Guided tissue regeneration with immobilized cytokine

骨形态发生蛋白2（BMP-2）在骨的生长和再生中起着重要的作用。纯化的重组人BMP-2（rhBMP-2）的产生导致其临床应用潜力的进步，然而，这种蛋白质是可溶的，植入后很快就会分散。因此rhBMP-2不能对BMP信号通路产生长时间的刺激作用。为了克服这个问题，我们报道了rhBMP-2与琥珀酰化I型去端胶原的固定化作用。固定化rhBMP-2与细胞表面的受体结合而不被内化到细胞内，因此能够长时间地将信号传递到细胞内，实现高效的促进细胞增殖。我们通过（a）组织学观察，（B）碱性磷酸酶（ALP）活性，（c）BMP细胞内信号传导级联结果表明：（1）未固定化和固定化rhBMP-2均能提高ST 2细胞的碱性磷酸酶（ALP）活性，MC 3 T3和C2 C12细胞。然而，通过固定化rhBMP-2处理观察到显著更高的ALP活性。(2)未固定化rhBMP-2刺激后Smad 1/5/8的磷酸化水平从刺激后15 min开始升高，6 h后下降至对照组水平，而固定化rhBMP-2可延长Smad 1/5/8的磷酸化水平，48 h后仍能检测到其作用。因此，由于固定化rhBMP-2可增强和延长rhBMP-2的作用，固定化rhBMP-2是提高骨诱导的有效方法。

Bone morphogenetic protein 2(BMP-2) plays an important role in bone growth and regeneration. The creation of purified recombinant human BMP-2 (rhBMP-2) has led to advances in its potential for clinical use, however this protein is soluble and disperses soon after implantation. So rhBMP-2 can't have long time stimulation to BMP signaling cascade. To overcome this problem we reported the effects of immobilization of rhBMP-2 to succinylated type I atelocollagenThe mechanism of immobilized rhBMP-2 is based on the theories of covalent bonding between rhBMP-2 and succinylated type I atelocollagen. The immobilized rhBMP-2 binds with the receptor on the cell surface without being internalized into the cell, so it is able to transduce signals into the cell over a long period of time, achieving high efficiency in promoting cell proliferation.We investigated the effect of immobilized rhBMP-2 by the way of (a) histological findings, (b) alkaline phosphatase (ALP) activity, (c) BMP intracellular signaling cascade (Smad1, Smad5 and phospho Smad1/5/8) expression, and (d) type I collagen, osteonectin, osteopontin, ostocalcin, and Smad1/5/8 mRNA expression.Our results showed that (1) nonimmobilized and immobilized rhBMP-2 also increased the alkaline phosphatase (ALP) activity in ST2, MC3T3 and C2C12 cells. However significantly higher ALP activity was observed by the immobilized rhBMP-2 treatment. (2) Phosphorylation of Smad1/5/8 after nonimmobilized rhBMP-2 stimulation increased from 15 min after stimulation and decrease to the control revel after 6hrs, however the phosphorylation was prolonged by immobilized rhBMP-2 and its effect was detectable even after 48hrs.So we conclude that because immobilized rhBMP-2 can emphasize and prolonged the effect of rhBMP-2, immobilizing rhBMP-2 is an efficient method to increase bone induction.