCs,并培养、扩增;将第4代骨髓MSCs应用骨诱导培养液(10-8mol/L地塞米松+10mmol/L甘油磷酸钠+ 50mg/L维生素C)培养,骨向诱导。
2.诱导后的成骨细胞应用细胞形态学、细胞活力检测、细胞生长及增殖曲线、ALP活性检测、矿化结节茜素红染色、I型胶原免疫组化与ALP鉴定。
3.于兔左侧股骨髁制作一0.6×1.2cm的穿透性松质骨骨缺损,植入钛合金(Ti-6Al-4V)植入体于骨缺损的中央,在体外将同种异体骨髓MSCs与CHA复合构建组织工程化骨,修复植入体周围骨缺损,同法右侧骨缺损处仅植入CHA为对照组,术后4、8、12周行X射线检查、生物力学测试、放射性核素骨扫描、脱钙骨组织学HE染色,观察其骨缺损修复效果。
4.并于术后4、8、12周行X射线检查、剖面观察、植入体剪切强度测定、植入体表面扫描电镜、植入体骨界面能谱分析、不脱钙骨组织学Van Gieson苦味酸-品红染色及图像分析系统计算植入体骨接触率,观察组织工程化骨对植入体-骨界面骨整合的影响。
5.行犬人工全髋关节置换术,制作股骨骨缺损模型,随机分为三组,分别用rhBMP-2+自体骨髓MSCs +CHA、CHA+自体骨髓MSCs、CHA修复骨缺损,术后3个月处死动物,行X射线检查、生物力学测试、不脱钙骨组织学Van Gieson苦味酸-品红染色及图像分析系统分析计算假体骨接触率。
结果:
1.应用密度梯度离心结合贴壁培养法成功的分离、培养、扩增兔骨髓MSCs,培养3天后细胞的形态由圆形转为梭形;MSCs生长曲线分为生长潜伏期、对数生长期和平台期;MSCs表面标志物SH3呈阳性。
2.骨诱导后细胞培养5-8天,细胞形态逐渐变为多角形,呈现聚集生长。诱导后细胞ALP活性较未诱导组显著性增高。骨向诱导后第三代细胞ALP染色、I型胶原免疫组化染色均呈阳性,茜素红染色可见典型的矿化结节。
3.体外构建组织工程化骨修复植入体周围松质骨缺损结果:术后4、8、12周大体观察与剖面观察,实验组随时间的延长可见骨修复。X线表现为CHA颗粒吸收,被新生骨取代。生物力学结果示术后12周股骨髁最大压力载荷、载荷/应变比值均较对照组高,最大应变位移较对照组低(P<0.01)。放射性核素骨扫描实验组不同时间点ROI平均计数均较对照组高,有显著性差异(P<0.01)。实验组术后8周内ROI平均计数为快速增长期,术后8周开始进入缓慢增长期,12周达到峰值。组织学观察,实验组术后4周植入区内CHA颗粒之间有岛状新生骨组织;术后8周植入区内可见支架材料部分吸收,遗留空间被新生骨组织取代,骨缺损基本得到修复;术后12周可见明显编织骨、板层骨及骨小梁形成,CHA大部分吸收,但仍可见到未吸收的HA颗粒。对照组术后4、8周无新生骨,术后12周有少量的新生骨。
4.组织工程化骨对植入体-骨界面骨整合的影响:术后4、8、12周大体观察与剖面观察,见实验组随时间的延长骨缺损得到修复,植入体周围无间隙。X线检查见CHA颗粒吸收,被新生骨取代,植入体周围为新生骨,植入体位置良好,而对照组植入体周围主要为高密度的CHA颗粒。生物力学结果示不同时间点植入体骨界面剪切强度较对照组增高(P<0.01)。扫描电镜观察,可见实验组植入体表面孔隙内无定形物质随时间的变化而逐渐增加。能谱分析术后4周实验组Ca、P元素百分含量较对照组增高,8、12周Ca元素百分含量较对照组高(P<0.05),P元素百分含量与对照组比较无显著性差异;Ca/P比值随时间的延长逐渐增高,于术后12周达2.02(P<0.01)。不脱钙骨组织学观察,术后4周对照组植入体-骨界面为厚而疏松的软组织,实验组界面为结缔组织纤维膜,新生骨偶见;术后8周对照组植入体-骨界面为结缔组织纤维膜,厚薄不一,界面较松,周围可见少量新生骨组织,实验组界面有新骨沉积,植入体表面有部分点状骨接触;术后12周对照组植入体-骨界面为结缔组织纤维膜,界面有少量点状骨接触,实验组界面主要为骨组织,部分界面可见连续性骨接触,甚至呈环形包绕。术后4周对照组与实验组植入体骨接触率为0,术后8周、12周植入体骨接触率均高于对照组(P<0.01)。
5. rhBMP-2促进组织工程化骨修复假体周围骨缺损及假体-骨界面骨整合:术后3个月X线表现BMP组新骨生成明显,CHA颗粒完全吸收,假体稳定;细胞组有大量的新骨形成,但仍有CHA颗粒影,假体稳定;对照组新骨生成不明显,假体周围有透亮带。对照组、细胞组、BMP组假体骨界面剪切强度无显著性差异。不脱钙骨组织学观察,BMP组假体骨界面为骨组织充填,部分界面可见连续性骨接触,仅见少量纤维组织,新生骨以成熟的板层骨为主,与假体结合紧密。细胞组假体骨界面主要为骨组织充填,可见较多的骨接触,有部分纤维结缔组织。对照组假体骨界面为纤维结缔组织,边缘有少量编织骨,骨接触较少。图形分析计算对照组、细胞组、BMP组假体界面骨接触率(%)分别为15.47±8.05、38.78±19.40、67.23±18.36(P<0.01)。
结论:
1.本研究成功的分离、培养了兔骨髓MSCs,经骨向诱导后表达高水平的ALP活性与I型胶原,并能矿化细胞外基质,证实已分化为成熟的成骨细胞。
2.骨髓MSCs骨向诱导后与CHA复合可修复植入体周围松质骨骨缺损,移植物血管化、骨代谢活性前8周为快速发展阶段,8-12周进入缓慢发展渐趋成熟阶段。
3.骨髓MSCs骨向诱导后与CHA复合修复植入体周围骨缺损,促进新骨生成,改善植入体-骨界面骨整合的作用。
4.植入体骨界面能谱分析示随着时间的延长Ca、P元素百分比含量有增高趋势,Ca/P比值随着时间的延长逐渐接近正常骨组织,表明早期为新生骨形成阶段,逐渐转为骨改建与成熟阶段。
5. BMP促进组织工程化骨修复人工关节周围骨缺损,改善早期假体骨界面骨整合率。
Background:
In the 1960s, Branemark proposed the conception of osseointegration that there is no soft tissue between rebuilding bone and prosthesis under microscope, that bone contacts tightly with prosthesis, and that the loading taken on by prosthesis can pass and disperse constantly into bone by the bone-prosthesis contact. Osseointegration is regarded as the ideal state after arthroplasty.
Long-time satisfactory result of biological prosthesis replacement depends on the tight bone- prosthesis contact, favorable bone growth and bone growing time. Many patients who undergo an operation for prosthesis replacement or revision often suffer from bone defect around prosthesis. Bone grafts become a necessary method to recover the construction and stability of bone. According to the size and property of the bone defect, we usually adopt auto graft or allograft to treat bone defects in clinic. Due to the shape, size and quantity of bone, auto graft can not achieve the request of bone defect, especially of gigantic or special bone defect. The use of auto graft often is limited. Since Sloof etc firstly reported that bone defect of acetabular can be repaired by packing bone graft with variant granulation bone in 1984 and Ling etc used the same technique to treat bone defect of femur in 1993, the technique has been widely used in the operation for prosthesis replacement or revision. Because variant bone has no osteoblasts and much lower activity of bone induction, to achieve real creeping, substiution and remolding of new bone needs much more time. Allograft usually has many complications, such as absorption, collapse of grafting bone, and asceptic loosening of prosthesis.
In the 1980s, the emergence of tissue engineering provides us a brand-new thought and method in repairing bone defect. Tissue engineering has three important elements, namely seed cell, bioactivity factor, and scaffold material. Although there are many reports on repair of segmental defect of long bone and skull bone defect by use of tissue engineering bone, untill now any reports about repairing periprosthetic bone defect with tissue engineering bone and the effects of tissue engineering bone on osseointegration of the bone-impant interface have not been found in articles at home and abroad.
Aim:
1. To establish method of abstraction, cultivation and bone induction of mesenchymal stem cell (MSCs).
2. To construct the tissue engineering bone with MSCs and coral hydroxyapatite (CHA) in vitro, and observe the effects on repair of bone defect around implant.
3. To observe the effects of tissue engineering bone on osseointegration of the bone-implant interface.
4. To study the capability of BMP to boost tissue engineering bone to repair periprosthetic bone defect, promote bone growth around prosthesis and the osseointegration of the bone-prosthesis interface.
Method:
1. BMSCs were segregated by Ficol(l1.073g/ml)density gradient centrifugation and the adherent culture method, then cultivated and multiplied. The 4th generation MSCs were induced by bone induction culture fluid (10-8mol/L DEX+10mmol/L Sodium Glycerophosphate + 50mg/L Vitamin C).
2. Osteoblasts derived from BMSCs were verified by cell morphology, cell vigor detection, cell growth curve, ALP activity examination, alizarin Bordeaux dyeing of mineralization nodes , immunohistochemistry of collagen I and Gomori dyeing of ALP.
3. A penetrating transverse bone defect (0.6cm×1.2cm) was created in bilateral femoral condyles respectively in rabbits. The titanium alloy implants were implanted in centre of bone defects. Tissue engineering bone constructed with osteblasts induced from allogeneic induced BMSCs and CHA in vitro was embedded into defect around the impant in left as experimental group and only CHA was embedded into the defect in right as control group. The effects on repairing bone defect around implant were studied by X-ray examination, biomechanical examination, radionuclide bone imaging by ECT, decalcificated bone histology with HE dyeing.
4. The osseointegration of the bone-implant interface was observed by X-ray examination, section observation, push-out test of implant, scanning electron microscope, spectrum analysis, undecalcified bone histology with Van Gieson carbazotic acid–solferino dyeing, bone-to-implant contact calculated by datum from image analysis.
5. Total hip replacement with femoral bone defects was operated in right hip of dog. Animals were divided to three groups, called BMP group, cell group and CHA group The bone defects were imlpanted at random by the compound of rhBMP-2 and tissue engineering bone, tissue engineering bone and CHA respectively. Animals sacrificed at three months after operation. The effects of BMP on osseointegration of the bone-prosthesis interface and repairing bone defect around prosthesis were studied by X-ray examination, biomechanical examination, undecalcified bone histology with Van Gieson carbazotic acid–solferino dyeing as well as bone-to- prosthesis contact calculated by datum from image analysis.
Results:
1. BMSCs were segregated, cultivated and multiplied successfully by way of density gradient centrifugation and adherent culture. The round shape of cells turned to fusiform after culture for three days. Growth curve of BMSCs was divided into detention phase, logarithmic growth phase and platform phase. The surface market SH3 of BMSCs took on positive.
2. The shape of cells cultivated in bone induction culture fluid turned to polygon gradually. Cells began to growt in cluster after culture for 5-8 days. ALP activity was significantly higher in experimental group than that in control group(P<0.01). Both collegan I immunohistochemistry and ALP Gomori dyeing presented positive and alizarin Bordeaux dyeing of the 3th generation BMSCs after bone induction showed typical mineral nodus .
3. Cancelous bone defect was repaired by tissue engineering bone constructed in vitro. As time going by, bone repair was observed in experimental group by the way of general observation and section observation at 4th, 8th and 12th week postoperatively. Biomechanical results showed that both the maximum pressure load and ratio of load to straining in experimental group were significantly higher than that in control group; The maximum strain- displacement in experimental group was lower than that in control group(P<0.01). Radionuclide bone imaging showed that mean of ROI in experimental group increased significantly higher than that in control group at different time points. Mean of ROI increased at quicker epacme within 8 weeks and began to increase at slower epacme at 8th week and achieved peak amplitude at 12th week postoperately. Histology study demonstrated that little new bone around CHA granulation in experimental group was found at 4th week; Scaffold materials were absorbed partially and replaced by new bone tissue, and bone defects were repaired basically by some new bone which rebuild maturely in partial domain in experimental group at 8th week; The obvious woven bone, lamellar bone and bone trabecula with blood sinus were found, but unabsorbed CHA granulation still existed in experimental group at 12th week. Little new bone around CHA granulation in controll group was found at 8th and12th week.
4. The effects of tissue engneering bone on osseointegration of the bone-implant interface were observed. As time going by, the implants were embedded well by new bone in experimental group in general observation at 8th and 12th week postoperatively. X-ray showed that CHA granulations were absorbed and replaced by new bone and that the position of implant surrounded by new bone was good in experimental group; Gaps around implants in control group were found. Biomechanical examination showed that the shear-strength of bone-implant intreface in experimental group was significantly higher than that in control group at different time points. Scanning electron microscope notified that amount of amorphous material on pores of the implant surface increased gradually with time going by in experimental group. The percent content of calcium and phosphate was significantly higher in experimental group than that in control group at 4th week postoperatively; The percent content of calcium was significantely higher in experimental group than that in control group, but the percent content of phosphate increased unsignificantely at 8th, 12th week; The ratio calcium of and phosphate increased gradually with time going by and reached to 2.02 at 12th week postoperatively(P<0.01). Histology study: (1) At 4th week postoperatively, the thick and lax soft tissue on the implant surface and CHA granulation without new bone were found in control group; In experimental group the connective tissue membrane with little new bone on the implant surface was presented. (2) At 8th week postoperatively, uneven loosening connective tissue membrane with little new bone on the implant surface was found in control group; New bone formation with point bone-implant contact at interface existed in experimental group. (3) At 12th week postoperatively, the connective tissue fibrous membrane with little point bone-implant contact was presented in control group; In experimental group bone tissue with continuous and encircled bone-implant contact was found at the interface. The bone-to-implant contact (BIC) was zero both in experimental group and control group at 4th week postoperatively. The bone-to-implant contact in experimental group and control group was 15.36±3.57%, 6.09±1.46% at 8th week, and 46.54±6.89%, 17.70±6.32% at 12th week respectively. The difference between two groups had statistical sense.
5. Repair of bone defect around prosthesis and the effects of rhBMP-2 and tissue engineering bone on osseointegration of the bone-prosthesis interface were observed. X-ray study: Lots of new bone with stable femoral prosthesis was found and CHA granulation absorbed completely in BMP group. Although massive new bone formed with stable prosthesis, the shape of CHA granulation still was presented in cell group; New bone formation was not obvious with radiolucent zone around prosthesis in CHA group. Biomechanical study: The shear-strength of bone-implant interface was 1.868±0.351, 1.269±0.432, 0.588±0.226 respectively in BMP group, cell group and CHA group; The difference had no statistical significance. Histology study: New bone tissue mainly with mature lamellar bone and little fibrous tissue and continuous bone-prosthesis contact at the interface were found in BMP group; New bone tissue partially with fibrous connective tissue and point bone-prosthesis contact at the interface were presented in cell group; Fibrous connective tissue with little lamellar bone and verge and slight bone- prosthesis contact were presented in HA group. Image analysis: The bone-to-implant contact(BIC) was 67.23±18.36、38.78±19.40、15.47±8.05 respectively in BMP group, cell group and control group(P<0.01).
Conclusion:
1. BMSCs can be segregated and cultivated successfully. Cells express high level of ALP activity and collagen I and capability of mineralizing intracellular matrix after bone induction. This demonstrates that BMSCs have differentiated to mature osteoblasts.
2. Cancellous bone defect around the implant can be repaired by tissue engineering bone constructed with BMSCs and CHA.Vascularization and bone metabolic activity of bone graft is at stage of quick development within 8 weeks and enter into stage of ripeness and slow development at period of 8th to 12th week. CHA granulation still chould be found at 12th week in histology.
3. Tissue engineering bone constructed with BMSCs andCHA can repair the bone defect around implant and promote the osseointegration on the bone-implant interface.
4. Microanalysis of implant surface shows that percent contents of calcium and phosphate have the trend to increase and the ratio of calcium to phosphate gradually approaches the data of normal bone tissue with time going by in experimental period. This demonstrates that earlier period is the stage of new bone formation and gradually turns into stage of bone rebuilding and ripeness.
5. BMP improves the efects of tissue engineering bone on the repair of bone defect around prosthesis and the osseointegration on the bone-prosthesis interface.
引文
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