Regional variations in the viscoelastic compressive properties of the temporomandibular joint disc and implications toward tissue engineering

Evidence from experimental and finite element studies have shown that the temporomandibular joint disc is heavily loaded during normal physiological function. Several studies have been carried out to investigate the response of the disc to tensile and static compressive forces. However, there is limited information that elucidates the dynamic characteristics of the disc under in vivo loading conditions. These investigations assessed the effect of physiologically relevant applied strain amplitudes and frequencies to determine regional mechanical properties of the disc. Cyclic tests on porcine TMJ discs were carried out over a period of 15 cycles, and the resultant compressive moduli and energy dissipation properties of the disc were reported. Results showed that modulus values were more dependent on strain amplitude than on frequency, and modulus values exhibited a strong regional variation. Clear hysteresis loops were evident in each set of testing parameters, and the only statistically significant regional variation in energy dissipation was between the central and medial regions. From these investigations, a more detailed understanding of the spatial mechanical properties of the TMJ disc has been achieved under physiologically relevant loading conditions. Combined with studies on other loading modalities of the disc, these results will serve as a benchmark for future TMJ disc tissue engineering endeavors.     

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system

A novel approach was utilized to grow in vitro mineralized bone tissue using lighter-than-water, polymeric scaffolds in a high aspect ratio rotating bioreactor. We have adapted polymer microencapsulation methods for the formation of hollow, lighter-than-water microcarriers of degradable poly(lactic-co-glycolic acid). Scaffolds were fabricated by sintering together lighter-than-water microcarriers from 500 to 860 microm in diameter to create a fully interconnected, three-dimensional network with an average pore size of 187 microm and aggregate density of 0.65 g/mL. Motion in the rotating bioreactor was characterized by numerical simulation and by direct measurement using an in situ particle tracking system. Scaffold constructs established a near circular trajectory in the fluid medium with a terminal velocity of 98 mm/s while avoiding collision with the bioreactor wall. Preliminary cell culture studies on these scaffolds show that osteoblast-like cells readily attached to microcarrier scaffolds using controlled seeding conditions with an average cell density of 6.5 x 10(4) cells/cm(2). The maximum shear stress imparted to attached cells was estimated to be 3.9 dynes/cm(2). In addition, cells cultured in vitro on these lighter-than-water scaffolds retained their osteoblastic phenotype and showed significant increases in alkaline phosphatase expression and alizarin red staining by day 7 as compared with statically cultured controls.     

组织工程化旋转生物反应器研究进展

概述了组织工程化水平旋转生物反应器的工作原理、培养环境、应用现状和发展趋势。水平旋转生物反应器为体外培养动物细胞保持其正常形态、结构、功能和遗传特性提供了一种新手段,得天独厚的微重力、高效物质传递和低剪应力环境、多孔立体网状支架材料、在线监测和控制细胞三维生长等优势,为离体细胞重建组织、实现人工构建组织和器官有望成为现实。     

组织工程化旋转生物反应器研究进展

概述了组织工程化水平旋转生物反应器的工作原理、培养环境、应用现状和发展趋势。水平旋转生物反应器为体外培养动物细胞保持其正常形态、结构、功能和遗传特性提供了一种新手段，得天独厚的微重力、高效物质传递和低剪应力环境、多孔立体网状支架材料、在线监测和控制细胞三维生长等优势，为离体细胞重建组织、实现人工构建组织和器官有望成为现实。     

A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc

Tissue-engineered temporomandibular joint (TMJ) discs offer a viable treatment option for patients with severe joint internal derangement. To date, only a handful of TMJ tissue engineering studies have been carried out and all have incorporated the use of synthetic scaffold materials. These current scaffolds have shown limited success in recapitulating morphological and functional aspects of the native disc tissue. The present study is the first to investigate the potential of a xenogeneic scaffold for use in tissue engineering the TMJ disc. The effects of decellularization agents on the disc's mechanical properties were assessed using three common decellularization protocols: Triton X-100, sodium dodecyl sulfate (SDS) and an acetone/ethanol solution. Decellularized scaffolds were subsequently characterized through cyclic mechanical testing at physiologically relevant frequencies to determine which chemical agent most accurately preserved the native tissue properties. Results have shown that porcine discs treated with SDS most closely matched the energy dissipation capabilities and resistance to deformation of the native tissue. Treatments using Triton X-100 caused the resultant tissue to become relatively softer with inferior energy dissipation capabilities, while treatment using acetone/ethanol led to a significantly stiffer and dehydrated material. These findings support the potential of a porcine-derived scaffold decellularized by SDS as a xenograft for TMJ disc reconstruction.     

组织工程生物反应器的生物力学

<正>组织工程,是指用工程科学和生命科学的原理和方法,制备组织和器官替代物,以恢复、维持或改善人体组织、器官的功能,是一个发展迅速、意义深远的生物医学工程应用领域。目前,组织工程化皮肤产品已正式进入临床应用,培育的组织工程骨骼、软骨、血管、皮肤以及神经组织正在进行体内实验,组织工程肝脏、胰脏、乳房、心脏、手指、角膜等也可以在实验室里构建生长。组织工程已形成一个发展中的产业。但是目前组织工程距离广泛应用于临床、成为社会经济新的增长点还有相当长的路要走。阻碍组织工程发展和临床应用的主要因素至少包括两点:(1)对调控组织的功能化培养的特定物理-生物化学因素知之还少;(2)高昂的生产成本和缺乏商业化的功能性组织工程产品。     

Seeding techniques and scaffolding choice for tissue engineering of the temporomandibular joint disk

Temporomandibular joint (TMJ) disk removal, or diskectomy, is a detrimental yet necessary surgery for patients with extremely displaced disks. Tissue engineering is an enticing methodology for improvement of the postoperative outcome of diskectomy. Unfortunately, the field of tissue engineering of the TMJ disk is only in the early stages of development. The initial objective of this investigation was to study the cellular response of TMJ disk cells in alginate culture. However, a marked decrease in cell population and the lack of detection of extracellular matrix (ECM) products did not support the use of alginate culture. The second objective was then to attempt TMJ disk cell culture in polyglycolic acid (PGA) nonwoven meshes. However, as suitable seeding methods for TMJ disk cells on PGA had not been determined, three techniques were selected for study: spinner flask, orbital shaker, and a novel pelleting technique. PGA constructs maintained cellularity throughout the culture period, and scaffolds seeded with the spinner flask produced about 35 microg of collagen per construct. Thus, as evidenced by the production of a major extracellular component, PGA nonwoven meshes seeded with TMJ disk cells, using a spinner flask, may be a first positive culturing step in tissue engineering the TMJ disk.     

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5× p < 0.01), calcium deposition (5.5×, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.     

Soft tissue mechanics of the temporomandibular joint

Direct measurement of temporomandibular joint (TMJ) tissue deformation requires animal experimentation. Most of the available data pertain to the mechanical strain on the bone surfaces around the joint. However, bone is rarely the first joint tissue to show injury, being affected after damage to collagenous tissues such as the disc or capsule. Capsular ligaments guide or limit movement, while the intra-articular disc may also distribute joint loads. However, these tissues are difficult to visualize dynamically and not suitable for strain gage attachment, so in vivo deformations are poorly understood. Using pigs as the best nonprimate model for human TMJ function, we implanted differential variable reluctance transducers to measure antero-posterior strain in the lateral aspect of the intra-articular disc. The results were compared to previously published data on the TMJ capsule. Passive manipulation in anesthetized animals indicated that opening, protrusion, and contralateral movements caused the disc to elongate. On the contrary, closing, retrusion and ipsilateral movements caused disc shortening. These strains are opposite to those observed in the capsule and are expected on anatomical grounds. Surprisingly, disc strain during mastication differed from that during manipulation. The disc elongated during jaw closure, more on the retruding balancing side (16% +/- 1) than on the working side (8% +/- 2). This anomalous behavior may reflect compressive loading, such that the disc elongates as a result of the Poisson effect rather than condylar movement. Because the capsule also elongates during the power stroke, especially on the balancing side, both disc and capsule are maximally loaded on the same side at the same moment.     

A comparison of human umbilical cord matrix stem cells and temporomandibular joint condylar chondrocytes for tissue engineering temporomandibular joint condylar cartilage

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.     

Network of telocytes in the temporomandibular joint disc of rats

The phenotypes of the temporomandibular joint (TMJ) disc cells range from fibroblasts to chondrocytes. There are relatively few reported studies using transmission electron microscopy (TEM) to determine the ultrastructural features of these cells. It was hypothesized that at least a subpopulation of TMJ stromal cells could be represented by the telocytes, cells with telopodes. In this regard a TEM study was performed on rat TMJ samples. Collagen-embedded networks were found built-up by cells with telopodes with subplasmalemmal caveolae, moderate content in matrix secretory organelles and well-represented intermediate filaments. Appositions of cell bodies were found. Prolongations of such cells were closely related to nerves and microvessels. Our study indicates that the TMJ disc attachment seems equipped with telocytes capable of stromal signaling. However, further studies are needed to assess whether the telocytes belong to a renewed cell population derived from circulating precursors.     

正常及关节盘移位后颞下颌关节滑膜OPG/OPGL的表达及意义

目的： 观察正常及关节盘移位后颞下颌关节滑膜内护骨素OPG及其配体OPGL的表达及意义。方法： 32只日本大耳白兔，25只建立颞下颌关节盘前移位的动物模型，术后1、2、4、8、12周处死，5只行模拟手术作为假手术对照，另2只不行手术作为空白对照。取颞下颌关节标本，HE染色观察镜下结构， S-P免疫组化法检测滑膜中OPG和OPGL的表达与分布。结果： 各实验组动物术后体重无减轻，伤口愈合良好，大体及显微镜下观察关节盘明显前移位。对照组OPG主要表达于滑膜下层血管内皮细胞及部分滑膜衬里细胞，滑膜内 OPGL无表达或鲜有表达。关节盘移位后，仅1、2周各1个样本滑膜下层少量OPGL表达，其它样本未见明显OPGL阳性细胞；OPG表达强度无减弱。结论： 正常颞下颌关节滑膜内OPG表达明显强于OPGL，对正常状态骨软骨代谢平衡的维持有重要意义；关节盘移位后OPG和OPGL无明显变化，保证了关节盘移位后骨软骨代谢能维持平衡。     

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.     

关节盘移位对颞下颌关节内应力分布的影响

目的 通过对不同关节盘移位的数值模拟,探究各种移位情况下颞下颌关节（temporomandibular joint,TMJ）内各结构的应力分布规律。方法 依据CT图像,建立包含下颌骨、全牙列、关节盘和关节软骨的正常TMJ三维有限元模型;参考关节盘前、后、外、内移位的临床特征,建立对应的4个模型。关节盘与关节软骨间考虑接触,用缆索元模拟下颌韧带和关节盘附着,施加正中咬合荷载。结果 前移位将导致关节盘中带产生过高的压应力,达到3.23 MPa;后、内、外移位时关节盘的整体应力水平比前移位和正常TMJ高;各种移位都使关节结节后斜面的应力值大幅度增加,但对髁突关节面的影响却不大。结论 各种移位都将导致关节盘和关节结节后斜面产生过高的应力,且后、内、外移位更为危险,更容易造成关节结构和功能的损伤。     

Design of a high-throughput flow perfusion bioreactor system for tissue engineering

Flow perfusion culture is used in many areas of tissue engineering and offers several key advantages. However, one challenge to these cultures is the relatively low-throughput nature of perfusion bioreactors. Here, a flow perfusion bioreactor with increased throughput was designed and built for tissue engineering. This design uses an integrated medium reservoir and flow chamber in order to increase the throughput, limit the volume of medium required to operate the system, and simplify the assembly and operation.     

心肌组织工程生物反应器研究进展

组织工程的研究主要围绕种子细胞、生物材料和组织构建这三个基本要素而展开。组织构建技术是组织工程研究的核心。组织工程生物反应器是一种体外构建人体组织的系统装置。心肌组织工程在替代和维持梗塞的心肌组织功能,并进而治愈疾病以最大限度地挽救病人生命方面可能发挥巨大作用。主要介绍了国内外工程化心肌组织体外构建技术,特别是用于构建工程化心肌组织的心肌组织工程生物反应器研究方面的进展。     

Three-dimensional-construct bioreactor conditioning in human tendon tissue engineering

Human tendon tissue engineering attempts to address the shortage of autologous tendon material arising from mutilating injuries and diseases of the hand and forearm. It is important to maximize the tissue-engineered construct's (TEC's) biomechanical properties to ensure that the construct is in its strongest possible state before reimplantation. In this study, we sought to determine the bioreactor treatment parameters that affect these properties. Using small- and large-chamber three-dimensional-construct bioreactors (SCB and LCB, respectively), we applied cyclic axial load to TECs comprising reseeded human flexor and extensor tendons of the hand. First, small-sample pilot studies using the LCB were performed on matched-paired full-length flexor tendons to establish proof of concept. Next, large-sample studies using the SCB were performed on matched-paired extensor tendon segments to determine how reseeding, load duty cycle, load magnitude, conditioning duration, and testing delay affected ultimate tensile stress (UTS) and elastic modulus (EM). We found that compared with reseeded matched-paired controls under dynamic-loading at 1.25?N per TEC for 5 days, (1) acellular TECs had lower UTS (p=0.04) and EM (p<0.01), (2) unloaded TECs had lower UTS (p=0.01) and EM (p=0.02), (3) static-loaded TECs had lower UTS (p=0.01) and EM (p<0.01), (4) TECs conditioned for 3 days had lower UTS (p=0.03) and EM (p=0.04), and (5) TECs conditioned for 8 days had higher UTS (p=0.04) and EM (p=0.01). However, TECs conditioned at higher loads (2.5?N per TEC) and lower loads (0.625?N per TEC) possessed similar UTS (p=0.83 and p=0.89, respectively) and EM (p=0.48 and p=0.89, respectively) as controls stimulated with 1.25?N per TEC. After cycle completion, there is attrition of UTS (p=0.03) and EM (p=0.04) over a 2-day period. Our study showed that the material properties of human allograft TECs can be enhanced by reseeding and dynamic-conditioning. While conditioning duration has a significant effect on material properties, the load magnitude does not. The issue of attrition in biomechanical properties with time following cycle completion must be addressed before bioreactor preconditioning can be successfully introduced as a step in the processing of these constructs for clinical application.     

组织工程生物反应器的力学环境特点

组织工程学是一门以细胞生物学和工程学为基础,应用工程学和生命科学的原理,开发器官缺损患者所需代替物,并构建和保持或增强其组织功能性的一门交叉学科。生物反应器作为组织工程中非常重要的工具,目前主要是从生物力学问题、三维空间培养问题、传质问题、培养的环境条件问题(pH、氧张力等)和物理因素(电场、磁场、应力场)等方面开展其研究。本文作者主要从生物力学角度介绍组织工程生物反应器研究的最新进展,重点探讨组织工程生物反应器的力学环境。