The effect of surface demineralization of cortical bone allograft on the properties of recombinant adeno-associated virus coatings.
The effect of surface demineralization of cortical bone allograft on the properties of recombinant adeno-associated virus coatings.
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皮质骨同种异体移植物表面脱矿对重组腺相关病毒涂层特性的影响。
DOI：
10.1016/j.biomaterials.2008.06.007
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发表时间：
2008
期刊：
影响因子：
14
通讯作者：
Schwarz,EdwardM
中科院分区：
文献类型：
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作者：
Yazici,Cemal;Yanoso,Laura;Xie,Chao;Reynolds,DavidG;Samulski,RJude;Samulski,Jade;Yannariello-Brown,Judith;Gertzman,ArthurA;Zhang,Xinping;Awad,HaniA;Schwarz,EdwardM
冻干重组腺相关病毒（rAAV）包被的结构性同种异体移植物已成为一种产生坏死皮质骨的方法，其宿主因子将在手术后持续数周，以促进血运重建、骨整合和重塑。然而，一个主要的限制是无孔皮质表面，其在冷冻干燥之前阻止rAAV涂层的均匀分布。为了克服这一点，我们开发了一种去矿化方法，以增加表面吸收，同时保持同种异体移植物的结构完整性。与矿化对照相比，由人股骨同种异体移植物环制成的脱矿骨晶片（DBW）的表面积涂层百分比显著增加21.1%（73.6±3.9% vs 52.5±2.6%; p<0.001）。将rAAV-荧光素酶（rAAV-Luc）包被的DBW与单层C3 H10 T12细胞在培养物中共孵育导致荧光素酶水平峰值，其与可溶性rAAV-Luc对照没有显著差异（p>0.05），尽管峰值分别出现在60小时和12小时。为了评估rAAV-Luc包被的DBW在体内的转导效率，我们首先用含有107、109或1010个颗粒的同种异体移植物进行剂量响应，所述同种异体移植物通过手术植入小鼠的四头肌中，并在第1、3、5、7、10、14和21天通过体内生物发光成像（BLI）进行测定。结果证明了剂量反应，其中用1010 rAAV-Luc颗粒包被的DBW在第3天达到峰值基因表达水平，其持续到第21天，并且在整个这段时间内显著大于107剂量（p<0.01）。矿化与用1010 rAAV-Luc颗粒包被的DBW的直接比较未能证明体内转导动力学或效率的任何显著差异。因此，人皮质骨同种异体移植物的表面脱矿增加了其对均匀rAAV涂层的吸收，而不影响载体转导效率。
Freeze-dried recombinant adeno-associated virus (rAAV) coated structural allografts have emerged as an approach to engender necrotic cortical bone with host factors that will persist for weeks following surgery to facilitate revascularization, osteointegration, and remodeling. However, one major limitation is the nonporous cortical surface that prohibits uniform distribution of the rAAV coating prior to freeze-drying. To overcome this we have developed a demineralization method to increase surface absorbance while retaining the structural integrity of the allograft. Demineralized bone wafers (DBW) made from human femoral allograft rings demonstrated a significant 21.1% (73.6±3.9% versus 52.5±2.6%; p<0.001) increase in percent surface area coating versus mineralized controls. Co-incubation of rAAV-luciferase (rAAV-Luc) coated DBW with a monolayer of C3H10T1/2 cells in culture led to peak luciferase levels that were not significantly different from soluble rAAV-Luc controls (p>0.05), although the peaks occurred at 60h and 12h, respectively. To assess the transduction efficiency of rAAV-Luc coated DBW in vivo, we first performed a dose response with allografts containing 107, 109or 1010particles that were surgically implanted into the quadriceps of mice, and assayed by in vivo bioluminescence imaging (BLI) on days 1, 3, 5, 7, 10, 14, and 21. The results demonstrated a dose response in which the DBW coated with 1010rAAV-Luc particles achieved peak gene expression levels on day 3, which persisted until day 21, and was significantly greater than the 107dose throughout this time period (p<0.01). A direct comparison of mineralized versus DBW coated with 1010rAAV-Luc particles failed to demonstrate any significant differences in transduction kinetics or efficiency in vivo. Thus, surface demineralization of human cortical bone allograft increases its absorbance for uniform rAAV coating, without affecting vector transduction efficiency.