摘要
研究背景
颅骨缺损是神经外科中的一个常见问题,其形成的原因主要见于烧伤、创伤、肿瘤等。修复骨缺损,重建其生物学特性是神经外科急需解决的重要课题之一。颅骨缺损的修复主要包括修复材料的选择和修复方式的优化。为了避免传统的材料所带来的问题,人们开发了许多种人工骨修复材料。其中,“诱导因子+载体缓释系统”模式已成为骨组织工程学研究的一个热点。一种良好的诱导型人工骨材料应具有以下特性:①良好的生物相容性及生物降解性;②骨传导及与其他活性分子复合共同诱导骨发生;③能负载大量细胞,支持骨细胞生长和分化;④合适的机械强度与可塑形性;⑤原料来源广,生产价格廉,便于消毒保存。
纵观国内外骨组织工程材料研究可发现,以下几种天然或人工材料逐渐被大多数研究者认可。例如:骨形态发生蛋白(bone morphogenetie protein,BMP)是一种多功能生长因子,其特征是有较强诱导成骨活性,且无种属特异性;胶原(collagen, COL)是骨基质的主要有机成分,COL与BMP复合可延缓BMP释放,可降低BMP的诱导剂量,令其维持较长的作用;羟基磷灰石(Hydroxyapatite,HA)是一种生物相容性和免疫原性都很理想的骨修复材料,纳米级羟基磷灰石(Nano-sized hydroxyapatite,n-HA)因其晶体尺径与人体骨矿中磷灰石相当,骨修复效果更佳;磷酸钙骨水泥(Calcium Phosphate Cement,CPC)具有可塑型性、吸收性和等温自固化等优良性能。到目前为止,上述各种材料单独使用或复合应用,均因存在塑形性、孔隙率、机械强度等不同问题而至实际成骨效果欠佳。
为了找到一种可供临床用来修复骨缺损的组织工程材料,我们经过反复实验对CPC进行改良。合成α-磷酸三钙(α-TCP)体系的CPC作为附加载体(Accessional Delivery,AD),利用仿生矿化技术将天然骨胶原与无机晶核(HA)自组装构建成纳米级核心载体(Nucleus Delivery,ND),借助AD与ND的内在联系与特性有机地构建成双载体系统(Double Delivery System,DDS);以提取天然活性蛋白为诱导核心与DDS进行有机组合,在满足材料强度的前提下在DDS上预制出活性生长通道(Active Growth Channel,AGC)的空间结构。把AD、ND和bBMP复合物制备成bBMP/ND/AD释放系统,用真空负压技术将释放体系有效地填充至AGC预制空间,最终制备出在成份、空间构型和生物活性上与正常人骨极其相似的人造骨主体部分,试图解决现有众多单一载体材料的缺陷。基于这种认识,我们进行了以下方面的工作:(1)活性蛋白异位诱导成骨评估;(2)载体材料的物性评估;(3)诱导型人工骨材料修复兔颅骨缺损实验。
一、活性蛋白异位诱导成骨实验
目的:检验自制备的牛骨形态发生蛋白(bovine bone morphogenetic proteins,,bBMPs)异位诱导成骨性。
方法:取昆明小鼠40只,随机分成2组,将bBMPs材料植入小鼠右大腿后内侧肌袋内,术后2周、3周、4周、8周分别行放射学检查、组织学方法评价骨组织生长情况。
结果:术后4周x线片显示材料植入区有小条状密度增高,8周见小块状密度增高。组织学显示2周有大量细胞集聚;3周有较多的骨细胞,有骨基质生成;4周有明显骨样组织形成;8周见明显完整的骨组织形成。而对照组没有上述结果。
结论:本研究提取纯化的bBMPs在小鼠肌袋中具有良好得生物相容性和异位诱导成骨能力。
二、载体材料的制备与物性评估
目的:检测核心载体(ND)、附加载体(AD)以及双载体系统(DDS)的微观结构、机械强度与可塑性等。
方法:①湿法制备ND,经X线衍射(XRD)、电子显微镜扫描(SEM)测其粒度与微观形态;
②马弗炉煅烧法制备α-磷酸三钙(α-TCP)体系的CPC(AD),用国产多功能压力测试机检测其机械强度,XRD测其TCP纯度,能谱检测钙磷比,SEM观察微观结构;③DDS检测方法同上。
结果:①ND材料:ND的粒度为98纳米(nanometer,nm),与人骨接近;样品经SEM显示微观结构呈花瓣样,具有HA与胶原的共同特征。
②AD材料:材料水化72h时平均承压力5000牛顿/cm3(约170Mpa),水化7d压力为8800牛顿/cm3;受试者工作曲线(ROC)显示结果呈正态分布;XRD测定其TCP含量为92.7%(标样的TCP含量为94%,Fluka公司产品,编号:50553);材料水化14d后,原料成份已全部水化成HA,XRD显示材料中未见有害基团,孔隙率为45%,能谱分析其成份中钙磷比为1.52;随着水化时间的延长(24h、72h、7d、14d),SEM显示该材料的结晶先为针状,最终形成板状,呈低结晶状态。
③DDS:三组材料样品分别加入0.25%、0.5%、1%ND后,压力测试显示加入0.25% ND的复合材料压力下降至4200牛顿/cm3左右;动态水化过程XRD测试显示添加0.25%ND,水化产物未见明显异常;材料的孔隙率为55%,钙磷比1.6,SEM显示其微观形态与ND相同。
结论:本研究所制备的AD材料,添加ND会造成复合材料承压能力下降,压力下降数值与ND加入量呈正相关。加0.25%ND复合材料的压力下降程度符合本实验研究作为支撑(载体)材料的压力强度。复合材料在保持水化产物的稳定条件下,钙磷比更接近人骨,机械强度下降、孔隙率增加,有利于材料降解,是一种理想的骨缺损修复材料。
三、诱导型人工骨材料修复兔颅骨缺损实验
目的:观察诱导型人工骨材料在修复兔颅骨缺损中的作用效果。
方法:选取健康成年新西兰大白兔51只,随机分成实验材料组(复合材料,M组)、实验对照组(同种异体冻干骨,B组)和空白对照组(N组);M组材料采用固化成型的圆“纽扣”状样品,预制含释放系统(ND、bBMP)的活性生长空间(AGC);制备双侧颅顶骨8mm的骨膜与骨缺损模型,分别植入相应材料后固定。术后按2周、4周、8周、12周、16周时程分别取材,进行大体观察、外周血液检验、X线影像检查、荧光双标示踪、组织学与免疫组化观察,比较各组材料修复兔颅骨缺损效果。
结果:①大体观察:手术后,各组均有3-4只动物术后头顶缝线处轻度肿胀,在3天内消失;2周时,M组与B组植入材料在缺损表面清晰可见,材料与宿主骨之间有组织长入,N组缺损表面有薄层组织覆盖。4周时,M组表面见薄层组织覆盖,但材料仍可见;从顶骨内面见一侧材料的AGC位置有透明黄色组织微突;8周时, M组表面有外骨痂出现,其它各组少量结缔组织覆盖。12周和16周各组骨缺损表面均有组织交织因而不易分辨。
②外周血液检查:手术前与术后1周、2周,M组、B组、N组动物的血LYM%值结果统计分析显示:M组LYM%分别与B组、N组间的差异有统计学意义。而三组动物手术前与取材时的血总蛋白、谷丙转氨酶、尿素氮、碱性磷酸酶、钙离子等结果统计分析显示,各组间以及同组5个时间点之间的差异均没有显著意义。
③X线影像观察:术后2周M组材料密度高于周围松质骨,植入材料清浙可见;4周时可见左侧材料上已有骨痂形成,8周时轮廓已分辨不清,材料中央区密度较4周时有所下降,12周材料中央区密度下降明显,接近周围组织,边缘均有连续性骨痴,材料降解。16周时材料降解明显,已与周边连成一体B组术后2周与周围松质骨近似,8周周围有骨痴生长,边界不清;16周B组中央已部分吸收,边缘硬化。N组2-8周缺损区明显,12周、16周边缘模糊似有新骨,但骨缺损区明显大于B组。
④骨磨片组织形态观察:术后8周、12周,M组4倍光镜下可见缺损区边缘已不规则,内部出现较多不规则蓝色骨小梁和类骨质,其中可见暗红色钙质沉着,提示为新生骨小梁;而B组材料不完整,呈小片分散状,仅边缘有少量新生骨小梁与类骨质。N组大部缺损仍存在,中心部位无新生骨小梁。
⑤骨磨片组织荧光双标观察:术后8周、12周、16周,M组植入区边缘和中央可见较多绿色和金黄色荧光带;有散点状、不规则环形、“双轨征”,提示有新骨长入,且生长活跃;绿、黄荧光带间距接近20微米(2种示踪济相隔7天),提示新骨生长速度较快。B组荧光以边缘区为主,缺损区中央可见1-2条状荧光,较M组少。N组只在边缘区见少量荧光。
⑥组织形态学观察:
M组术后2周植入材料区大量细胞聚集为主;4周边缘区有新骨生长,活性生长空间(AGC)内见骨样组织、小血管和大量细胞聚集,材料已部分降解吸收,剩余人工骨裂解成颗粒状晶体散在其中;8周时材料AGC部位骨细胞增殖活跃,新骨相互连接成片状;12周缺损区新骨进一步重建,纵切面见新骨样组织形成“框”形架,呈向“框”架内延伸;16周见AGC区形成髓腔样结构,周围大量新生骨样组织和血管。
B组术后各时程新骨形成量较M组少,缺损区材料逐渐分离成片状,4周以后宿主骨缺损端见新生骨组织;N组4周后边缘见少量新骨,但缺损中央未见新骨生长,代之以大量纤维结缔组织填充。各组各时间点均未见植入区周围有炎性细胞聚集。
⑦免疫组织化学观察:实验中取M组三个时间点(4周、8周、12周)取样,免疫组化分析M组材料与缺损交界区BMP2含量变化,结果显示三个时间点在边界区均有内源性BMP2分布,同时在材料中央孔区均有外源性BMP2着色。利用Motic Images Advanced3.2图像分析软件,对各组样本免疫组化结果通过灰度扫描进行半定量分析,数据经统计学处理显示:三个时间点的BMP2含量的灰度值均无显著差异。
结论:
①在体实验显示该复合材料生物相容性较好,能复合活性骨生长因子缓慢释放;
②在复合材料中AGC的构建使诱导成骨呈多点生长趋势,有利缩短新骨生长距离,加速骨缺损修复;
③复合材料植入机体后可降解并持续促进新骨生长,诱导成骨效果强于同种异体冻干骨。
Background
Skull defects is a common problem in neurosurgery, its causes are mainly including burns, trauma, cancer, and so on. To repair bone defects and rebuild its biological characteristics is one of the important issues to be need in neurosurgery. The repair of the skull defect, including the choice of repair materials and optimization the methods. In order to avoid the problems of the traditional materials ,many kinds of artificial bone materials for repair have been developed, Among them,"inducing factor + Carrier-delivery system "model already become a hot point in bone injure and bone tissue engineering research.
A ideal bone-induced artificial material should have the following characteristics:①Good biocompatibility and biodegradability;②Osteoacusis and other active molecules induced osteogenesis together;③Be able to load a large number of cells , to support bone cell growth and differentiation;④Appropriate mechanical strength and plastic;⑤R aw materials have a wide source , low price and are easy to disinfect and save.
Based on the research progress of bone tissue engineering materials at home and abroad ,The following types of natural or artificial materials have been gradually approved by many researchers,
For example: bone morphogenetic protein (BMP) is a multifunctional growth factor, characterized by a stronger-induced osteoblast activity, and no species-specific; collagen (COL) is the main organic components in the bone matrix, the compound of COL and BMPs can delay the release of BMP and reduce the induction dose of BMPs, maintain a longer effect; Hydroxyapatite (HA) is a ideal bone repair materials with biocompatibility and immunogenicity, nano-hydroxyapatite (n-HA) have a better repair effect for crystal size is equal with the hydroxyapatite in bone in human body, calcium phosphate cement (CPC) has good properties in Plasticity , absorbency and isothermal auto-solidification.however, A separate application of the above-mentioned material shows different problem including plasticity, porosity and mechanical strength etc, in addition, no an ideal composite materials have been used in the practice at present.
In order to find more effective tissue-engineered bone defect repair materials for clinical application, we carried out improvements on the CPC through a series of experiment. synthesized the CPC based onα-tricalcium phosphate (α-TCP) system as an Accessional Delivery (AD),Natural bone collagen and hydroxyapatite (HA) self-assembled into a nanometer Nucleus Delivery (ND) by the bio-mimetic mineralization technology. With the properties of ND and AD , the inherent relation between ND and AD , we constructed organically the Double Delivery System(DDS); Extracted the natural active protein as a core for induction and prefabricate the space of Active Growth Channel(AGC) in the DDS with precision and stability under the premise of enough material strength .Prepared the bBMP / ND / AD delivery system with AD, ND and bBMP complex, Applying the vacuum-negative pressure vacuum technology fill the space of AGC with the release system effectively for the construction of AGC. Finally obtain the main part of the artificial bone which its composition, space configuration and biological activity are extremely similar to normal bone. Based on above thinking, to solve the defects of the existing material vector, we have carried out the following work.
1. Evaluation of ectopic induced osteogenesis activity of active protein
Objective: To determinate the ectopic induced osteogenic activity of self-maded bovine bone morphogenetic protein
Methods: 40 Kunming mice were randomly divided into 2 groups, bBMPs materials implanted into lateral inner muscle bag of the right thigh, carry out radiology examination respectively in 2 weeks, 3 weeks, 4 weeks and 8 weeks after operation, Evaluate the state of bone tissue growth by histology methods
Results: x-ray show the density increased with small bar in material implanted area in 4 weeks after operation, the density show a small blocky increase in 8 weeks,. Histology show that a large number of cells gather in 2 weeks; more bone cells and bone matrix generation in 3 weeks, clear formation of bone-like tissue in 4 weeks; significantly formation of bone tissue in 8 weeks but the control group did not show above results
Conclusion: The extracted and purificated bBMPs in this research show a good biocompatibility and ectopic osteoinduction ability in the mouse muscle bag.
2. Preparation and physical property evaluation of Delivery Material
Objective: Detection for micro-structure, mechanical strength and plasticity of the ND, AD and DDS
Method:①prepared the ND by wet method, measured its size and morphology by X-ray diffraction (XRD), scanning electron microscopy (SEM);②prepared CPC (AD) based onα-TCP (α-TCP) system by muffle calcination process, detected its mechanical strength with domestic multi-function pressure testing machine, measured the purity of TCP by XRD, detected the calcium/phosphorus ratio by energy spectrum, observed the microstructure by SEM;③DDS detection was same as above. Results:①ND: Particle size of ND is 98 nanometers close to the human bone; SEM show micro-structure of samples was like petals, have a same feature with collagen and HA
②AD: The average hold pressure has reached 5,000 Newton / cm3 when material hydrated for 72h, the 8800 Newton / cm3 when material hydrated for 7d; Receiver operating curve (ROC) showed that the results were normal distribution; the content of TCP is 92.7% (standard samples are 94%,from Fluka company,NO:50553) by XRD detection; after 24h hydration, the materials transformed to HA, XRD showed that materials had no the harmful group, the porosity of the material was 45% , energy spectrum showed that calcium/phosphorus ratio was 1.52; With the prolong of hydration time (24h, 72h, 7d, 14d), SEM revealed that the first crystallization of the material were needle-like, eventually the crystallization formed plate,in low-crystalline state,
③DDS: three groups respectively added 0.25%, 0.5%, 1% ND, pressure detection showed that the pressure of the composite material added 0.25% ND had went down to 4200 Newton / cm3; XRD of dynamic hydration process showed that hydration products had no obvious abnormalities when added 0.25% ND; the porosity of the material was 55%, calcium/phosphorus ratio was 1.6, SEM shows that micro-structure similar to ND.
Conclusion: AD materials prepared in this study, the pressure-carrying capacity of the composite materials reduced when ND was added, the pressure drop in value had a positive correlation with the addition of ND. The pressure drop in value when 0.25% ND was added meet the requirements of pressure and strength as delivery material in this study. Under the conditions of maintaining the stability of the hydration product, calcium/phosphorus ratio of the composite materials is more close to the human bone, mechanical strength decrease and porosity increase, in favor of material degradation, is an ideal bone defect repair materials.
3. Rabbit skull defects repair experiment with the induced artificial bone material
Objective: To observe the effect of the induced artificial bone material in the rabbit skull defect repairing procedure
Methods: 51 healthy adult New Zealand white rabbits were divided into experimental materials group (composite material, M group), the experimental control group (freeze-dried bone, B group) and the blank control group ( N group); the materials of M group were "buttons" shaped samples by solidification to make, prefabricate Active Growth Channel(AGC) with release system (ND, bBMP); prepared the periosteum and bone defects models with 8mm size in bilateral parietal bone respectively, implanted corresponding materials and fixed, drawed materials respectively according to the time points of 2 weeks,4 weeks, 8 weeks,12 weeks and 16 weeks after the operation,carried out the general observation, blood tests, X-ray inspection, the fluorescent double marker detection, histology and immunohisto- chemical methods for compare evaluation of every group material in the rabbit skull defect repairing procedure.
Results:
①General observation:
after the operation, there were 3 or 4 animals every group with mild swelling in overhead suture line, disappeared in 3 days; After removed the material, implanted material was clearly visible on defect surface in 2 weeks in M Group and B Group, with the tissue growth between host bone and material, there were the thin tissue cover defect surface in N group.
There were the thin tissue coverage from the above observation in M group in 4 weeks, but material was still visible; there were transparent yellow tissue in AGC site on one side of material surface when observed from inside surface of parietal bone; in 8 weeks the callus can be seen on the surface of materials in M group, a small amount of connective tissue coverage can be seen in other groups. In 12 weeks and 16 weeks the bone defects surface were packed by tissue and were not easily distinguishable in every group.
②Blood tests:
In preoperative and postoperative 1 week, 2 weeks, blood LYM% value of M group, B group and N group animals statistical analysis showed that: the differences between LYM% of M group respectively with the B group, N group have statistics significance. The statistical analysis of total protein, alanine aminotransferase, urea nitrogen, alkaline phosphatase and calcium iron of the three groups animals in preoperative and in the time of blood drawn showed the differences between every group or five time point of the same group have no statistics significance .
③X-ray observation:
The density of the material in M group is higher than trabecular bone around material in postoperative 2 week, the implanted material was clearly visible ; Callus formation seems to seen on the left side of the material in 4 weeks, Vague outline of material had been indistinct in 8 weeks, the density of central district of material has declined than 4 weeks, and the decline is more obvious in 12-week, close to the surrounding tissue, there were continuous callus in the edges of the material, degradation had been observed in material in 12 weeks. the degradation of material is more obvious in 16 weeks, and material had blended into bone tissues around implants .
The density of the material in B group is similar to trabecular bone around the material in postoperative 2 week, Callus formation can be seen in the edges of the material and vague outline of material had been indistinct in 8 weeks, the absorption had been seen in the central part of freeze-dried bone, bone sclerosis had been observed in the edges in16 weeks. The defect were obvious in N group in 2-8 weeks, The edge of defect became fuzzy and may be the growth of new bone in 12 weeks and 16 weels,, but the area of bone defect were bigger than B group.
④Observation on morphometry of undecalcified bone tissue:
The edge of defects had been irregular under the light microscope(4X) in postoperative 8weeks and 12 weeks in M group, The many irregular blue trabecule and osteoid can be observed in internal part of material,and the dark red calcium deposit show the growth of new bone; But B group material is incomplete, scattered to scrape, only in the edge of freeze-dried bone a small amount of new trabecule and osteoid can be observed. In N group ,most defects still existed, no new bone structure found in central district of defects.
⑤Observation with fluorescence dual-labeling technique of undecalcified bone tissue:
The many green and yellow fluorescent band can be seen in the implanted site in M group in postoperative 8weeks,12 weeks and 16 weeks, with the features of scattered spots, irregular ring shape and "two-track" sign. showed there were active growth of new bone. The spacing between green and yellow fluorescent band was close to 20 meters(the time interval between injection of two fluorescence tracer was 7 days),showed the growth speed of new bone was very quick. The fluorescence mainly located in marginal areas of defects in B group,in center of defects 1-2 fluorescent bands can be seen,the amount of bands was less than the M group. In N Group, the fluorescence was very few and only can be seen in marginal areas of defects.
⑥observation on histology and morphology:
A large number of cells gathered in implanted material area in M group in 2 weeks, there were new bone growth in marginal areas in 4 weeks, The bone-like tissue found in AGC, small blood vessels and a large number of cells gathered, the material had degraded and absorbed partially. the remaining material split into particles. The growth of bone cells were very rapid in AGC and new bone connected mutually into the new sheet in 8 weeks; The new bone had a further reconstruction in defect in 12 weeks, the formation of new bone showed " frame " in longitudinal section, and new bone extended into inside of“frame”;The formation of marrow -Like structure could be seen in 16 weeks, surrounded by a large number of new bone tissue and blood vessels.
The amount of new bone was less in every time point in B group than M group after operation, the material in defects areas gradually separated into sheet; the new bone could be seen in defects after 4 weeks. A small amount of new bone could be found in marginal areas of defects afer 4 weeks, no growth of new bone had been found but a large number of connective tissue fibers filled in the central areas of defects. No inflammatory cells had been found gathered around the defects in each group at different time points .
⑦Observation on immunohistochemistry
Set three check time point (4 weeks, 8 weeks, 12 weeks) in M group and drawed samples, analyzed the changes of BMP2 contents at the junction of material and defect in M group by immunohistochemical technology. The results showed that there were endogenous BMP2 distribution at the junction at three time point, meanwhile, there were exogenous BMP2 in holes located central areas of materials. the semi-quantitative analysis of gray levels of immunohistochemical results had been carried out by Motic Images Advanced3.2 image analysis software. Statistical analysis shows that the gray levels of BMP2 was no significant difference in the three time points
Conclusion:①Animal experiments show the complex materials have good biocompatibility,can be organically combined with bone growth factor , beneficial to slowly release of growth factor.②Because of the construction of AGC in complex material, there are multi-point growth in the process of bone induction, favorable to shorten the growth distance of new bone, accelerate bone defect repairing.③The material can degradate after implanted into body and promote the growth of new bone continuously, the capacity of bone induction is stronger than the allogeneic freeze-dried bone.
引文
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