Magnetic Resonance Studies Of Cells, Organs And Animals

用于软骨细胞和组织磁共振显微成像和光谱分析的生物反应器系统 继发于创伤性损伤或退行性关节疾病的关节软骨修复是一个重要的治疗挑战。尽管在理解这种高度流行疾病的发病机制方面取得了重大进展，但还没有得到广泛接受的疾病修正干预措施。开发一种灵活可靠的与MRI兼容的软骨中空纤维生物反应器(HFBR)系统用于新生软骨的生长，有可能为治疗方法做出贡献。首先，可以在这样的系统中深入研究促进细胞发育成高质量软骨的条件，该系统可以完全控制发育中的新生软骨对生长因子、底物成分、溶解的O2和CO2浓度、温度和其他环境因素的暴露。虽然在生物体中从细胞原位发育软骨，包括软骨细胞和潜在的骨髓基质细胞，将在重要方面与生物反应器的条件不同，但体外研究将能够为从细胞发育有功能的新软骨指出合适的条件。其次，在生物反应器中生长高质量的软骨可能会产生用于实际移植的组织来源。最后，也是最普遍的，不管最终软骨修复和再生过程的细节如何，监测组织质量的能力显然是重要的。虽然关节镜活检提供了这样的数据，允许评估组织的生化和组织状态，但使用非侵入性评估方法显然更可取。MRI作为一种无创性测量软骨厚度、体积和局部病理的工具越来越被人们所接受，而MRI非侵入性评估软骨质量的能力目前是一个活跃的研究课题。在一个允许详细的MRI评估的系统中，一个高度可控的系统用于产生具有广泛不同性质的软骨，这将代表着这一努力的明显进步。最后，我们注意到，与MRI兼容的生物反应器为当前和未来的治疗剂和干预措施提供了一个灵活的试验台。总而言之，作为一种细胞系统，HFBR与其他3D培养系统一样，具有支持透明软骨类型的能力。因此，可以评估生长条件和治疗方法对透明软骨组织的影响，而不是纤维软骨。作为一个组织系统，HFBR允许真正的宏观生长。因此，细胞-基质相互作用和基质屏障对底物输送和代谢产物外流的影响比单层系统更真实地表达出来。最后，作为生长条件和试剂的试验台，HFBR提供了对基质和灌流条件的完全控制。 我们已经成功地证明了从HFBR中的鸡胸骨细胞培养出的软骨将发展并保持透明的表型；MRI的形态测量与组织学相关；MRI对局部T1、T2、扩散和MT的测量与胶原、蛋白多糖和水合的生化分析相关。因此，无创性MRI测量提供了有关软骨基质成分的可靠信息。我们进一步证明，可以通过引入生物活性化合物来改变HFBR中的软骨生长，并且尽管这些干预产生了更大的组织特征动态范围，但MRI衍生参数和上述生化结果之间的相关性仍然保持不变。我们还利用pH、无机磷(PI)和三磷酸腺苷(ATP)的31P核磁共振测量来证明生物反应器中发育的软骨在典型的4周生长期内保持代谢稳定。此外，我们工作的一个主要焦点是证明MRI测量的基质固定密度与动态和平衡压缩模数的测量之间存在相关性。MRI获得的FCD值与S-GAG含量相关，但与胶原含量无关。这些相关性被发现，即使在软骨素酶的存在下经历了发育的组织中也是如此，软骨素酶作为基质蛋白多糖的分解代谢剂。因此，在对照组织和经历了类似于骨关节炎的退变的组织中，FCD的无创性MRI评估已经被证明在HFBR的动态条件下提供了关于软骨基质组成的可靠信息。

A Bioreactor System for Magnetic Resonance Microimaging and Spectroscopy of Chondrocytes and Tissue Repair of articular cartilage secondary to either traumatic injury or degenerative joint disease represents an important therapeutic challenge. In spite of significant progress in understanding the pathogenesis of this highly prevalent disease, there are no well-accepted disease-modifying interventions. The development of a flexible and reliable MRI-compatible cartilage hollow fiber bioreactor (HFBR) system for neocartilage growth has the potential to contribute to therapeutic approaches. First, conditions promoting the development of high-quality cartilage from cells can be studied intensively in such a system, which provides full control over exposure of the developing neocartilage to growth factors, substrate composition, dissolved O2 and CO2 concentrations, temperature, and other environmental factors. While in situ development of cartilage from cells, including both chondrocytes and, potentially, bone marrow stromal cells, in an organism will differ in important ways from the bioreactor conditions, in vitro studies will be able to point the way to appropriate conditions for development of functioning neocartilage from cells. Second, growth of high-quality cartilage in the bioreactor may result in a source of tissue for actual transplantation. Finally, and most generally, regardless of the specifics of eventual cartilage repair and regeneration procedures, the ability to monitor tissue quality will be of clear importance. While arthroscopic biopsies provide such data, permitting assessment of the biochemical and histologic state of the tissue, it is clearly more desirable to utilize noninvasive assessment methods. MRI is becoming increasingly accepted as a noninvasive tool for the measurement of cartilage thickness and volume and of localized pathology while the ability of MRI to noninvasively assess cartilage quality is currently a topic of active research. The availability of a highly controllable system for generating cartilage with widely varying properties in a system permitting detailed MRI assessment would represent a clear advance in this effort. Finally, we note that the MRI-compatible bioreactor provides a flexible test-bed for current and future therapeutic agents and interventions. In summary, as a cellular system, the HFBR shares with other 3D culture systems the ability to support the hyaline cartilage type. Thus, one can evaluate the effect of growth conditions and therapeutics on hyaline cartilage tissue rather than fibrocartilage. As a tissue system, the HFBR permits true macroscopic growth. Thus, cell-matrix interactions and the effects of the matrix barrier to substrate delivery and metabolic product efflux are represented much more realistically than in monolayer systems. Finally, as a test bed for growth conditions and agents, the HFBR provides full control of substrate and perfusion conditions. We have successfully demonstrated that cartilage grown from chick sternal cells in the HFBR will develop and maintain the hyaline phenotype; that morphologic measurements with MRI correlate with tissue histology; and that MRI measurements of local T1, T2, diffusion and MT correlate with biochemical assays of collagen, proteoglycans and hydration. Thus, noninvasive MRI measures provide reliable information about cartilage matrix composition. We have further demonstrated that cartilage growth in the HFBR can be modified by introduction of biologically active compounds, and that the correlations between MRI-derived parameters and biochemical results noted above are maintained in spite of the greater dynamic range of tissue characteristics resulting from these interventions. We have also utilized 31P NMR measurements of pH, inorganic phosphate (Pi) and ATP to demonstrate that the developing cartilage in the bioreactor remains metabolically stable over the typical 4 week growth period. A major focus of our work has, in addition, been to demonstrate that MRI measurements of matrix fixed density correlate with measurements of dynamic and equilibrium compressive moduli. The MRI-derived FCD values correlate with S-GAG content but not with collagen content. These correlations were found to persist even in tissue which has undergone development in the presence of chondroitinase, acting as a catabolic agent on matrix proteoglycans. Noninvasive MRI evaluation of FCD therefore has been shown to provide reliable information about cartilage matrix composition under the dynamic conditions of the HFBR in both control tissue and in tissue which has undergone degeneration analogous to that seen in osteoarthritis.