基于可注射性水凝胶支架材料的心肌组织工程研究
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摘要
缺血性心脏疾病严重威胁人类的生命安全与健康。冠状动脉阻塞引起心肌梗死(MI),伴随心肌细胞大量死亡,损失的心肌被没有收缩功能的疤痕组织所替代最终导致心力衰竭。目前针对缺血性心脏病的治疗措施,如药物治疗、冠状动脉介入治疗、冠状动脉旁路术等,虽然能在一定程度上缓解患者症状,但均无法根治。心脏移植是晚期心衰患者唯一的治疗方式,但存在器官来源短缺等问题。因此,迫切需要新的方法来修复受损心肌组织以重建心脏的功能。
随着组织工程研究的不断深入,运用组织工程技术研制可供移植的工程化心肌组织为心脏缺血性疾病的治疗带来了新的希望。近十余年,心肌组织工程进展迅速,主要包括两种策略：三维立体工程化心肌组织体外再造与移植策略(以心脏创可贴的方式移植)与可注射心肌组织工程策略。可注射心肌组织工程是指将细胞和／或生长因子等与可注射性支架材料复合直接注射到心肌损伤部位进行损伤的修复,由于其对患者造成的创伤较小而更易于临床应用。目前,可注射心肌组织已成为心肌组织工程研究的热点之一。
干细胞是心肌组织工程研究中应用最为广泛的种子细胞来源之一。其中,具有无限增殖能力和多向分化潜能的ESC由于能够向心肌细胞高效分化、与宿主心肌整合并存在电信号传导等优势具有广阔的应用前景。然而,ESC体内移植后面临引发免疫排斥反应等问题严重限制了其在移植治疗器官损伤中的应用。通过治疗性克隆策略获得患者特异性的nt-ESC有望解决ESC体内移植后引发免疫排斥反应这一难题。然而,nt-ESC是否与普通受精卵来源ESC一样,能够在心梗部位移植后具有修复作用,目前尚未见报道。
温敏性壳聚糖水凝胶是可注射性心肌组织工程研究中一种优良的可注射性支架材料。我们实验室之前的研究表明,以温敏性壳聚糖水凝胶为载体携带ESC心梗部位移植能显著提高移植细胞在注射区域的滞留率及存活率,增加梗死区域心室壁厚度并显著改善心梗动物的心脏功能。证明了温敏性壳聚糖水凝胶携带ESC构建可注射性工程化心肌组织修复心肌梗死的可行性。然而,目前尚未见以温敏性壳聚糖水凝胶为可注射性载体,携带nt-ESC进行动物心梗部位移植与修复的研究报道。
可注射性支架材料另一个重要作用是能够对其携带的治疗性因子在体内起缓释作用。通过生长因子诱导心梗区域再血管化对缺血性心脏疾病的治疗具有重要意义。碱性成纤维细胞生长因子(bFGF)是最重要的促血管生长因子之一。然而,由于注射到心脏部位后体内扩散率高且半衰期短,bFGF的修复作用并不总是能够按照需要成功实现。以生物材料作为载体将携带的bFGF持续释放到梗死心肌中有望对组织再血管化起促进作用。目前尚未见以温敏性壳聚糖水凝胶为载体携带bFGF心梗部位移植修复心肌梗死的研究报道。
除天然的生物材料外,人工合成的生物材料由于其力学性能与降解速度更加可控且加工性能和价格都比天然材料占有优势,近年来在可注射性心肌组织工程研究中的应用迅速增多。低聚乙二醇延胡索酸酯(OPF)水凝胶具有良好的组织相容性、生物可降解性以及可注射性,能够携带多种细胞与生物活性因子进行体内移植,已广泛应用于骨、软骨、角膜组织工程。然而,目前尚未见以OPF水凝胶为载体应用于可注射性心肌组织工程修复心肌梗死的研究。
本研究首先合成温敏性壳聚糖水凝胶与OPF水凝胶,并对其生物学性能进行评价。在此基础上,以温敏性壳聚糖水凝胶作为载体携带nt-ESC心梗部位移植,对其心梗损伤修复能力进行深入研究,并对nt-ESC和F-ESC的心梗修复能力进行比较。其次,以温敏性壳聚糖水凝胶作为载体携带bFGF心梗部位移植,研究其作为携带与缓释生长因子的可注射性支架材料的可行性及心梗治疗效果。最后,在成功合成OPF水凝胶的基础上,以其为载体携带eGFP标记的ESC进行心梗部位移植,研究其心梗损伤修复能力并探讨其作为可注射性心肌组织工程支架材料的可行性。
本论文主要包含以下四部分研究内容：
第一部分：可注射性水凝胶支架材料的制备与生物学评价
可注射性水凝胶支架材料是携带细胞和／或生长因子的载体,是可注射性组织工程化心肌研制的重要组成部分。
实验一：温敏性壳聚糖水凝胶的制备及生物学评价。以氯化壳聚糖、p-甘油磷酸钠为原料,羟乙基纤维素为交联剂制备了温敏性壳聚糖水凝胶。将nt-ESC与该水凝胶复合于体外培养后,应用AO/PI染色观察nt-ESC在水凝胶中的存活情况。此外,将温敏性壳聚糖水凝胶注射于大鼠心梗边缘区,不同时间点取材,通过组织学染色检测其组织相容性及体内生物降解性。结果表明：制备的温敏性壳聚糖水凝胶具有温度敏感性,其在室温条件下为液态,当温度升高到37℃时形成凝胶。生物学评价结果说明其具有良好的细胞相容性、组织相容性及可生物降解性。
实验二：低聚乙二醇延胡索酸酯(OPF)水凝胶的制备及生物学评价。以不同分子量的OPF与PEG-DA为原料,APS/TEMED为催化剂,制备了一系列不同分子量的OPF水凝胶。通过比较成胶时间筛选出具有适宜成胶时间的OPF水凝胶。在此基础上,研究了水凝胶不同交联度(1：2,1：5,1：10)对其溶胀比以及降解时间的影响,筛选出具有适宜降解时间的OPF水凝胶。随后,将其与ESC在体外复合并培养,通过组织学染色分析其细胞相容性；并将OPF水凝胶注射到大鼠心梗边缘区,不同时间点取材,通过组织学染色评价其体内组织相容性及生物降解性能。
结果表明,10K OPF与2K PEG-DA合成的OPF水凝胶成胶时间最短,7-8分钟即可形成凝胶；交联度为1：2的OPF水凝胶体外降解时间较长；组织学染色结果表明ESC细胞在OPF水凝胶中培养1天后存活状况良好；体内移植后其心肌内降解时间为4-6周。本研究筛选出的以10K OPF与2K PEG-DA按照1：2交联度合成的OPF水凝胶适合用于可注射性心肌组织工程的研究。
第二部分：温敏性壳聚糖水凝胶携带nt-ESC心梗移植的可注射性心肌组织工程研究
本研究以温敏性壳聚糖水凝胶为可注射性载体,nt-ESC为种子细胞,体外构建可注射性工程化心肌并进行大鼠心梗部位移植。移植后对移植细胞的24小时滞留率、4周存活率、心梗面积、左心室室壁厚度、心梗部位微血管密度、nt-ESC在心梗部位分化情况等指标进行检测,并利用心脏超声对心梗大鼠的心脏功能进行测定。此外,以温敏性壳聚糖水凝胶为载体携带F-ESC进行大鼠心梗部位移植,4周后利用心脏超声检测心梗大鼠的心脏功能并对nt-ESC和F-ESC的心梗修复能力进行比较。
结果表明,温敏性壳聚糖水凝胶作为nt-ESC载体,能显著提高移植细胞在注射区域的滞留及存活。移植后4周,可以显著改善心梗大鼠的心脏功能、减少心梗面积、增加室壁厚度并提高微血管密度。心功能检测结果表明nt-ESC同F-ESC一样,具有相同的心梗修复能力。免疫荧光结果表明nt-ESC移植到大鼠心脏4周后,可以分化为cTnT阳性细胞、SMA阳性细胞以及vWAg阳性细胞。目前尚未见应用温敏性壳聚糖水凝胶携带nt-ESC进行可注射性心肌组织工程的研究报道。
第三部分：温敏性壳聚糖水凝胶携带碱性成纤维细胞生长因子(bFGF)心梗移植的可注射性心肌组织工程研究
本研究以温敏性壳聚糖水凝胶作为载体携带bFGF注射移植到大鼠心肌梗死部位。移植后4周,利用心脏超声对心肌梗死大鼠的心脏功能进行测定；对取材的心脏标本进行组织学与免疫组织化学的染色,对心梗面积、心梗部位胶原沉积程度以及心梗部位微血管密度等指标进行检测。以期探索温敏性壳聚糖水凝胶作为可注射性支架材料携带bFGF心梗移植的可行性。
结果表明,单独bFGF注射不能有效改善心梗的心脏功能以及组织形态学特征。温敏性壳聚糖水凝胶携带bFGF注射不但能显著改善心梗大鼠的心脏功能,而且能减小心梗面积,减弱纤维化程度并增加心梗区域微血管的密度。目前尚未见以温敏性壳聚糖水凝胶为载体携带bFGF心梗部位移植修复心肌梗死的研究报道。
第四部分：低聚乙二醇延胡索酸酯(OPF)水凝胶携带ESC心梗移植的可注射性心肌组织工程研究
在本研究中,为了探讨OPF水凝胶作为可注射性支架携带细胞修复梗死心肌的可行性,以OPF水凝胶为可注射性载体,eGFP-labeled mESC为种子细胞,体外构建可注射性组织工程化心肌并进行大鼠心梗部位移植。对移植细胞的24小时滞留率进行检测,以及检测移植后4周心梗大鼠的心脏功能,并对ESC在心梗部位分化情况标进行了检测。
结果表明,OPF水凝胶作为可注射性水凝胶支架能显著提高移植细胞在注射区域的24小时滞留率。心脏超声结果表明这种基于OPF水凝胶材料的可注射性组织工程心肌能够改善心梗大鼠的心脏功能。免疫荧光检测结果发现以OPF水凝胶为载体注射到心梗心脏中的ESC能够分化为心肌细胞、血管内皮细胞以及血管平滑肌细胞。目前尚未有应用OPF水凝胶携带ESC进行可注射性心肌组织工程的研究报道。
综上所述,本研究首先成功制备与筛选了温敏性壳聚糖水凝胶与OPF水凝胶。研究发现以温敏性壳聚糖水凝胶为载体携带nt-ESC构建的可注射性工程化心肌组织具有良好的心肌损伤修复能力,为nt-ESC进一步应用于细胞移植治疗提供实验依据。此外,以温敏性壳聚糖水凝胶为载体携带bFGF心梗移植并取得了较好的修复效果,这不但对bFGF进一步应用于心梗临床治疗具有促进作用,而且进一步验证了温敏性壳聚糖水凝胶作为可注射性心肌组织工程支架材料携带生长因子的可行性。最后,以OPF水凝胶为载体携带ESC心梗移植验证了OPF水凝胶作为心肌组织工程中可注射性支架材料的可行性。这种基于可注射性水凝胶支架材料的心肌组织工程研究对未来缺血性心脏病治疗具有重要意义。
Cardiac Tissue Engineering Based on Injectable Hydrogel as scaffolds
Heart failure, especially myocardial infarction (MI), is one of the main causes of morbidity and mortality in the world. Myocardial infarction results in the loss of irreplaceable contractile elements. The necrotic tissue is removed by macrophages and replaced with granulation tissue, resulting in a collagenous scar. During this process, the infarcted wall thins, the left ventricular (LV) chamber dilates, and interstitial fibrosis and cardiomyocyte hypertrophy appear in the noninfarcted region of the ventricle. These changes are linked with the cardiac dysfunction that leads to heart failure. Current therapeutic approaches have limited effects in attenuating disease progression. The only successful treatment is heart transplantation. Yet it is used for late-stage patients only and constrained by the shortage of organ supply. Therefore, it is desirable to develop alternative strategies to repair hearts with infarction to ameliorate both patient prognosis and life quality.
The emerging field of tissue engineering may offer promising alternatives. The ultimate goal in cardiac tissue engineering is to generate biocompatible, non-immunogenic heart muscles with morphological and functional properties of natural myocardium. For this purpose, about two distinguishable tissue engineering modalities have been established over the last decade. They include:1) in vitro engineered cardiac tissue approach--cell culturing on a biomaterial scaffold in vitro and tissue implantation onto the epicardial surface; 2) injectable cardiac tissue engineering approach--the use of an injectable biomaterial to deliver cells directly into the infarcted wall to increase cell survival. Injectable biomaterials can also be utilized in acellular approaches to support the LV wall and avoid the negative remodeling after an MI, or for the controlled delivery of therapeutic genes and proteins to ischemic myocardium. The approach of injectable cardiac tissue engineering is clinically appealing because it is more minimally invasive than that of in vitro-engineered tissue or an epicardial patch implantation.
Stem cells have been studied in an effort to finding the best source for cardiac regeneration and cardiac tissue engineering. Compared with adult stem cells, ES cells have many advantages in direct differentiation into cardiomyocytes. They are able to integrate with the host heart to improve electrical conduction. Therefore in theory, ES cells could potentially provide cardiomyocytes for cell therapy to regenerate functional myocardium. However, the immunological rejection after the ES cells transplantation makes the application of cell-replacement strategies difficult. The immunological rejection can be avoided by using patient-specific cells derived from the nuclear transferred embryonic stem (nt-ESC) cells. In theory, the nt-ESC cells carried the same genome as the donor somatic cells. After directed induction, the differentiated cells could rescue the damaged tissues without immune rejection. However, the persistence of abnormalities in cloned animals has doubted whether SCNT-derived ES cells may pose risks in contrast to fertilization-derived ones in their therapeutic applications. So far, no report focused on their comparison in infarcted heart tissue repairs. The results from the studies shall provide more sufficient support to the notion that the ES cells derived from cloned blastocysts have a strong therapeutic potential.
Chitosan is one of the most promising scaffolds in the injectable cardiac tissue engineering. The temperature-responsive chitosan hydrogel was a suitable injectable scaffold for stem cell transplantation. Our previous study showed that chitosan was a suitable injectable scaffold for ESC to form neovasculature when transplanted into the infarcted heart. However, hardly has any topic addressed whether the temperature-responsive chitosan hydrogel could be used as a carrier to deliver nt-ESC to the infarcted heart.
The injectable biomaterials should also be utilized for controlled delivery of therapeutic genes and proteins to ischemic myocardium in order to improve the microenvironment of infarcted region and facilitate the living conditions of transplanted cells. The basic fibroblast growth factor (bFGF) is a potent angiogenic protein which elicits angiogenesis and linkage to the extant vascular network. However, angiogenesis induced by growth factors has not been always successful. One reason for this difficulty is the high diffusibility. Another relates to the too short half-life time during which growth factors retain their biological activity in vivo. The angiogenic activity in vivo was enhanced by the sustained release of bFGF via using biomaterials as scaffolds. However, no report addressed whether the temperature-responsive chitosan hydrogel could be used as a carrier of slow-release bFGF in the infarcted heart.
In addition to the natural biomaterials, the synthetic biomaterials were developed for injectable cardiac tissue engineering for its advantages. The oligo(poly(ethylene glycol) fumarate)(OPF) hydrogel is an injectable biomaterial with good biocompatibility and biodegradability. The encapsulation of cell populations and particulate drug delivery systems within the hydrogels demonstrated the potential of injectable hydrogel formulations for cartilage, bone, and lens tissue engineering applications. However, we have not investigated whether the OPF hydrogel could be used as a carrier of ESC for the treatment of the infarcted heart.
This study can be divided into four parts:
Part 1:The preparation and evaluation of injectable hydrogel
In the Experiment One, the temperature responsive chitosan hydrogel were formed by mixing chitosan, GP and hydroxyethyl cellulose. nt-ESC survived well in the chitosan hydrogel. Histopathology staining was performed to evaluate the biocompatibility and biodegradability of chitosan hydrogel in vivo. Results showed that the degrading time of chitosan in myocardium is about 4 weeks. The biocompatibility and biodegradability of Chitosan hydrogel are good in the myocardium.
In the Experiment Two, OPF hydrogel were prepared by combining OPF with different molecular weight with poly(ethylene glycol)-diacrylate (PEG-DA) for crosslinking ratios (w/w) of PEG-DA to OPF of 1:2,1:5,1:10 respectively, and APS/TEMED solution were used as catalyst. The ESC survived well in the OPF hydrogel. Histopathology staining was performed to evaluate the biocompatibility and biodegradability of OPF hydrogel in vivo. Results showed that the degrading time of chitosan in myocardium is about 4-6 weeks. The biocompatibility and biodegradability of Chitosan hydrogel are good in the myocardium. The OPF hydrogel prepared by using 10K OPF and 2K PEG-DA with 1:2 crosslinking ratios is suitable for the application in cardiac tissue engineering.
Part 2:Both the Transplantation of Somatic Cell Nuclear Transfer-and Fertilization-Derived-mouse Embryonic Stem Cells with Temperature-responsive Chitosan Hydrogel Improve Myocardial Performance in Infarcted Rat Hearts.
As the scaffold, chitosan hydrogel was co-injected with nt-ESC into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were used to determine the structure and functional consequences of transplantation. The myocardial performance in SCNT-and fertilization-derived-mESC transplantation with chitosan hydrogel was compared, too. The result showed that both the 24h-cell retention and 4-week graft size were significantly greater in the nt-ESC+chitosan group than that of nt-ESC+PBS group. The nt-ESC cells might differentiate into cardiomyocytes in vivo. The heart function improved significantly in the chitosan+nt-ESC group compared with others 4 weeks after transplantation. In addition, the arteriole/venule densities within the infarcted area improved significantly in the chitosan+nt-ESC group. There was no difference in the myocardial performance in SCNT-and fertilization-derived-mESC transplantation with chitosan hydrogel. The NTES cells with chitosan hydrogel are proved with therapeutic potential to improve the function of infarcted heart. Thus the method of in situ injectable tissue engineering is promising clinically.
Part 3:Improved Myocardial Performance in Infarcted Rat Heart by Co-injection of Basic Fibroblast Growth Factor with Temperature-Responsive Chitosan Hydrogel
In this study, temperature-responsive chitosan hydrogel was prepared and injected intramyocardial into the left ventricular wall of rat infarction models alone or together with bFGF. The detailed histological analysis and echocardiography were used to determine the structure and functional consequences 4 weeks after injection. The results showed that the heart function improved significantly in the chitosan+ bFGF group compared with that of PBS+bFGF group in LVEF and LVFS 4 weeks after transplantation. In addition, the arteriole densities within the infarcted area improved significantly in the chitosan+bFGF group compared with that of PBS+ bFGF group 4 weeks after transplantation. The infarct size and fibrotic area was decreased significantly in the chitosan+bFGF group when compared to PBS+bFGF group. No significant difference existed between the PBS and PBS+bFGF groups. The co-injection of bFGF with temperature-responsive chitosan hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling, and cardiac function. Our finding suggests a new approach to improve infarcted repairs to prevent adverse remodeling after MI.
Part 4:Cardiac Tissue Engineering based on Injectable OPF Hydrogels and ESC for the treatment of MI.
In this study, OPF hydrogel was co-injected with eGFP-labeled ESC into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were used to determine the structure and functional consequences of transplantation. The result showed that the 24h-cell retention was significantly greater in the ESC+OPF group than that of ESC+PBS group. The ESC cells might differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vivo. The heart function improved significantly in the OPF+ESC group compared with others 4 weeks after transplantation. Thus the method of in situ injectable tissue engineering by using OPF hydrogel as injectable scaffold is promising clinically.
In summary, the results from this study indicate that temperature-responsive chitosan hydrogel and OPF hydrogel were potential injectable scaffolds that can be used to deliver stem cells and growth factors to infarcted myocardium. The injectable cardiac tissue engineering in conjunction with current treatment modalities may help reduce mortality and improve the quality of life in MI patients.
随着组织工程研究的不断深入,运用组织工程技术研制可供移植的工程化心肌组织为心脏缺血性疾病的治疗带来了新的希望。近十余年,心肌组织工程进展迅速,主要包括两种策略：三维立体工程化心肌组织体外再造与移植策略(以心脏创可贴的方式移植)与可注射心肌组织工程策略。可注射心肌组织工程是指将细胞和／或生长因子等与可注射性支架材料复合直接注射到心肌损伤部位进行损伤的修复,由于其对患者造成的创伤较小而更易于临床应用。目前,可注射心肌组织已成为心肌组织工程研究的热点之一。
干细胞是心肌组织工程研究中应用最为广泛的种子细胞来源之一。其中,具有无限增殖能力和多向分化潜能的ESC由于能够向心肌细胞高效分化、与宿主心肌整合并存在电信号传导等优势具有广阔的应用前景。然而,ESC体内移植后面临引发免疫排斥反应等问题严重限制了其在移植治疗器官损伤中的应用。通过治疗性克隆策略获得患者特异性的nt-ESC有望解决ESC体内移植后引发免疫排斥反应这一难题。然而,nt-ESC是否与普通受精卵来源ESC一样,能够在心梗部位移植后具有修复作用,目前尚未见报道。
温敏性壳聚糖水凝胶是可注射性心肌组织工程研究中一种优良的可注射性支架材料。我们实验室之前的研究表明,以温敏性壳聚糖水凝胶为载体携带ESC心梗部位移植能显著提高移植细胞在注射区域的滞留率及存活率,增加梗死区域心室壁厚度并显著改善心梗动物的心脏功能。证明了温敏性壳聚糖水凝胶携带ESC构建可注射性工程化心肌组织修复心肌梗死的可行性。然而,目前尚未见以温敏性壳聚糖水凝胶为可注射性载体,携带nt-ESC进行动物心梗部位移植与修复的研究报道。
可注射性支架材料另一个重要作用是能够对其携带的治疗性因子在体内起缓释作用。通过生长因子诱导心梗区域再血管化对缺血性心脏疾病的治疗具有重要意义。碱性成纤维细胞生长因子(bFGF)是最重要的促血管生长因子之一。然而,由于注射到心脏部位后体内扩散率高且半衰期短,bFGF的修复作用并不总是能够按照需要成功实现。以生物材料作为载体将携带的bFGF持续释放到梗死心肌中有望对组织再血管化起促进作用。目前尚未见以温敏性壳聚糖水凝胶为载体携带bFGF心梗部位移植修复心肌梗死的研究报道。
除天然的生物材料外,人工合成的生物材料由于其力学性能与降解速度更加可控且加工性能和价格都比天然材料占有优势,近年来在可注射性心肌组织工程研究中的应用迅速增多。低聚乙二醇延胡索酸酯(OPF)水凝胶具有良好的组织相容性、生物可降解性以及可注射性,能够携带多种细胞与生物活性因子进行体内移植,已广泛应用于骨、软骨、角膜组织工程。然而,目前尚未见以OPF水凝胶为载体应用于可注射性心肌组织工程修复心肌梗死的研究。
本研究首先合成温敏性壳聚糖水凝胶与OPF水凝胶,并对其生物学性能进行评价。在此基础上,以温敏性壳聚糖水凝胶作为载体携带nt-ESC心梗部位移植,对其心梗损伤修复能力进行深入研究,并对nt-ESC和F-ESC的心梗修复能力进行比较。其次,以温敏性壳聚糖水凝胶作为载体携带bFGF心梗部位移植,研究其作为携带与缓释生长因子的可注射性支架材料的可行性及心梗治疗效果。最后,在成功合成OPF水凝胶的基础上,以其为载体携带eGFP标记的ESC进行心梗部位移植,研究其心梗损伤修复能力并探讨其作为可注射性心肌组织工程支架材料的可行性。
本论文主要包含以下四部分研究内容：
第一部分：可注射性水凝胶支架材料的制备与生物学评价
可注射性水凝胶支架材料是携带细胞和／或生长因子的载体,是可注射性组织工程化心肌研制的重要组成部分。
实验一：温敏性壳聚糖水凝胶的制备及生物学评价。以氯化壳聚糖、p-甘油磷酸钠为原料,羟乙基纤维素为交联剂制备了温敏性壳聚糖水凝胶。将nt-ESC与该水凝胶复合于体外培养后,应用AO/PI染色观察nt-ESC在水凝胶中的存活情况。此外,将温敏性壳聚糖水凝胶注射于大鼠心梗边缘区,不同时间点取材,通过组织学染色检测其组织相容性及体内生物降解性。结果表明：制备的温敏性壳聚糖水凝胶具有温度敏感性,其在室温条件下为液态,当温度升高到37℃时形成凝胶。生物学评价结果说明其具有良好的细胞相容性、组织相容性及可生物降解性。
实验二：低聚乙二醇延胡索酸酯(OPF)水凝胶的制备及生物学评价。以不同分子量的OPF与PEG-DA为原料,APS/TEMED为催化剂,制备了一系列不同分子量的OPF水凝胶。通过比较成胶时间筛选出具有适宜成胶时间的OPF水凝胶。在此基础上,研究了水凝胶不同交联度(1：2,1：5,1：10)对其溶胀比以及降解时间的影响,筛选出具有适宜降解时间的OPF水凝胶。随后,将其与ESC在体外复合并培养,通过组织学染色分析其细胞相容性；并将OPF水凝胶注射到大鼠心梗边缘区,不同时间点取材,通过组织学染色评价其体内组织相容性及生物降解性能。
结果表明,10K OPF与2K PEG-DA合成的OPF水凝胶成胶时间最短,7-8分钟即可形成凝胶；交联度为1：2的OPF水凝胶体外降解时间较长；组织学染色结果表明ESC细胞在OPF水凝胶中培养1天后存活状况良好；体内移植后其心肌内降解时间为4-6周。本研究筛选出的以10K OPF与2K PEG-DA按照1：2交联度合成的OPF水凝胶适合用于可注射性心肌组织工程的研究。
第二部分：温敏性壳聚糖水凝胶携带nt-ESC心梗移植的可注射性心肌组织工程研究
本研究以温敏性壳聚糖水凝胶为可注射性载体,nt-ESC为种子细胞,体外构建可注射性工程化心肌并进行大鼠心梗部位移植。移植后对移植细胞的24小时滞留率、4周存活率、心梗面积、左心室室壁厚度、心梗部位微血管密度、nt-ESC在心梗部位分化情况等指标进行检测,并利用心脏超声对心梗大鼠的心脏功能进行测定。此外,以温敏性壳聚糖水凝胶为载体携带F-ESC进行大鼠心梗部位移植,4周后利用心脏超声检测心梗大鼠的心脏功能并对nt-ESC和F-ESC的心梗修复能力进行比较。
结果表明,温敏性壳聚糖水凝胶作为nt-ESC载体,能显著提高移植细胞在注射区域的滞留及存活。移植后4周,可以显著改善心梗大鼠的心脏功能、减少心梗面积、增加室壁厚度并提高微血管密度。心功能检测结果表明nt-ESC同F-ESC一样,具有相同的心梗修复能力。免疫荧光结果表明nt-ESC移植到大鼠心脏4周后,可以分化为cTnT阳性细胞、SMA阳性细胞以及vWAg阳性细胞。目前尚未见应用温敏性壳聚糖水凝胶携带nt-ESC进行可注射性心肌组织工程的研究报道。
第三部分：温敏性壳聚糖水凝胶携带碱性成纤维细胞生长因子(bFGF)心梗移植的可注射性心肌组织工程研究
本研究以温敏性壳聚糖水凝胶作为载体携带bFGF注射移植到大鼠心肌梗死部位。移植后4周,利用心脏超声对心肌梗死大鼠的心脏功能进行测定；对取材的心脏标本进行组织学与免疫组织化学的染色,对心梗面积、心梗部位胶原沉积程度以及心梗部位微血管密度等指标进行检测。以期探索温敏性壳聚糖水凝胶作为可注射性支架材料携带bFGF心梗移植的可行性。
结果表明,单独bFGF注射不能有效改善心梗的心脏功能以及组织形态学特征。温敏性壳聚糖水凝胶携带bFGF注射不但能显著改善心梗大鼠的心脏功能,而且能减小心梗面积,减弱纤维化程度并增加心梗区域微血管的密度。目前尚未见以温敏性壳聚糖水凝胶为载体携带bFGF心梗部位移植修复心肌梗死的研究报道。
第四部分：低聚乙二醇延胡索酸酯(OPF)水凝胶携带ESC心梗移植的可注射性心肌组织工程研究
在本研究中,为了探讨OPF水凝胶作为可注射性支架携带细胞修复梗死心肌的可行性,以OPF水凝胶为可注射性载体,eGFP-labeled mESC为种子细胞,体外构建可注射性组织工程化心肌并进行大鼠心梗部位移植。对移植细胞的24小时滞留率进行检测,以及检测移植后4周心梗大鼠的心脏功能,并对ESC在心梗部位分化情况标进行了检测。
结果表明,OPF水凝胶作为可注射性水凝胶支架能显著提高移植细胞在注射区域的24小时滞留率。心脏超声结果表明这种基于OPF水凝胶材料的可注射性组织工程心肌能够改善心梗大鼠的心脏功能。免疫荧光检测结果发现以OPF水凝胶为载体注射到心梗心脏中的ESC能够分化为心肌细胞、血管内皮细胞以及血管平滑肌细胞。目前尚未有应用OPF水凝胶携带ESC进行可注射性心肌组织工程的研究报道。
综上所述,本研究首先成功制备与筛选了温敏性壳聚糖水凝胶与OPF水凝胶。研究发现以温敏性壳聚糖水凝胶为载体携带nt-ESC构建的可注射性工程化心肌组织具有良好的心肌损伤修复能力,为nt-ESC进一步应用于细胞移植治疗提供实验依据。此外,以温敏性壳聚糖水凝胶为载体携带bFGF心梗移植并取得了较好的修复效果,这不但对bFGF进一步应用于心梗临床治疗具有促进作用,而且进一步验证了温敏性壳聚糖水凝胶作为可注射性心肌组织工程支架材料携带生长因子的可行性。最后,以OPF水凝胶为载体携带ESC心梗移植验证了OPF水凝胶作为心肌组织工程中可注射性支架材料的可行性。这种基于可注射性水凝胶支架材料的心肌组织工程研究对未来缺血性心脏病治疗具有重要意义。
Cardiac Tissue Engineering Based on Injectable Hydrogel as scaffolds
Heart failure, especially myocardial infarction (MI), is one of the main causes of morbidity and mortality in the world. Myocardial infarction results in the loss of irreplaceable contractile elements. The necrotic tissue is removed by macrophages and replaced with granulation tissue, resulting in a collagenous scar. During this process, the infarcted wall thins, the left ventricular (LV) chamber dilates, and interstitial fibrosis and cardiomyocyte hypertrophy appear in the noninfarcted region of the ventricle. These changes are linked with the cardiac dysfunction that leads to heart failure. Current therapeutic approaches have limited effects in attenuating disease progression. The only successful treatment is heart transplantation. Yet it is used for late-stage patients only and constrained by the shortage of organ supply. Therefore, it is desirable to develop alternative strategies to repair hearts with infarction to ameliorate both patient prognosis and life quality.
The emerging field of tissue engineering may offer promising alternatives. The ultimate goal in cardiac tissue engineering is to generate biocompatible, non-immunogenic heart muscles with morphological and functional properties of natural myocardium. For this purpose, about two distinguishable tissue engineering modalities have been established over the last decade. They include:1) in vitro engineered cardiac tissue approach--cell culturing on a biomaterial scaffold in vitro and tissue implantation onto the epicardial surface; 2) injectable cardiac tissue engineering approach--the use of an injectable biomaterial to deliver cells directly into the infarcted wall to increase cell survival. Injectable biomaterials can also be utilized in acellular approaches to support the LV wall and avoid the negative remodeling after an MI, or for the controlled delivery of therapeutic genes and proteins to ischemic myocardium. The approach of injectable cardiac tissue engineering is clinically appealing because it is more minimally invasive than that of in vitro-engineered tissue or an epicardial patch implantation.
Stem cells have been studied in an effort to finding the best source for cardiac regeneration and cardiac tissue engineering. Compared with adult stem cells, ES cells have many advantages in direct differentiation into cardiomyocytes. They are able to integrate with the host heart to improve electrical conduction. Therefore in theory, ES cells could potentially provide cardiomyocytes for cell therapy to regenerate functional myocardium. However, the immunological rejection after the ES cells transplantation makes the application of cell-replacement strategies difficult. The immunological rejection can be avoided by using patient-specific cells derived from the nuclear transferred embryonic stem (nt-ESC) cells. In theory, the nt-ESC cells carried the same genome as the donor somatic cells. After directed induction, the differentiated cells could rescue the damaged tissues without immune rejection. However, the persistence of abnormalities in cloned animals has doubted whether SCNT-derived ES cells may pose risks in contrast to fertilization-derived ones in their therapeutic applications. So far, no report focused on their comparison in infarcted heart tissue repairs. The results from the studies shall provide more sufficient support to the notion that the ES cells derived from cloned blastocysts have a strong therapeutic potential.
Chitosan is one of the most promising scaffolds in the injectable cardiac tissue engineering. The temperature-responsive chitosan hydrogel was a suitable injectable scaffold for stem cell transplantation. Our previous study showed that chitosan was a suitable injectable scaffold for ESC to form neovasculature when transplanted into the infarcted heart. However, hardly has any topic addressed whether the temperature-responsive chitosan hydrogel could be used as a carrier to deliver nt-ESC to the infarcted heart.
The injectable biomaterials should also be utilized for controlled delivery of therapeutic genes and proteins to ischemic myocardium in order to improve the microenvironment of infarcted region and facilitate the living conditions of transplanted cells. The basic fibroblast growth factor (bFGF) is a potent angiogenic protein which elicits angiogenesis and linkage to the extant vascular network. However, angiogenesis induced by growth factors has not been always successful. One reason for this difficulty is the high diffusibility. Another relates to the too short half-life time during which growth factors retain their biological activity in vivo. The angiogenic activity in vivo was enhanced by the sustained release of bFGF via using biomaterials as scaffolds. However, no report addressed whether the temperature-responsive chitosan hydrogel could be used as a carrier of slow-release bFGF in the infarcted heart.
In addition to the natural biomaterials, the synthetic biomaterials were developed for injectable cardiac tissue engineering for its advantages. The oligo(poly(ethylene glycol) fumarate)(OPF) hydrogel is an injectable biomaterial with good biocompatibility and biodegradability. The encapsulation of cell populations and particulate drug delivery systems within the hydrogels demonstrated the potential of injectable hydrogel formulations for cartilage, bone, and lens tissue engineering applications. However, we have not investigated whether the OPF hydrogel could be used as a carrier of ESC for the treatment of the infarcted heart.
This study can be divided into four parts:
Part 1:The preparation and evaluation of injectable hydrogel
In the Experiment One, the temperature responsive chitosan hydrogel were formed by mixing chitosan, GP and hydroxyethyl cellulose. nt-ESC survived well in the chitosan hydrogel. Histopathology staining was performed to evaluate the biocompatibility and biodegradability of chitosan hydrogel in vivo. Results showed that the degrading time of chitosan in myocardium is about 4 weeks. The biocompatibility and biodegradability of Chitosan hydrogel are good in the myocardium.
In the Experiment Two, OPF hydrogel were prepared by combining OPF with different molecular weight with poly(ethylene glycol)-diacrylate (PEG-DA) for crosslinking ratios (w/w) of PEG-DA to OPF of 1:2,1:5,1:10 respectively, and APS/TEMED solution were used as catalyst. The ESC survived well in the OPF hydrogel. Histopathology staining was performed to evaluate the biocompatibility and biodegradability of OPF hydrogel in vivo. Results showed that the degrading time of chitosan in myocardium is about 4-6 weeks. The biocompatibility and biodegradability of Chitosan hydrogel are good in the myocardium. The OPF hydrogel prepared by using 10K OPF and 2K PEG-DA with 1:2 crosslinking ratios is suitable for the application in cardiac tissue engineering.
Part 2:Both the Transplantation of Somatic Cell Nuclear Transfer-and Fertilization-Derived-mouse Embryonic Stem Cells with Temperature-responsive Chitosan Hydrogel Improve Myocardial Performance in Infarcted Rat Hearts.
As the scaffold, chitosan hydrogel was co-injected with nt-ESC into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were used to determine the structure and functional consequences of transplantation. The myocardial performance in SCNT-and fertilization-derived-mESC transplantation with chitosan hydrogel was compared, too. The result showed that both the 24h-cell retention and 4-week graft size were significantly greater in the nt-ESC+chitosan group than that of nt-ESC+PBS group. The nt-ESC cells might differentiate into cardiomyocytes in vivo. The heart function improved significantly in the chitosan+nt-ESC group compared with others 4 weeks after transplantation. In addition, the arteriole/venule densities within the infarcted area improved significantly in the chitosan+nt-ESC group. There was no difference in the myocardial performance in SCNT-and fertilization-derived-mESC transplantation with chitosan hydrogel. The NTES cells with chitosan hydrogel are proved with therapeutic potential to improve the function of infarcted heart. Thus the method of in situ injectable tissue engineering is promising clinically.
Part 3:Improved Myocardial Performance in Infarcted Rat Heart by Co-injection of Basic Fibroblast Growth Factor with Temperature-Responsive Chitosan Hydrogel
In this study, temperature-responsive chitosan hydrogel was prepared and injected intramyocardial into the left ventricular wall of rat infarction models alone or together with bFGF. The detailed histological analysis and echocardiography were used to determine the structure and functional consequences 4 weeks after injection. The results showed that the heart function improved significantly in the chitosan+ bFGF group compared with that of PBS+bFGF group in LVEF and LVFS 4 weeks after transplantation. In addition, the arteriole densities within the infarcted area improved significantly in the chitosan+bFGF group compared with that of PBS+ bFGF group 4 weeks after transplantation. The infarct size and fibrotic area was decreased significantly in the chitosan+bFGF group when compared to PBS+bFGF group. No significant difference existed between the PBS and PBS+bFGF groups. The co-injection of bFGF with temperature-responsive chitosan hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling, and cardiac function. Our finding suggests a new approach to improve infarcted repairs to prevent adverse remodeling after MI.
Part 4:Cardiac Tissue Engineering based on Injectable OPF Hydrogels and ESC for the treatment of MI.
In this study, OPF hydrogel was co-injected with eGFP-labeled ESC into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were used to determine the structure and functional consequences of transplantation. The result showed that the 24h-cell retention was significantly greater in the ESC+OPF group than that of ESC+PBS group. The ESC cells might differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vivo. The heart function improved significantly in the OPF+ESC group compared with others 4 weeks after transplantation. Thus the method of in situ injectable tissue engineering by using OPF hydrogel as injectable scaffold is promising clinically.
In summary, the results from this study indicate that temperature-responsive chitosan hydrogel and OPF hydrogel were potential injectable scaffolds that can be used to deliver stem cells and growth factors to infarcted myocardium. The injectable cardiac tissue engineering in conjunction with current treatment modalities may help reduce mortality and improve the quality of life in MI patients.
引文
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[41]Scheinowitz M, Kotlyar AA, Zimand S, et al. Effect of basic fibroblast growth factor on left ventricular geometry in rats subjected to coronary occlusion and reperfusion. Isr Med Assoc J.2002; 4:109-113.
[42]Shao ZQ, Takaji K, Katayama Y, et al. Effects of Intramyocardial Administration of Slow-Release Basic Fibroblast Growth Factor on Angiogenesis and Ventricular Remodeling in a Rat Infarct Model. Circ J.2006; 70:471-477.
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[45]Fisher JP, Lalani Z, Mikos AG Effect of biomaterial properties on bone healing in a rabbit tooth extraction socket model.J Biomed Mater Res A.2004;68:428-38.
[46]Holland TA, Tabata Y, Mikos AG Dual growth factor delivery from degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds for cartilage tissue engineering.J Control Release.2005; 101:111-25.
[47]Guo X, Park H, Young S, Kretlow JD, van den Beucken JJ, Baggett LS, Tabata Y, Kasper FK, Mikos AG, Jansen JA. Repair of osteochondral defects with biodegradable hydrogel composites encapsulating marrow mesenchymal stem cells in a rabbit model. Acta Biomater.2010; 6:39-47.
[48]Park H, Guo X, Temenoff JS, Tabata Y, Caplan AI, Kasper FK, Mikos AG Effect of Swelling Ratio of Injectable Hydrogel Composites on Chondrogenic Differentiation of Encapsulated Rabbit Marrow Mesenchymal Stem Cells In Vitro. Biomacromolecules.2009; 10:541-546.
[49]Shin H, Temenoff JS, Mikos AG Osteogenic differentiation of rat bone marrow stromal cells cultured on Arg-Gly-Asp modified hydrogels without dexamethasone and beta-glycerol phosphate.Biomaterials.2005;26:3645-54.
[50]Mimi W. Zhang, Hansoo Park,Xuan Guo,Kenta Nakamura,Robert M. Raphael, F. Kurtis Kasper, Antonios G Mikos,Panagiotis A. Tsonis. Adapting Biodegradable Oligo(Poly(Ethylene Glycol) Fumarate) Hydrogels for Pigment Epithelial Cell Encapsulation and Lens Regeneration. Tissue Eng Part C.2009, doi: 10.1089/ten.tec.2009.0162
[51]C. D. Hoemann, J. Sun, A. Legare et al. Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle. OsteoArthritis and Cartilage.2005,13,318
[52]Brambrink T, Hochedlinger K, Bell G, and Jaenisch R. ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. Proc Natl Acad Sci U S A.103,933,2006.
[53]Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res.1979; 44:503-512.
[54]Menard C, Hagege AA, Agbulut O, et al.Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium:a preclinical study. Lancet.2005; 366:1005-1012
[55]Xue, T. et al. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes:insights into the development of cell-based pacemakers. Circulation.2005,111:11-20.
[56]Temenoff JS, Park H, Jabbari E, Sheffield TL, Lebaron RG, Ambrose CG, Mikos AG. In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels. J Biomed Mater Res A.2004; 70:235-244.
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