McAlinden Audrey, Hudson David M, Fernandes Aysel A, Ravindran Soumya, Fernandes Russell J
Department of Orthopaedic Surgery, Washington University School of Medicine, St Louis, MO, USA.
Department of Cell Biology & Physiology, Washington University School of Medicine, St Louis, MO, USA.
Matrix Biol Plus. 2021 Jul 5;12:100077. doi: 10.1016/j.mbplus.2021.100077. eCollection 2021 Dec.
For next generation tissue-engineered constructs and regenerative medicine to succeed clinically, the basic biology and extracellular matrix composition of tissues that these repair techniques seek to restore have to be fully determined. Using the latest reagents coupled with tried and tested methodologies, we continue to uncover previously undetected structural proteins in mature intervertebral disc. In this study we show that the "embryonic" type IIA procollagen isoform (containing a cysteine-rich amino propeptide) was biochemically detectable in the annulus fibrosus of both calf and mature steer caudal intervertebral discs, but not in the nucleus pulposus where the type IIB isoform was predominantly localized. Specifically, the triple-helical type IIA procollagen isoform immunolocalized in the outer margins of the inner annulus fibrosus. Triple helical processed type II collagen exclusively localized within the inter-lamellae regions and with type IIA procollagen in the intra-lamellae regions. Mass spectrometry of the α1(II) collagen chains from the region where type IIA procollagen localized showed high 3-hydroxylation of Proline-944, a post-translational modification that is correlated with thin collagen fibrils as in the nucleus pulposus. The findings implicate small diameter fibrils of type IIA procollagen in select regions of the annulus fibrosus where it likely contributes to the organization of collagen bundles and structural properties within the type I-type II collagen transition zone.
为了使下一代组织工程构建体和再生医学在临床上取得成功，必须全面确定这些修复技术试图恢复的组织的基本生物学特性和细胞外基质组成。使用最新的试剂并结合经过验证的方法，我们不断在成熟椎间盘内发现以前未检测到的结构蛋白。在本研究中，我们发现“胚胎型”IIA原胶原异构体（含有富含半胱氨酸的氨基前肽）在小牛和成熟公牛尾椎间盘的纤维环中均可通过生化方法检测到，但在主要定位IIB异构体的髓核中未检测到。具体而言，三螺旋IIA原胶原异构体免疫定位在内层纤维环的外缘。三螺旋加工后的II型胶原仅定位于板层间区域，而IIA原胶原定位于板层内区域。对IIA原胶原定位区域的α1(II)胶原链进行质谱分析，结果显示脯氨酸-944具有较高的3-羟基化水平，这种翻译后修饰与髓核中细胶原纤维相关。这些发现表明，IIA原胶原的小直径纤维存在于纤维环的特定区域，可能有助于I型- II型胶原过渡区内胶原束的组织和结构特性。
