摘要
第一部分：脊柱转移性肿瘤骨折风险的预测---结合定量CT和Analyze MD软件预测骨折风险值的应用研究
背景与目的
美国每年有超过一百万新增癌症患者,其中约2／3将会发生骨转移,脊柱为最常见的骨转移部位。转移性脊柱肿瘤患者由于脊柱结构完整性的破坏,临床上可以出现疼痛和脊髓神经受压的症状。据统计,约有超过30%的骨转移癌患者会因合并有病理性骨折而需要保守或外科处理。
脊柱骨折风险的预测及早期干预措施,是近年来讨论研究的热点。脊柱转移性肿瘤所致的病理性骨折因其病变的特殊解剖部位,常常合并有脊髓受压,而且骨折后病变椎体的机械强度亦不足以维持人体正常的活动。所以脊柱转移性肿瘤患者一旦发生病理性骨折,往往后果严重,多需要开放性手术治疗,从而极大的增加了医疗费用与医疗环节,同时也严重影响了转移癌患者晚期的生活质量。因此,对转移性脊柱肿瘤患者进行准确的骨折风险预测,进而对骨折高危患者行积极有效的预防性干预措施,将具有重要的临床意义。
对于转移性脊柱肿瘤骨折风险的预测必须考虑到两方面的因素：病变的密度特性和脊柱病损的几何形状。定量计算机断层扫描(QCT)可以同时显示肿瘤病变和残余椎体结构的骨质密度和几何特点,结合组合柱理论,可以较为准确的预测病变脊椎的负荷量。
目前,对于以QCT影像为基础的椎体模量和强度的计算,以及后续的分析过程不但耗时,而且需要专业人员操作。因此,设计一种自动的、操作简便以及重复性好的软件系统对于脊椎力学结构的预测分析具有重要的临床意义。由Mayo Clinic医学影像中心(BIR)设计的影像分析软件系统：Analyze MD是一种功能性的以数据为基础的分析系统。该系统能够识别医疗数位影像传输协定和定量CT,建立并优化特定的应用界面。在本研究中,我们选择Analyze MD系统作为我们的软件应用平台,经过简单的操作后,对病变脊椎骨质密度和骨结构进行自动测量,继而推算出其轴向负荷的能力,最终估算出病变椎体的骨折风险值(Fracture Risk Value,FRV)。其中, FRV被定义为病变椎体理论正常轴向刚度与实际病变椎体轴向刚度的比值。它代表病变椎体机械强度下降的程度,当比值较高时,即存在骨折的高风险。通过对Mayo Clinic308例脊柱转移性肿瘤患者的回顾性对照研究,对FRV预测患者骨折发生的诊断价值进行客观的评价。这种结合QCT和先进的数字化影像分析系统(Analyze MD)对脊柱转移性肿瘤病变椎体进行骨折风险的预测,国际上尚未见类似的报到,属于创新性的研究。
资料与方法
1.病例选择
在经美国Mayo Clinic临床研究机构审查委员会(Institutional Review Board, IRB)审查通过之后,我们对2001-2005年间Mayo Clinic308例临床诊断为脊柱转移瘤的患者进行回顾性分析。
病人的入选标准有：(1)必须满足至少有两次连续的病变椎体的CT检查,而且两次CT检查的时间不长于6个月；(2)本研究所评价的病变椎体限于胸腰椎,颈椎和骶椎转移性病变未被入选；(3)初次CT检查时,影像学显示病变椎体尚未发生病理性骨折；(4)病变椎体的上下相邻节段椎体在两次CT检查时,均未见明显转移性病变；(5)初次CT检查确诊为脊柱转移性肿瘤后,除继续针对原发性肿瘤行必要的化疗或(和)放疗外,患者未行病变椎体的手术治疗(包括开放性手术或椎体成形术等)；而且在保守治疗方面,除动态观察患者病情变化外,患者未针对性的使用拐杖、轮椅或佩戴支具等,因为这些都将明显减少病变椎体的正常负荷和骨折的发生,从而对最后的结果造成误差。
二次CT影像下病变椎体病理性骨折的诊断标准为：在冠状面或矢状面上,一侧椎体高度较对侧下降超过15%,或椎体高度较正常椎体高度(上下相邻正常椎体高度平均值)下降超过15%。根据二次CT的影像诊断结果,将最终符合入选标准的病变椎体分为骨折组和非骨折组。
2.病变椎体的骨折风险值(FRV)推算
在确定符合标准的入选病例后,由影像科负责将所有病例的初次CT影像学资料传入Analyze MD分析系统。作者在该软件系统中打开病人的CT影像学资料,并选择病变椎体,以及上下相邻的正常椎体。操作完成后,由Analyze MD软件系统即时分析计算出该病变椎体的骨折风险值。
3.统计学分析
对于骨折组与非骨折组之间的临床资料的统计学比较,运用t检验、和卡方检验。临床资料的比较包括：患者的年龄、性别、临床症状、病变椎体的节段、部位以及原发肿瘤的诊断等。对所有病变椎体所计算出的FRV值,首先经过柯尔莫诺夫-斯米尔诺夫检验确定变量复合正态分布。然后,在骨折组和非骨折组之间,运用双样本t检验。最后确定FRV的最佳临界值,及其灵敏度、特异度和准确度。
结果
在本研究中,共有73例患者、116个病变椎体符合入选标准。其中39例患者为男性,34名患者为女性,所有患者年龄范围在31～82岁,平均58.7±10.9岁。73例患者原发性肿瘤的类型为：肾癌12例,肺癌10例,乳腺癌9例,前列腺癌7例,其他35例。所有116个病变椎体中,胸椎病变57例,腰椎病变59例,经二次CT结果诊断为骨折41例,非骨折75例。
骨折组和非骨折组患者临床资料的单因素比较显示,两组患者的年龄、性别、病变部位等均无显著性差异(P＞0.05)
通过SPSS统计软件绘制FRV预测骨折的受试者工作曲线(Receiver operator characteristic curve, ROC曲线)。在ROC曲线中,确定FRV的最佳临界值为1.45。在该临界值下,FRV的灵敏度为92.7%；特异度为64.0%；准确度为74.1%。
结论
结合QCT和Analyze MD图像软件分析系统,我们可以在简易的操作界面下,方便迅速的计算出脊柱转移瘤患者病变椎体的骨折风险值FRV。而且回顾性的对照分析显示,FRV是一项独立的预测骨折风险的指标,在预测骨折特别是胸腰椎病理性骨折方面具有良好的灵敏度和特异度。该方法临床实用价值高,可以为转移性脊柱肿瘤患者的临床诊治提供较为准确客观的参考依据。
第二部分：脊柱转移瘤病理性骨折的预防性治疗---一种新型可注射生物材料的合成、表征及交联特性的基础研究
背景与目的
高骨折风险的转移性脊柱肿瘤的病变椎体一旦发生骨折,往往后果严重,患者预后极差,因此有必要对其进行积极的预防性干预措施。近几十年来脊柱转移瘤的治疗取得了巨大进展,总的治疗策略包括：化疗、放疗、手术治疗或综合治疗。化疗和放疗对于肿瘤的控制以及疼痛缓解都有一定效果,却不能增加病变脊椎的稳定性；手术治疗虽效果明确,可以即时增加病变脊椎的稳定性,但同时也存在许多可能的并发症；各种保守治疗的疗效尚不确切,而且亦不能从根本上增加病变脊椎的稳定性,更不能延缓疾病本身的进展。
经皮椎体成形术是一种在影像定位引导下的微创治疗技术。它通过穿刺针将骨水泥(聚甲基丙烯酸甲酯PMMA)注入病变椎体,以达到缓解疼痛及恢复椎体高度和强度等目的。该术式具有创伤小、恢复快、临床效果显著等特点,尤其适用于放疗效果不佳、有明显疼痛而又不能耐受开放性手术的大多数转移性脊柱肿瘤患者。有研究表明椎体成形术后绝大多数患者临床疼痛症状得到显著缓解,生活质量明显改善。PMMA是目前临床上使用最为广泛的可注射型骨移植替代物之一,尤其多用于体内的负重部位。在注入骨缺损部位后,骨水泥快速原位交联固化并提供即时的力学支撑作用。但是,PMMA骨水泥也同时存在着一些不足之处。例如,PMMA骨水泥交联固化后,其压缩模量远远大于松质骨的压缩模量,而且不可降解。再如,PMMA骨水泥的临床可操作时间较短以及交联固化时产生的热量过高而易导致邻近组织特别是脊髓等重要结构的热损伤等。这些方面在一定程度上限制了PMMA骨水泥的临床应用。
聚丙烯延胡索酸酯(PPF)是一种新型的可注射型组织工程材料,在骨科临床具有非常广阔的应用前景。该生物材料是一种线性的不饱和聚酯结构,其长链结构中的延胡索酸双键,可以进行原位交联固化。有研究表明其交联固化时间变动于1至121分钟之间,交联固化时的最高温度约为48℃,大大低于PMMA。虽然大量的以PPF为基础的生物材料的应用研究正在开展,但是对于PPF材料结构本身,仍存在很多的局限性。PPF结构中碳碳单键的密度较低,链结构缺乏足够的活动度和旋转性。再者,由于PPF交联固化需要引入交联剂来提供自由基,而交联固化后未参与反应的交联剂单体可能引起细胞毒性等一系列负作用。
聚己内酯(PCL)是一种FDA通过的可生物降解的聚合物,它具有优良的生物组织相容性和结构的灵活性。许多复合有PCL的共聚物明显改善了其原聚合物的理化性质和交联特性。
为了综合PPF和PCL的优势特性,我们设计合成出了一种新型的共聚物：聚丙烯延胡索酸酯复合聚己内酯共聚物(PPF-co-PCL),并对其理化性质进行了全面的表征。另外还通过引入各种变量,对该共聚物交联固化后的各种特性进行了较为全面的分析与比较。所分析的共聚物的交联特性有：交联固化时的最高温度、凝胶时间,交联固化后的抗压模量和抗压韧度,以及交联固化后的共聚物的体外细胞毒性和体外降解过程等,以期为该共聚物的进一步深入研究奠定必要的实验基础。对该种新型共聚物的设计合成,尤其是对其交联固化后各种特性的全面分析,国际上尚未见有类似有关报道,属于创新性研究。
材料与方法
1.材料
聚己内酯(PCL)购自美国Sigma-Aldrich公司,分子量分别为530、1250和2000 g／mol。实验中其他所用到的所有试剂亦均购自美国Sigma-Aldrich公司。
2.实验设计
在该实验设计中,共引入三个变量：共聚物前体PPF和PCL的分子量以及PCL在共聚反应中的进给比。结合三种不同的变量,我们共合成了十八种不同的共聚物。共聚物合成后,通过加入引发剂和促进剂进行交联固化反应,并测量交联固化过程中的最高交联温度、凝胶时间以及交联固化后共聚物的生物力学特性等。所有的实验标本均为三倍样本,测量的结果以均数加减标准差表示。
3.PPF的合成
PPF为两步法合成,第二步的聚合反应的时间设定为1小时或5小时,从而生成两种不同分子量的PPF聚合物(约为700和2000 g／mol),聚合物分子量经凝胶色谱分析仪测量确定。
4.PPF-co-PCL的合成
共聚物PPF-co-PCL的合成以PPF和PCL为前体,以三氧化二锑作为催化剂。最后反应获得的PPF-co-PCL共聚物通过溶解析出的方法进一步纯化,最后得到粘性胶状物或蜡样固体。
5.共聚物各种特性的表征
5.1凝胶色谱分析(GPC)
对于交联固化前的共聚物通过凝胶色谱分析法(GPC)测量其分子量及其多分散性指数。
5.2傅里叶变换红外光谱分析(FT-IR)
傅里叶变换红外光谱分析由Nicolet 550型光谱仪分析完成。
5.3核磁共振光谱分析(NMR)
质子核磁共振光谱分析(1H-NMR)由Varian Mercury Plus NMR光谱仪完成(1H=300.1 MHz)。
5.4差示扫描量热分析(DSC)
差示扫描量热分析由美国TA公司Q1000型差示扫描量热分析仪完成,DSC曲线中的玻璃化转变温度(Tg)定义为玻璃转化过程曲线中的中点温度。
6.共聚物PPF-co-PCL的交联固化
共聚物交联固化参考我实验室对聚合物交联固化的统一方案完成。为了研究促进剂对该共聚物交联固化各性状的影响,在共聚物交联固化过程中,我们还选用了另外两种剂量的促进剂溶液。
7.交联固化时的最高温度：
共聚物交联固化时的温度变化用热偶计测量并记录。将记录的时间零基点定义为交联反应混合物中最后加入促进剂溶液的时间。
8.交联物凝胶时间
共聚物交联固化的凝胶时间由流变仪测量。将记录的时间零基点亦定义为交联反应混合物中最后加入促进剂溶液的时间,而共聚物交联固化的凝胶时间点定义为在混合物粘度变化的曲线中,粘度值突然升高时的时间点。
9.共聚物交联固化后的生物力学特性
将共聚物交联固化后的柱形标本,在312型材料测试系统上测量材料的生物力学指标,记录描绘出应力-应变曲线。材料的压缩模量为应力-应变曲线中的第一段线性部分的斜率值。压缩韧度为标本被压溃之前,反映在应力-应变曲线下的面积。
10.共聚物交联固化后的体外细胞毒性实验
用MTS比色法检测交联固化后的共聚物标本对人类成骨样细胞的细胞毒性,并与共聚物的前体PPF以及传统的聚甲基丙烯酸甲酯PMMA骨水泥的细胞毒性相比较。
11.共聚物交联固化后的体外降解实验
将共聚物交联固化后的标本浸泡于PBS缓冲液,并置于37℃温箱,分别于第4、第8和第16周后,取出标本,真空干燥,测量标本降解后的残余质量,以计算出标本的失重质量分数,并与共聚物的前体PPF的失重质量分数相比较。标本测重后,在312型材料测试系统上测量材料的生物力学指标,计算标本的压缩模量。
12.统计学分析
所有测量结构均为三组测试值,最终结果以均数加减标准差表示。统计学分析方法为单因素或三因素方差分析(ANOVA),然后行邓肯多重范围检验,统计学计算过程由SAS版9.1.3软件包计算完成,计算结果P值小于0.05时定义为有统计学差异。
结果
1.共聚物的性状表征
所有PPF-co-PCL共聚物的分子量在3141±48至6607±144 g／mol之间,合成的共聚物的多分散性指数在2.6±0.2至4.0±0.3之间。共聚物FT-IR光谱分析显示为其两种前体的组合光谱结果。通过1H NMR光谱分析计算出的PCL比例与实际的PCL进给比对应良好。DSC测量曲线显示,所有共聚物有单一的玻璃化温度,温度值范围为-39.7±2.4至-15.0±1.6℃之间,该温度值与共聚反应中PCL进给比呈明显负相关(P＜0.01)。
2.交联固化时的最高温度
所有合成的共聚物的交联固化时的最高温度范围在38.2±0.3至47.2±0.4℃之间,该温度值与PCL前体的分子量呈正相关。
3.交联固化的凝胶时间
所有交联共聚物的凝胶时间波动于4.2±0.2和8.5±0.7分钟之间,它与PCL前体的分子量呈负相关。所有合成的共聚物的凝胶时间均大于PMMA交联固化时的凝胶时间,而均明显小于其PPF前体的凝胶时间(P＜0.01)。共聚物交联固化时,随着促进剂剂量的减少,凝胶时间明显延长(从6分钟延长至超过60分钟)。
4.生物力学特性
交联固化后的共聚物的压缩模量值在44±1.8和142±7.4 MPa之间,并与其PPF前体的分子量大小呈正相关,而与其PCL前体的进给比呈负相关,个别PPF-co-PCL共聚物的压缩韧度明显大于其PPF前体。所有测量计算出的共聚物以及其PPF前体的压缩韧度,其值在4.1±0.3至17.1±1.3KJ／m3之间。
5.共聚物的细胞毒性
研究结果表明,在第1、第3和第7天的细胞毒性实验中,合成的PPF-co-PCL共聚物以及PMMA骨水泥与对照组相比,均未出现明显的细胞毒性反应,而在细胞培养的第7天,共聚物前体PPF组的细胞活性与对照组相比略有下降,但是并无明显统计学差异(P＞0.05)。
6.共聚物的体外降解
共聚物交联固化后的体外降解实验结果显示,共聚物的体外降解率与前体PCL的进给比呈正相关。而PPF和PCL前体的分子量对共聚物的降解率无明显影响。降解后的共聚物的压缩模量随时间变化的曲线显示,所有共聚物的压缩模量在交联固化的前四周内,随时间的延长,压缩模量明显升高；而在四周以后,其压缩模量值趋于稳定。
结论
研究中合成的新型共聚物PPF-co-PCL交联固化时的最高温度、凝胶时间以及交联固化后的生物力学性能和降解率等可以通过调节其合成过程中的变量值而作相应改变,从而达到适应不同临床需要的目的。引入PCL结构的共聚物在交联固化时反应更加完全,从而减少了单纯PPF交联固化时,因反应不完全而残留的单体所可能导致的细胞毒性。PPF-co-PCL共聚物整合了其PPF和PCL前体的各自优点,具有更加优异的性能特征。它作为一种新型的可注射型、原位交联聚合的生物材料,具有极其广阔的应用前景。
Part 1:Fracture Risk Analysis of Metastatic Spine Tumors--Analysis of Fracture Risk by Combining QCT and Analyze MD software
Background and Objective
At least one million new cases of cancer are diagnosed each year in the United States, skeletal metastases occur in 2/3 of them, most commonly to the spine. Failure of the spine's structural integrity from metastatic disease can lead to both pain and neurologic deficit. Fractures that require treatment occur in over 30% of bony metastases.
Fracture risk analysis and prophylactic stabilization of spine are the hot topics recently. Prophylactic cement augmentation of a vertebral body infiltrated with tumor is an entirely different entity. An osteoporotic vertebral compression fracture does not cause spinal cord compression, whereas a vertebral body that fractures due to tumor infiltration often causes neurologic symptoms and signs with potential devastating consequences. Therefore, it is vital important to exactly predict the fracture risk of the metastatic spine and apply prophylactic spinal stabilization so that patients could remain ambulant and continent.
Methods to predict fracture risk in metastatic vertebral disease must measure changes in both the material properties and the bone geometry within the vertebral body. Quantitative computerized tomography is able to delineate the density and geometry of both the tumor and the remaining bone and accurately predict, via composite beam theory, the vertebra's load carrying capacity
Currently, manual input of the QCT image-based strength and modulus data, and their subsequent analysis is a time consuming and user dependent process. The development of an automated, user friend, and reproducible software package would enable the vertebral load carrying capacity determination to occur in a clinical setting. The image analysis software program, Analyze MD, developed at the Mayo Clinic College of Medicine's Biomedical Imaging Resource (BIR) includes a functional data-base management system that recognizes digital imaging and communications in medicine (DICOM) and QCT images. Application-specific interfaces can be developed and optimized for specific clinical applications. We have chosen Analyze as our software platform for the automation of bone mineral density and bone structure determination, then calculate the load-bearing capacity of the lesion spine, and finally calculate the Fracture Risk Value (FRV). The risk for impending fracture was defined as the theoretic normal axial rigidity divided by the axial rigidity of lesion vertebra. The risk of fracture increases with the increase of FRV.
Finally, we retrospectively studied 308 existing Mayo cancer patients with spinal metastatic disease and evaluated the significance of FRV calculated from Analyze MD. This is a creative study to predict the fracture risk of metastatic spine lesion by combining the QCT results and novel Analyze MD software.
Materials and Methods
1. Study Cohort
The appropriate IRB approval has been accomplished. Then we retrospectively studied 308 existing Mayo cancer patients with spinal metastatic disease between 2001 and 2005. Patient inclusion criteria include: (1) patient had two consecutive spine CT scans done in less than a six month period; (2) we only investigated the metastatic lesion of thoracic and lumbar spine, metastatic lesions of the cervical and sacral spine were not counted in; (3) no pathologic fracture was detected on the first CT images; (4) the investigated spinal lesion was a separated lesion whose adjacent upper and lower segment were relatively normal on radiographic images; (5) after the diagnosis of spinal metastasis from the first radiographic images, patient didn't have some further operative treatment (like open surgery and internal fixation or percutaneous vertebroplasty) other than subsequent radiotherapy or chemotherapy for the primary tumor; no conservative treatment had been provided other than observation by the treating physician; patient they had used crutches or were advised to substantially decrease their activities for more than a few weeks were excluded, since this modification inactivity alone would have decreased the fracture risk.
The initial CT scans will be compared to the follow-up CT scans for the presence of new pathologic fractures, a fracture will be defined by criteria used for osteoporotic vertebral fractures or vertebral endplate fractures. These criteria include either a 15% loss of height from one side of the vertebra compared to the other in the frontal or sagittal plane, or a 15% loss of vertebral height compared to adjacent vertebrae. Finally, all the eligible cases were divided into two groups:fractured group and non-fractured group.
2. FRV analysis with QCT and Analyze MD
After confirmation of all the eligible cases, all the radiographic images of the patients were transferred into the Analyze MD system by radiology department of Mayo Clinic. The author opened the radiographic data of the patient from the Analyze MD system, and selected the interested metastatic vertebral body together with its adjacent upper and lower spinal segments. Once the vertebral body was selected, the Analyze MD system can automatically calculate final FRV of the metastatic vertebral body through some empirical formulas.
3. Statistical analysis
Univariate analysis, with the Student t test and chi-square test as appropriate, was used to compare demographic data, including age, gender, primary tumor diagnosis, site of the defect, and affected segment, between the fracture and non-fracture groups. Calculated values of FRV were assessed for normality with use of the Kolmogorov-Smirnov test, and no significant skewness was detected. Therefore, the two-sample Student t test was used to compare the fracture and non-fracture groups. Sensitivity, specificity, and accuracy were calculated to determine the diagnostic performance of FRV. For all comparisons, a two-tailed P< 0.05 was considered significant. The data were analyzed with use of the SPSS software package (version 13.0; SPSS, Chicago, Illinois).
Results
In out study,73 eligible cases in total were included. Thirty nine patients were male and thirty four were female, and their ages ranged from thirty one to eighty two years (mean and standard deviation,58.7±10.9 years). Twelve patients had a primary tumor of renal cell cancer, ten lung cancers,9 breast cancers,7 prostate cancers and 35 others. Fifty seven lesions were located in the thoracic spine; fifty nine in the lumbar spine.
The results of the univariate comparisons of the demographic data between the fracture and non-fracture groups showed no significant difference between these two groups (P>0.05).
FRV derived with quantitative computed tomography and Analyze MD system differed significantly (P<0.01) between the fracture and non-fracture groups. At the cut-off value of 1.45, FRV attained the 92.7% sensitive and 64.0% specific and 74.1% accuracy for predicting fracture.
Conclusion
By combining the QCT and the powerful Analyze MD software image analysis system, we can easily accommodate current clinical CT imaging and calculate the FRV by validating an automated program. Our retrospective study indicated that the calculated FRV is an excellent independent criterion to predict the fracture risk of a metastatic spinal lesion especially in thoracic and lumbar spines with high sensitivity and specificity. We believe that this method can provide accurate objective criteria for planning treatment of metastatic spinal lesions and monitoring treatment response.
Part 2:The Experimental Study of Prophylactic Treatment of Spinal Metastasis--Synthesis, Characterization, and Crosslinking Properties of a Novel Injectable Biomaterial
Background and Objective
Prophylactic treatment of a vertebral body infiltrated with tumor is very necessary. Great progresses have been made in the treatment of spinal metastasis recently. Treatments include chemotherapy, radiotherapy, surgery and combined therapy. Chemotherapy and radiotherapy are useful to decrease the symptoms, however, they can not increase the stability of spine. Surgery has many complications, and the effects of conservative treatments are in issue, and they can not be used to increase the stability of spine, or halt the progress of disease.
Vertebroplasty is a minimal invasive treatment guided under radiographic images. PMMA bone cement was injected into the lesion vertebral body to relieve pain and stabilize the vertebral body. It has several strong points of less invasive, early recovery and great clinical effect. It is especially useful in the treatment of most spinal metastases which are not eligible for open surgery. PMMA is the most common used injectable bone cement especially in some load-bearing areas. After injection, PMMA polymerizes in situ and provides instant mechanical support. However, several drawbacks also exist in PMMA. PMMA has a significantly higher modulus than trabecular bone and is non-degradable. Additionally, PMMA has a relatively short working time for preparation and injection, and exhibits high exothermic heat release during curing that is not ideal for use as injectable bone substitute.
A promising candidate material of this type is poly(propylene fumarate) (PPF), an unsaturated linear polyester that can be modified or cross-linked through its fumarate double bonds. The curing time has ranged from 1 to 121 min, depending on the ratio of initiator, monomer or macromer, and PPF. The maximum temperature during PPF cross-linking has been 48℃, which is much less than that of PMMA. Although many efforts have been made to explore the applications of PPF-based materials, there are still many important limitations of this material. The propylene glycol in each repeating unit provides only one free rotating carbon-carbon bond that contributes to the rigidity of the PPF polymer chain. In addition, a cross-linker is needed to form cross-linked PPF networks via redox initiation, which may lead to cytotoxicity associated with unreacted cross-linking monomers.
Polycaprolactone (PCL) is a FDA-approved biodegradable polymer with excellent biocompatibility and flexibility. A variety of copolymers based on PCL have been made to enhance the applications and crosslinking properties of such material.
In an attempt to combine the favorable properties of PPF and PCL, our laboratory recently designed and synthesized a new injectable copolymer PPF-co-PCL composed of PPF and PCL. The chemical and physical properties of the uncrosslinked copolymers have been characterized. In this study, we further evaluated the handling and mechanical properties of the crosslinked copolymer by varying parameters. Maximum crosslinking temperature, gelation time, mechanical properties, cytotoxicity and in vitro degradation of the crosslinked copolymers were evaluated. This is a creative study to characterize and evaluate the novel polymerized copolymer as an injectable bone substitute.
Materials and Methods
1. Materials
PCL diols with nominal molecular weights of 530,1250 and 2000 g/mol were purchased from Aldrich (Milwaukee, WI). All the other chemicals in the present study were also purchased from Aldrich.
2. Experimental design
The three variables we investigated were:PPF Mn, PCL precursor Mn, and PCL feed ratio. Eighteen copolymers were synthesized and characterized. The copolymers were crosslinked by adding benzoyl peroxide and dimethly toluidine. The maximum crosslinking temperature, gelation time, mechanical properties and biocompatability were measured. All experiments were conducted in triplicates, and data is expressed as means +/- standard deviations.
3. Synthesis of PPF
PPF was synthesized as described previously. The polymerization reaction was run at 150℃for 1 and 5 h, producing PPF with Mn around 700 and 2000 g/mol, respectively, as measured by gel permeation chromatography (GPC).
4. Synthesis of PPF-co-PCL
The PPF-co-PCL copolymer was synthesized from PPF and PCL with antimony trioxide added as a catalyst. The resulting PPF-co-PCL copolymer was purified by solution precipitation forming a viscous melt or wax-like solid.
5. Copolymer Characterizations
5.1 GPC
The uncrosslinked copolymer's molecular weight and molecular weight distribution were characterized by gel permeation chromatography (GPC).
5.2 FT-IR
Fourier transform infrared spectroscopy (FT-IR) spectra were obtained on a Nicolet 550 spectrometer.
5.3 NMR
Proton nuclear magnetic resonance (1H-NMR) spectra were acquired on a Varian Mercury Plus NMR spectrometer (1H=300.1 MHz).
5.4 DSC
Differential scanning calorimetry (DSC) was measured on a TA Instruments Q1000 differential scanning calorimeter. The glass transition temperature (Tg) was determined by using the midpoint temperature of the glass transition process.
6. Crosslinking of PPF-co-PCL
As related previously, a typical crosslinking procedure was performed. Additionally, two other different amounts of accelerator solution were also used when crosslinking copolymer 1.
7. Maximum temperature
The temperature profile was recorded through a thermocouple. Time zero was defined when accelerator was added into the mixture.
8. Gelation time
The gelation time was measured on a rheometer. Time zero was defined when accelerator was added into the mixture, and the gelation time was defined when the viscosity of the composite suddenly increased as shown on rheometer.
9. Mechanical properties
The crosslinking cylinder specimens were analyzed using a 312 Materials Testing System mechanical testing machine. The compressive modulus was calculated as the slope of the initial linear portion of the stress-strain curve. The compressive toughness was calculated as the area under the stress-strain curve before sample failure.
10. In vitro Cytotoxicity evaluation
The in vitro cytotoxicity of parts of our PPF-co-PCL formulations together with PPF and PMMA were investigated using MTS Assay. Three time points (1 day,3 days, and 7 days) were chosen.
11. In vitro Degradation
The crosslinked specimens were dipped into the PBS solution. Three time points (1 day,3 days, and 7 days) were chosen to check the weight loss of the specimen. Specimens were also analyzed using a 312 Materials Testing System mechanical testing machine.
12. Statistical analysis
All experiments were conducted in triplicate and the data were expressed as means±standard deviation (SD). One or three factors analysis of variance (ANOVA), followed by Duncan's multiple range test, were performed with SAS version 9.1.3 software to identify statistical difference at a significance level of P<0.05.
Results
Copolymer Characterizations
PPF-co-PCL Mn ranged from 3141±48 to 6607±144 g/mol. The polydispersity ranged from 2.6±0.2 to 4.0±0.3. FT-IR of copolymer showed a combination of spectra from its two components. PCL composition in the copolymer was calculated in 1H NMR and showed a good correlation with the actual PCL feed ratio. All the copolymers have a single glass transition temperature which fell between -39.7±2.4 and -15.0±1.6℃. They decreased significantly with increasing PCL feed ratio (P<0.01).
Maximum crosslinking temperature
The maximum crosslinking temperature of crosslinked copolymer fell between 38.2±0.3 and 47.2±0.4℃. It increased with increasing PCL precursor's molecular weight.
Gelation time
Gelation time of the crosslinked copolymer fell between 4.2±0.2 and 8.5±0.7 min. It decreased with increasing PCL precursor's molecular weight. All the gelation times of crosslinked copolymers were longer than that of PMMA, and significantly shorter than those of their PPF precursors (P<0.01). When decreasing accelerator, the gelation time greatly increased (from 6 min to over 60 min).
Mechanical properties
The compressive modulus ranged between 44±1.8 and 142±7.4 MPa. It increased with increasing PPF precursor's molecular weight and decreasing PCL feed ratio. Copolymer 13 had a much greater compressive toughness than its PPF precursor (PPF2000).The compressive toughness of all crosslinked copolymers and PPF (PPF700 and PPF2000) fell between 4.1±0.3 and 17.1±1.3 KJ/m3.
Cytotoxicity
No cytotoxic response was demonstrated from the different formulations of PPF-co-PCL and PMMA bone cement. On the 7 days time point, there was a slight decrease trend of cell viability in PPF samples compared with the control group. However no statistical difference was shown.
In vitro Degadation
Our in vitro degradation experiment showed that the copolymer degraded faster with increasing PCL feed ratio. No significant difference of weight loss was found from different formulations varying in PPF or PCL precursor's molecular weights. The compressive modulus of all degraded copolymers was tested at different time points after crosslinking. The compressive modulus of all these copolymers kept increasing the end of the fourth week after setting. It subsequently became stable.
Conclusion
Our results show that the maximum crosslinking temperature, the gelation time, the mechanical properties and degradation rate of crosslinked copolymer PPF-co-PCL could be modulated by varying its composition for different applications. Incorporation of PCL in the copolymer composition led to a fully crosslinked copolymer network. By integrating both virtues of its PPF and PCL precursors, PPF-co-PCL appeared to be an optimal injectable, in situ polymerizable biomaterial with a more favorable characteristic.
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