Optimization of Human Tendon Tissue Engineering Using Bioreactors

使用生物反应器优化人体肌腱组织工程

• 批准号: 7861379
• 负责人: James Chang
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7861379
• 关键词: Aging; Allografting; Articular Range of Motion; Autologous; Biocompatible; Biocompatible Materials; Biomedical Engineering; Biopsy; Bioreactors; Blast Injuries; Cadaver; Cartilage; Cells; Cicatrix; Clinical; Collagen; Connective Tissue; Custom; Defect; Degenerative polyarthritis; Dermal; Digit structure; Disease; Effectiveness; Evolution; Fibroblasts; Fingers; Flexor; Freezing; Goals; Gunshot wound; Hand; Hand Injuries; Hand functions; Hematoxylin and Eosin Staining Method; Histocompatibility Testing; Histology; Hour; Human; In Vitro; Incidence; Injury; Lead; Limb structure; Lower Extremity; Mechanics; Methods; Microinjections; Microscopy; Military Personnel; Modeling; Motor; Muscle; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Patients; Ploidies; Postoperative Period; Production; Proliferating; Protocols documentation; Reconstructive Surgical Procedures; Research; Rest; Rheumatoid Arthritis; Risk; Rupture; Seeds; Silicones; Skin; Soldier; Source; Specimen; Staging; Stem cells; Stress; Stretching; Structure; Surgeon; Suspension substance; Suspensions; System; Techniques; Tendon Injuries; Tendon structure; Tensile Strength; Testing; Time; Tissue Banking; Tissue Banks; Tissue Engineering; Tissues; Translating; Translational Research; Triton; Tritons; Upper Extremity; Veterans; Work; Wrist joint; abstracting; base; bone; clinical application; density; disability; efficacy testing; functional restoration; human tissue; improved functioning; injured; novel; reconstruction; repaired; retinal rods; scaffold; tissue culture; vehicular accident

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.

描述（由申请人提供）： 项目摘要/摘要本研究的目的是将先前在兔模型中进行屈肌腱组织工程的工作转化为人类临床病例。具体来说，目的和目标是： 1）基于兔子的工作优化人类屈肌腱脱细胞化技术； 2）用候选细胞接种脱细胞人类肌腱，以创建组织工程肌腱结构； 3）通过使用新型大型组织生物反应器施加循环剪切力和应变力，最大限度地提高组织工程肌腱结构的体外强度和活力； 4) 通过使用这些组织工程肌腱结构在严重的手部损伤病例中进行肌腱重建，转化为选择的人类临床病例。人体屈肌腱将被解剖并保存在培养物中。使用 SDS、Triton x-100 和冻融循环的条件将有所不同，直到建立最小化抗原性同时保持结构完整性的最佳方案。有效性将取决于组织学、荧光细胞染色和 DNA 含量。结构强度将通过张力测定法确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。接种将包括细胞悬浮液、显微注射和超声处理的组合。肌腱构建体将在培养物中保存 7 天。细胞接种功效将通过 H&E 显微镜、细胞染色以及胶原蛋白 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。在 5 天的过程中，肌腱结构将承受每个 1.25N 的单轴拉伸力。初始循环参数为 1 循环/分钟，机械加载和休息交替 1 小时。经过生物反应器处理或静态孵育后，样本将进行拉伸测试，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 肌腱是一个冗余系统，通常不进行重建。这提供了一个独特的机会来测试组织工程肌腱移植物的功效，同时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进行重建，FDS 将使用新型组织工程肌腱移植物进行重建。结果将包括术后活动范围、活检组织学以及是否需要进行修复手术。这些技术开发出来后，退伍军人管理局的外科医生可以切除一小部分肌腱（或其他细胞源），然后在患者病情稳定的情况下让细胞在培养物中增殖。然后，来自组织库的尸体同种异体移植肌腱可以被脱细胞并接种患者自己的细胞。当进行四肢重建时，将可以获得大量生物相容性肌腱。这项生物工程研究可以转化为直接临床应用，以满足受伤士兵的重大需求。 公共卫生相关性： 项目叙述 这项转化研究将优化生产组织工程肌腱的技术。这些技术开发出来后，军队和退伍军人管理局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），然后在患者病情稳定时让这些细胞在组织培养物中增殖。然后，来自人体组织库的尸体同种异体移植肌腱可以被脱细胞并接种患者自己的细胞。当进行四肢的最终重建时，将可以获得大量生物相容性肌腱材料。因此，生物工程研究的最新进展可能会转化为直接的临床应用，以满足受伤士兵的独特而重大的需求。此外，老年退伍军人患类风湿性关节炎和骨关节炎的发病率增加，导致肌腱退化和手部功能下降。在这些情况下，组织工程屈肌腱移植物也可用于重建。