【作者】 杨健；

【导师】 费文勇； 卢耀甲；

【作者基本信息】 扬州大学 ， 临床医学硕士（专业学位）， 2024， 硕士

【摘要】 肩袖损伤(Rotator cuff tear,RCT)是临床上常见的运动系统损伤之一,发病主要集中于中老年人群,通常导致患者肩关节无力、疼痛、活动受限,严重影响患者的生活质量。外科手术是治疗肩袖损伤的主要手段。但损伤肩袖在术后的恢复过程十分漫长,其中较大肩袖撕裂和巨大肩袖撕裂的患者,面临着更高的再撕裂风险。这主要是由于肩袖止点包含四层组织结构:纤维结缔组织、非钙化的纤维软骨、钙化的纤维软骨、骨组织,分层渐进的结构能够有效传递并缓冲应力。但是这一特殊组织结构在手术后大多被无序的疤痕组织取代,难以完全恢复,因此术后肩袖腱骨界面的力学性能短时间难以达到正常水平。到目前为止,仍没有找到能够快速促进肩袖腱骨界面完全恢复的治疗手段。我们的临床研究表明,单纯手术修复对于巨大肩袖撕裂患者的治疗效果不能达到预期,因此当前需要一种能够促进巨大肩袖撕裂愈合的辅助治疗手段。组织工程是一种结合运用支架材料、生长因子和种子细胞的科学,能够从结构、组份上模拟体内微环境,辅助特定复杂结构修复再生,这为肩袖腱骨界面修复提供了一种新的思路。针对上述情况,本课题开发了一种基于甲基丙烯酸化透明质酸(HAMA)/海藻酸(Alg)的双层仿生BMP缓释3D打印水凝胶,其中负载有脂肪间充质干细胞(ADSCs)。骨形态发生蛋白-2(BMP-2)和Ⅰ型胶原蛋白(COLⅠ)被分别加入HAMA/Alg生物墨水中,然后通过3D打印来构建仿生腱骨界面的梯度结构以诱导ADSCs分别进行成骨和成肌腱分化。针对生长因子高活性但易失活的特点,我们通过氨基酸3,4-二羟基苯丙氨酸(DOPA)将透明质酸改性为HA-DOPA(HAD)后对BMP-2进行黏附,以用于递送和缓释生长因子。研究结果证明双层仿生BMP缓释3D打印水凝胶在促进撕裂肩袖腱骨愈合方面的疗效。体外实验数据证实了梯度支架系统具有区域特异性诱导分化能力,能够模拟腱骨界面的有序结构以促进干细胞的定向分化。在体内实验中,我们利用大鼠急性肩袖损伤修复模型确定了梯度支架结合干细胞策略促进腱骨界面再生、增强腱骨界面力学性能的作用。综上所述,这项研究确定了双层仿生BMP缓释3D打印水凝胶联合ADSCs对肩袖腱骨界面愈合的治疗贡献,并证明了仿生梯度组织工程材料在肩袖撕裂临床修复中的潜力。第一章关节镜下肩袖修补术后不同肩袖撕裂大小患者治疗效果的临床探查及移植物开发分析目的:比较关节镜下肩袖修补术对不同程度肩袖撕裂患者的治疗效果。方法:(1)临床上回顾性分析了自2018年6月至2023年2月来自江苏省苏北人民医院收治的32例肩袖损伤患者。所有患者术前均经过体格检查、X线及MRI检查确诊为肩袖损伤,然后进行关节镜下肩袖修补术治疗。所有患者术前、术后均进行了肩部视觉模拟评分(VAS)、肩关节功能评分(Constant)和美国肩肘外科协会评分(ASES)。其中中、小肩袖撕裂16例,较大、巨大肩袖撕裂16例。结果:肩袖撕裂越小,术后疼痛缓解越快,肩关节功能恢复越好。反之,肩袖撕裂越大,术后恢复越困难,手术预后越差。组织工程策略在较大、巨大肩袖撕裂患者治疗中是必要的。结论:肩袖撕裂越小,肩关节镜下肩袖修补术后肩关节功能恢复越快,但是在面对巨大撕裂的时候,单纯手术治疗后的肩关节功能恢复相对较为困难,需要更多的时间。面对肩袖较大撕裂和巨大撕裂患者时,需要组织工程等辅助治疗手段。第二章双层仿生BMP缓释3D打印水凝胶的构建及生物相容性研究目的:研究HAMA、HAD的制备合成、生物墨水HAMA/Alg/COLⅠ(HAC)和HAMA/Alg/HAD/BMP-2(HAHB)的打印性能性和光固化性,探究水凝胶支架的物理特性、生长因子递送功能。方法:(1)制备HAMA和HAD,通过核磁共振验证产物结构。合成HA,HAC,HAHB生物墨水。(2)流变仪分析生物墨水的流变学特征。(3)通过3D打印机将生物墨水制备成水凝胶支架,并通过光学显微镜、扫描电镜观察水凝胶支架的表面结构、微观结构特点。(4)使用流变仪检测HA、HAC、HAHB及HAC/HAHB双层水凝胶支架的力学性能。(5)使用ELISA法检测HAMA/Alg/BMP-2(HAB)和HAHB水凝胶中BMP-2的释放特性。(6)检测冻干HA、HAC、HAHB及HAC/HAHB水凝胶支架在浸泡于模拟体液(SBF)中的溶胀特性。(7)检测冻干称重的HA、HAC、HAHB及HAC/HAHB水凝胶支架浸泡于SBF中的降解特性。(8)体内实验中使用HAC/HAHB水凝胶支架浸提液,通过细胞活死染色和CCK-8法检测水凝胶支架对ADSCs活性和增殖的影响。结果:(1)核磁共振结果提示HAD和HAMA的成功合成。(2)HAC和HAHB墨水均具备剪切变稀的特性,并且可以进行光固化,在加入了0.02 M Ca Cl2进行第一步交联后,两者的均具备了更好的适印性。(3)光学显微镜下HAC和HAHB墨水打印的支架形态并无明显差异,扫描电镜扫描冻干支架显示HAHB的内部孔隙相比HAC支架更小,结构更为致密。(4)随着COLⅠ和HAD加入HA墨水体系中,支架的强度增加,HAD对支架强度的提升最大。HAHB溶胀率更高,但是各组水凝胶均在24小时内达到溶胀平衡。HAD能够减慢水凝胶的降解速度,COLⅠ也有相同的作用。(5)相比HA,加入HAD可以减少BMP-2首日爆发释放量,同时在37℃环境下能够减少BMP-2的失活现象。(6)HAHB水凝胶支架的溶胀率最高,加入HAD后水凝胶的亲水性增加。(7)从长期来看,HAD和COLⅠ的加入减缓了水凝胶的降解速度。(8)HAC/HAHB双层水凝胶没有表现出细胞毒性,对ADSCs增殖没有明显影响。结论:HAC和HAHB生物墨水具有良好的打印性能性和光固化性,可以适用于3D打印,制作成的水凝胶支架相比单纯HA支架具备更好的力学性能。HAHB能够实现BMP-2缓释,同时也能够减少BMP-2的失活。双层水凝胶支架表现出了良好的生物相容性。第三章双层仿生BMP缓释3D打印水凝胶用于肩袖腱骨界面修复的研究目的:探索双层仿生BMP缓释3D打印水凝胶在体外对ADSCs的诱导作用,研究其联合ADSCs在大鼠急性肩袖损伤模型中的修复效果。方法:(1)将HAC和HAHB单层支架分别与ADSCs一同培养于Transwell体系内进行诱导分化培养,通过RT-q PCR法检测水凝胶支架对于ADSCs的诱导分化作用。(2)体内实验中,建立大鼠急性肩袖损伤模型,并随机分组为:单纯手术修复组、支架植入组、支架+ADSCs组。分别于修复4周、8周后对修复腱骨界面进行切片,进行HE、Masson、番红固绿、天狼星红染色以及相关病理评分,并进行生物力学检测。8周时对腱骨界面进行Micro-CT检测。结果:(1)HAHB层增强了ADSCs中OPN、OCN的表达,HAC层增强了ADSCs中TNMD的表达,同时,HAHB和HAC也对ADSCs中COL2A1和SOX9的表达有增强作用。(2)HE、Masson、番红固绿、天狼星红染色结果显示单纯手术修复组纤维血管增生更多、细胞形态无序,力学性能差。经过HAC/HAHB水凝胶支架联合ADSCs干预后,修复后的腱骨界面获得更好的组织学评分,结构更接近正常组织,生物力学性能更好。Micro-CT显示HAC/HAHB水凝胶支架联合ADSCs干预的腱骨界面中骨组织获得了更好的修复效果。结论:体外实验中HAC及HAHB水凝胶分别能够诱导ADSCs向成肌腱和成骨方向分化,同时均对ADSCs成软骨分化有一定的促进作用。体内实验中HAC/HAHB水凝胶支架联合ADSCs策略成功促进了损伤肩袖的愈合,加快了腱骨界面有序4层结构的修复再生。更多还原

【Abstract】 Rotator cuff tear(RCT)is one of the most common sports system injuries in clinical practice,and its incidence is mainly concentrated in middle-aged and old-aged people,which usually leads to shoulder weakness,pain,and activity limitation,and seriously affects the quality of life of patients.The rotator cuff has poor self-healing ability and usually requires surgical treatment.Although surgical interventions can accurately reposition the torn rotator cuff tissue and effectively relieve pain.However,the prognosis of surgical treatment alone is influenced by a number of factors,such as age and osteoporosis.Even with effective postoperative rehabilitation,recovery from rotator cuff injuries can be lengthy,especially in patients with large rotator cuff tears and giant rotator cuff tears,and there is still a risk of re-tear.This is mainly due to the fact that the rotator cuff stop contains four layers of tissue:fibrous connective tissue,non-calcified fibrocartilage,calcified fibrocartilage,and bone tissue,and the gradual layering of these layers is effective in transmitting and cushioning stresses.However,this special tissue structure is mostly replaced by disorganized scar tissue after surgery,which is difficult to be fully recovered,and therefore the mechanical properties of the rotator cuff tendon-bone interface are difficult to reach the normal level for a short period of time after surgery.So far,there is no therapeutic method that can quickly promote the complete recovery of the rotator cuff tendon bone interface.Tissue engineering,a science that combines the use of scaffold materials,growth factors and seed cells,can mimic the in vivo microenvironment in terms of structure and composition to assist the repair and regeneration of specific complex structures,which provides a new way of thinking about the repair of the rotator cuff tendon bone interface.In this study,we focused on the theme of tendon-bone interface regeneration,analyzed the differences in the therapeutic efficacy of surgical treatment alone in the face of rotator cuff tears of different sizes,and explored the necessity of adjunctive therapeutic means,while developing a bilayer bionic BMP slow-release 3D-printed hydrogel based on methacrylated hyaluronic acid(HAMA)/alginic acid(Alg),which was loaded with adipose-derived mesenchymal stem cells(ADSCs).Bone morphogenetic protein-2(BMP-2)or collagen type I(COL I)was incorporated into the HAMA/Alg bioink,respectively,and then 3D printed to construct gradient structures resembling the tendon-bone interface to induce osteogenic and tendinogenic differentiation of ADSCs,respectively.To address the easy degradation and high efficiency of growth factors,we bonded hyaluronic acid and with BMP-2 via the amino acid3,4-dihydroxyphenylalanine(DOPA)for delivery and slow release of growth factors.Our study suggests that surgical repair alone is far less effective in treating patients with giant rotator cuff tears than patients with much smaller tears.Adjunctive therapies that can promote healing of giant rotator cuff tears are necessary.The results of the study similarly demonstrate the potential of bilayer bionic BMP slow-release 3D printed hydrogels in promoting healing of torn rotator cuff tendon bones.In vitro studies have demonstrated that the gradient scaffold system has the ability to induce differentiation in a region-specific manner,mimicking the ordered structure of the tendon-bone interface to promote targeted differentiation of stem cells.In in vivo experiments,we utilized a rat acute rotator cuff injury repair model to determine the role of gradient scaffolds combined with stem cell strategies to promote tendon-bone interface regeneration and enhance tendon-bone interface mechanical properties.In summary,this study identifies the therapeutic contribution of bilayer bionic BMP slow-release 3D printed hydrogel in combination with ADSCs for rotator cuff tendon-bone interface healing and demonstrates the potential of bionic gradient tissue engineering materials in the clinical repair of rotator cuff tears.Chapter 1 Clinical Investigation of Treatment Outcomes and Analysis of Graft Development in Patients with Different Rotator Cuff Tear Sizes After Arthroscopic Rotator Cuff RepairOBJECTIVE: To compare the therapeutic effects of arthroscopic rotator cuff repair in patients with different sizes of rotator cuff tears.METHODS:(1)Thirty-two patients with rotator cuff injuries admitted from Jiangsu Provincial Subei People’s Hospital from June 2018 to August 2023 were retrospectively analyzed clinically.All patients were diagnosed with rotator cuff injury by physical examination,X-ray and MRI examination before surgery,and then treated with arthroscopic rotator cuff repair.All patients underwent preoperative and postoperative visual analog shoulder scoring(VAS),shoulder function scoring(Constant)and American Society for Shoulder and Elbow Surgery scoring(ASES).There were 16 cases of medium and small rotator cuff tears and 16 cases of large and massive rotator cuff tears.RESULTS: The smaller the rotator cuff tear,the faster the pain relief after surgery and the better the recovery of shoulder function.Conversely,the larger the rotator cuff tear,the more difficult the postoperative recovery and the worse the surgical prognosis.Tissue engineering strategies are necessary in the treatment of patients with larger,massive rotator cuff tears.CONCLUSION: The smaller the rotator cuff tear,the faster the functional recovery of the shoulder joint after arthroscopic rotator cuff repair,but in the face of a huge tear,the functional recovery of the shoulder joint after surgical treatment alone is relatively more difficult and requires more time.When facing patients with larger rotator cuff tears and giant tears,auxiliary treatments such as tissue engineering are needed.Chapter 2 Construction and Biocompatibility Study of Bilayer Bionic BMP Slow-release 3D Printed HydrogelsOBJECTIVE: To study the preparation and synthesis of HAMA and HAD,the printability and light-curing of bioinks HAC and HAHB,and to investigate the physical properties,growth factor delivery function of hydrogel scaffolds.METHODS:(1)HAMA and HAD were prepared and the products were analyzed by NMR.HAMA/Alg(HA),HAMA/Alg/COL Ⅰ(HAC),HAMA/Alg/HAD/BMP-2(HAHB)bioinks were synthesized.(2)Rheometer was used to analyze the rheological characteristics of the bioinks.(3)The bioink was prepared into hydrogel scaffolds by 3D printer,and the surface structure and microstructural characteristics of hydrogel scaffolds were observed by optical microscope and scanning electron microscope.(4)The mechanical properties of HA,HAC,HAHB and HAC/HAHB hydrogel scaffolds were examined using a rheometer.(5)Detect the release characteristics of BMP-2 in HAMA/Alg/BMP-2(HAB)and HAHB hydrogels using ELISA.(6)Detect the swelling characteristics of lyophilized HA,HAC,HAHB and HAC/HAHB hydrogel scaffolds when immersed in simulated body fluid(SBF).(7)Detection of degradation characteristics of lyophilized weighed HA,HAC,HAHB and HAC/HAHB hydrogel scaffolds immersed in SBF.(8)HAC/HAHB hydrogel scaffold immersion was used in in vivo experiments,and the effects of hydrogel scaffolds on ADSCs activity and proliferation were detected by cell live-dead staining and CCK-8 assay.RESULTS:(1)The NMR results showed the successful synthesis of HAD and HAMA.(2)Both HAC and HAHB inks have the characteristics of shear thinning and can be light-cured,and after adding 0.02 M Ca Cl2 for the first step of cross-linking,both have better printability.(3)The morphology of scaffolds printed with HAC and HAHB inks did not differ significantly under light microscopy,and TEM scanning of the lyophilized scaffolds showed that the internal pores of HAHB were smaller and the structure was more dense compared to HAC scaffolds.(4)The strength of the scaffolds increased with the addition of COL Ⅰ and HAD to the HA ink system,with HAD providing the greatest enhancement to the scaffold strength.The swelling rate of HAHB was higher,but the hydrogels of all groups reached the equilibrium of swelling within 24 h.HAD was able to slow down the degradation rate of the hydrogels,and COL Ⅰ also had the same effect.(5)Compared with HA,the addition of HAD could reduce the first day burst release of BMP-2,and also reduce the inactivation of BMP-2 at 37°C.(6)HAHB hydrogel scaffolds had the highest swelling rate,and the hydrophilicity of the hydrogel increased after the addition of HAD.(7)In the long term,the addition of HAD and COL Ⅰ increased slowed down the degradation of the hydrogel.(8)HAC/HAHB bilayer hydrogels did not exhibit cytotoxicity and had no significant effect on ADSCs proliferation.CONCLUSION: HAC and HAHB bioinks have good printability and light-curing properties,which can be suitable for 3D printing,and the fabricated hydrogel scaffolds have better mechanical properties compared with the HA scaffolds alone.HAHB was able to achieve a slow-release of BMP-2 as well as a reduction in the inactivation of BMP-2.The bilayer hydrogel scaffold showed good biocompatibility.Chapter 3 Double-layer Bionic BMP Slow-release3D-printed Hydrogel for Rotator Cuff Tendon-bone Interface RepairOBJECTIVE: To explore the induction of ADSCs by bilayer bionic BMP slow-release3D-printed hydrogel in vitro,and to study the repair effect of its combined ADSCs in the acute rotator cuff injury model in rats.METHODS:(1)HAC and HAHB monolayer scaffolds were co-cultured with ADSCs in Transwell system for induced differentiation culture,and the induced differentiation effect of hydrogel scaffolds on ADSCs was detected by RT-q PCR.(2)In the in vivo experiments,an acute rotator cuff injury model was established in rats and randomly grouped into the following groups: simple surgical repair group,scaffold implantation group,and scaffold-stem cell implantation group.Sections of the repaired tendon-bone interface were made after 4 and 8 weeks of repair,respectively,and were stained with HE,Masson,senna solid green,Sirius red and related pathologic scores,and biomechanical tests were performed.Micro-CT was performed on the tendon-bone interface at 8 weeks.RESULTS:(1)The HAHB layer enhanced the expression of OPN and OCN in ADSCs,and the HAC layer enhanced the expression of TNMD in ADSCs,meanwhile,HAHB and HAC also had an enhancing effect on the expression of COL2A1 and SOX9 in ADSCs.(2)The staining results of HE,Masson,senna solid green,and Sirius red staining showed more fibrovascular proliferation,disordered cell morphology,and poor mechanical properties in the surgical repair group alone.After the intervention of HAC/HAHB hydrogel scaffold combined with ADSCs,the repaired tendon-bone interface obtained better histological scores,the structure was closer to the normal tissue,and the biomechanical properties were better.Micro-CT showed that the bone tissues in the tendon-bone interface intervened with HAC/HAHB hydrogel scaffold combined with ADSCs obtained a better repair effect.CONCLUSION: In vitro,HAC and HAHB hydrogels were able to induce ADSCs differentiation in the tendonogenic and osteogenic directions,respectively,and both of them had a certain promotion effect on the chondrogenic differentiation of ADSCs.In the in vivo experiments,the HAC/HAHB hydrogel scaffold combined with ADSCs strategy successfully promoted the healing of injured rotator cuff and accelerated the repair and regeneration of the ordered 4-layer structure at the tendon-bone interface.更多还原