组织工程软骨生物反应器的设计

目的 根据流体力学原理优化反应器内腔结构,并设计一套组织工程软骨生物反应器.方法 采用计算机仿真的方法对反应器内腔流场进行仿真计算,通过流场分析确定内腔结构,最终构建一套完整的生物反应器系统.结果 确定了生物反应器的内腔结构,生物反应器由控制系统和细胞培养室两部分构成,能置于培养箱中对软骨细胞材料复合物进行动态培养.结论 反应器内腔的结构是合理的.整个生物反应器系统运行可靠.     

组织工程软骨生物反应器的设计

目的 根据流体力学原理优化反应器内腔结构,并设计一套组织工程软骨生物反应器.方法 采用计算机仿真的方法对反应器内腔流场进行仿真计算,通过流场分析确定内腔结构,最终构建一套完整的生物反应器系统.结果 确定了生物反应器的内腔结构,生物反应器由控制系统和细胞培养室两部分构成,能置于培养箱中对软骨细胞材料复合物进行动态培养.结论 反应器内腔的结构是合理的.整个生物反应器系统运行可靠.     

组织工程软骨实验生物反应器的设计

目的:设计一套计算机控制的组织工程软骨实验用灌流型生物反应器。方法:通过计算机控制时间及流量,利用蠕动泵、密闭的硅胶管、玻璃管、储液瓶等串联设计一套无菌的灌流型软骨组织工程用生物反应器。结果:生物反应器由控制系统和培养系统组成,运行良好,能置于培养箱中对软骨细胞材料复合物进行动态培养。结论:反应器设计合理。整个生物反应器系统运行可靠。     

一种新型血管生物反应器的研究与开发

目的 根据流体力学的分析改进支撑PGA-细胞材料复合物的硅胶管,设计一种应用于体外培养组织工程血管的生物反应器.方法 利用计算机仿真分析的方法对硅胶管内腔的压力场进行仿真计算,通过计算分析确定硅胶管的结构,进而开发一种新型血管生物反应器.结果 确定了硅胶管的尺寸结构以及培养室内相应的辅助结构,使整个反应器系统能够对PGA-细胞材料复合物进行动态培养.结论 改进后的硅胶管结构合理,整个血管生物反应器系统运行稳定.     

生物反应器系统在软骨组织工程中的应用

关节软骨的退行性变是当今社会主要的健康问题之一。据文献报道关节软骨的再生能力有限,到现在为止还没有一种十持久而且有效的治疗方法来替代关节软骨。组织工程是一个非常有潜力的领域,虽然要建立一套常规治疗软骨缺损的组织工程理论还有很多障碍,但是为治疗软骨损伤为目的的组织工程已经能够快速可靠的培养出适合的软骨组织。生物反应器和一些相关的设备为组织工程提供了一个快速且有效的方法。事实上,在体内生理刺激影响着软骨的功能,所以我们要设计一些特别的生物反应器来对在体外培养的关节软骨施加模拟应力的传导,像流体剪切力,流体静压力,周期压力,和一些混合力。本篇综述总结了一些软骨细胞反应器系统在细胞刺激和培养中的应用。     

衍生肌腱支架材料的细胞相容性研究

目的 探讨衍生肌腱支架材料（tendon derivation biomaterials，TDBM）与肌腱细胞的细胞相容性及肌腱细胞在此三维支架上培养的生物学行为，为肌腱组织工程新型支架材料的应用提供依据。方法将肌腱细胞与TDBM体外复合培养，设置单纯肌腱细胞培养为对照组，进行形态学观察，并检测细胞增殖、细胞周期、细胞DNA倍体水平及肌腱细胞凋亡率。通过^3H-脯氨酸（^3H-Proline）掺入试验了解材料对肌腱细胞胶原合成的影响。结果 肌腱细胞在TDBM上呈梭形生长，TDBM组细胞第2天进入对数增长期，倍增时间为3d，而单纯肌腱细胞培养对照组倍增时间为3．75d。TDBM组与单纯肌腱细胞培养对照组比较，^3H-Proline掺入值差异无统计学意义（P〉0．05），说明细胞功能未受影响。与支架材料复合培养的肌腱细胞DNA指数为0．96，增殖指数较对照组高10．1％，提示肌腱细胞在三维胶原支架上生长速度快，增殖能力强。结论TDBM具有良好的细胞相容性，可作为肌腱细胞的有效载体应用于组织工程化肌腱的构建。     

转壁生物反应器中动态分化组织工程软骨的实验研究

目的 :在转壁生物反应器动态环境中分化骨髓间充质干细胞 (bMSC) ,制备立体组织工程软骨。方法 :穿刺吸取成年兔骨髓 ,分离扩增bMSC ,复合于纤维蛋白凝胶制成圆柱状三维组织 (细胞密度2 0× 10 7/ml) ,置于转壁生物反应器中进行分化培养 ,同时设单纯静止培养组。 5周后观测大体形态和组织学形态、Ⅰ和Ⅱ型胶原免疫组织化学表达 ,测量分化组织的细胞活力。结果 :动态培养可以有效保持材料大体形态 ,甲苯胺兰染色阳性 ,无明显Ⅰ型胶原表达 ,大量表达Ⅱ型胶原 ,人工组织的细胞活力显著高于单纯静止培养方法。结论 :转壁生物反应器培养明显改善组织工程软骨的活力 ,有利于进行三维材料体外软骨分化。     

周期性流体应力刺激对组织工程软骨分化影响的实验研究

目的 研究周期性流体应力刺激对组织工程软骨体外分化的影响,确立良好的动态培养方案。方法 穿刺吸取成年兔骨髓,密度梯度离心法分离扩增骨髓间充质干细胞(Bone marrow derived mesenchymal stem cell,MSC)。以2.0×10~7/ml密度复合于纤维蛋白胶,制成圆柱形人工组织。实验组材料经受周期为0.2Hz流体应力刺激,于转壁生物反应器中培养2周,对照组静止培养,两组均于条件培养基内诱导软骨组织形成。2周后观察大体形态、和组织学形态、Ⅰ型和Ⅱ型胶原免疫组织化学表达,测量细胞活力和胶原、蛋白多糖含量等生化指标。结果 应力刺激组材料的大体形态完整,而对照组材料破碎回缩。实验和对照组均可以诱发材料中MSC分化成为软骨细胞,但流体刺激组表达更高水平的Ⅱ型胶原和蛋白多糖,细胞活力明显高于对照静止培养组(P<0.01)。结论 周期性流体应力刺激明显促进MSC体外软骨分化,转壁生物反应器培养优于单纯静止培养。     

胰腺癌肝转移体外模型的构建

目的使用旋转细胞培养系统(rotating cell culture system，RCCS)模拟微重力环境，建立胰腺癌肝转移的体外模型。方法将人肝组织块和人胰腺癌细胞株BxPC-3置于旋转细胞培养系统中混合培养，在额定时间点取肝组织块送病理，HE染色观察肝组织结构和胰腺癌细胞在肝组织块中生长情况。结果混合培养第1天人肝组织块周围和管腔内即有胰腺癌细胞粘附并聚集；第3天可见肝组织块中有散在癌细胞浸润；第7天可见微小转移灶形成；混合培养第10天胰腺癌肝转移灶、肝细胞形态和肝小叶结构可保持完好，并可见癌组织块形成。结论使用旋转细胞培养系统模拟微重力环境，构建胰腺癌肝转移体外模型，可以用于胰腺癌肝转移过程和机制的研究，为研究胰腺癌细胞和肝组织问的相互关系提供新的方法。     

医用合成材料假内膜体外性能评价方法的研究

在植入材料表面接种一层血管内皮细胞（ＥＣ）即假内膜，以改善医用合成材料的血液相容性，是降低植入物栓发生率的一种巧妙而有效的方法，为确定体外培养的假内膜是否能在体内动态环境中保持完整性及ＥＣ的生物活性，本实验建立了一套方法，通过模拟体内流体力学应力条件建立体外动态环境，对在合成材料表面（聚氨酯和乳胶）培养的牛主动脉ＥＣ假内膜层进行流体力这刺激，通过其形态学及生化学方面的改变，即选用细胞脱落率和前列腺     

自体肌腱细胞介导修复肌腱缺损的实验研究

目的　探讨以自体肌腱细胞为种子细胞的组织工程化肌腱体内形成。方法　取自体肌腱细胞经体外培养、扩增 ,与可吸收生物材料聚羟基乙酸 (polyglycolicacid ,PGA)混合培养形成细胞 生物材料复合物 ,将细胞 生物材料复合物移植于手术缺损的家猪趾浅屈肌腱处 ,并设单纯PGA组作为对照组。术后 6周取材 ,对标本进行大体观察、组织学和生物力学检测和评价再生组织。结果　实验组新生组织在大体、组织学和胶原排列等方面与正常肌腱相似 ,对照组新生组织细胞、胶原排列较为紊乱。生物力学显示其最大拉力、最大应力和弹性模量 ,实验组比对照组分别提高 3 6.1% (t =3 5 6、P <0 .0 5 )、2 7.4%(t =2 94、P <0 .0 5 )和 15 .0 % (t =2 62、P <0 .0 5 )。结论　应用组织工程技术以自体肌腱细胞可以修复肌腱缺损     

Cyclic mechanical stimulation rescues achilles tendon from degeneration in a bioreactor system

Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy‐like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis, and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25 Hz, 8 h/day) in a bioreactor, the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early‐stage pathological changes by increased collagen type I production, decreased collagen degradation and cell apoptosis. The ex vivo model developed in this study allows systematic study on the effect of mechanical stimulation on tendon biology. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1888–1896, 2015.     

Functional tissue engineering for tendon repair: A multidisciplinary strategy using mesenchymal stem cells,bioscaffolds, and mechanical stimulation

Over the past 8 years, our group has been continuously improving tendon repair using a functional tissue engineering (FTE) paradigm. This paradigm was motivated by inconsistent clinical results after tendon repair and reconstruction, and the modest biomechanical improvements we observed after repair of rabbit central patellar tendon defects using mesenchymal stem cell‐gel‐suture constructs. Although possessing a significantly higher stiffness and failure force than for natural healing, these first generation constructs were quite weak compared to normal tendon. Fundamental to the new FTE paradigm was the need to determine in vivo forces to which the repair tissue might be exposed. We first recorded these force patterns in two normal tendon models and then compared these peak forces to those for repairs of central defects in the rabbit patellar tendon model (PT). Replacing the suture with end‐posts in culture and lowering the mesenchymal stem cell (MSC) concentration of these constructs resulted in failure forces greater than peak in vivo forces that were measured for all the studied activities. Augmenting the gel with a type I collagen sponge further increased repair stiffness and maximum force, and resulted in the repair tangent stiffness matching normal stiffness up to peak in vivo forces. Mechanically stimulating these constructs in bioreactors further enhanced repair biomechanics compared to normal. We are now optimizing components of the mechanical signal that is delivered in culture to further improve construct and repair outcome. Our contributions in the area of tendon functional tissue engineering have the potential to create functional load‐bearing repairs that will revolutionize surgical reconstruction after tendon and ligament injury. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1–9, 2008     

Expression and mapping of lubricin in canine flexor tendon.

Matrix composition and the biomechanical environment are intimately interdependent in most connective tissues. Lubricin has many distinct biological functions, including lubrication, antiadhesion, and cytoprotection in cartilage, tendons, and other tissues. This study investigated the distribution of lubricin in the canine flexor digitorum profundus (FDP) tendon by immunohistochemistry. Lubricin was found both on the tendon surface and at the interface of collagen fiber bundles within the tendon, where the cells are subjected to shear force in addition to tension and compression. The expression of lubricin in regions of the canine flexor tendon that differ in mechanical or nutritional environment was also investigated using RT-PCR. Six N-terminal splicing variants were identified from six distinct anatomical regions of flexor tendon. The variants with larger sizes were noted in regions subjected to significant shear and compressive forces. Lubricin is ubiquitous in the FDP tendon, with variations in distribution and splicing that appear to correspond to discrete anatomic locations that differ by mechanical or nutritional environment.     

Chondroitin‐6‐sulfate incorporation and mechanical stimulation increase MSC‐collagen sponge construct stiffness

Using functional tissue engineering principles, our laboratory has produced tendon repair tissue which matches the normal patellar tendon force‐displacement curve up to 32% of failure. This repair tissue will need to withstand more strenuous activities, which can reach or even exceed 40% of failure force. To improve the linear stiffness of our tissue engineered constructs (TECs) and tissue engineered repairs, our lab is incorporating the glycosaminoglycan chondroitin‐6‐sulfate (C6S) into a type I collagen scaffold. In this study, we examined the effect of C6S incorporation and mechanical stimulation cycle number on linear stiffness and mRNA expression (collagen types I and III, decorin and fibronectin) for mesenchymal stem cell (MSC)‐collagen sponge TECs. The TECs were fabricated by inoculating MSCs at a density of 0.14 × 10⁶ cells/construct onto pre‐cut scaffolds. Primarily type I collagen scaffold materials, with or without C6S, were cultured using mechanical stimulation with three different cycle numbers (0, 100, or 3,000 cycles/day). After 2 weeks in culture, TECs were evaluated for linear stiffness and mRNA expression. C6S incorporation and cycle number each played an important role in gene expression, but only the interaction of C6S incorporation and cycle number produced a benefit for TEC linear stiffness. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1092–1099, 2010     

组织工程化肌腱研究进展

对组织工程化肌腱领域中目前研究的主要成果进行综述，着重阐述了肌腱细胞外基质替代物、肌腱细胞生物学性质及肌腱细胞与细胞外基质材料复合研究中的主要问题。认为，研制适于肌腱细胞生长、粘附和发挥功能的细胞外基质材料；建立生长、增殖可调控的肌腱细胞系；在模拟体内力学条件下，进行肌腱细胞三维培养，将是研究具有特定修复功能的组织工程化肌腱的重要问题。     

Biomechanical evaluation of metacarpophalangeal joint prosthesis designs.

A laboratory biomechanical analysis of metacarpophalangeal joint prosthesis designs was done with fresh cadaver finger rays. The center of rotation, range of motion, tendon excursion, and fingertip force were determined on the specimens before and after implanting Swanson, Niebauer, Steffee II, St. Georg-Buchholz, Schultz, and modified Strickland prostheses. Their biomechanical behavior varied considerably and none duplicated the normal metacarpophalangeal (MP) joint. Each has design characteristics that may be clinically advantageous as well as disadvantageous. Irrespective of the design, the studies done cannot be divorced from the following factors: (1) implant material properties--silicone rubber implants buckled with tendon loading; this deformity created a significant flexor mechanical advantage and an extensor mechanical disadvantage; (2) implant fixation--freely movable implant stems dampened part of the applied load; braided suture provided inadequate immediate fixation; (3) implantation technique--the articulated prostheses can be technically unforgiving; errors in technique resulted in alteration of their biomechanical behavior.     

自体组织工程化肌腱预制的初步研究

目的：应用组织工程技术研究组织工程化肌腱体内形成的可行性。方法：取成年家鸡的趾深屈肌腱，用酶消化法分离，培养肌腱细胞，将在体外扩增到一定浓度的肌腱细胞接种到聚羟基乙酸（PGA）上，形成细胞－材料复合物，体外培养5d后，将此复合物回植至自体右翼皮下，左侧以单纯PGA作为对照，培养后第3，4，6，8周取材，从大体，组织学等方面进行分析。结果；术后8周见组织工程化肌腱呈白色，有光泽，组织学见胶原组织平行排列，但仍可见未降解的PGA及少量炎性细胞，对照组则无任何组织形成，结论：自体肌腱细胞与生物材料复合后在免疫功能正常的自体动物体内能够再生出肌腱样组织，新生的肌腱样组织在大体，组织学等方面均与正常肌腱相似。     

基于有限元的组织工程肌腱支架材料的力学分析

目的 对组织工程生物可降解支架材料进行力学分析.方法 建立了一种组织工程肌腱支架材料的三维模型,用有限元数值分析方法,对所建模型在不同载荷情况下的受力状况进行了计算分析.结果 相对于该材料表面的狭长槽而言,最大应力发生在狭长槽两端面的上方的两个角.该材料在所加力大于0.01牛时产生明显变形,当拉力达到0.05牛顿时已超过材料的力学承受极限.结论 有限元的分析方法是合理、可行的,对细胞如何在材料的分布提供理论依据.     

Smad8/BMP2‐engineered mesenchymal stem cells induce accelerated recovery of the biomechanical properties of the achilles tendon

Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2‐engineered MSCs in a full‐thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), non‐engineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (>70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon‐like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2‐engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long‐term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short‐term functional recovery in the treatment of tendon injuries. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1932–1939, 2012