Yazici Cemal, Yanoso Laura, Xie Chao, Reynolds David G, Samulski R Jude, Samulski Jade, Yannariello-Brown Judith, Gertzman Arthur A, Zhang Xinping, Awad Hani A, Schwarz Edward M
The Center for Musculoskeletal Research, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Biomaterials. 2008 Oct;29(28):3882-7. doi: 10.1016/j.biomaterials.2008.06.007. Epub 2008 Jun 30.
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 10(7), 10(9) or 10(10) particles 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 10(10) rAAV-Luc particles achieved peak gene expression levels on day 3, which persisted until day 21, and was significantly greater than the 10(7) dose throughout this time period (p<0.01). A direct comparison of mineralized versus DBW coated with 10(10) rAAV-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.
冻干重组腺相关病毒（rAAV）包被的结构性同种异体骨已成为一种利用宿主因子促进坏死皮质骨生长的方法，这些宿主因子在手术后数周内持续存在，以促进血管再生、骨整合和重塑。然而，一个主要限制是无孔的皮质表面，这使得在冻干前rAAV包被无法均匀分布。为了克服这一问题，我们开发了一种脱矿质方法，以增加表面吸收率，同时保持同种异体骨的结构完整性。由人股骨干同种异体骨环制成的脱矿质骨片（DBW）与矿化对照相比，表面积包被百分比显著增加了21.1%（73.6±3.9%对52.5±2.6%；p<0.001）。在培养中，将rAAV-荧光素酶（rAAV-Luc）包被的DBW与单层C3H10T1/2细胞共孵育，导致荧光素酶水平峰值与可溶性rAAV-Luc对照无显著差异（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，我们首先对含有10^7、10^9或10^10个颗粒的同种异体骨进行剂量反应实验，将其手术植入小鼠股四头肌，并在第1、3、5、7、10、14和21天通过体内生物发光成像（BLI）进行检测。结果显示出剂量反应，其中包被10^10个rAAV-Luc颗粒的DBW在第3天达到基因表达峰值水平，并持续到第21天，且在整个时间段内显著高于10^7剂量（p<0.01）。对矿化骨与包被10^10个rAAV-Luc颗粒的DBW进行直接比较，未发现体内转导动力学或效率有任何显著差异。因此，人皮质骨同种异体骨的表面脱矿质增加了其对均匀rAAV包被的吸收率，而不影响载体转导效率。
