Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation
Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation
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DOI：
10.1089/biores.2014.0057
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发表时间：
2015-07-01
影响因子：
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通讯作者：
Asahina, Izumi
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文献类型：
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作者：
Umebayashi, Mayumi;Sumita, Yoshinori;Asahina, Izumi
到目前为止，体内基因传递的治疗方法还没有建立在骨工程学上，尽管它的潜在用途已经被提出。在临床应用中，需要开发一种有效的条件，在没有任何转染剂或病毒载体的情况下在体内转移基因。在本研究中，为了便于这一策略的临床应用，特别是针对萎缩的骨修复，我们简单地研究了用含有一定量编码成骨蛋白的质粒(P)DNA的胶原蛋白制成的基因激活基质(GAM)是否能够增强大鼠的颅骨。将携带BMP4(PBMP4)或Runx2(PRunx2)基因的AcGFP载体0.02、0.1或1 mg分别与2%的牛胶原蛋白和β-磷酸三钙颗粒混合制成GAM。在制造GAMs之前，为了确定所产生的pDNA的生物学活性，我们证实了在培养过程中，转导pBMP4或pRunx2的MC3T3-E1细胞表达了绿色荧光蛋白，并提高了碱性磷酸酶的活性。然后，GAMs被冷冻干燥并移植到颅骨上的嵌套式放置。移植后2周，仅在含有1 mg AcGFP载体的GAM中可检测到表达GFP的细胞。然后，在4周时，在含有1毫克编码BMP4或Runx2的pDNA的GAMs中发现了显著的骨形成，而在0.02或0.1 mg的GAMS中没有发现显著的骨形成。这些新形成的骨组织周围有骨钙素染色的区域，直到移植后8周，骨组织明显增多。相反，在不含成骨蛋白基因的GAMs中观察到最少的骨形成。同时，将GAMs移植到颅骨缺损处，在8周时，含有1 mg pBMP4或pRunx2的标本也能检测到骨形成。因此，当含有一定数量的编码成骨蛋白的质粒载体时，即使用于垂直强化，基于胶原的GAM也可以可靠地形成工程化骨。本研究为GAM在骨工程中的临床应用奠定了基础。
To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, we simply investigated whether manufactured gene-activated matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, or 1 mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and beta-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBMP4 or pRunx2 at 8 weeks as well. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic proteins. This study supports facilitating the clinical application of GAM for bone engineering.