Department of Bioengineering, Temple University, Philadelphia, PA, USA.
Analyst. 2017 Apr 10;142(8):1320-1332. doi: 10.1039/c6an02167k.
Articular cartilage degeneration causes pain and reduces the mobility of millions of people annually. Regeneration of cartilage is challenging, due in part to its avascular nature, and thus tissue engineering approaches for cartilage repair have been studied extensively. Current techniques to assess the composition and integrity of engineered tissues, including histology, biochemical evaluation, and mechanical testing, are destructive, which limits real-time monitoring of engineered cartilage tissue development in vitro and in vivo. Near infrared spectroscopy (NIRS) has been proposed as a non-destructive technique to characterize cartilage. In the current study, we describe a non-destructive NIRS approach for assessment of engineered cartilage during development, and demonstrate correlation of these data to gold standard mid infrared spectroscopic measurements, and to mechanical properties of constructs. Cartilage constructs were generated using bovine chondrocyte culture on polyglycolic acid (PGA) scaffolds for six weeks. BMP-4 growth factor and ultrasound mechanical stimulation were used to provide a greater dynamic range of tissue properties and outcome variables. NIR spectra were collected daily using an infrared fiber optic probe in diffuse reflectance mode. Constructs were harvested after three and six weeks of culture and evaluated by the correlative modalities of mid infrared (MIR) spectroscopy, histology, and mechanical testing (equilibrium and dynamic stiffness). We found that specific NIR spectral absorbances correlated with MIR measurements of chemical composition, including relative amount of PGA (R = 0.86, p = 0.02), collagen (R = 0.88, p = 0.03), and proteoglycan (R = 0.83, p = 0.01). In addition, NIR-derived water content correlated with MIR-derived proteoglycan content (R = 0.76, p = 0.04). Both equilibrium and dynamic mechanical properties generally improved with cartilage growth from three to six weeks. In addition, significant correlations between NIRS-derived parameters and mechanical properties were found for constructs that were not treated with ultrasound (PGA (R = 0.71, p = 0.01), water (R = 0.74, p = 0.02), collagen (R = 0.69, p = 0.04), and proteoglycan (R = 0.62, p = 0.05)). These results lay the groundwork for extension to arthroscopic engineered cartilage assessment in clinical studies.
关节软骨退变每年都会导致数百万人疼痛和活动能力下降。软骨的再生具有挑战性，部分原因是其无血管的特性，因此人们广泛研究了用于软骨修复的组织工程方法。目前评估工程组织的组成和完整性的技术，包括组织学、生化评估和机械测试，都是破坏性的，这限制了对体外和体内工程软骨组织发育的实时监测。近红外光谱（NIRS）已被提议作为一种非破坏性技术来表征软骨。在本研究中，我们描述了一种用于评估工程软骨发育的非破坏性 NIRS 方法，并证明了这些数据与金标准中红外光谱测量以及构建体的机械性能之间存在相关性。软骨构建体使用牛软骨细胞在聚乙醇酸（PGA）支架上培养六周生成。BMP-4 生长因子和超声机械刺激用于提供更大的组织特性和结果变量的动态范围。使用漫反射模式的红外光纤探头每天收集近红外光谱。培养三周和六周后收获构建体，并通过中红外（MIR）光谱、组织学和机械测试（平衡和动态刚度）的相关方式进行评估。我们发现，特定的近红外光谱吸收与化学组成的 MIR 测量相关，包括 PGA（R = 0.86，p = 0.02）、胶原（R = 0.88，p = 0.03）和糖胺聚糖（R = 0.83，p = 0.01）的相对含量。此外，近红外衍生的水含量与 MIR 衍生的糖胺聚糖含量相关（R = 0.76，p = 0.04）。从三周到六周，随着软骨生长，平衡和动态机械性能通常会提高。此外，对于未用超声处理的构建体，还发现了 NIRS 衍生参数与机械性能之间的显著相关性（PGA（R = 0.71，p = 0.01）、水（R = 0.74，p = 0.02）、胶原（R = 0.69，p = 0.04）和糖胺聚糖（R = 0.62，p = 0.05））。这些结果为将其扩展到临床研究中的关节内工程软骨评估奠定了基础。
