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Effect of Long-Term Osmotic Loading Culture on Matrix Synthesis from Intervertebral Disc Cells

机译：长期渗透负荷培养对椎间盘细胞基质合成的影响

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The intervertebral disc is a highly hydrated tissue that acts to absorb and distribute large complex loads placed on the spine. Diurnal loading and disc degeneration causes significant changes in water volume and proteoglycan content, which alters the internal osmotic environment. Short-term osmotic loading alters disc cell gene expression; however, the long-term effect of osmotic loading on disc cell matrix synthesis is not well understood. The objective of this study was to determine the effect of long-term osmotic loading on matrix turnover and proliferation by juvenile and adult cells from the nucleus pulposus (NP) and the cartilaginous endplate (EP). Matrix synthesis was evaluated using pellets and a 3D agarose system, which has been used for developing engineered tissues. Intervertebral discs were acquired from juvenile and adult cows. Cells were acquired through enzymatic digestion and expanded in culture. Pellets were formed through centrifugation, and constructs were created by encapsulating cells within 2% w/v agarose hydrogel. Pellets and constructs were cultured up to 42 days in chemically defined medium with the osmolality adjusted to 300, 400, or 500 mOsm/kg. EP cells were evaluated as a chondrocyte comparison to chondrocyte-like NP cells. Pellet and agarose cultures of juvenile NP and EP cells demonstrated similarities with respect to cell proliferation and functional mechanical properties. Cell proliferation decreased significantly with increased osmotic loading. The final compressive Young's modulus of juvenile NP cells was 10–40× greater than initial properties (i.e., day 0) and was greater than the final Young's modulus of adult NP and juvenile EP constructs. In juvenile NP constructs, there were no significant differences in GAG content with respect to osmotic loading. However, GAG synthesis and mechanical properties were greatest for the 400 mOsm/kg group in adult NP constructs. Taken together, the results presented here suggest a tradeoff between cell proliferation and matrix production under osmotic loading conditions. In conclusion, culturing disc cells in an osmotic environment that best mimics the healthy disc environment (400 mOsm/kg) may be ideal for balancing cell proliferation, matrix production, and mechanical properties of engineered disc tissues.
机译：椎间盘是高度水化的组织，其作用是吸收和分布置于脊柱上的大的复杂负荷。昼夜负荷和椎间盘退变会引起水量和蛋白聚糖含量的显着变化，从而改变内部渗透环境。短期渗透负荷改变椎间盘细胞基因表达;然而，渗透负荷对盘状细胞基质合成的长期影响尚不十分清楚。这项研究的目的是确定长期渗透负荷对髓核（NP）和软骨终板（EP）的成年和成年细胞基质转化和增殖的影响。使用沉淀和3D琼脂糖系统评估了基质合成，该系统已用于开发工程组织。从幼年和成年母牛获得椎间盘。通过酶消化获得细胞，并在培养中扩增。通过离心形成药丸，并通过将细胞封装在2％w / v琼脂糖水凝胶中来创建构建体。将小丸和构建体在化学成分确定的培养基中培养长达42天，并将重量克分子渗透压调节至300、400或500 mOsm / kg。将EP细胞评价为与软骨细胞样NP细胞相比的软骨细胞。少年NP和EP细胞的沉淀和琼脂糖培养在细胞增殖和功能机械特性方面显示出相似性。随着渗透负荷的增加，细胞增殖显着降低。少年NP细胞的最终杨氏模量比初始特性（即第0天）大10–40倍，并且比成年NP和少年EP结构的最终杨氏模量大。在少年NP结构中，相对于渗透负荷，GAG含量没有显着差异。但是，对于成人NP构建体中的400 mOsm / kg组，GAG合成和机械性能最大。两者合计，这里呈现的结果表明在渗透负荷条件下细胞增殖与基质产生之间的权衡。总之，在最能模拟健康椎间盘环境（400 mOsm / kg）的渗透环境中培养椎间盘细胞可能是平衡细胞增殖，基质产生和工程椎间盘组织机械特性的理想选择。

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