控释性可注射牙槽骨修复材料的生物学性能研究
摘要
【目的】
本研究从新型牙槽骨修复材料的体外细胞毒性、MC3T3-E1成骨细胞的相容性、软组织和骨组织对材料的反应及其用于牙周组织再生的效果四个方面对该控释性可注射可生物降解牙槽骨修复材料进行了生物学评价，从而为进一步的研究奠定基础。研究共分四个部分：第一部分是用浸提液法评价新型控释性可注射可生物降解牙槽骨修复材料中各组份、交联产物及其降解产物对L929细胞的毒性，以评价其生物安全性；第二部分用直接接触法评价MC3T3-E1成骨细胞在该牙槽骨修复材料上的生长情况和功能的表达，以深入分析该材料的骨细胞相容性和体外骨引导性：第三部分则检测该材料植入兔体内时的软组织和硬组织反应，以评价其在体内的生物相容性、生物可降解性和体内骨引导性；第四部分则将含有四环素(tetracycline，TTC)的牙槽骨修复材料用于实验动物牙周炎模型牙槽骨缺损区的修复，以检测四环素药物在牙周袋内的缓释情况和对牙周组织再生效果的的实际影响，从而评价其在体内的生物学有效性。
第一部分新型可注射可生物降解牙槽骨修复材料的体外细胞毒性研究
【材料与方法】将不饱和聚磷酸酯／β-磷酸三钙(unsaturated polyphosphoester／β-tricalcium phosphate，UPPE／β-TCP)复合物各组份分别在细胞培养液中浸提，正常培养液作为对照组，使用AlamarBlue法评价上述各组份不同浓度的浸提液对小鼠成纤维细胞(L-929)的体外细胞毒性；同时对不同浓度的已交联UPPE聚合物浸提液、UPPE／β-TCP复合物浸提液及交联复合物体外快速降解产物稀释液的细胞毒性分别进行评价，对实验结果进行单因素方差分析及Tukey's HSD多重比较。【结果】β-TCP的细胞毒性与对照组的差异无显著性意义(P＞0．05)，其余UPPE／β-TCP复合物各组份最高浓度组的细胞毒性均较强，与对照组的差异具有显著性意义(P＜0．05)，但其细胞毒性随着浸提液浓度的降低而降低；已交联的UPPE聚合物和UPPE／β-TCP复合物的细胞毒性与对照组的差异无显著性意义(P＞0．05)；交联复合物降解产物的细胞毒性同样表现为剂量依赖性，其中两组高浓度降解液的细胞毒性较强，与对照组的差异具有显著性意义(P＜0．05)。
第二部分四环素控释性可生物降解牙槽骨修复材料的体外MC3T3-E1细胞培养研究
【材料与方法】采用直接接触法评价四环素控释性可注射牙槽骨修复材料的骨细胞相容性。将β-TCP、UPPE聚合物、UPPE／β-TCP复合物及含1％(w／w)TTC的UPPE／β-TCP(UPPE／β-TCP+1％TTC)复合物分别与小鼠胚胎颅顶骨MC3T3-E1细胞(MC3T3-E1)共同培养，AlamarBlue法评价上述材料对MC3T3-E1细胞的体外细胞毒性，吖啶橙-溴化乙锭双荧光染色(Acridine orange／ethidium bromide staining assay，AO／EB)法在荧光显微镜下检测材料表面MC3T3-E1细胞的存活和死亡情况，电子显微镜(scanning electron microscopy，SEM)下观察MC3T3-E1细胞在材料表面的形态、粘附和生长情况；另外，通过测量碱性磷酸酶(alkaline phosphatase，ALP)的活性检测MC3T3-E1细胞在材料表面的分化和功能表达，对实验结果进行单因素方差分析及Turkey's HSD多重比较。【结果】AlamarBlue法检测MC3T3-E1细胞的毒性结果表明β-TCP、UPPE聚合物、UPPE／β-TCP复合物对小鼠胚胎颅顶骨MC3T3-E1细胞的毒性与阴性对照组的差异无显著性意义，添加1％TTC后对复合物的体外细胞毒性无不良影响(P＞0．05)；荧光显微镜观察结果表明各种材料表面的活细胞密度值相近，死细胞数量均较少；电子扫描显微镜观察发现MC3T3-E1细胞可在所有材料表面附着和增值，并伸展呈现为多角形，细胞突起互相连接；β-TCP、UPPE聚合物、UPPE／β-TCP复合物的MC3T3-E1细胞碱性磷酸酶活性检测值均与阴性对照组相近，其间的差异无统计学意义(P＞0．05)，而UPPE／β-TCP+1％TTC复合物稍高于阴性对照组，其差异有统计学意义(P＜0．05)。
第三部分四环素控释性可注射可生物降解牙槽骨修复材料的软组织和骨组织反应评价
【材料与方法】选用25只新西兰大白兔，按2 weeks(w)、4 w、8 w、12 w、24w的植入期分为五组，将β-TCP、UPPE聚合物、UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物分别植入其皮下；同时在其股骨内侧髁制作骨缺损区模型，分别注射UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物，对软组织和骨组织样本进行组织病理学和组织形态学评价，并进行统计学分析。【结果】各组材料的纤维组织囊厚度随植入期的延长而增加，在8 w时最大，以后无明显改变，表明所形成的纤维组织囊已完全成熟；但UPPE聚合物在第8 w时开始出现降解，纤维组织向材料内部浸润生长，未见明显的炎性细胞浸润；所有植入体周围的软组织囊中均未发现成骨细胞、骨细胞或骨组织。在整个植入期内，UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物植入体的骨组织反应基本一致，均可见骨组织与材料的界面处无纤维组织囊插入，骨缺损区内无炎性细胞存在，新生骨组织与材料结合较好。植入4 w后，复合体中与材料结合良好，无纤维结缔组织囊形成，在材料发生吸收而改建的区域，可见成骨细胞和活跃的新骨形成、新生血管；8 w之后，大部分植入体仍存在于骨缺损区，而部分植入体成为“独岛”被完全整合进入新生的骨组织中；12 w时骨组织有广泛的重建；24 w后，植入体主体部分消失，骨组织内生生长和改建获得更加活跃，在新生骨和骨髓样组织中可见大量材料碎片，并可见成骨样细胞。组织形态学分析表明8 w时β-TCP、UPPE聚合物和UPPE／β-TCP复合物组的软组织评级得分低于第2 w，其间的差异具有统计学意义(P＜0．05)；各时期UPPE／β-TCP+1％TTC复合物组软组织得分的差别无统计学意义(P＞0．05)。UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物植入骨缺损区后的吸收率随植入时间的延长而逐渐增加，在8 w以前其吸收率相似，组间的差异无统计学意义(P＞0．05)，而12 w后UPPE／β-TCP+1％TTC复合物组的吸收率高于UPPE／β-TCP复合物，其间的差异具有统计学意义(P＜0．05)，24 w后其吸收率均在60％以上。
第四部分四环素控释性可注射可生物降解牙槽骨修复材料用于牙周组织再生的动物研究
【材料与方法】选用15只雄性、健康的杂种犬，在双侧上颌尖牙的近中根面放置不锈钢网6 w以形成实验动物牙周炎模型，随后在上颌尖牙近中牙槽骨上制作大小形状基本相同的三壁骨内袋缺损，将含有1％、5％、10％TTC的UPPE／β-TCP复合物直接注射入骨质缺损区，未植入任何材料的一侧作为空白对照组。分别在术后3 days(d)、7 d和14 d用滤纸提取术区的龈沟液，使用高效液相色谱法测定(high-performance liquid chromatography，HPLC)滤纸上的TTC的含量，评价UPPE／β-TCP+TTC复合物植入动物体内后的药物缓释性能；在术后16 w处死动物，常规制备组织病理学样本，对牙周组织的愈合情况进行组织学和组织形态学评价，对实验结果进行单因素方差分析和组间多重比较。【结果】在牙周骨缺损内注射三种含不同浓度TTC的UPPE／β-TCP复合物3 d、7 d和14 d后，所检测的龈沟液中TTC浓度均高于抑制牙周主要致病菌所需要的TTC最低抑菌浓度，在14 d时各材料药物缓释性能间的差异无统计学意义(P＞0．05)。在植入复合物的牙周骨缺损区内均可见有新骨和新牙骨质形成等牙周组织的愈合表现，无明显的炎症反应特征，部分UPPE／β-TCP+TTC复合物为新生骨组织所替代，且新生骨组织、新生牙骨质和新生牙周结缔组织附着的宽度与空白对照侧间的差异均有统计学意义(P＜0．05)。
【结论】
虽然UPPE／β-TCP复合物各组份及其体外降解产物表现出一定的细胞毒性，但已交联的UPPE聚合物和UPPE／β-TCP复合物则具有良好的细胞相容性，因此UPPE／β-TCP复合物仍有望成为理想的可注射、可生物降解的骨缺损修复材料。四环素控释性可注射牙槽骨修复材料具有较好的骨细胞生物相容性，对MC3T3-E1细胞的生长无不利影响，且有利于其粘附形成较高的细胞密度和活性，并表达分化的MC3T3-E1细胞功能，具有良好的体外骨诱导性。可注射牙槽骨修复材料UPPE／β-TCP复合物在动物体内具有良好的软组织和骨组织生物相容性，在骨质缺损区可逐渐降解并为新生骨组织所替代。TTC控释性可注射牙槽骨修复材料UPPE／β-TCP+TTC复合物不但在实验动物体内有较好的药物缓释性能，而且可为牙槽骨缺损区的愈合提供稳定的空间，促进了牙周组织的愈合，有望用于牙槽骨再生的修复治疗。
PART ONE In Vitro Cytotoxicity of a Novel Injectable and BiodegradableAlveolar Bone Substitiute
Objective Currently, the unsaturated polyphosphoester (UPPE) polymer is beinginvestigated as an injectable and biodegradable system for alveolar bone repair in thetreatment of periodontal diseases. The incorporation of beta-tricalcium phosphate(β-TCP) particles into the UPPE polymer was previously shown to significantlyincrease the material's mechanical properties. Moreover, in vitro experimentsdemonstrated that the UPPE/β-TCP composite was capable of release of tetracyclinefor over 2 weeks (w). This study aimed to investigate the in vitro cytotoxicity of eachindividual component, the resulting cross-linked network, and the eventualdegradation products of the UPPE/β-TCP composite. Methods Each individualcomponent of the UPPE/β-TCP composite was immersed in culture media andincubated, respectively. The culture media without extracts severed as the control.Using cell line L929 as model cells, the individual cytotoxicity of each constituentwas evaluated by an AlamarBlue viability assay. The effects of the leachable productsfrom the cross-linked UPPE polymer and UPPE/β-TCP composite, the in vitrodegradation products obtained from the complete breakdown of the cross-linkedcomposite under accelerated conditions on the cytotoxicity were also investigated.Single factor analysis of variance and Tukey's HSD multiple comparison tests wereused to determine statistical significance of results. Resultsβ-TCP demonstratedcell viability comparable to the control (P＞0.05). The other components of the UPPE/β-TCP composite exhibited significantly higher cytotoxicity at the highestconcentration (P＜0.05), and dilutions of these extracts produced an improvement incell viability. Once cross-linked, however, the cytotoxicity of the leachable productsfrom the cross-linked UPPE polymer and UPPE/β-TCP composite was comparable tothat of the control (P＞0.05) because most of the individual constituents may beincorporated into the cross-linked network. The degradation products of thecross-linked composite displayed a dose-dependent cytotoxic response with only thetwo highest concentration solutions demonstrating cell viability significantly lowerthan the control (P＜0.05). Conclusion The results suggest that although there areconcerns regarding the biocompatibility of the novel UPPE/β-TCP composite, it holdsgreat promise for use as an injectable and biodegradable alveolar bone substitute inthe treatment of periodontal diseases.
PART TWO Investigation of MC3T3-E1 Cell Behavior on the Surface of aBiodegradable Alveolar Bone Substitiute Containing Tetracycline
Objecitve The early osseointegration of bone substitutes is an important factorfor their clinical success. The challenge in the engineering of bone substitute'ssurfaces is to attract, above all, osteoblasts that produce a bone extracellular matrix,which will ensure a high bone-implant contact. Cell adhesion is one of the initialstages for subsequent proliferation and differentiation of osteoblastic cells producingbone tissue. The aim of this present study is to investigate the behavior of osteoblasticMC3T3-E1 cells on four different bone grafts surfaces in vitro:β-TCP, UPPE polymer,UPPE/β-TCP composite and UPPE/β-TCP containing 1% tetracycline(UPPE/β-TCP+1% TTC) composite. Methods MC3T3-E1 cells were culturedeither on theβ-TCP, UPPE polymer, UPPE/β-TCP composite and UPPE/β-TCP+1%TTC composite discs or in the absence of material (plastic) for 4, 8 and 15 days (d).The cell viability of each group was measured as optical density by an AlamarBlueviability assay, and normalized to cell culture medial (the control) in order to demonstrate differences in cell viability on the different substrates. After 1 or 14 dincubations of the cells on the four materials or the control, the cellswere double-stained with Acridine orange/Ethidium bromide (AO/EB) for 5 minutes(min) and viewed by epifluorescence microscopy to simultaneously examine of bothlive and dead cells on the materials. The morphology of osteoblastic cells MC3T3-E1after culturing for 2 d on the different substrates was examined using a scanningelectron microscope (SEM). The differentiation function of osteoblastic cellsMC3T3-E1 was assessed by measuring alkaline phosphatase (ALP) activity. Singlefactor analysis of variance and Tukey's HSD multiple comparison tests were used todetermine statistical significance of results. Results For all substrates, the resultsshow an increase in cell viability indicating cell proliferation with culture time. After4 d of culture, the cell viability was slightly inferior for all groups compared with thecontrol, but there was no statistical differences between groups (P＞0.05). The dataappeared similar for the UPPE/β-TCP composite after 8 and 15 d of culture, andslightly superior for theβ-TCP or UPPE/β-TCP+1% TTC composite after 15 dcompared with the culture. Nevertheless, the statistical analysis did not showsignificant differences between groups (P＞0.05). The cell viability remainedslightly lower in UPPE compared with the control. Visual examination revealed thatthe density of live cells adherent to each material was similar after 2 and 15 d ofculture. Live cells, stained green, appeared to have adhered and attained a normalpolygonal morphology on all materials. Dead cells (stained red) were very few on allthree materials. SEM micrographs show similar cell attachment on the four materials.ALP activity increased with culture time whatever the substrate was and appeared tobe at its maximum after 21 d. Conculsion These data show that a UPPE/β-TCPcontaining 1% TTC composite is biocompatible and supports the adhesion, spreading,proliferation and viability of osteoblast cells.
PART THREE In Vivo Bone and Soft Tissue Response to an Injectable,Biodegradable Alveolar Bone Substitiute Containing Tetracycline
Objective Earlier investigations have characterized some of the physical andbiological properties of UPPE/β-TCP composite. It is worth noting that sometimes thebone substitute is not only in contact with bone, but also with the surrounding softtissues. To ensure the safety and effectiveness of this technique, this study wasdesigned to assess in vivo soft and bone tissue response to to the UPPE/β-TCPcomposite and UPPE/β-TCP+1% TTC composite after implantation using a rabbitmodel. Methods Twenty-five male New Zealand White rabbits were used asexperimental animals. For the insertion of the subcutaneous implants, fourssubcutaneous pockets at the dorsum of the rabbits were created. The implants (β-TCP UPPE polymer, UPPE/β-TCP composite and UPPE/β-TCP+1% TTC) wereinserted in these pockets. UPPE/β-TCP composite and UPPE/β-TCP+1% TTC wereinjected in circular defects as created in the femoral condyles of rabbits and were leftin place for 2, 4, 8, 12 and 24 w. The specimens were evaluated morphologically(histology, and histomorphometry). Single factor analysis of variance and Tukey'sHSD multiple comparison tests were used to determine statistical significance ofresults. Results The histologic evaluation in vivo after 2 w showed a mild infiltrateof inflammatory cells for most of the groups. UPPE/β-TCP+1% TTC compositeexhibited no signal of inflammatory response. After 4 w. the tissue showed nearnormal pattern for all the groups. The soft tissue capsule had a non-uniformdistribution in thickness, which increased most significantly between 4 w and 12 wafter implantation. After 8 weeks, the capsule thickness did not change much. None ofpolymorphic cells, osteoblast cells or bone cells adjacent to the implant were observed.The surface of theβ-TCP, UPPE/β-TCP composite and UPPE/β-TCP+1% TTCcomposite themselves remained substantially intact without noticeable cracking,chipping or dissolution. However, at 8 w, UPPE polymer showed extensive surfaceerosion and superficial fragmentation that was surrounded by a few inflammatory cells. Gross examination of retrieved implant/bone composite samples indicated thatthe UPPE/β-TCP composite and UPPE/β-TCP+1% TTC composite did not evokeinflammatory response, necrosis or fibrous encapsulation in surrounding bony tissues.Histological examination revealed excellent composite/host bone bonding. At 4 w, theresorption induced voids between terminals of bone defects and implants were largelyfilled with new bone. Composite resorption, new blood vessels, osteocytes, osteonsand osteoblast-like cells lining up with active new bone were observed at remodelingsites. At 12 w, a new bone network was developed within femoral defect, whilecomposite became islands incorporated in the new bone. At 24 w, bone ingrowth andremodeling activities became so extensive that the interface between residual cementand new bone became less identifiable. In general, the resorption ratio valuesincreased with implantation time. Conclusion TC addition optimizedbiocompatibility of the UPPE/β-TCP composite, contributing to anti-inflammatoryresponse during the early phases of the wound healing process. These results showedthat UPPE/β-TCP composite holds promise for use as a syntheticbiodegradable scaffolds for tissue engineering.
PART FOUR The Influence of Novel Injectable Alvelor Bone Substitutes onRegeneration of Periodontonal Defects with Experimental Periodontitis in Dogs
Objective Earlier studies showed that an injectable UPPE/β-TCP composite wasbiocompatible and promoted histocompatible healing of bone tissue in rabbits. Thesemisolid consistency of UPPE/β-TCP composite containing TTC shows sustainedand almost constant in vitro drug release in phosphate buffer, pH 7.4 at 37℃, for upto 14 days. The purposes of this present study were (1) to assess the in vivo releaseof drug in the periodontal pockets from the UPPE/β-TCP composite containing 1%,5% or 10% (wt/wt) TTC and (2) to evaluate influence of the formulation onregeneration of periodontal defects with experimental periodontitis in dogs.
Methods Experimental periodontitis was induced by placing stainless-steel mesh on the mesial side of maxillary canines for 6 w in fifteen adult, healthy dogs.Subsequently, intrabony defects were resized so as to be standard, and UPPE/β-TCPcomposite containing 1%, 5% and 10% TTC were directly injected in theexperimental bone defects. Non-grafted defects on the contralateral side served ascontrols. The retention time of the antibiotic product was determined indirectly bymeasuring the concentration of tetracycline in gingival crevicular fluid (GCF) usinghigh-performance liquid chromatography (HPLC) at 3, 7, and 14 d after the placementof the UPPE/β-TCP + TTC composites. Sixteen weeks after surgery, the animals weresacrificed and histologic specimens were prepared. Periodontal tissue healing wasevaluated histologically and histometrically. Results The TTC concentrations inthe GCF well above the minimum inhibitory concentration (MIC) of most periodontalpathogens were seen at 3, 7, and 14 d following the injection of theUPPE/β-TCP+TTC composite formulations. Healing of periodontal tissues, in termsof bone and cementum formation, was consistently observed in theUPPE/β-TCP+TTC composite-applied sites. UPPE/β-TCP+TTC composites werepartly replaced by new bone. New cementum and periodontal ligament-like tissuewere observed between UPPE/β-TCP+TTC composites and the root surface. Newbone (P＜0. 05), new cementum (P＜0.05), and new connective tissue attachment andadhesion (P＜0.05) were significantly enhanced in the experimental sites.
Conclusion The UPPE/β-TCP+TTC composite formulations can be retained in theperiodontal pocket for a prolonged period, releasing a sufficient amount oftetracycline to eliminate pathogenic bacteria. The composites containing TTCprovides stable wound healing and enhanced periodontal regeneration in periodontaldefects in dogs with experimental periodontitis. The local application of injectablebioerodible UPPE/β-TCP+TTC composites appears to be promising in the context ofperiodontal treatment.
本研究从新型牙槽骨修复材料的体外细胞毒性、MC3T3-E1成骨细胞的相容性、软组织和骨组织对材料的反应及其用于牙周组织再生的效果四个方面对该控释性可注射可生物降解牙槽骨修复材料进行了生物学评价，从而为进一步的研究奠定基础。研究共分四个部分：第一部分是用浸提液法评价新型控释性可注射可生物降解牙槽骨修复材料中各组份、交联产物及其降解产物对L929细胞的毒性，以评价其生物安全性；第二部分用直接接触法评价MC3T3-E1成骨细胞在该牙槽骨修复材料上的生长情况和功能的表达，以深入分析该材料的骨细胞相容性和体外骨引导性：第三部分则检测该材料植入兔体内时的软组织和硬组织反应，以评价其在体内的生物相容性、生物可降解性和体内骨引导性；第四部分则将含有四环素(tetracycline，TTC)的牙槽骨修复材料用于实验动物牙周炎模型牙槽骨缺损区的修复，以检测四环素药物在牙周袋内的缓释情况和对牙周组织再生效果的的实际影响，从而评价其在体内的生物学有效性。
第一部分新型可注射可生物降解牙槽骨修复材料的体外细胞毒性研究
【材料与方法】将不饱和聚磷酸酯／β-磷酸三钙(unsaturated polyphosphoester／β-tricalcium phosphate，UPPE／β-TCP)复合物各组份分别在细胞培养液中浸提，正常培养液作为对照组，使用AlamarBlue法评价上述各组份不同浓度的浸提液对小鼠成纤维细胞(L-929)的体外细胞毒性；同时对不同浓度的已交联UPPE聚合物浸提液、UPPE／β-TCP复合物浸提液及交联复合物体外快速降解产物稀释液的细胞毒性分别进行评价，对实验结果进行单因素方差分析及Tukey's HSD多重比较。【结果】β-TCP的细胞毒性与对照组的差异无显著性意义(P＞0．05)，其余UPPE／β-TCP复合物各组份最高浓度组的细胞毒性均较强，与对照组的差异具有显著性意义(P＜0．05)，但其细胞毒性随着浸提液浓度的降低而降低；已交联的UPPE聚合物和UPPE／β-TCP复合物的细胞毒性与对照组的差异无显著性意义(P＞0．05)；交联复合物降解产物的细胞毒性同样表现为剂量依赖性，其中两组高浓度降解液的细胞毒性较强，与对照组的差异具有显著性意义(P＜0．05)。
第二部分四环素控释性可生物降解牙槽骨修复材料的体外MC3T3-E1细胞培养研究
【材料与方法】采用直接接触法评价四环素控释性可注射牙槽骨修复材料的骨细胞相容性。将β-TCP、UPPE聚合物、UPPE／β-TCP复合物及含1％(w／w)TTC的UPPE／β-TCP(UPPE／β-TCP+1％TTC)复合物分别与小鼠胚胎颅顶骨MC3T3-E1细胞(MC3T3-E1)共同培养，AlamarBlue法评价上述材料对MC3T3-E1细胞的体外细胞毒性，吖啶橙-溴化乙锭双荧光染色(Acridine orange／ethidium bromide staining assay，AO／EB)法在荧光显微镜下检测材料表面MC3T3-E1细胞的存活和死亡情况，电子显微镜(scanning electron microscopy，SEM)下观察MC3T3-E1细胞在材料表面的形态、粘附和生长情况；另外，通过测量碱性磷酸酶(alkaline phosphatase，ALP)的活性检测MC3T3-E1细胞在材料表面的分化和功能表达，对实验结果进行单因素方差分析及Turkey's HSD多重比较。【结果】AlamarBlue法检测MC3T3-E1细胞的毒性结果表明β-TCP、UPPE聚合物、UPPE／β-TCP复合物对小鼠胚胎颅顶骨MC3T3-E1细胞的毒性与阴性对照组的差异无显著性意义，添加1％TTC后对复合物的体外细胞毒性无不良影响(P＞0．05)；荧光显微镜观察结果表明各种材料表面的活细胞密度值相近，死细胞数量均较少；电子扫描显微镜观察发现MC3T3-E1细胞可在所有材料表面附着和增值，并伸展呈现为多角形，细胞突起互相连接；β-TCP、UPPE聚合物、UPPE／β-TCP复合物的MC3T3-E1细胞碱性磷酸酶活性检测值均与阴性对照组相近，其间的差异无统计学意义(P＞0．05)，而UPPE／β-TCP+1％TTC复合物稍高于阴性对照组，其差异有统计学意义(P＜0．05)。
第三部分四环素控释性可注射可生物降解牙槽骨修复材料的软组织和骨组织反应评价
【材料与方法】选用25只新西兰大白兔，按2 weeks(w)、4 w、8 w、12 w、24w的植入期分为五组，将β-TCP、UPPE聚合物、UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物分别植入其皮下；同时在其股骨内侧髁制作骨缺损区模型，分别注射UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物，对软组织和骨组织样本进行组织病理学和组织形态学评价，并进行统计学分析。【结果】各组材料的纤维组织囊厚度随植入期的延长而增加，在8 w时最大，以后无明显改变，表明所形成的纤维组织囊已完全成熟；但UPPE聚合物在第8 w时开始出现降解，纤维组织向材料内部浸润生长，未见明显的炎性细胞浸润；所有植入体周围的软组织囊中均未发现成骨细胞、骨细胞或骨组织。在整个植入期内，UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物植入体的骨组织反应基本一致，均可见骨组织与材料的界面处无纤维组织囊插入，骨缺损区内无炎性细胞存在，新生骨组织与材料结合较好。植入4 w后，复合体中与材料结合良好，无纤维结缔组织囊形成，在材料发生吸收而改建的区域，可见成骨细胞和活跃的新骨形成、新生血管；8 w之后，大部分植入体仍存在于骨缺损区，而部分植入体成为“独岛”被完全整合进入新生的骨组织中；12 w时骨组织有广泛的重建；24 w后，植入体主体部分消失，骨组织内生生长和改建获得更加活跃，在新生骨和骨髓样组织中可见大量材料碎片，并可见成骨样细胞。组织形态学分析表明8 w时β-TCP、UPPE聚合物和UPPE／β-TCP复合物组的软组织评级得分低于第2 w，其间的差异具有统计学意义(P＜0．05)；各时期UPPE／β-TCP+1％TTC复合物组软组织得分的差别无统计学意义(P＞0．05)。UPPE／β-TCP复合物和UPPE／β-TCP+1％TTC复合物植入骨缺损区后的吸收率随植入时间的延长而逐渐增加，在8 w以前其吸收率相似，组间的差异无统计学意义(P＞0．05)，而12 w后UPPE／β-TCP+1％TTC复合物组的吸收率高于UPPE／β-TCP复合物，其间的差异具有统计学意义(P＜0．05)，24 w后其吸收率均在60％以上。
第四部分四环素控释性可注射可生物降解牙槽骨修复材料用于牙周组织再生的动物研究
【材料与方法】选用15只雄性、健康的杂种犬，在双侧上颌尖牙的近中根面放置不锈钢网6 w以形成实验动物牙周炎模型，随后在上颌尖牙近中牙槽骨上制作大小形状基本相同的三壁骨内袋缺损，将含有1％、5％、10％TTC的UPPE／β-TCP复合物直接注射入骨质缺损区，未植入任何材料的一侧作为空白对照组。分别在术后3 days(d)、7 d和14 d用滤纸提取术区的龈沟液，使用高效液相色谱法测定(high-performance liquid chromatography，HPLC)滤纸上的TTC的含量，评价UPPE／β-TCP+TTC复合物植入动物体内后的药物缓释性能；在术后16 w处死动物，常规制备组织病理学样本，对牙周组织的愈合情况进行组织学和组织形态学评价，对实验结果进行单因素方差分析和组间多重比较。【结果】在牙周骨缺损内注射三种含不同浓度TTC的UPPE／β-TCP复合物3 d、7 d和14 d后，所检测的龈沟液中TTC浓度均高于抑制牙周主要致病菌所需要的TTC最低抑菌浓度，在14 d时各材料药物缓释性能间的差异无统计学意义(P＞0．05)。在植入复合物的牙周骨缺损区内均可见有新骨和新牙骨质形成等牙周组织的愈合表现，无明显的炎症反应特征，部分UPPE／β-TCP+TTC复合物为新生骨组织所替代，且新生骨组织、新生牙骨质和新生牙周结缔组织附着的宽度与空白对照侧间的差异均有统计学意义(P＜0．05)。
【结论】
虽然UPPE／β-TCP复合物各组份及其体外降解产物表现出一定的细胞毒性，但已交联的UPPE聚合物和UPPE／β-TCP复合物则具有良好的细胞相容性，因此UPPE／β-TCP复合物仍有望成为理想的可注射、可生物降解的骨缺损修复材料。四环素控释性可注射牙槽骨修复材料具有较好的骨细胞生物相容性，对MC3T3-E1细胞的生长无不利影响，且有利于其粘附形成较高的细胞密度和活性，并表达分化的MC3T3-E1细胞功能，具有良好的体外骨诱导性。可注射牙槽骨修复材料UPPE／β-TCP复合物在动物体内具有良好的软组织和骨组织生物相容性，在骨质缺损区可逐渐降解并为新生骨组织所替代。TTC控释性可注射牙槽骨修复材料UPPE／β-TCP+TTC复合物不但在实验动物体内有较好的药物缓释性能，而且可为牙槽骨缺损区的愈合提供稳定的空间，促进了牙周组织的愈合，有望用于牙槽骨再生的修复治疗。
PART ONE In Vitro Cytotoxicity of a Novel Injectable and BiodegradableAlveolar Bone Substitiute
Objective Currently, the unsaturated polyphosphoester (UPPE) polymer is beinginvestigated as an injectable and biodegradable system for alveolar bone repair in thetreatment of periodontal diseases. The incorporation of beta-tricalcium phosphate(β-TCP) particles into the UPPE polymer was previously shown to significantlyincrease the material's mechanical properties. Moreover, in vitro experimentsdemonstrated that the UPPE/β-TCP composite was capable of release of tetracyclinefor over 2 weeks (w). This study aimed to investigate the in vitro cytotoxicity of eachindividual component, the resulting cross-linked network, and the eventualdegradation products of the UPPE/β-TCP composite. Methods Each individualcomponent of the UPPE/β-TCP composite was immersed in culture media andincubated, respectively. The culture media without extracts severed as the control.Using cell line L929 as model cells, the individual cytotoxicity of each constituentwas evaluated by an AlamarBlue viability assay. The effects of the leachable productsfrom the cross-linked UPPE polymer and UPPE/β-TCP composite, the in vitrodegradation products obtained from the complete breakdown of the cross-linkedcomposite under accelerated conditions on the cytotoxicity were also investigated.Single factor analysis of variance and Tukey's HSD multiple comparison tests wereused to determine statistical significance of results. Resultsβ-TCP demonstratedcell viability comparable to the control (P＞0.05). The other components of the UPPE/β-TCP composite exhibited significantly higher cytotoxicity at the highestconcentration (P＜0.05), and dilutions of these extracts produced an improvement incell viability. Once cross-linked, however, the cytotoxicity of the leachable productsfrom the cross-linked UPPE polymer and UPPE/β-TCP composite was comparable tothat of the control (P＞0.05) because most of the individual constituents may beincorporated into the cross-linked network. The degradation products of thecross-linked composite displayed a dose-dependent cytotoxic response with only thetwo highest concentration solutions demonstrating cell viability significantly lowerthan the control (P＜0.05). Conclusion The results suggest that although there areconcerns regarding the biocompatibility of the novel UPPE/β-TCP composite, it holdsgreat promise for use as an injectable and biodegradable alveolar bone substitute inthe treatment of periodontal diseases.
PART TWO Investigation of MC3T3-E1 Cell Behavior on the Surface of aBiodegradable Alveolar Bone Substitiute Containing Tetracycline
Objecitve The early osseointegration of bone substitutes is an important factorfor their clinical success. The challenge in the engineering of bone substitute'ssurfaces is to attract, above all, osteoblasts that produce a bone extracellular matrix,which will ensure a high bone-implant contact. Cell adhesion is one of the initialstages for subsequent proliferation and differentiation of osteoblastic cells producingbone tissue. The aim of this present study is to investigate the behavior of osteoblasticMC3T3-E1 cells on four different bone grafts surfaces in vitro:β-TCP, UPPE polymer,UPPE/β-TCP composite and UPPE/β-TCP containing 1% tetracycline(UPPE/β-TCP+1% TTC) composite. Methods MC3T3-E1 cells were culturedeither on theβ-TCP, UPPE polymer, UPPE/β-TCP composite and UPPE/β-TCP+1%TTC composite discs or in the absence of material (plastic) for 4, 8 and 15 days (d).The cell viability of each group was measured as optical density by an AlamarBlueviability assay, and normalized to cell culture medial (the control) in order to demonstrate differences in cell viability on the different substrates. After 1 or 14 dincubations of the cells on the four materials or the control, the cellswere double-stained with Acridine orange/Ethidium bromide (AO/EB) for 5 minutes(min) and viewed by epifluorescence microscopy to simultaneously examine of bothlive and dead cells on the materials. The morphology of osteoblastic cells MC3T3-E1after culturing for 2 d on the different substrates was examined using a scanningelectron microscope (SEM). The differentiation function of osteoblastic cellsMC3T3-E1 was assessed by measuring alkaline phosphatase (ALP) activity. Singlefactor analysis of variance and Tukey's HSD multiple comparison tests were used todetermine statistical significance of results. Results For all substrates, the resultsshow an increase in cell viability indicating cell proliferation with culture time. After4 d of culture, the cell viability was slightly inferior for all groups compared with thecontrol, but there was no statistical differences between groups (P＞0.05). The dataappeared similar for the UPPE/β-TCP composite after 8 and 15 d of culture, andslightly superior for theβ-TCP or UPPE/β-TCP+1% TTC composite after 15 dcompared with the culture. Nevertheless, the statistical analysis did not showsignificant differences between groups (P＞0.05). The cell viability remainedslightly lower in UPPE compared with the control. Visual examination revealed thatthe density of live cells adherent to each material was similar after 2 and 15 d ofculture. Live cells, stained green, appeared to have adhered and attained a normalpolygonal morphology on all materials. Dead cells (stained red) were very few on allthree materials. SEM micrographs show similar cell attachment on the four materials.ALP activity increased with culture time whatever the substrate was and appeared tobe at its maximum after 21 d. Conculsion These data show that a UPPE/β-TCPcontaining 1% TTC composite is biocompatible and supports the adhesion, spreading,proliferation and viability of osteoblast cells.
PART THREE In Vivo Bone and Soft Tissue Response to an Injectable,Biodegradable Alveolar Bone Substitiute Containing Tetracycline
Objective Earlier investigations have characterized some of the physical andbiological properties of UPPE/β-TCP composite. It is worth noting that sometimes thebone substitute is not only in contact with bone, but also with the surrounding softtissues. To ensure the safety and effectiveness of this technique, this study wasdesigned to assess in vivo soft and bone tissue response to to the UPPE/β-TCPcomposite and UPPE/β-TCP+1% TTC composite after implantation using a rabbitmodel. Methods Twenty-five male New Zealand White rabbits were used asexperimental animals. For the insertion of the subcutaneous implants, fourssubcutaneous pockets at the dorsum of the rabbits were created. The implants (β-TCP UPPE polymer, UPPE/β-TCP composite and UPPE/β-TCP+1% TTC) wereinserted in these pockets. UPPE/β-TCP composite and UPPE/β-TCP+1% TTC wereinjected in circular defects as created in the femoral condyles of rabbits and were leftin place for 2, 4, 8, 12 and 24 w. The specimens were evaluated morphologically(histology, and histomorphometry). Single factor analysis of variance and Tukey'sHSD multiple comparison tests were used to determine statistical significance ofresults. Results The histologic evaluation in vivo after 2 w showed a mild infiltrateof inflammatory cells for most of the groups. UPPE/β-TCP+1% TTC compositeexhibited no signal of inflammatory response. After 4 w. the tissue showed nearnormal pattern for all the groups. The soft tissue capsule had a non-uniformdistribution in thickness, which increased most significantly between 4 w and 12 wafter implantation. After 8 weeks, the capsule thickness did not change much. None ofpolymorphic cells, osteoblast cells or bone cells adjacent to the implant were observed.The surface of theβ-TCP, UPPE/β-TCP composite and UPPE/β-TCP+1% TTCcomposite themselves remained substantially intact without noticeable cracking,chipping or dissolution. However, at 8 w, UPPE polymer showed extensive surfaceerosion and superficial fragmentation that was surrounded by a few inflammatory cells. Gross examination of retrieved implant/bone composite samples indicated thatthe UPPE/β-TCP composite and UPPE/β-TCP+1% TTC composite did not evokeinflammatory response, necrosis or fibrous encapsulation in surrounding bony tissues.Histological examination revealed excellent composite/host bone bonding. At 4 w, theresorption induced voids between terminals of bone defects and implants were largelyfilled with new bone. Composite resorption, new blood vessels, osteocytes, osteonsand osteoblast-like cells lining up with active new bone were observed at remodelingsites. At 12 w, a new bone network was developed within femoral defect, whilecomposite became islands incorporated in the new bone. At 24 w, bone ingrowth andremodeling activities became so extensive that the interface between residual cementand new bone became less identifiable. In general, the resorption ratio valuesincreased with implantation time. Conclusion TC addition optimizedbiocompatibility of the UPPE/β-TCP composite, contributing to anti-inflammatoryresponse during the early phases of the wound healing process. These results showedthat UPPE/β-TCP composite holds promise for use as a syntheticbiodegradable scaffolds for tissue engineering.
PART FOUR The Influence of Novel Injectable Alvelor Bone Substitutes onRegeneration of Periodontonal Defects with Experimental Periodontitis in Dogs
Objective Earlier studies showed that an injectable UPPE/β-TCP composite wasbiocompatible and promoted histocompatible healing of bone tissue in rabbits. Thesemisolid consistency of UPPE/β-TCP composite containing TTC shows sustainedand almost constant in vitro drug release in phosphate buffer, pH 7.4 at 37℃, for upto 14 days. The purposes of this present study were (1) to assess the in vivo releaseof drug in the periodontal pockets from the UPPE/β-TCP composite containing 1%,5% or 10% (wt/wt) TTC and (2) to evaluate influence of the formulation onregeneration of periodontal defects with experimental periodontitis in dogs.
Methods Experimental periodontitis was induced by placing stainless-steel mesh on the mesial side of maxillary canines for 6 w in fifteen adult, healthy dogs.Subsequently, intrabony defects were resized so as to be standard, and UPPE/β-TCPcomposite containing 1%, 5% and 10% TTC were directly injected in theexperimental bone defects. Non-grafted defects on the contralateral side served ascontrols. The retention time of the antibiotic product was determined indirectly bymeasuring the concentration of tetracycline in gingival crevicular fluid (GCF) usinghigh-performance liquid chromatography (HPLC) at 3, 7, and 14 d after the placementof the UPPE/β-TCP + TTC composites. Sixteen weeks after surgery, the animals weresacrificed and histologic specimens were prepared. Periodontal tissue healing wasevaluated histologically and histometrically. Results The TTC concentrations inthe GCF well above the minimum inhibitory concentration (MIC) of most periodontalpathogens were seen at 3, 7, and 14 d following the injection of theUPPE/β-TCP+TTC composite formulations. Healing of periodontal tissues, in termsof bone and cementum formation, was consistently observed in theUPPE/β-TCP+TTC composite-applied sites. UPPE/β-TCP+TTC composites werepartly replaced by new bone. New cementum and periodontal ligament-like tissuewere observed between UPPE/β-TCP+TTC composites and the root surface. Newbone (P＜0. 05), new cementum (P＜0.05), and new connective tissue attachment andadhesion (P＜0.05) were significantly enhanced in the experimental sites.
Conclusion The UPPE/β-TCP+TTC composite formulations can be retained in theperiodontal pocket for a prolonged period, releasing a sufficient amount oftetracycline to eliminate pathogenic bacteria. The composites containing TTCprovides stable wound healing and enhanced periodontal regeneration in periodontaldefects in dogs with experimental periodontitis. The local application of injectablebioerodible UPPE/β-TCP+TTC composites appears to be promising in the context ofperiodontal treatment.
引文
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