【作者】 方建军；

【导师】 崔磊；

【作者基本信息】 上海大学 ， 材料学， 2013， 博士

【摘要】 先天性骨畸形、缺陷及创伤等导致的骨和软骨缺损，是目前临床医学面临的主要难题。组织工程技术为组织缺损修复提供了一种全新的治疗途径。骨和软骨组织工程要求为缺损或病变部位提供足够数量且具有适当表型的细胞。多孔微载体不仅能满足这一要求，而且能通过注射方式修复组织缺损，可免除外科手术创伤，减轻病人痛苦。可注射微载体用于体内组织再生研究尚处于起步阶段。本论文在国内外研究现状基础上，提出以合成聚肽—聚L-谷氨酸(PLGA)和聚L-谷氨酸苄酯(PBLG)为主要原料，通过冷冻相分离技术、双乳液法及生物模拟矿化手段制备不同类型的多孔可注射细胞微载体，并尝试将其用于组织工程领域。以PLGA、壳聚糖(CS)为原料，采用冷冻相分离技术制备CS多孔微球，通过静电作用在CS微载体表面吸附PLGA，构建CS/PLGA聚电解质复合物(PEC)微球。研究冷冻温度、CS浓度对CS微球孔径和孔隙率影响，发现CS浓度由1%增加到3%，产物由非球形向球形转变；CS浓度为2%、2.5%和3%时，可获得微球，随着浓度增加，孔径逐渐变小，孔隙率逐渐降低；当冷冻温度为20oC，CS浓度为2%(w/v)时，制备的CS微球孔径为47.5±5.4μm，且具有较好形貌。分析了PLGA吸附量及其吸附分布，结果表明PLGA吸附量高达113μg/mg，且能均匀分布于PEC微球中，其吸附对微载体孔结构没有明显影响。通过FT-IR、TGA、Zeta电位等研究手段分析了PEC微载体形成机理，证明PEC微球由CS与PLGA通过静电相互作用形成。通过激光共聚焦显微镜及扫描电子显微镜观察对比分析CS和PEC微载体1、3、5、7d的软骨细胞生长情况，发现随着时间延长，PEC比CS微载体中软骨细胞增殖更快，且分泌更多细胞外基质，同时能更好维持细胞表型。通过MTT法和Real-time PCR定量分析14d内细胞生长情况和14d时软骨基因表达，发现PLGA的引入对微载体细胞黏附、增殖及相关基因表达均有促进作用；通过共聚焦显微镜对软骨细胞/PEC复合物进行多层扫描，发现软骨细胞可在PEC微载体内部生长。裸鼠皮下等量注射CS和PEC微载体细胞悬液，体内培养8周后取材，发现两种微载体均可形成新生软骨。PEC组形成的软骨组织湿重为55.3±7.1mg，比CS组重39.3%。同时，免疫组化和甲苯胺蓝染色分析显示，PEC组形成的软骨组织具有更多的Ⅱ型胶原(COL Ⅱ)和糖胺多糖(GAG)沉积。生化定量分析显示，GAG/湿重、GAG/DNA、COL Ⅱ/湿重及COL Ⅱ/DNA分别为47.6±6.1μg/mg、62.3±7.1μg/μgDNA、99.5±16.1μg/mg和130.2±17.1μg/μgDNA，分别比CS组含量高40.4%、60.9%、52.1%和74.5%。以PBLG为原料构建可注射多孔微载体。对比分析不同致孔剂对PBLG微球孔结构影响，确定以明胶作为温敏性致孔剂。考察致孔剂含量和搅拌速度等对PBLG微球的制备影响，发现当明胶含量为6.5%，搅拌速度为400rpm时，微球具有较大孔径与较高孔隙率，分别为41±3.8μm和79±4.3%。评价了PBLG微载体体外PBS缓冲液浸泡降解性能，结果显示体外降解18周后，PBLG微球破碎，随着分子量降低，降解速率加快，当分子量为1.2×10~5，其18周降解率为63.7%，分子量为3.0×10~5，18周降解率为40.5%。C57小鼠皮下注射分子量为3.0×10~5的PBLG微球，研究体内降解性能，HE染色、masson染色及SEM观察结果显示，PBLG微球在体内具有良好的组织相容性和生物可降解性，8周内微球已完全破碎，大部分材料在体内被降解吸收。与体外降解结果比较，发现PBLG微载体在体内降解速率明显加快。通过MTT法评价PBLG微载体细胞毒性，发现软骨细胞在体外培养3、7d后，PBLG微载体细胞可利用率与TCPS对照组相似，近100%，无细胞毒性。定性分析软骨细胞在PBLG微载体中生长情况，发现随着时间延长，细胞数目增多、细胞外基质分泌量增加。通过MTT法和Real-time PCR定量分析PBLG微载体14d内细胞生长情况和14d时软骨基因表达，发现PBLG微球支持细胞黏附和增殖，并维持软骨细胞表型。通过激光共聚焦显微镜对软骨细胞/PBLG复合物进行多层扫描，发现PBLG微载体孔结构适合软骨细胞在其内部生长。软骨细胞接种到1mg PBLG微载体，体外培养2d，将1mL软骨细胞/微载体悬液注射至裸鼠皮下，体内培养4、8、12周后取材，发现形成的软骨组织湿重分别为30±3.5、60±5.1和108±10.1mg，且8周时比无材料软骨细胞对照组重114%，番红-O与甲苯胺蓝染色呈阳性。生化定量分析显示，4、8、12周形成软骨组织的GAG/干重分别为0.296±0.02、0.391±0.012和0.443±0.041g/g，COL Ⅱ/干重分别为0.385±0.01、0.414±0.01和0.433±0.03g/g。尝试用生物模拟矿化法制备羟基磷灰石(HA)/PBLG多孔复合微载体，并分析HA形成过程。SEM和TGA分析结果表明，随着浸泡时间延长，无机物沉积量增加。EDS分析发现，5、8d时沉积的无机粒子钙磷比值分别为19.02和10.78，随着时间延长，钙磷比增加，11、14、17d时分别为2.09、2.03和1.669，接近理论钙磷比1.666；FITR和XRD结果表明，无机粒子在11d才出现HA特征吸收峰，在14和17d峰强度增加；TEM观察发现，11d后沉积的无机粒子晶体形貌为纳米针状结构，类似于天然骨中HA结构。研究结果表明最初析出的无机粒子不是HA，当延长浸泡时间，磷酸根离子逐步取代融合，进而产生HA，并在11d出现明显HA晶体结构，17d获得完善。评价模拟体液浸泡11d时HA/PBLG复合微载体细胞生长情况，结果显示微载体支持成骨诱导前后脂肪干细胞黏附与增殖，表明HA/PBLG复合微球是一种潜在的成骨组织工程材料。更多还原

【Abstract】 Bone and cartilage tissue damage caused by defects, injuries or other types ofdamage remain the main obstacles for repair of challenging defects. Recently, tissueengineering (TE) open new perspectives for treatment of tissue damage. A majorproblem in TE is the availability of a suffcient number of cells with the appropriatephenotype for delivery to damaged or diseased cartilage and bone. The microcarrierssystem offers an attractive method for cell amplifcation and enhancement ofphenotype expression. Moreover, the microcarriers allow for easy manipulation orminimally invasive procedures by surgeons, thereby reducing complications andimproving patient comfort and satisfaction. However, injectable microcarrier for invivo tissue regeneration research is still in its infancy. In the present work, on thebasis of current status at home and abroad, porous microcarriers based on syntheticpolypeptide of Poly-L-glutamate acids (PLGA) and Poly-benzyl-L-glutamate (PBLG)were prepared by freezing phase separation technique, double emulsion method andbiomimetic mineralization, respectively. Furthermore, we also tried theirapplications in tissue engineering.Chitosan microspheres were firstly developed by freezing phase separationtechnique. Then, a novel kind of porous PLGA/chitosan polyelectrolyte complex(PEC) microsphere was developed through the electrostatic interaction betweenPLGA and chitosan. The effects of freezing temperature and CS concentration onpore size and porosity of CS microsphere were investigated, respectively, and foundthat the morphologies of products transite from the non-spherical to spherical withthe CS concentration increased from1%to3%. When the CS concentration waselevated to be at2%,2.5%,3%(w/v), porous microspheres could be generated, andincreasing the amount of chitosan in the preparation of microspheres resulted insmaller open pores and porosities. When chitosan concentration was fixed at2% (w/v), and freezing temperature at20°C, chitosan microsphere with optical poresize of47.5±5.4μm was developed. The amount and distribution of PLGAassembled on chitosan microsphere were investigated. The results showd that a largeamount of PLGA (110.3μg/mg) was homogeneously absorbed within PECmicrospheres and not significantly changed the pore structure. Fourier transforminfrared spectroscopy, thermal gravimetric analysis and zeta-potential analyzerrevealed that the PEC microspheres were successfully prepared through electrostaticinteraction.Attachment as well as proliferation of chondrocyte loaded on CS and PECmicrocarriers were examined by confocal laser scanning microscopy and SEM at1,3,5,7days after seeding, respectively. Compared with the microspheres fabricatedby chitosan, the porous PEC microspheres were shown to effciently promotechondrocyte attachment and proliferation and were found to produce significantmore chondrogenic matrix and to retain phenotype expression. Cell numbers withinthe microspheres at1,3,5,7and14days post-seeding were quantified by MTTassay and the expression of aggrecan and collagen type Ⅱ genes was detected byRT-PCR analysis. It can be found that the presence of PLGA helps to retain thechondrocyte phenotype. To further answer whether seeded cells would infiltrate andsurvive in the inner region of microspheres, harboring cells were subjected toconfocal microscope observation after7days. The results showed that the inoculatedcells could sufficiently infiltrate into the most inner region of PEC microspheres.Microspheres fabricated by chitosan/PLGA or chitosan alone were mixed withchondrocytes and injected subcutaneously in nude mice, respectively. After8weeks,harvested specimens from either PEC groups or chitosan alone group showedcartilage-like tissue. By immunohistochemical and toluidine blue staining,respectively, it was found that typical lacunae inhabited by chondrocytes, withabundant deposition of collagen type Ⅱ and proteoglycans in the neo-generatedcartilage. Furthermore, the average wet weight (55.3±7.1mg) of tissue formed from the PEC microspheres/chondrocytes was39.3%higher than that formed from thechitosan microspheres/chondrocytes. Biochemical quantification showed that tissuegenerated from PEC microspheres/chondrocytes had significantly higher GAG/wetweight (47.6±6.1μg/mg), GAG/DNA ratios(62.3±7.1μg/μg), collagen Ⅱ/wet-weight(99.5±16.1μg/mg)and the collagen Ⅱ/DNA ratios (130.2±17.1μg/μg), which weresignificantly higher than those of the chitosan alone group of40.4%,60.9%,52.1%and74.5%, respectively.Injectable porous micromicrocarriers were prepared based on PBLG. Theeffects of diffirent kinds of porogens on the pore size and porosity of PBLGmicrosphere were investigated, and found that when used gelatin as anthermosensitive porogen, porous PBLG microspheres could be generated. Theeffects of porogen amounts and stirred speeds on the the size and structure of PBLGmicrosphere were also investigated. The results showed that when the gelatin contentwas fixed at6.5%, and stirring speeds400rpm, we are easily to produce highlyporous large PBLG microspheres with an average pore size of41±3.8μm and aporosity of79±4.3%. The in vitro degradability of porous microcarriers wasinvestigated with certain incubation time in phosphate buffered saline (PBS) at37°C.After18weeks of incubation, the porous microspheres disintegrated into smallpieces, and as the molecular weights of PBLG microcarriers increased, thedegradation rate reduced. when the PBLG microspheres with a molecular weight of1.2×10~5lost63.7%of the weight and with a molecular weight of3.0×10~5lost40.5%of their weights, respectively. Porous PBLG microcarriers with a molecular weightof3.0×10~5were injecteded subcutaneously in C57rats with the purpose of studyingrelated to the degradation in vivo. The results showd by HE, masson and SEMimages indicated that PBLG microspheres possess good biocompatibility andcapacity to be biodegradable to naturally-occurring biological products. Overall, thescaffolds were well degraded by the host rats and no abnormal conditions wereobserved during the8weeks of implantation. The signifcant difference between in vitro and in vivo degradation in the remainning weights of the PBLG microcarriersat the end of8weeks refects that the degradation rate of PBLG microcarriers can beaccelerated by particular enzyme of living systems.In order to screen the ultimate cytotoxicity of the prepared porous PBLGmicrocarriers, MTT tests were carried out by means of assessing the cell viability ofCCK8cells cultured with3and7days. Chondrocytes culture on tissue culturepolystyrene (TCPS) without microspheres served as the control. The results showedthat the viability levels are similar, reaching the maximum viability of approximately100%, indicating all of these microspheres were found not to be toxic to cells.Attachment as well as proliferation of chondrocyte loaded on CS and PECmicrocarriers were examined by confocal laser scanning microscopy and SEM at1,3,5,7days after seeding, respectively, and found that with longer culture time, themicrospheres could attach more cells and produce more chondrogenic matrix. Cellnumbers within the microspheres at1,3,5,7and14days post-seeding werequantified by MTT assay and the expression of aggrecan and collagen type Ⅱ geneswas detected by RT-PCR analysis. the porous PEC microspheres were shown toeffciently promote chondrocyte attachment and proliferation and were found toproduce significant more chondrogenic matrix and to retain phenotype expression.Chondrocytes on PBLG microcarriers were examined after cultivation of1,3,5,7days by confocal microscope observation. The results implies that initially attachedcells on the surface gradually migrated towards inner region and cells couldsufficiently infiltrate into the most inner region of PBLG microspheres.Chondrocyte-seeded PBLG microcarriers were mixed with chondrocytes andinjected subcutaneously in nude mice. The inject specimens of chondrocyte-seededporous PBLG composites resulted in new cartilage-like tissue formation in vivo ininject sites at4,8and12weeks after injection subcutaneously. Byimmunohistochemical and toluidine blue staining, respectively, it was found thattypical lacunae inhabited by chondrocytes, with abundant deposition of collagen type Ⅱ and proteoglycans in the neo-generated cartilage. The average tissue wetweight (108±10.1mg) formed from the12weeks group was78.6%higher than thatformed from the8weeks group (60±5.1mg), and263.3%higher than that formedfrom the4weeks group (30±3.5mg). Furthermore, the average wet weight of8weeks group was114%higher than that formed from the control group. Biochemicalquantification showed that tissue generated from PBLG microspheres/chondrocyteshad significantly higher GAG/dried weight of0.296±0.02,0.391±0.012and0.443±0.041g/g, and higher collagen Ⅱ/dried-weight of0.385±0.01,0.414±0.01and0.433±0.03g/g at4,8and12weeks after injection subcutaneously, respectively.The bonelike mineral, carbonate apatite, was successfully used to functionalizeporous PBLG microspheres by a biomimetic mineralization method. Moreover, thetime of the bonelike apatite formation on PBLG was proposed. The results showedby SEM and TGA indicated that the amount of mineral phase increased graduallywith increasing incubation time. EDS spectra of the apatite coating on PBLGmicrospheres showed that the Ca/P ratio of apatite mineral was19.02formicrospheres incubated for5days and10.78for those incubated for8days. Whenincreased the incubation time, the Ca/P ratio enhanced. When the incubation wasfixed at11,14and17d, the Ca/P ratio of apatite mineral was2.09,2.03and1.669,respectively. These values are similar to Ca/P ratio of biological apatite of5:3. Thechemical structure of the mineral coating was provided by FT-IR and XRD spectra.It was found that mineralized PBLG microspheres displayed characteristic peaksassociated with HA when the microspheres incubated for11days, and thecharacteristic peaks were enhanced at14and17days. Based on TEM analyses, thecrystal structure of apatite particles on PBLG microspheres incubated for11dayswas detected to be nano-needle, which were similar to the apatite in natural bone.The above results indicated that the initial inorganic particles are not HA. Whenprolonged the incubation time, phosphate ions wereion of gradually replaced andintegrated to produce HA. When the microspheres incubated for11days, mineralized PBLG microspheres displayed characteristic crystal structure related toHA, which were perfected at17days. Adipose derived stem cells were cultured onHA/PBLG microcarriers incubated for11days and were induced to undergoosteogenic differentiation in the presence of HA/PBLG microcarriers for bone tissueengineering. The results showed that HA/PBLG microcarriers is favorable for thegrowth of adipose derived stem cells before and after osteogenic. The observedresults proved that HA/PBLG microcarriers may be promising scaffolding materialsfor bone tissue engineering and regeneration.更多还原