Optimization of Human Tendon Tissue Engineering Using Bioreactors

描述（由申请人提供）： 项目摘要/摘要本研究的目的是将以前在兔模型中对屈肌腱组织工程的研究转化为人类临床病例。具体而言，目标和目的是：1）基于在兔中的工作优化人屈肌腱的去细胞化技术; 2）将候选细胞接种到去细胞化的人肌腱以创建组织工程化肌腱构建物; 3）使用新型大规模组织生物反应器通过施加循环剪切力和应变力来最大化体外组织工程化肌腱构建物的强度和存活力; 4）将这些组织工程化肌腱构建物应用于严重手外伤患者的肌腱重建，并转化为人类临床病例。将解剖人屈肌腱并保存在培养物中。使用SDS、Triton x-100和冻融循环的条件将不断变化，直到建立最大限度地减少抗原性同时保持结构完整性的最佳方案。将通过组织学、荧光细胞染色和DNA含量确定有效性。结构强度将通过张力测定法测定极限拉伸强度和弹性模量。肌腱细胞、真皮成纤维细胞和脂肪源性干细胞的原代培养物将在培养物中扩增，并以2x106个细胞/cc的密度接种。接种将由细胞悬浮液、显微注射和超声处理的组合组成。肌腱结构将在培养物中保持7天。细胞接种效力将通过H & E显微镜、细胞染色和胶原蛋白I和III的定量分析来确定。将使用提供单轴肌腱应变的定制组织生物反应器。肌腱结构将在5天的过程中接受1.25 N的单轴拉伸力。初始循环参数为1个循环/分钟，机械负载和休息交替1小时。在生物反应器处理或静态孵育后，将对样本进行拉伸试验，以比较极限拉伸应力和弹性模量。在患有严重上肢损伤的退伍军人患者中，两组屈肌腱均缺失[指深屈肌（FDP）和指浅屈肌（FDS）]。FDS肌腱是一个通常不重建的冗余系统。这提供了一个独特的机会，以测试组织工程肌腱移植物的有效性，并将额外的手术时间和患者风险降至最低。FDP将使用正常肌腱移植物重建，FDS将使用新的组织工程肌腱移植物重建。结局将包括术后活动度、活检组织学和翻修手术需求。在这些技术开发出来后，VA外科医生可以切除一小部分肌腱（或其他细胞来源），然后在患者稳定的同时让细胞在培养物中增殖。然后，可以将来自组织库的尸体同种异体肌腱去细胞化，并接种患者自己的细胞。当进行肢体重建时，大量的生物相容性肌腱将是可用的。这项生物工程研究可以转化为直接的临床应用，以满足受伤士兵的重要需求。 公共卫生相关性： 该转化研究将优化生产组织工程肌腱的技术。在这些技术开发出来后，军队和退伍军人事务部的外科医生可以切除一小部分剩余的肌腱（或其他细胞来源），然后让这些细胞在组织培养中增殖，同时病人稳定下来。然后，可以将来自人体组织库的尸体同种异体肌腱去细胞化，并用患者自己的细胞接种。当进行最终的肢体重建时，大量的生物相容性肌腱材料将是可用的。因此，生物工程研究的最新进展可以转化为直接的临床应用，以满足受伤士兵的独特和重要需求。此外，老年退伍军人类风湿性关节炎和骨关节炎的发病率增加，导致肌腱退化和手功能下降。组织工程屈肌腱移植物也将是有用的重建在这些情况下。

DESCRIPTION (provided by applicant): Project Summary/Abstract The objective of this research is to translate previous work on flexor tendon tissue engineering in the rabbit model to human clinical cases. Specifically, the goals & objectives are to: 1) Optimize techniques for acellularization of human flexor tendons based on work in the rabbit; 2) Seed acellularized human tendons with candidate cells to create tissue-engineered tendon constructs; 3) Maximize tissue-engineered tendon construct strength and viability in vitro by applying cyclic shear and strain forces using a novel large-scale tissue bioreactor; and 4) Translate to select human clinical cases by using these tissue-engineered tendon constructs for tendon reconstruction in severe cases of mutilating hand injuries. Human flexor tendons will be dissected and preserved in culture. Conditions using SDS, Triton x-100, and freeze-thaw cycles will be varied until the optimal protocol to minimize antigenicity while maintaining structural integrity is established. Effectiveness will be determined by histology, fluorescent cytostaining, and DNA content. Structural strength will be determined by tensiometry for ultimate tensile strength and elastic modulus. Primary cultures of tenocytes, dermal fibroblasts, and adipoderived stem cells will be expanded in culture and seeded at a density of 2x106 cells/cc. Seeding will consist of combinations of cell suspension, microinjection, and ultrasonication. The tendon constructs will be kept in culture for 7 days. Cell seeding efficacy will be determined by H&E microscopy, cytostaining, and quantitative analysis of collagen I & III. A custom tissue bioreactor providing uniaxial tendon strain will be used. The tendon constructs will be subjected to a uniaxial stretch force 1.25N each over a 5 day course. The initial cycle parameters will be 1cycle/min in alternating 1 hour periods of mechanical loading and rest. After bioreactor treatment or stationary incubation, the specimens will undergo tensile testing to compare ultimate tensile stress and elastic modulus. In Veteran patients with severe upper extremity injuries, both sets of flexor tendons are missing [flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS)]. The FDS tendons are a redundant system that is usually not reconstructed. This provides a unique opportunity to test efficacy of the tissue-engineered tendon grafts with minimal additional operative time and risk to the patient. The FDPs will be reconstructed using normal tendon grafts and the FDSs will be reconstructed using the new tissue-engineered tendon grafts. Outcomes will consist of postoperative range of motion, histology on biopsies, and need for revision surgery. After these techniques are developed, VA surgeons could remove a small portion of tendon (or other cell source) and then allow cells to proliferate in culture while the patient is stabilized. Then, cadaver allograft tendons from a tissue bank can be acellularized and seeded with the patient's own cells. When reconstruction of the extremity is undertaken, large amounts of biocompatible tendon would be available. This bioengineering research may be translated to direct clinical applications for a significant need in injured soldiers. PUBLIC HEALTH RELEVANCE: Project Narrative This translational research will optimize techniques to produce tissue engineered tendons. After these techniques are developed, military and VA surgeons could remove a small portion of remaining tendon (or other cell source) and then allow these cells to proliferate in tissue culture while the patient is stabilized. Then, cadaver allograft tendons from a human tissue bank can be acellularized and seeded with the patient's own cells. When definitive reconstruction of the extremity is undertaken, large amounts of biocompatible tendon material would be available. Therefore, recent advances in bioengineering research may be translated to direct clinical applications for a unique and significant need in injured soldiers. Furthermore, aging veterans have increased incidence of rheumatoid arthritis and osteoarthritis which lead to degeneration of tendons and decreased hand function. Tissue engineered flexor tendon grafts would also be useful for reconstruction in these cases.