DSs/rh-BMP-2/CS复合微球的制备及成骨活性研究
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摘要
骨缺损是长期困扰骨科临床有效治疗的难题,骨肿瘤、严重的骨创伤均可能引起骨缺损,因此各种骨修复材料相继推出,主要包括自体骨、异体骨和人工骨等类型。自体骨吸收快,效果好,但是受来源问题的限制；异体骨、异种骨则因免疫排斥反应在临床应用受到限制,而且其存在传染疾病的风险。人工骨凭借具备取材广泛、易加工、生物相容性好、价格低廉、能消毒、利于临床操作等优点,成为目前人工骨修复材料的主流。
人工骨修复材料主要包括无机材料和有机材料两类,前者以羟基磷灰石(hydroxylapatite,HA)和磷酸三钙(tricalcium phosphate,TCP)为代表,后者则以聚乳酸、聚乙醇酸或两者的共聚物为代表。无机材料相对于有机材料,机械强度好,其中珊瑚羟基磷灰石(Coralline Hydroxyapatite, CHA)更是因来源广泛、生物相容性好、骨传导作用好、对人体无害等特点而受到临床应用的青睐。尽管人工骨已经应用于临床并取得了较好的效果,但是植入体内的人工骨往往不能满足临床需要,导致植骨失败。这是因为人工骨(直径>5mm)植入人体后,人工骨中心部位营养物质供应不足,氧分压低,不能满足人工骨中心部位的种子细胞生长、增殖、分化和成骨的需要,导致种子细胞停止生长甚至死亡,使成骨效应丧失,其根本原因在于组织工程骨骨诱导效应不强；另外,人工骨的成骨效应欠佳也是制约人工骨发展的一个重要原因。为此,国内外研究了三种方法解决人工骨的骨化活性：一种是采用联合细胞培养,将培养的细胞与人工骨复合,将不同类型的细胞进行混合培养,通过细胞间存在着精细的相互调控关系来促进细胞的生长分化。常用来培养的细胞有成纤维细胞,平滑肌细胞和成骨细胞。联合细胞培养的优点是活细胞能促进成骨效应,但操作繁琐,培养周期长,临床不易于推广；另外一种方法就是采用显微外科手术来促进人工骨的骨诱导效应,目前主要有预购带血管蒂筋膜瓣包裹人工骨、带血管蒂肌瓣包裹人工骨。预购复合组织瓣具有血供可靠,人工骨存活能力强,抗感染能力强等优点,但缺点也很明显,就是先需行预购手术,增加了病人的痛苦；还有一种方法就是采用细胞生长因子来促进人工骨骨诱导效应,方法简便,临床易于推广。相对来说,采用细胞生长因子促进人工骨骨化活性具有显著地临床应用优势,已经成为该领域研究的热点,近期关于细胞生长因子促进人工骨骨诱导效应的文章发表在国际相关研究领域的顶尖杂志上。目前用于促进人工骨骨化生长因子有骨形态发生蛋白(bone morphogenetic protein,BMPs),血管内皮细胞生长因子(vascular endothelial growth factor,VEGF),转化生长因子-β(transforming growth factor-P,TGF-p)等。而对于骨诱导效应的研究,主要集中在BMPs上,目前研究表明BMPs具有明显的成骨效应。但是因半衰期短,容易代谢流失,不能维持足够的浓度。目前,通过缓释载体负载活性生长因子可有效地解决这一问题。
近年来,对负载活性生长因子缓释载体的研究正受到越来越广泛的重视和关注,从传统载体到各种载药微球载体,目标是延长生长因子的缓释时间,精确控制释放浓度,拓展其适用范围。但是,对于怎样利用缓释微球来调控活性细胞因子按照临床要求进行释放,以最大限度的促进人工骨成骨诱导活性,是需要解决的主要问题。近年来微球控释技术的发展和人工可降解材料及其复合材料的不断涌现,为活性细胞因子的可控缓释和组织工程化人工骨成骨诱导活性的提高提供了前所未有的机遇和条件。将微球控释系统应用于活性生长因子的缓释,为外源性生长因子的开发与利用开辟了新的研究空间。针对人工骨成骨诱导活性缓释微球的研究,国内外很多学者利用高分子合成与结构控制技术制备了骨形态发生蛋白缓释微球,取得良好的成骨效应,但是在微球结构控制、微球分子和生长因子之间的相互作用对生长因子释放过程的影响以及如何通过微球结构实现对生长因子释放过程的调控尚需要深入研究。特别是为了提高载生长因子复合微球促进成骨诱导活性,必须深入研究负载生长因子的聚合物微球促进骨化活性的影响因素,这些包括：微球种类、微球结构、生长因子种类以及微球和生长因子之间的相互作用机制、生长因子的释放过程控制等。
因此本项目构建了一种重组人骨形态发生蛋白的聚合物复合微球,取得了良好的骨诱导效应。由于性能优异的聚合物微球载体,是让细胞生长因子按照临床需要控释的关键。本项目选用壳聚糖(CS)为合成载体微球的模型聚合物,壳聚糖是天然多糖中唯一的碱性多糖,其分子2位上有游离氨基,通过醛氨缩合反应使之形成键桥固化微球,可将药物固定在其骨架中,其微球具有无毒、生物相容性好、生物可降解、控制药物释放、增加药物局部滞留、提高药物的生物利用度等特点。但是重组人骨形态发生蛋白-2/壳聚糖复合微球载药率较低(33.437±2.290μg/mg),究其原因,主要是重组人骨形态发生蛋白与壳聚糖之间的作用主要是静电作用,在微球的制备过程中,无法大量的结合。因此我们引入硫酸葡聚糖(DS)作为中间载体,硫酸葡聚糖与骨形态发生蛋白有肝素结合位点,能广泛结合,制备的硫酸葡聚糖／重组人骨形态发生蛋白-2/壳聚糖微球具有更好的载药率和稳定释放效应。目前,有关负载活性生长因子缓释载体的研究中,临床使用性能研究较多,而有关生长因子和载体之间相互作用机制研究较少,而这是决定活性生长因子缓释过程的主要因素。同时缓释体在促进成骨活性过程中的影响因素也需要系统研究。
有鉴于此,本项目采用壳聚糖和硫酸葡聚糖制备单一和复合微球,利用共振光散射光谱和荧光光谱法研究生长因子(重组人骨形态发生蛋白-2)和微球载体聚合物(壳聚糖、硫酸葡聚糖)之间的相互作用机理及其对重组人骨形态发生蛋白-2释放过程的影响,阐明重组人骨形态发生蛋白-2释放过程中的热力学和动力学控制过程及复合微球缓释机制；研究微球粒子形态结构、粒子尺寸及分布控制方法以及他们对包裹药物的负载率及释放过程的影响,揭示载药载体的功能化形态结构对重组人骨形态发生蛋白-2释放过程的调控；并通过细胞实验和动物实验,研究硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖复合微球释放过程及其骨诱导效应。本项目的研究成果对骨缺损的人工修复提供新思路和理论指导。
研究目的：
1.制备壳聚糖/硫酸葡聚糖纳米微球,对硫酸葡聚糖/壳聚糖纳米微球的聚电解质复合过程进行研究,探讨了pH值、离子浓度等因素对壳聚糖、硫酸葡聚糖之间的相互作用的影响,分析载蛋白空白微球的径粒范围,为制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖微球提供理论依据。
2.制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球,观察微球的形态,并对微球的径粒、分散度进行研究,计算蛋白包封率、载药率,绘制缓释曲线。
3.对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的聚电解质复合过程进行研究,探讨了pH值、离子浓度等因素对重组人骨形态发生蛋白-2、壳聚糖、硫酸葡聚糖之间的相互作用的影响,结合缓释曲线,分析缓释机制。
4.评估硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的生物安全性,为硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的临床应用及骨诱导活性研究提供理论依据。
5.设计细胞学实验,评估硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球体外对骨髓间充质干细胞的成骨诱导活性,并与空白微球组、游离重组人骨形态发生蛋白-2组组进行对比,从细胞学的角度评估两种微球成骨效应的异同。
6.统计学处理：计量资料的统计描述用均数±标准差表示,运用SPSS13.0统计软件进行分析,组间比较用单因素方差分析,方差齐时组间多重比较用LSD-t检验法,若方差不齐则应用Games-Howell法。P<0.05认为差异有统计学意义。
研究方法：
1.应用离子交联法制备壳聚糖/硫酸葡聚糖纳米微球,采用共振光散射法研究pH值、离子浓度等因素对硫酸葡聚糖、壳聚糖之间的相互作用的影响,应用扫描电镜和Zeta电位分析载蛋白空白微球的径粒范围,为复和硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖微球提供理论依据。
2.应用离子交联法制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球,采用扫描电镜和原子力显微镜观察微球的形态,并对微球的径粒、分散度进行研究,应用重组人骨形态发生蛋白-2试剂盒计算蛋白包封率、载药率,绘制缓释曲线。
3.采用共振光散射法对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的聚电解质复合过程进行研究,应用共振光散射法探讨pH值、离子浓度等因素对重组人骨形态发生蛋白-2、壳聚糖、硫酸葡聚糖之间的相互作用的影响,结合缓释曲线,分析缓释机制。
4.按照国家标准植入材料的生物安全评估实验对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的生物毒性进行评估,复苏并传代培养鼠成纤维细胞,进行硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球和硫酸葡聚糖/壳聚糖空白微球的细胞毒性实验,设置对照组,对其生物安全性进行评估。
5.设计细胞学实验,体外传代培养SD大鼠的骨髓间充质干细胞,加入硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球、硫酸葡聚糖/壳聚糖空白微球、重组人骨形态发生蛋白-2,通过MTT检测法研究硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球体外对骨髓间充质干细胞的成骨诱导活性,并与空白微球组、重组人骨形态发生蛋白-2组、阴性对照组进行对比分析。
研究结果：
1.应用离子交联法制备硫酸葡聚糖纳/聚糖纳米微球,扫描电镜和原子力显微镜下提示微球成球良好,形态规整,分散度好,平均径粒为210nm,硫酸葡聚糖/壳聚糖纳米微球复合溶液的RLS强度在外界条件刺激如改变溶液pH和添加金属离子时会发生急剧的变化。在常温状态下,硫酸葡聚糖纳和壳聚糖可结合为聚电解质复合物。
2.应用离子交联法成功制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖微球,CS/DS/rh-BMP-2纳米微球分布均匀,平均粒径为(217±8)nm,微球包封率和载药量分别为(85.6±3)%和(47.245±3.321)ug/mg。微球体外释放在2小时后出现一个突释放期,2天后释放度达高峰,之后缓慢下降,释放周期约28天。
3.应用共振光散射的方法研究CS、DSS、与rhBMP-2之间的关系,结果表明,CS与rhBMP-2之间的相互作用弱,主要作用力为范德华力,DSS与rhBMP-2之间存在肝素结合位点,有较强的相互作用。在制备的过程中,rhBMP-2与DSS优先结合,而在缓释过程中,与CS结合的rhBMP-2会优先释放。
4.按照国家标准植入材料的生物安全评估实验对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的生物毒性进行评估,复苏并传代培养鼠成纤维细胞,细胞毒性试验提示硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖生物相容性好,无明显细胞毒性。壳聚糖/硫酸葡聚糖纳空白微球载体的生物相容性可靠,无细胞毒性,是良好的蛋白载体。
5.体外传代培养SD大鼠的骨髓间充质干细胞,加入硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球、壳聚糖/硫酸葡聚糖纳空白微球、重组人骨形态发生蛋白-2进行培养,发现硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球体和游离重组人骨形态发生蛋白在实验浓度下对骨髓间充质干细胞的增殖效应促进不明显。对骨髓间充质干细胞的分化效应有明显的促进作用,培养3d内,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球提高骨髓间充质干细胞ALP含量的作用低于游离重组人骨形态发生蛋白-2,但5d后,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球有效控制重组人骨形态发生蛋白-2的释放,使得硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球促进骨髓间充质干细胞的分化的效应明显强于游离重组人骨形态发生蛋白-2组,有统计学意义(P<0.01)。光学显微镜下观察钙结节的生长情况发现7d后,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖组的钙结节生长情况明显好于游离重组人骨形态发生蛋白-2组,促进骨髓间充质干细胞分化作用明显。
结论
应用离子交联法制备硫酸葡聚糖纳/壳聚糖纳米微球,微球成球良好,形态规整,分散度好,并表现出了良好的缓释性能。利用共振光散射光谱和荧光光谱法研究生长因子(重组人骨形态发生蛋白-2)和微球载体聚合物(壳聚糖、硫酸葡聚糖)之间的相互作用机理及其对重组人骨形态发生蛋白-2释放过程的影响,阐明重组人骨形态发生蛋白-2释放过程中的热力学和动力学控制过程及复合微球缓释机制；研究微球粒子形态结构、粒子尺寸及分布控制方法以及他们对包裹药物的负载率及释放过程的影响,揭示载药载体的功能化形态结构对重组人骨形态发生蛋白-2释放过程的调控；并通过体外细胞毒性实验和细胞增殖分化实验证实,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖复合微球骨诱导效应满意。本项目的研究成果对骨缺损的人工修复提供新思路和理论指导。
Background
Bone defects have long been plagued by orthopedics in clinical effective treatment. Bone tumors, severe bone trauma may cause bone defects, bone repair materials have been invented by many scholars, including autologous bone, allograft bone and artificial bone. Since autologous bone can be resorpted quickly and obtain perfect clinical effect, It is limited by the source of the problem; Allogeneic bone, the xenograft are limited bone due to immune rejection in the clinical application, and the risk of infection is also one of the factors in restricting the development Artificial bone with have drawn wide, easy processing, good biocompatibility, low price, can be disinfected, conducive to clinical operation, etc., become the mainstream of the current artificial bone as repair materials.
The artificial bone mainly includes two types of inorganic and organic materials. The hydroxyapatite (hydroxylapatite, HA) and tricalcium phosphate (tricalcium phosphate, TCP) represent the former and the representative of the latter are polylactic acid, polyglycolic acid or bothby a copolymer. With respect to the organic material, Inorganic materials have good mechanical strength, in which coralline hydroxyapatite (Coralline Hydroxyapatite, CHA) has been paid great attention due to the wide variety of sources, biocompatibility, bone conduction, harmless to human body. Despite the artificial bone has been used in clinical and achieved good results, but the implanted artificial bone often can not be absorbed by the body, resulting in bone graft failure. This is because the artificial bone (diameter>5mm) implanted in the human body, lack of artificial center of the supply of nutrients and low oxygen partial pressure, can not meet the artificial center of the seed cell growth, proliferation, differentiation, and osteogenic need, resulting in the seed cells stop growing or even death, so that fundamental reason about loss of osteoblasts effect lies in the weak inductive effect of tissue engineering bone. In addition, poor bone osteogenic effect is also an important reason for constraining the development of artificial bone. To this end, three ways are used to solve the artificial bone ossification activity by the domestic and foreign researchers. Firstly, composition of the cultured cells and artificial bone by culturing different types of cells all together.The different types of cells in the mixed culture, can promote cell growth and differentiation by the mutual regulatory relationships among cells. Youcheng fibroblasts, smooth muscle cells, and osteoblasts are commonly used to cultured in a mixed state. The living cells which can promote osteogenic effect are advantages of the joint cell culture, but the operation is cumbersome, long culture period, not easy to promote in clinical; Another approach which can promote artificial bone inductive effect is micro-surgery, prefabricated fascia and muscle flap with vascular pedicle are mainly used to wrap bone for gaining adequate blood supply, New composite flap have many advantages such as blood for reliable, bone viability, resistance to infection,but the disadvantages are also obvious, pre-surgery would be required to increase the suffering of the patients. Another method is to use a cell growth factor to promote the artificial bone induction effect, the method is simple, easy to promote in clinical. Relatively speaking, cell growth factor, due to promote the activity of bone ossification significantly in clinical application, has become a hot spot of research in this field, articles on cell growth factor promotes the artificial bone induction effect have been published on top magazines in this field. The growth factors such as bone morphogenetic protein (bone morphogenetic protein, BMPs), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), and transforming growth factor-β (transforming growth factor-β, TGF-P) are currently used to promote bone ossification. For the study of bone induction effect, mainly concentrated in the BMPs, the present study shows that the BMPs have a significant osteogenic effect. However, due to the short half-life, easy metabolic loss, BMPs can not maintain a sufficient concentration. Currently, load the active growth factor through the sustained release carrier can effectively solve this problem.
In recent years, the study focused on sustained release carrier load active growth factor has being gained more and more attention and concern. From traditional carriers to a variety of drug-loaded microspheres carrier, the goal is to extend the sustained release of the growth factor, precisely control the release concentration, and expand its scope of application. But how to regulate the activity of cytokine release in accordance with the clinical requirements by preparing different sustained release microspheres, in order to maximize the promotion of artificial bone into bone-inducing activity and vascular activity, are the main issues that need to be addressed. The rapid development of controlled release technology and continued emergence of artificial biodegradable materials and their composites in recent years, provides an unprecedented opportunities and conditions for the controlled release of active cytokines and tissue-engineered artificial bone to promote osteoinductive activity. Microspheres controlled release system used in sustained release of the active growth factor, has opened a new research space for the development and utilization of exogenous growth factors. For the research on osteogenic inducing activity of sustained-release microspheres, many scholars at home and abroad have prepared sustained-release microspheres loaded the bone morphogenetic protein by the polymer synthesis and structure control technology, and the osteogenic effect is encouraging. However, the research on controlling the structure of sustained-release microspheres is not yet perfect. At the same time, how the interactions between microspheres molecules and growth factors affect the release process, how to regulate growth factor release by changing the structure of the microspheres, still need in-depth study. Specially, in order to improve the bone-inducing activity of composite microspheres loaded growth factor, the activity factors on how the microspheres promote ossification need to research deeply. The influencing factors include types of microspheres, the structure of the microspheres, the type of growth factor, and the interaction mechanism between the microspheres and growth factors, and the release process control on polymer microspheres load growth factors.
The project has constructed a kind of polymer composite microspheres loaded recombinant human bone morphogenetic protein and has gained a good bone inductive effect. Because the performance of a carrier which is a polymer microsphere, is the key to allow cell growth factor controlled release according to clinical need. Chitosan (CS) of the item was selected as polymer microspheres for the synthesis support model. Chitosan is a natural polysaccharide, only basic polysaccharide in its molecule two free amino groups, the condensation reaction of the aldehyde-ammonia to form keybridge curing microspheres, the drug can be fixed in the skeleton. The microspheres having advantages such as non-toxic, good biocompatibility, and biodegradable, controlled drug release, improvement of the local retention of the drugs, addition of the bioavailability of the drug and other characteristics. The drug loading rate of recombinant human bone morphogenetic protein-2/Chitosan Microspheres is relatively low (33.437±2.290μg/mg).The reason may be only electrostatic interaction occur between the recombinant human bone morphogenetic protein and chitosan during the preparation of the microspheres, As a result,they could not bind together in a large number. Because dextran there are some heparin binding sites among sulfate and bone morphogenetic protein therefore, we have introduced dextran sulfate (DS) into microsphere as an intermediate carrier, and we have prepared recombinant human bone morphogenetic protein-2/chitosan/sulfuric acid Portugalpolysaccharide microspheres and gained high loading efficiency and stability release effect. Currently, more studies focus on sustained-release carrier loaded active growth factor and the clinical performance of these microspheres, but research on the mechanism of interaction between the relevant growth factor and carrier remains few, which is the main factors to determine the release process of the active growth factor. The influencing factors about Sustained-release body in the process of bone activity in promoting bone also need a system study.
For this reason, the project has prepared single and composite microspheres with chitosan and dextran sulfate preparation. We have studied the interactions between growth factor (recombinant human bone morphogenetic protein-2) and the carrier polymer microspheres (the interaction mechanism between chitosan and dextran sulfate) using the resonance light scattering spectroscopy and fluorescence spectroscopy. At the same time, we have also and its recombinant human bone morphogenetic protein-2release process occurs clarify recombinant human bone morphogenetic protein-2release in thermodynamic and kinetic control process and compositemicrosphere sustained release mechanism; We also have explored the interactions between recombinant human bone morphogenetic protein-2and blank microspheres in the release process, furthermore, we have stated the thermodynamic and kinetic control process of recombinant human bone morphogenetic protein-2in the release process, and revealed the sustained release mechanism of the composite microsphere; We have studied the morphology, particle size and distribution control method of single and composite microsphere particles, as well as their impact on the the wrapped load rate of protein and release process. We have also revealed the regulation of the carrier morphology on controlling release of recombinant human bone morphogenetic protein-2; At the same time, we have studied the release process and its osteoinductive effect of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres. The research results of the project would provide new ideas and theoretical guidance manual repair of bone defects.
Objectives:
1. Preparation nanosphere of chitosan/dextran sulfate, study the polyelectrolyte complex process of dextran sulfate/chitosan nanospheres, investegate the interaction effect of the pH value, ion concentration of chitosan, sulfate dextranimpact, analysis of the interaction between the sugar contained protein blank microsphere diameter grain, provide a theoretical basis for the preparation of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.
2. Preparation of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres, observe morphology of the microspheres and microspheres. Drive tablets the dispersity of study, to calculate the protein encapsulation efficiency, drug loading ratedraw the sustained-release curve.
3. Study polyelectrolyte complex process of Dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, studies of the pH value, ion concentration of recombinant human bone morphogenetic protein-2, chitosanthe influence of the interaction between the sugars, dextran sulfate combined release curves, analysis of the sustained-release mechanism.
4. Assess Biological safety of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres, provide a theoretical basis for clinical applications and Osteoinductive activity studies of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.
5. Design cytology experiments, evaluate induction of osteogenic activity of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres in vitro on bone marrow mesenchymal stem cells, control with blank microsphere group, free of recombinant human bone morphogeneticprotein-2group, the groups were compared to assess similarities and differences of the two microspheres osteogenic effect, from the perspective of cytology.
6. Statistical analysis:statistical description of the measurement data are expressed as mean±standard deviation, using SPSS13.0statistical software for analysis, the groups were compared using one-way ANOVA, multiple comparisons with LSD-t test method homogeneity of variance between the two groups,If heterogeneity of variance applications Games-Howell method. P<0.05was considered statistically significant.
Methods:
1. Prepare chitosan/dextran sulfate Nanosphere by the ionic crosslinking, study the pH and ion concentration on the interaction between dextran sulfate, chitosan, by resonance light scattering method, analyze contained protein blank microsphere diameter grain range by scane electron microscopy and Zeta potential, provide a theoretical basis for complex and dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.
2. Prepare dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres by ionic crosslinking, observe microspheres morphology using scanning electron microscopy and atomic force microscopy, study microsphere diameter grain and dispersitystudy of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres. Calculate protein encapsulation efficiency, drug loading efficiency, draw sustained-release curve recombinant human bone morphogenetic protein-2kit.
3. Study complex poly electrolyte process of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres by Resonance light. To explore the pH, ion concentration of recombinantthe human bone morphogenetic protein-2, chitosan, dextran sulfate interaction between the combination of sustained-release curve analysis of slow-release mechanism in the application of resonance light scattering method.
4. Assess biosafety of dextran sulfate/recombinant human bone morphogenetic protein-2/the chitosan nanospheres by experimental implant materials in accordance with the national standards biological toxicity assessment, recovery and subcultured rat fibroblasts sulfatedthe glucan/recombinant human bone morphogenetic protein-2/chitosan microspheres and dextran sulfate/chitosan blank microspheres cytotoxic experiment, a control group to assess their biological security.
5. Design cytological experiments, to culture SD rat bone marrow mesenchymal stem cells in vitro by adding dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, dextran sulfate/chitosan blankmicrospheres, recombinant human bone morphogenetic protein-2by the MTT assay dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres in vitro bone marrow mesenchymal stem cells into bone-inducing activity, and blank microsphere group, recombinant human bone morphogenetic protein-2group, negative control group were analyzed.
The results:
1. Prepare dextran sulfate satisfied/chitosan microspheres in application of ionic crosslinking method, observe microspheres in scanning electron microscopy and atomic force microscopy, discover microsphere which is prompted into a ball, in a regular shape, at a good dispersity, average grain diameter about210nm, the sugar/chitosan nanospheres composite solution RLS intensity will change very rapidly by changing sulfuric acid glucan outside unconditioned stimulus, such as changing the pH of the solution, and the addition of metal ions,. State at room temperature, dextran sulfate sodium and chitosan can be combined in shape of a polyelectrolytecomplexes.
2. Successfully prepare dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres in application of ionic crosslinking method. CS/DS/rh-BMP-2nanospheres evenly distributed, with an average particle size (217±8) nm, entrapment efficiency and drug loading of the microspheres were (85.6±3)%and47.245±3.321ug/mg. Microspheres release two hours after the a sudden release period in vitro, two days after the release reached a peak, followed by a slow decline in the release cycle is about28days.
3. To study CS, DSS, and the relationship between the rhBMP-2in application of resonance light scattering method, the results show that the weak interaction between CS rhBMP-2, the main force for the van der Waals force, DSS with rhBMP-2exists between the heparin binding site, there is a strong interaction. In the process of preparation, rhBMP-2with DSS first combination in the sustained-release process, in combination with CS rhBMP-2will be preferentially released.
4. Assess biosafety of experimental implant materials in accordance with the national standards on dextran sulfate/recombinant human bone morphogenetic protein-2/the chitosan nanospheres, recovery and subcultured rat fibroblast cells, cytotoxicThe test indicated that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan biocompatible, no significant cytotoxicity. Biocompatibility of chitosan/sodium dextran sulfate blank microspheres carrier reliable, non-cytotoxic is good protein carrier.
5. culture SD rat bone marrow mesenchymal stem cells In vitro, add dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, chitosan/dextran sulfate satisfied blank microspheres, restructuring cultivation of human bone morphogenetic protein-2, and found that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres and free recombinant human bone morphogenetic protein in the experimental concentration could not promote effect on the proliferation of bone marrow mesenchymal stem cells obviously. Sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres could promote the differentiation of mesenchymal stem cells significantly. In the bone marrow effects cultivate3d Sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres improve the role of bone marrow-derived mesenchymal stem cells ALP content below free recombinant human bone morphogenetic protein-2, but after5d, dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres effective control of recombinant human bone morphogenetic protein-2release, so that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres could promote the differentiation of bone marrow mesenchymal stem cells.It was significantly stronger than free recombinant human bone morphogenetic protein-2group, there was significant difference (P<0.01). To observe the growth of calcium nodules on optical microscope found7d, dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan calcium nodules growth significantly better than free recombinant human bone morphogenetic protein-2group, promote bone marrow obvious role of mesenchymal stem cell differentiation.
Conclusions:
we have prepared by dextran sulfate satisfied/chitosan microspheres in application of ionic crosslinking method. The microspheres have many advantages such as into a ball, regular shape, disperse well, a good release properties. At the same time, we have also studied recombinant human bone morphogenetic protein-2release process occurs clarify recombinant human bone morphogenetic protein-2release in thermodynamic and kinetic control process and compositemicrosphere sustained release mechanism; We also have explored the interactions between recombinant human bone morphogenetic protein-2and blank microspheres in the release process, furthermore, we have stated the thermodynamic and kinetic control process of recombinant human bone morphogenetic protein-2in the release process, and revealed the sustained release mechanism of the composite microsphere; We have studied the morphology, particle size and distribution control method of single and composite microsphere particles, as well as their impact on the the wrapped load rate of protein and release process. We have also revealed the regulation of the carrier morphology on controlling release of recombinant human bone morphogenetic protein-2; At the same time, we have studied the release process and its osteoinductive effect of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres. The research results of the project would provide new ideas and theoretical guidance manual repair of bone defects.
人工骨修复材料主要包括无机材料和有机材料两类,前者以羟基磷灰石(hydroxylapatite,HA)和磷酸三钙(tricalcium phosphate,TCP)为代表,后者则以聚乳酸、聚乙醇酸或两者的共聚物为代表。无机材料相对于有机材料,机械强度好,其中珊瑚羟基磷灰石(Coralline Hydroxyapatite, CHA)更是因来源广泛、生物相容性好、骨传导作用好、对人体无害等特点而受到临床应用的青睐。尽管人工骨已经应用于临床并取得了较好的效果,但是植入体内的人工骨往往不能满足临床需要,导致植骨失败。这是因为人工骨(直径>5mm)植入人体后,人工骨中心部位营养物质供应不足,氧分压低,不能满足人工骨中心部位的种子细胞生长、增殖、分化和成骨的需要,导致种子细胞停止生长甚至死亡,使成骨效应丧失,其根本原因在于组织工程骨骨诱导效应不强；另外,人工骨的成骨效应欠佳也是制约人工骨发展的一个重要原因。为此,国内外研究了三种方法解决人工骨的骨化活性：一种是采用联合细胞培养,将培养的细胞与人工骨复合,将不同类型的细胞进行混合培养,通过细胞间存在着精细的相互调控关系来促进细胞的生长分化。常用来培养的细胞有成纤维细胞,平滑肌细胞和成骨细胞。联合细胞培养的优点是活细胞能促进成骨效应,但操作繁琐,培养周期长,临床不易于推广；另外一种方法就是采用显微外科手术来促进人工骨的骨诱导效应,目前主要有预购带血管蒂筋膜瓣包裹人工骨、带血管蒂肌瓣包裹人工骨。预购复合组织瓣具有血供可靠,人工骨存活能力强,抗感染能力强等优点,但缺点也很明显,就是先需行预购手术,增加了病人的痛苦；还有一种方法就是采用细胞生长因子来促进人工骨骨诱导效应,方法简便,临床易于推广。相对来说,采用细胞生长因子促进人工骨骨化活性具有显著地临床应用优势,已经成为该领域研究的热点,近期关于细胞生长因子促进人工骨骨诱导效应的文章发表在国际相关研究领域的顶尖杂志上。目前用于促进人工骨骨化生长因子有骨形态发生蛋白(bone morphogenetic protein,BMPs),血管内皮细胞生长因子(vascular endothelial growth factor,VEGF),转化生长因子-β(transforming growth factor-P,TGF-p)等。而对于骨诱导效应的研究,主要集中在BMPs上,目前研究表明BMPs具有明显的成骨效应。但是因半衰期短,容易代谢流失,不能维持足够的浓度。目前,通过缓释载体负载活性生长因子可有效地解决这一问题。
近年来,对负载活性生长因子缓释载体的研究正受到越来越广泛的重视和关注,从传统载体到各种载药微球载体,目标是延长生长因子的缓释时间,精确控制释放浓度,拓展其适用范围。但是,对于怎样利用缓释微球来调控活性细胞因子按照临床要求进行释放,以最大限度的促进人工骨成骨诱导活性,是需要解决的主要问题。近年来微球控释技术的发展和人工可降解材料及其复合材料的不断涌现,为活性细胞因子的可控缓释和组织工程化人工骨成骨诱导活性的提高提供了前所未有的机遇和条件。将微球控释系统应用于活性生长因子的缓释,为外源性生长因子的开发与利用开辟了新的研究空间。针对人工骨成骨诱导活性缓释微球的研究,国内外很多学者利用高分子合成与结构控制技术制备了骨形态发生蛋白缓释微球,取得良好的成骨效应,但是在微球结构控制、微球分子和生长因子之间的相互作用对生长因子释放过程的影响以及如何通过微球结构实现对生长因子释放过程的调控尚需要深入研究。特别是为了提高载生长因子复合微球促进成骨诱导活性,必须深入研究负载生长因子的聚合物微球促进骨化活性的影响因素,这些包括：微球种类、微球结构、生长因子种类以及微球和生长因子之间的相互作用机制、生长因子的释放过程控制等。
因此本项目构建了一种重组人骨形态发生蛋白的聚合物复合微球,取得了良好的骨诱导效应。由于性能优异的聚合物微球载体,是让细胞生长因子按照临床需要控释的关键。本项目选用壳聚糖(CS)为合成载体微球的模型聚合物,壳聚糖是天然多糖中唯一的碱性多糖,其分子2位上有游离氨基,通过醛氨缩合反应使之形成键桥固化微球,可将药物固定在其骨架中,其微球具有无毒、生物相容性好、生物可降解、控制药物释放、增加药物局部滞留、提高药物的生物利用度等特点。但是重组人骨形态发生蛋白-2/壳聚糖复合微球载药率较低(33.437±2.290μg/mg),究其原因,主要是重组人骨形态发生蛋白与壳聚糖之间的作用主要是静电作用,在微球的制备过程中,无法大量的结合。因此我们引入硫酸葡聚糖(DS)作为中间载体,硫酸葡聚糖与骨形态发生蛋白有肝素结合位点,能广泛结合,制备的硫酸葡聚糖／重组人骨形态发生蛋白-2/壳聚糖微球具有更好的载药率和稳定释放效应。目前,有关负载活性生长因子缓释载体的研究中,临床使用性能研究较多,而有关生长因子和载体之间相互作用机制研究较少,而这是决定活性生长因子缓释过程的主要因素。同时缓释体在促进成骨活性过程中的影响因素也需要系统研究。
有鉴于此,本项目采用壳聚糖和硫酸葡聚糖制备单一和复合微球,利用共振光散射光谱和荧光光谱法研究生长因子(重组人骨形态发生蛋白-2)和微球载体聚合物(壳聚糖、硫酸葡聚糖)之间的相互作用机理及其对重组人骨形态发生蛋白-2释放过程的影响,阐明重组人骨形态发生蛋白-2释放过程中的热力学和动力学控制过程及复合微球缓释机制；研究微球粒子形态结构、粒子尺寸及分布控制方法以及他们对包裹药物的负载率及释放过程的影响,揭示载药载体的功能化形态结构对重组人骨形态发生蛋白-2释放过程的调控；并通过细胞实验和动物实验,研究硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖复合微球释放过程及其骨诱导效应。本项目的研究成果对骨缺损的人工修复提供新思路和理论指导。
研究目的：
1.制备壳聚糖/硫酸葡聚糖纳米微球,对硫酸葡聚糖/壳聚糖纳米微球的聚电解质复合过程进行研究,探讨了pH值、离子浓度等因素对壳聚糖、硫酸葡聚糖之间的相互作用的影响,分析载蛋白空白微球的径粒范围,为制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖微球提供理论依据。
2.制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球,观察微球的形态,并对微球的径粒、分散度进行研究,计算蛋白包封率、载药率,绘制缓释曲线。
3.对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的聚电解质复合过程进行研究,探讨了pH值、离子浓度等因素对重组人骨形态发生蛋白-2、壳聚糖、硫酸葡聚糖之间的相互作用的影响,结合缓释曲线,分析缓释机制。
4.评估硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的生物安全性,为硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的临床应用及骨诱导活性研究提供理论依据。
5.设计细胞学实验,评估硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球体外对骨髓间充质干细胞的成骨诱导活性,并与空白微球组、游离重组人骨形态发生蛋白-2组组进行对比,从细胞学的角度评估两种微球成骨效应的异同。
6.统计学处理：计量资料的统计描述用均数±标准差表示,运用SPSS13.0统计软件进行分析,组间比较用单因素方差分析,方差齐时组间多重比较用LSD-t检验法,若方差不齐则应用Games-Howell法。P<0.05认为差异有统计学意义。
研究方法：
1.应用离子交联法制备壳聚糖/硫酸葡聚糖纳米微球,采用共振光散射法研究pH值、离子浓度等因素对硫酸葡聚糖、壳聚糖之间的相互作用的影响,应用扫描电镜和Zeta电位分析载蛋白空白微球的径粒范围,为复和硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖微球提供理论依据。
2.应用离子交联法制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球,采用扫描电镜和原子力显微镜观察微球的形态,并对微球的径粒、分散度进行研究,应用重组人骨形态发生蛋白-2试剂盒计算蛋白包封率、载药率,绘制缓释曲线。
3.采用共振光散射法对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的聚电解质复合过程进行研究,应用共振光散射法探讨pH值、离子浓度等因素对重组人骨形态发生蛋白-2、壳聚糖、硫酸葡聚糖之间的相互作用的影响,结合缓释曲线,分析缓释机制。
4.按照国家标准植入材料的生物安全评估实验对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的生物毒性进行评估,复苏并传代培养鼠成纤维细胞,进行硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球和硫酸葡聚糖/壳聚糖空白微球的细胞毒性实验,设置对照组,对其生物安全性进行评估。
5.设计细胞学实验,体外传代培养SD大鼠的骨髓间充质干细胞,加入硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球、硫酸葡聚糖/壳聚糖空白微球、重组人骨形态发生蛋白-2,通过MTT检测法研究硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球体外对骨髓间充质干细胞的成骨诱导活性,并与空白微球组、重组人骨形态发生蛋白-2组、阴性对照组进行对比分析。
研究结果：
1.应用离子交联法制备硫酸葡聚糖纳/聚糖纳米微球,扫描电镜和原子力显微镜下提示微球成球良好,形态规整,分散度好,平均径粒为210nm,硫酸葡聚糖/壳聚糖纳米微球复合溶液的RLS强度在外界条件刺激如改变溶液pH和添加金属离子时会发生急剧的变化。在常温状态下,硫酸葡聚糖纳和壳聚糖可结合为聚电解质复合物。
2.应用离子交联法成功制备硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖微球,CS/DS/rh-BMP-2纳米微球分布均匀,平均粒径为(217±8)nm,微球包封率和载药量分别为(85.6±3)%和(47.245±3.321)ug/mg。微球体外释放在2小时后出现一个突释放期,2天后释放度达高峰,之后缓慢下降,释放周期约28天。
3.应用共振光散射的方法研究CS、DSS、与rhBMP-2之间的关系,结果表明,CS与rhBMP-2之间的相互作用弱,主要作用力为范德华力,DSS与rhBMP-2之间存在肝素结合位点,有较强的相互作用。在制备的过程中,rhBMP-2与DSS优先结合,而在缓释过程中,与CS结合的rhBMP-2会优先释放。
4.按照国家标准植入材料的生物安全评估实验对硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球的生物毒性进行评估,复苏并传代培养鼠成纤维细胞,细胞毒性试验提示硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖生物相容性好,无明显细胞毒性。壳聚糖/硫酸葡聚糖纳空白微球载体的生物相容性可靠,无细胞毒性,是良好的蛋白载体。
5.体外传代培养SD大鼠的骨髓间充质干细胞,加入硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球、壳聚糖/硫酸葡聚糖纳空白微球、重组人骨形态发生蛋白-2进行培养,发现硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球体和游离重组人骨形态发生蛋白在实验浓度下对骨髓间充质干细胞的增殖效应促进不明显。对骨髓间充质干细胞的分化效应有明显的促进作用,培养3d内,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球提高骨髓间充质干细胞ALP含量的作用低于游离重组人骨形态发生蛋白-2,但5d后,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球有效控制重组人骨形态发生蛋白-2的释放,使得硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖纳米微球促进骨髓间充质干细胞的分化的效应明显强于游离重组人骨形态发生蛋白-2组,有统计学意义(P<0.01)。光学显微镜下观察钙结节的生长情况发现7d后,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖组的钙结节生长情况明显好于游离重组人骨形态发生蛋白-2组,促进骨髓间充质干细胞分化作用明显。
结论
应用离子交联法制备硫酸葡聚糖纳/壳聚糖纳米微球,微球成球良好,形态规整,分散度好,并表现出了良好的缓释性能。利用共振光散射光谱和荧光光谱法研究生长因子(重组人骨形态发生蛋白-2)和微球载体聚合物(壳聚糖、硫酸葡聚糖)之间的相互作用机理及其对重组人骨形态发生蛋白-2释放过程的影响,阐明重组人骨形态发生蛋白-2释放过程中的热力学和动力学控制过程及复合微球缓释机制；研究微球粒子形态结构、粒子尺寸及分布控制方法以及他们对包裹药物的负载率及释放过程的影响,揭示载药载体的功能化形态结构对重组人骨形态发生蛋白-2释放过程的调控；并通过体外细胞毒性实验和细胞增殖分化实验证实,硫酸葡聚糖/重组人骨形态发生蛋白-2/壳聚糖复合微球骨诱导效应满意。本项目的研究成果对骨缺损的人工修复提供新思路和理论指导。
Background
Bone defects have long been plagued by orthopedics in clinical effective treatment. Bone tumors, severe bone trauma may cause bone defects, bone repair materials have been invented by many scholars, including autologous bone, allograft bone and artificial bone. Since autologous bone can be resorpted quickly and obtain perfect clinical effect, It is limited by the source of the problem; Allogeneic bone, the xenograft are limited bone due to immune rejection in the clinical application, and the risk of infection is also one of the factors in restricting the development Artificial bone with have drawn wide, easy processing, good biocompatibility, low price, can be disinfected, conducive to clinical operation, etc., become the mainstream of the current artificial bone as repair materials.
The artificial bone mainly includes two types of inorganic and organic materials. The hydroxyapatite (hydroxylapatite, HA) and tricalcium phosphate (tricalcium phosphate, TCP) represent the former and the representative of the latter are polylactic acid, polyglycolic acid or bothby a copolymer. With respect to the organic material, Inorganic materials have good mechanical strength, in which coralline hydroxyapatite (Coralline Hydroxyapatite, CHA) has been paid great attention due to the wide variety of sources, biocompatibility, bone conduction, harmless to human body. Despite the artificial bone has been used in clinical and achieved good results, but the implanted artificial bone often can not be absorbed by the body, resulting in bone graft failure. This is because the artificial bone (diameter>5mm) implanted in the human body, lack of artificial center of the supply of nutrients and low oxygen partial pressure, can not meet the artificial center of the seed cell growth, proliferation, differentiation, and osteogenic need, resulting in the seed cells stop growing or even death, so that fundamental reason about loss of osteoblasts effect lies in the weak inductive effect of tissue engineering bone. In addition, poor bone osteogenic effect is also an important reason for constraining the development of artificial bone. To this end, three ways are used to solve the artificial bone ossification activity by the domestic and foreign researchers. Firstly, composition of the cultured cells and artificial bone by culturing different types of cells all together.The different types of cells in the mixed culture, can promote cell growth and differentiation by the mutual regulatory relationships among cells. Youcheng fibroblasts, smooth muscle cells, and osteoblasts are commonly used to cultured in a mixed state. The living cells which can promote osteogenic effect are advantages of the joint cell culture, but the operation is cumbersome, long culture period, not easy to promote in clinical; Another approach which can promote artificial bone inductive effect is micro-surgery, prefabricated fascia and muscle flap with vascular pedicle are mainly used to wrap bone for gaining adequate blood supply, New composite flap have many advantages such as blood for reliable, bone viability, resistance to infection,but the disadvantages are also obvious, pre-surgery would be required to increase the suffering of the patients. Another method is to use a cell growth factor to promote the artificial bone induction effect, the method is simple, easy to promote in clinical. Relatively speaking, cell growth factor, due to promote the activity of bone ossification significantly in clinical application, has become a hot spot of research in this field, articles on cell growth factor promotes the artificial bone induction effect have been published on top magazines in this field. The growth factors such as bone morphogenetic protein (bone morphogenetic protein, BMPs), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), and transforming growth factor-β (transforming growth factor-β, TGF-P) are currently used to promote bone ossification. For the study of bone induction effect, mainly concentrated in the BMPs, the present study shows that the BMPs have a significant osteogenic effect. However, due to the short half-life, easy metabolic loss, BMPs can not maintain a sufficient concentration. Currently, load the active growth factor through the sustained release carrier can effectively solve this problem.
In recent years, the study focused on sustained release carrier load active growth factor has being gained more and more attention and concern. From traditional carriers to a variety of drug-loaded microspheres carrier, the goal is to extend the sustained release of the growth factor, precisely control the release concentration, and expand its scope of application. But how to regulate the activity of cytokine release in accordance with the clinical requirements by preparing different sustained release microspheres, in order to maximize the promotion of artificial bone into bone-inducing activity and vascular activity, are the main issues that need to be addressed. The rapid development of controlled release technology and continued emergence of artificial biodegradable materials and their composites in recent years, provides an unprecedented opportunities and conditions for the controlled release of active cytokines and tissue-engineered artificial bone to promote osteoinductive activity. Microspheres controlled release system used in sustained release of the active growth factor, has opened a new research space for the development and utilization of exogenous growth factors. For the research on osteogenic inducing activity of sustained-release microspheres, many scholars at home and abroad have prepared sustained-release microspheres loaded the bone morphogenetic protein by the polymer synthesis and structure control technology, and the osteogenic effect is encouraging. However, the research on controlling the structure of sustained-release microspheres is not yet perfect. At the same time, how the interactions between microspheres molecules and growth factors affect the release process, how to regulate growth factor release by changing the structure of the microspheres, still need in-depth study. Specially, in order to improve the bone-inducing activity of composite microspheres loaded growth factor, the activity factors on how the microspheres promote ossification need to research deeply. The influencing factors include types of microspheres, the structure of the microspheres, the type of growth factor, and the interaction mechanism between the microspheres and growth factors, and the release process control on polymer microspheres load growth factors.
The project has constructed a kind of polymer composite microspheres loaded recombinant human bone morphogenetic protein and has gained a good bone inductive effect. Because the performance of a carrier which is a polymer microsphere, is the key to allow cell growth factor controlled release according to clinical need. Chitosan (CS) of the item was selected as polymer microspheres for the synthesis support model. Chitosan is a natural polysaccharide, only basic polysaccharide in its molecule two free amino groups, the condensation reaction of the aldehyde-ammonia to form keybridge curing microspheres, the drug can be fixed in the skeleton. The microspheres having advantages such as non-toxic, good biocompatibility, and biodegradable, controlled drug release, improvement of the local retention of the drugs, addition of the bioavailability of the drug and other characteristics. The drug loading rate of recombinant human bone morphogenetic protein-2/Chitosan Microspheres is relatively low (33.437±2.290μg/mg).The reason may be only electrostatic interaction occur between the recombinant human bone morphogenetic protein and chitosan during the preparation of the microspheres, As a result,they could not bind together in a large number. Because dextran there are some heparin binding sites among sulfate and bone morphogenetic protein therefore, we have introduced dextran sulfate (DS) into microsphere as an intermediate carrier, and we have prepared recombinant human bone morphogenetic protein-2/chitosan/sulfuric acid Portugalpolysaccharide microspheres and gained high loading efficiency and stability release effect. Currently, more studies focus on sustained-release carrier loaded active growth factor and the clinical performance of these microspheres, but research on the mechanism of interaction between the relevant growth factor and carrier remains few, which is the main factors to determine the release process of the active growth factor. The influencing factors about Sustained-release body in the process of bone activity in promoting bone also need a system study.
For this reason, the project has prepared single and composite microspheres with chitosan and dextran sulfate preparation. We have studied the interactions between growth factor (recombinant human bone morphogenetic protein-2) and the carrier polymer microspheres (the interaction mechanism between chitosan and dextran sulfate) using the resonance light scattering spectroscopy and fluorescence spectroscopy. At the same time, we have also and its recombinant human bone morphogenetic protein-2release process occurs clarify recombinant human bone morphogenetic protein-2release in thermodynamic and kinetic control process and compositemicrosphere sustained release mechanism; We also have explored the interactions between recombinant human bone morphogenetic protein-2and blank microspheres in the release process, furthermore, we have stated the thermodynamic and kinetic control process of recombinant human bone morphogenetic protein-2in the release process, and revealed the sustained release mechanism of the composite microsphere; We have studied the morphology, particle size and distribution control method of single and composite microsphere particles, as well as their impact on the the wrapped load rate of protein and release process. We have also revealed the regulation of the carrier morphology on controlling release of recombinant human bone morphogenetic protein-2; At the same time, we have studied the release process and its osteoinductive effect of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres. The research results of the project would provide new ideas and theoretical guidance manual repair of bone defects.
Objectives:
1. Preparation nanosphere of chitosan/dextran sulfate, study the polyelectrolyte complex process of dextran sulfate/chitosan nanospheres, investegate the interaction effect of the pH value, ion concentration of chitosan, sulfate dextranimpact, analysis of the interaction between the sugar contained protein blank microsphere diameter grain, provide a theoretical basis for the preparation of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.
2. Preparation of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres, observe morphology of the microspheres and microspheres. Drive tablets the dispersity of study, to calculate the protein encapsulation efficiency, drug loading ratedraw the sustained-release curve.
3. Study polyelectrolyte complex process of Dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, studies of the pH value, ion concentration of recombinant human bone morphogenetic protein-2, chitosanthe influence of the interaction between the sugars, dextran sulfate combined release curves, analysis of the sustained-release mechanism.
4. Assess Biological safety of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres, provide a theoretical basis for clinical applications and Osteoinductive activity studies of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.
5. Design cytology experiments, evaluate induction of osteogenic activity of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres in vitro on bone marrow mesenchymal stem cells, control with blank microsphere group, free of recombinant human bone morphogeneticprotein-2group, the groups were compared to assess similarities and differences of the two microspheres osteogenic effect, from the perspective of cytology.
6. Statistical analysis:statistical description of the measurement data are expressed as mean±standard deviation, using SPSS13.0statistical software for analysis, the groups were compared using one-way ANOVA, multiple comparisons with LSD-t test method homogeneity of variance between the two groups,If heterogeneity of variance applications Games-Howell method. P<0.05was considered statistically significant.
Methods:
1. Prepare chitosan/dextran sulfate Nanosphere by the ionic crosslinking, study the pH and ion concentration on the interaction between dextran sulfate, chitosan, by resonance light scattering method, analyze contained protein blank microsphere diameter grain range by scane electron microscopy and Zeta potential, provide a theoretical basis for complex and dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.
2. Prepare dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres by ionic crosslinking, observe microspheres morphology using scanning electron microscopy and atomic force microscopy, study microsphere diameter grain and dispersitystudy of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres. Calculate protein encapsulation efficiency, drug loading efficiency, draw sustained-release curve recombinant human bone morphogenetic protein-2kit.
3. Study complex poly electrolyte process of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres by Resonance light. To explore the pH, ion concentration of recombinantthe human bone morphogenetic protein-2, chitosan, dextran sulfate interaction between the combination of sustained-release curve analysis of slow-release mechanism in the application of resonance light scattering method.
4. Assess biosafety of dextran sulfate/recombinant human bone morphogenetic protein-2/the chitosan nanospheres by experimental implant materials in accordance with the national standards biological toxicity assessment, recovery and subcultured rat fibroblasts sulfatedthe glucan/recombinant human bone morphogenetic protein-2/chitosan microspheres and dextran sulfate/chitosan blank microspheres cytotoxic experiment, a control group to assess their biological security.
5. Design cytological experiments, to culture SD rat bone marrow mesenchymal stem cells in vitro by adding dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, dextran sulfate/chitosan blankmicrospheres, recombinant human bone morphogenetic protein-2by the MTT assay dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres in vitro bone marrow mesenchymal stem cells into bone-inducing activity, and blank microsphere group, recombinant human bone morphogenetic protein-2group, negative control group were analyzed.
The results:
1. Prepare dextran sulfate satisfied/chitosan microspheres in application of ionic crosslinking method, observe microspheres in scanning electron microscopy and atomic force microscopy, discover microsphere which is prompted into a ball, in a regular shape, at a good dispersity, average grain diameter about210nm, the sugar/chitosan nanospheres composite solution RLS intensity will change very rapidly by changing sulfuric acid glucan outside unconditioned stimulus, such as changing the pH of the solution, and the addition of metal ions,. State at room temperature, dextran sulfate sodium and chitosan can be combined in shape of a polyelectrolytecomplexes.
2. Successfully prepare dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres in application of ionic crosslinking method. CS/DS/rh-BMP-2nanospheres evenly distributed, with an average particle size (217±8) nm, entrapment efficiency and drug loading of the microspheres were (85.6±3)%and47.245±3.321ug/mg. Microspheres release two hours after the a sudden release period in vitro, two days after the release reached a peak, followed by a slow decline in the release cycle is about28days.
3. To study CS, DSS, and the relationship between the rhBMP-2in application of resonance light scattering method, the results show that the weak interaction between CS rhBMP-2, the main force for the van der Waals force, DSS with rhBMP-2exists between the heparin binding site, there is a strong interaction. In the process of preparation, rhBMP-2with DSS first combination in the sustained-release process, in combination with CS rhBMP-2will be preferentially released.
4. Assess biosafety of experimental implant materials in accordance with the national standards on dextran sulfate/recombinant human bone morphogenetic protein-2/the chitosan nanospheres, recovery and subcultured rat fibroblast cells, cytotoxicThe test indicated that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan biocompatible, no significant cytotoxicity. Biocompatibility of chitosan/sodium dextran sulfate blank microspheres carrier reliable, non-cytotoxic is good protein carrier.
5. culture SD rat bone marrow mesenchymal stem cells In vitro, add dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, chitosan/dextran sulfate satisfied blank microspheres, restructuring cultivation of human bone morphogenetic protein-2, and found that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres and free recombinant human bone morphogenetic protein in the experimental concentration could not promote effect on the proliferation of bone marrow mesenchymal stem cells obviously. Sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres could promote the differentiation of mesenchymal stem cells significantly. In the bone marrow effects cultivate3d Sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres improve the role of bone marrow-derived mesenchymal stem cells ALP content below free recombinant human bone morphogenetic protein-2, but after5d, dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres effective control of recombinant human bone morphogenetic protein-2release, so that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres could promote the differentiation of bone marrow mesenchymal stem cells.It was significantly stronger than free recombinant human bone morphogenetic protein-2group, there was significant difference (P<0.01). To observe the growth of calcium nodules on optical microscope found7d, dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan calcium nodules growth significantly better than free recombinant human bone morphogenetic protein-2group, promote bone marrow obvious role of mesenchymal stem cell differentiation.
Conclusions:
we have prepared by dextran sulfate satisfied/chitosan microspheres in application of ionic crosslinking method. The microspheres have many advantages such as into a ball, regular shape, disperse well, a good release properties. At the same time, we have also studied recombinant human bone morphogenetic protein-2release process occurs clarify recombinant human bone morphogenetic protein-2release in thermodynamic and kinetic control process and compositemicrosphere sustained release mechanism; We also have explored the interactions between recombinant human bone morphogenetic protein-2and blank microspheres in the release process, furthermore, we have stated the thermodynamic and kinetic control process of recombinant human bone morphogenetic protein-2in the release process, and revealed the sustained release mechanism of the composite microsphere; We have studied the morphology, particle size and distribution control method of single and composite microsphere particles, as well as their impact on the the wrapped load rate of protein and release process. We have also revealed the regulation of the carrier morphology on controlling release of recombinant human bone morphogenetic protein-2; At the same time, we have studied the release process and its osteoinductive effect of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres. The research results of the project would provide new ideas and theoretical guidance manual repair of bone defects.
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[1]Martinez A, Arana P, Fernandez A, Olmo R, Teijon C, Blanco MD. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems.J Microencapsul.2013 Mar 14.
[2]杨强,彭江,卢世璧,等.软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究[J].中华骨科杂志,2011,31(8)：904-910.
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[4]Jun SH, Lee EJ, Jang TS, Kim HE, Jang JH, Koh YH.Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering.J Mater Sci Mater Med.2013 Mar;24(3):773-82.
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[9]鲁小云,郭常安,阎作勤,等.壳聚糖-明胶支架/滑膜干细胞的相容性[J].中华实验外科杂志,2011,28(7)：1026-1028.11.Palazzo B, Gallo A, Casillo A, Nitti P, Ambrosio L, Piconi C.Fabrication. characterization and cell cultures on a novel composite chitosan-nano-hydroxyapatite scaffold.Int J Immunopathol Pharmacol.2011 Jan-Mar;24(1 Suppl 2):73-8.
[10]Huang D, Zuo Y, Zou Q, Wang Y, Gao S, Wang X, Liu H, Li Y.Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering.J Biomed Mater Res B Appl Biomater.2012 Jan;100(1):51-7.
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[1]杨强,彭江,卢世璧,等.软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究[J].中华骨科杂志,2011,31(8)：904-910.
[2]贾静,邓志华,刘燕,等.壳聚糖纳米粒介导的人类端粒酶反转录酶启动子连接的自杀基因耦联更昔洛韦靶向抗肝癌研究[J].肿瘤研究与临床,2011,23(7)：438-442.
[3]鲁小云,郭常安,阎作勤,等.壳聚糖-明胶支架/滑膜干细胞的相容性[J].中华实验外科杂志,2011,28(7)：1026-1028.
[4]金捷,周少怀,裴胜利,等.壳聚糖复合骨髓间充质干细胞修复兔骨缺损的研究[J].中华实验外科杂志,2011,28(7)：1042-1044.
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[8]Jun SH, Lee EJ, Jang TS, Kim HE, Jang JH, Koh YH.Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering.J Mater Sci Mater Med.2013 Mar;24(3):773-82.
[9]Nitta SK, Numata K.Biopolvmer-based nanoparticles for drug/gene delivery and tissue engineering.Int J Mol Sci.2013 Jan 14; 14(1):1629-54.
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[1]Cheburu CN, Stoica B, Neamtu A, Vasile C.Biocompatibility testing of chitosan hydrogels. Rev Med Chir Soc Med Nat Iasi.2011 Jul-Sep;115(3):864-70.
[2][Antunes JC, Pereira CL, Molinos M, Ferreira-da-Silva F, Dessi M, Gloria A, Ambrosio L, Goncalves RM, Barbosa MA.Layer-by-layer self-assembly of chitosan and poly(y-glutamic acid) into polyelectrolyte complexes. Biomacromolecules.2011 Dec 12;12(12):4183-95.6.
[3]Aziz MA, Cabral JD, Brooks HJ, Moratti SC, Hanton LR.Antimicrobial properties of a chitosan dextran-based hydrogel for surgical use.Antimicrob Agents Chemother.2012 Jan;56(1):280-7.
[4]Kumar S, Kim H, Gupta MK, Dutta PK, Koh J.A new chitosan-thymine conjugate:Synthesis, characterization and biological activity.
[5]Int J Biol Macromol.2011 Oct 1.
[6]Pattnaik S, Nethala S, Tripathi A, Saravanan S, Moorthi A, Selvamurugan N.Chitosan scaffolds containing silicon dioxide and zirconia nano particles for bone tissue engineering.Int J Biol Macromol.2011 Dec 1;49(5):1167-72.
[7]Pati F, Adhikari B, Dhara S.Collagen Intermingled Chitosan-Tripolyphosphate Nano/Micro Fibrous Scaffolds for Tissue-Engineering Application.J Biomater Sci Polym Ed.2011 Sep 29.
[8]Assaad E, Blemur L, Lessard M, Mateescu MA.Polyelectrolyte Complex of Carboxymethyl Starch and Chitosan as Protein Carrier:Oral Administration of Ovalbumin J Biomater Sci Polym Ed.2011 Sep 29.
[9]Lindhorst D, Tavassol F, Von See C, Schumann P, Laschke M.W, Harder Y, Bormann K.-H, Essig H, Kokemuller H., Kampmann A, Voss A, Mulhaupt R, Menger M.D, Gellrich N.-C, Riicker M. Effects of VEGF loading on scaffold-confined vascularization Journal of Biomedical Materials Research-Part A,2010,95:3 A (783-792).
[10]Costa-Pinto A.R., Reis R.L. and Neves N.M. Scaffolds based bone tissue engineering:The role of chitosanTissue Engineering-Part B: Reviews 2011 17:5(331-347).
[11]Martinez A, Arana P, Fernandez A, Olmo R, Teijon C, Blanco MD. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems.J Microencapsul.2013 Mar 14.
[12]杨强,彭江,卢世璧,等.软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究[J].中华骨科杂志,2011,31(8)：904-910.
[13]Mohajel N, Najafabadi AR, Azadmanesh K, Vatanara A, Amini M, Moazenil E, Rahimi A, Gilani K.Drying of a plasmid containing formulation:chitosan as a protecting agent.Daru.2012 Sep 10;20(1):29.
[14]Jun SH, Lee EJ, Jang TS, Kim HE, Jang JH, Koh YH.Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering.J Mater Sci Mater Med.2013 Mar;24(3):773-82.
[15]Nitta SK, Numata K.Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering.Int J Mol Sci.2013 Jan 14; 14(1):1629-54.
[16]贾静,邓志华,刘燕,等.壳聚糖纳米粒介导的人类端粒酶反转录酶启动子连接的自杀基因耦联更昔洛韦靶向抗肝癌研究[J].肿瘤研究与临床,2011,23(7)：438-442.
[17]Cheburu CN, Stoica B, Neamtu A, Vasile C.Biocompatibility testing of chitosan hydrogels. Rev Med Chir Soc Med Nat Iasi.2011 Jul-Sep;115(3):864-70.
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[19]鲁小云,郭常安,阎作勤,等.壳聚糖-明胶支架/滑膜干细胞的相容性[J].中华实验外科杂志,2011,28(7)：1026-1028.
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[11]杨强,彭江,卢世璧,等.软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究[J].中华骨科杂志,2011,31(8)：904-910.
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[1]杨强,彭江,卢世璧,等.软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究[J].中华骨科杂志,2011,31(8)：904-910.
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[8]Jun SH, Lee EJ, Jang TS, Kim HE, Jang JH, Koh YH.Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering.J Mater Sci Mater Med.2013 Mar;24(3):773-82.
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