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首页> 外文期刊>BMC Biotechnology >Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis
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Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

机译：Perlecan域1重组蛋白聚糖增强BMP-2活性和成骨作用

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Background Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans ( GAGs ), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs , and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo . Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs , tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro . In vitro , the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs . Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse?). Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo , the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.
机译：背景技术许多生长因子（例如骨形态发生蛋白（BMP）-2）已显示与称为硫酸乙酰肝素（HS）糖胺聚糖（GAGs）的硫酸盐脱羧酶的聚合物相互作用，这种蛋白在整个过程中均存在于基质和细胞表面蛋白聚糖上身体。 HS GAG和一些硫酸化程度更高的硫酸软骨素（CS）通过在与受体的GF相互作用中充当辅因子或辅受体来调节细胞功能，并且已证明HS或CS GAG对于甚至在成骨谱系中也能诱导信号传导和GF活性。但是，与重组蛋白不同，HS和CS GAG的异质性很大，这在很大程度上是由于翻译后添加硫酸盐基团然后沿着GAG聚合物的各个位置去除了硫酸盐基团。因此，我们研究了递送DNA前药在原位产生可溶的HS / CS蛋白聚糖是否可行，该蛋白会增强体内包括BMP-2在内的生长因子的活性。结果利用纯化的重组人perlecan结构域1（rhPln.D1）在HEK 293细胞中表达了HS和CS GAGs，证实了rhBMP-2活性的紧密结合和剂量增强。在体外，表达的rhPln.D1的特征是用硫酸化HS和CS GAGs修饰。通过在质粒磷酸三钙支架上冻干的单相一起递送的pln.D1表达质粒增强rhBMP-2的剂量达6周或更长时间，在大鼠上颌上从头开始产生的骨量最多增加了9倍与没有pln.D1质粒的对照位点相比，该模型更为合理。使用显着较低的BMP-2剂量，这种组合提供的上颌骨增大量和密度比胶原海绵（InFuse？）上的rhBMP-2高出5倍以上。结论重组HS / CS PG在结合和细胞检测中与BMP-2发生了强烈的功能相互作用，并且在体内pln.247表达质粒显着改善了BMP-2成骨活性对体内de的剂量效应。当在支架上以单相形式一起递送时，将产生新骨。 HS / CS PG的使用可能有助于增加GF疗法，并且基于质粒的方法在此已显示出很高的效果。

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