【作者】 肖玉梅；

【导师】 张兴栋；

【作者基本信息】 四川大学 ， 生物医学工程， 2007， 博士

【摘要】 工业、交通、体育事业等的高速发展及全球人口老龄化程度加剧，使因疾病、外伤或老龄所致的骨组织缺损、缺失或功能障碍日益成为威胁人类健康的重大问题，其治疗也成为目前临床所面临的重点和难点。对于骨组织的修复和治疗，临床上一般可采用自体骨移植和异体骨移植，但这两种方法都存在一些问题：自体骨移植不可避免地给患者带来二次损伤，且来源匮乏；而异体骨移植可能因免疫排斥反应而导致植入失效甚至威胁患者生命，所以临床上越来越广泛地采用人工制备的生物医学材料作为骨修复或替代材料。自然骨通过非常复杂的方式巧妙地将有机大分子与无机盐互相紧密地结合起来，实现了生物学和力学上的要求。从材料学的角度来看，人体骨可以看成是一种天然的无机物／高聚物复合材料。因此，根据骨的基本组成而制备的无机物／高聚物骨修复复合材料与单纯的陶瓷、金属材料或高分子材料相比，其物理性能和生物学性能均有不同程度的改善，显示了复合材料在应用上的优势。聚乳酸（PLA）因其具有良好的生物相容性、生物可降解性、易加工性、无毒性、无刺激性等优点而在手术缝合线、骨折内固定、药物控释系统以及组织工程支架材料等生物医学领域有着较为广泛的应用。然而其细胞亲和力差，酸性降解产物会使局部酸度过大而出现非感染性炎症，缺乏生物活性。磷灰石是构成自然骨无机质的主要成分，生物活性好，能与骨形成牢固的骨性结合。但其脆性大，塑形难，和人体骨组织力学性能不相容。将磷灰石与聚乳酸复合，可以将二者优良性能充分地结合起来。于是，磷灰石／聚乳酸复合骨修复材料得到了广泛的关注。骨的生物矿化过程实现了在分子水平上对其微结构的调控，通过有机大分子和无机矿物离子在界面处的相互作用和在分子水平上对晶体成核和生长的精细调控，将以胶原纤维为主的有机大分子与以骨磷灰石为主的无机盐紧密地结合起来，既加强了两相之间的界面结合，也调控了无机晶体的生长，实现了生物学和生物力学上的要求。这一过程的揭示为复合材料的制备提供了全新的思路，即通过有机物质从分子水平上调控无机晶体的生长来制备无机／有机高分子复合材料。本研究以仿自然骨结构为出发点，利用聚乳酸的调控作用，采用原位合成法通过聚合物调控纳米磷灰石的生长来获得两相间均匀复合、存在化学键合且具有优良生物活性及其它生物学性能的纳米磷灰石／聚乳酸复合材料。利用PLA分子终端的羧基和羟基以及PLA分子链中的羰基与Ca²⁺的相互作用来调控磷灰石晶体的生长，原位合成了n-HA／PLA复合材料。结果表明，在形成复合材料的过程中，PLA分子中的羰基、羟基以及离解的羧基可能成为磷灰石晶体的成核位点，使磷灰石在PLA表面成核、生长，从而调控磷灰石晶体的形态和生长方向，而PLA的空间结构阻止了磷灰石晶体的进一步晶化或生长。得到的复合材料具有较好的均一性，基本实现了纳米尺度的复合；纳米磷灰石在复合材料中的含量可以调节，两相间存在一定的化学性键合和分子间作用力。然而，该种方法虽然能够通过PLA调控HA的生长，但聚乳酸分子链中只有终端羧基，其分子链中羰基与Ca²⁺的作用力相对较弱，因而其调控能力是有限的，需要发展新的合成方法。以过氧化氢为氧化剂，经紫外诱导将过氧基团引入到聚乳酸颗粒表面。结果表明，单位聚乳酸表面可以引入的过氧键的量可以通过氧化时间来控制。当单体溶液的浓度为15％、照射剂量为3000×100μJ／cm²时，照射氧化50min时单位聚乳酸表面引入过氧键的量最多，大约为5.6×10^-2mmol／g，之后开始减少。随后，再在紫外照射下将甲基丙烯酸接枝聚合到聚乳酸表面，得到聚甲基丙烯酸修饰的聚乳酸材料（PMAA-PLA），其接枝量在氧化时间、照射剂量及单体浓度相同时，随接枝时间的增加而增加。而接枝时间相同时，氧化时间越长接枝量越大。PMAA-PLA表面的羧基在碱性条件下容易发生离解形成-COO^-离子，使PMAA-PLA微粒表面带上很高的负电荷。在磷酸氢二钠溶液中得到的PMAA-PLA为纳米微球，其平均粒度为133.1±2.3 nm，可以放置几周而没有什么变化。PMAA-PLA微球悬浮液的存在状态受到溶液pH值的影响，当pH=5时，微球的zeta电位的负值仍然很大（-79.8mV）；直到pH=3时，微球的zeta电位的绝对值才低于30mV，开始有PMAA-PLA沉淀析出。利用PMAA-PLA分子中原有的羧基、羟基和羰基以及引入的羧基来调控磷灰石的生长合成了纳米磷灰石／P-PLA复合材料，通过红外光谱、X-射线衍射、扫描电镜、透射电镜、粒度分布测定与zeta电位仪以及X射线光电子能谱等对复合材料进行分析，结果表明在合成n-HA／P-PLA复合材料的过程中，PMAA-PLA可以调控磷灰石晶体的成核和生长从而控制磷灰石晶体的形态、尺寸、生长方向以及其在有机相中的分布，得到的纳米磷灰石／P-PLA复合材料中n-HA与PMAA-PLA两相间可能存在化学键合和分子间相互作用力。当该复合材料再与聚乳酸复合得到n-HA／PLA复合材料后，强化了磷灰石和PLA两相间以及PLA之间的结合，也保证了纳米磷灰石晶体在复合材料中的均匀分布和纳米级的尺寸也保证了复合材料的生物活性。n-HA／PLA复合材料无论是在体内还是SBF溶液中，其表面都能形成骨磷灰石层，表明该复合材料具有良好的生物活性。同时，n-HA／PLA在生理环境中能够发生钙释放和钙沉积，进一步表明n-HA／PLA复合材料具有良好的生物活性和骨结合能力。体内外降解实验表明复合材料无论在体内还是体外都是可降解的，体内降解速率大于体外。体内外的降解基本上都是简单的本体水解，体内降解速率较快是由于血液流动、酶的催化及应力作用所致。PLA降解过程中产生的酸性环境可被n-HA中和或缓冲，减弱了复合材料内层的自催化作用。体外降解实验中，前2周分子量下降较为迅速，之后降解较为缓慢，但重量的损失滞后一些。溶液的pH值降低缓慢，与复合材料中存在弱碱性的纳米磷灰石有关。生物相容性评价实验结果表明：n-HA／PLA复合材料安全、无毒，无刺激性，无遗传毒性，无致敏作用，无任何不良组织反应，无排异反应，具有良好的生物活性和生物相容性。皮下植入2周时有纤维包囊形成，但随着植入时间的延长，纤维包囊逐渐变薄。复合材料骨内植入后，发现材料具有促进新骨形成的能力，可与骨组织形成骨性结合，新生骨组织在材料周围是以膜内成骨方式形成。植入4周后，在材料和骨组织的界面处以及骨缺损区有新骨生成、成骨细胞活跃。12周时，界面处有大量的新骨形成，骨缺损区已基本被新骨修复。至于本研究中得到的纳米磷灰石／聚乳酸复合材料长期植入的情况如何还需进一步考察。更多还原

【Abstract】 With the development of industry, transportation, sports and the aging of thepopulation, defection, losses and disfunction of bone tissues has gradually become aserious threat to public health and the clinical therapy to these diseases has attractedmuch research interest. Until now, autograft and allograft bone repair are used inclinics to restore bone defect. Autograft is the gold standard for bone repairing inclinics. But the disadvantages of autograft are obvious: limited resources, creatingsecond trauma and the possibility to induce donor tissue syndrome. Allograft maycause immuno-rejection and the risk to infect diseases from the donor. Therefore,artificial materials are used more and more in Clinics to replace bone defects.By organizing organic materials and inorganic salt in a sophisticated way, naturalbone obtains its structure and physical strength. From a viewpoint of materialsscience, bone is a kind of natural composite composed of inorganic materials andpolymers. Therefore, as a material to restore defective bone, inorganicmaterials/polymer composites are better than pure ceramics, metals and polymersbecause the improvement in physical properties and biological properties.Composite showes its advantage as bone substitute.Because of its excellent biocompatibility, biodegradability, feasibility ofprocessing, polylactic acid （PLA） has been used widely in the field of biomedicalmaterials field as surgical suture, fasten plate in bone fracture, drug controlled release system and scaffold in tissue engineering. However, it has the disadvantageof lack of bioactivity and poor cell compatibility, which results in non-infectiveinflammation due to the high local acidic concentration. Apatite is the main naturalinorganic component of bone with bioactivity, which combined with bone withstrong chemical bonding when implanted in bone tissue. But its disadvantage is thebrittleness, difficulty in processing and the incompatibility with bone in mechanicalproperty. If apatite is compounded with PLA, the composite will possess theirindividual advantages. Therefore, hydroxyapatie/poly（lactide）（HA/PLA） compositehas attracted much research interest.Mineralization of bone realizes the modulation of bone microstructure inmolecular level. By the interaction of organic materials and inorganic materials onthe interlace and the precise modulation of crystal nucleation and growth inmolecular level, organic materials （mainly collagen） bond with salt （mainly boneapatite）. The interface between them is strengthened and crystal growth is controlled,which satisfied the biological and biomechanical demand of bone. Theunderstanding of this process provides new ideas on the preparation of newcomposite, which is to prepare inorganic material/organic material composites bythe modulation of growth of inorganic crystal with organic materials in molecularlevel.In this study, in situ synthesis method was used to prepare nano-HA/PLA（n-HA/PLA） composite by the modulation effect of PLA based on the structure ofnatural bone, in which two phases were evenly distributed with chemical bonding inbetween. And the obtained composite possesses good bioactivity and otherbiological properties.Nano-HA/PLA was prepared with in situ synthesis by the interaction of theterminal carboxyl groups （-COOH） group and -OH group and carbonyl group insidePEA with calcium ions to modulate apatite growth. The results indicated that in theprocess of composite formation, the terminal COOH group, OH group and carbonylgroup in PLA might be the nucleation sites. Apatite nucleated and grew on the PLA surface and its morphology and growth direction were modulated by the PLA.Therefore, the further nucleation and growth of apatite was controlled by the 3-Dstructure of PLA. The composite obtained was homogenous with n-HA evenlydistributed in PLA matrix, which realized nano-sized HA composite. The content ofn-HA in the composite is controllable and chemical bonding and force of molecularinteraction was found between the two phases. However, the modulation ability ofPLA is limited because there is only terminal carboxyl group in PLA molecule andthe interaction between carbonyl in PLA and calcium ions is weak. New method isnedded to be developed.The peroxide groups were introduced onto PLA particle surfaces viaphotooxidization with H₂O₂ oxidant. The results showed that the peroxide groups onPLA particle surfaces could be controlled by oxidation time, and the maximumcontent of peroxide groups produced on the PLA surfaces was about 5.6×10^-2mmol/g with energy of 3000×100μJ/cm² after 50min of oxidation. After 50min. thecontent of peroxide groups decreased. Then the PMAA was grafted onto the PLAsurface via UV induced polymerization and PMAA-modified PLA （PMAA-PLA）was obtained. The graft weight increased with the grafting time under the sameoxidation time, irradiation dose and monomer concentration. When grafting timewas the same. the grafting weight increased with the oxidation time.The carboxyl groups （-COOH） on PMAA-PLA surface were easily ionized into-COO in alkaline aqueous solution, which resulted in PMAA-PLA microparticlesurface with high negative charge. PMAA-PLA nanosphere suspension with anaverage particle size of 133.1±2.3nm in Na₂HPO₄ aqueous solution was stable for afew weeks without any deposit. The absolute value of zeta potential of PMAA-PLAnanosphere suspension was affected by pH value of suspension system. When thepH value decreased to 5, PMAA-PLA nanosphere suspension still had high negativezeta potential （-79.8mV） with no deposit. Only when the pH decreased to 3.0. theabsolute value of zeta potential of PMAA-PLA nanosphere suspension was less than30mV and flocculent PMAA-PLA deposit could be observed. Nano-hydroxyapatite/poly（lactide） （n-HA/P-PLA） composites were prepared bythe carboxylic groups, -OH groups and carbonyl groups on the PLA molecules andintroduced carboxylic groups of PMAA on PMAA-PLA controlling the growth ofapatite crystals. Several analyses about FTIR, XRD, TEM, SEM and XPS suggestedthat the PMAA-PLA could manipulate the nucleation and growth of n-HA crystals,control the morphology, size and growth orientation of n-HA crystals as well as theirdistribution over the organic phase. There might be chemical bonds and moleculesinteraction in obtained n-HA/P-PLA composites between the two phases. Theobtained n-HA/PLA composites synthesized by mixing n-HA/P-PLA compositeswith PLA not only strengthened combination between PLA molecules and twophases of HA and PLA but also guaranteed homogenous n-HA crystals distributionin n-HA/PLA composites and bioactivity of obtained composite.Bone apatite could be formed on the surface of n-HA/PLA composites in vivo andin SBF, which indicated that n-HA/PLA composites had good bioactivity. Calciumrelease and calcium precipitation of n-HA/PLA composites in physiologicalenvironment further confirmed bioactivity and bone binding ability of n-HA/PLAcomposites.The in vivo and in vitro experimental results showed that the n-HA/PLAcomposites could be degraded by simple hydrolyzation and the degradation rate invivo was quicker than in vitro because of blood fluidity, enzymatic activity and stressaction. The acidic environment due to the degradation of PLA could be neutralizedor buffered by n-HA, which lowered the intrinsic catalysis inside the composites.Molecular weight decreased quickly in 2 weeks in in vitro experiment, then itdecreased slower, but the loss of weight was lagged a little. The pH value reducedslower, which might be arised by alkalescent n-HA.The results of biological evaluation in vivo suggested that the composites hadexcellent biocompatibility, biodegradability, bioactivity and osteogenicity. At 2weeks, there was a dense fibrous capsule surrounding the composites and the fibrouscapsule didn’t become thicker with time. At 4 weeks, the newly formed immature bone grew along the surface of the composite and bone directly contacted with thesamples without intervening fibrous tissue. In undecalcified specimens, osteoblastsand osteoclasts were observed between the composite surface and lamellar bone（woven bone）, and new bone formed which took on different structure andorientation from the old bone. The histological results of pure HA control rods wassimilar with that of the composite rods. After 1-2 weeks of implantation, new bonewrapping the implant was clearly visible. The investigation of long timeimplantation about the obtained n-HA/PLA composites needed to be studied further.更多还原