Molecular Heparan Sulfate Delivery in Guided Tissue Regeneration

描述（由申请人提供）：蛋白质聚糖和碳水化合物聚合物（如硫酸肝素）在生物过程中的作用正在得到更好的理解，但这一新的前沿领域才刚刚开始探索。组织再生也是一个新兴的前沿领域。这项研究一旦完成，将对蛋白多糖和组织再生领域做出重要贡献，对牙周再生具有特定的临床影响。Perlecan，最初被命名为硫酸肝素蛋白聚糖-2，现在已知是所有基底膜的重要组成部分（与胶原型IV和层粘连蛋白一起）。硫酸肝素修饰的Perlecan在血管生成或新生血管中的作用有强有力的科学支持和理论依据。据报道，Perlecan的硫酸肝素可促进细胞粘附，促进增殖和/或分化，并结合和传递生长因子。硫酸肝素修饰重组Perlecan D1 （rD1）在多种细胞类型中的表达已被证实。体外实验表明，rD1与成纤维细胞生长因子结合，促进细胞粘附。在牙周再生，屏障膜是一个重要的辅助程序，旨在恢复口腔的形式和功能。在这些引导组织再生（GTR）过程中，屏障膜位于手术皮瓣和下面的再生部位之间。然而，GTR手术中存在不必要的高失败率，这是由于皮瓣在膜和再生部位的初级闭合时愈合失败，通常导致再生部位暴露于口腔环境中。该I期申请的目的是：1)确定在临床可接受的参数范围内，用Perlecan结构域1表达构建体加载常用屏障膜的有效方法；2)比较Perlecan D1转基因传递系统负载的膜与假负载的膜产生的Perlecan结构域1构建体的体外细胞表达和细胞反应。3)在动物模型中比较加载与假加载手术膜放置产生的体内反应，以实现2¿intention愈合。膜将通过体外建立的参数进行预处理，然后在牙龈皮瓣下固定膜暴露的确定区域。愈合将在临床和组织学上进行测量，其中上皮的暴露膜覆盖率将是主要结果。硫酸肝素和Perlecan D1递送以支持组织再生的充分支持原理，加上这些初步数据和Agenta的相关专利，使Agenta能够独特地实现该拟议项目的目标。

DESCRIPTION (provided by applicant): The role of proteoglycans and carbohydrate polymers such as heparan sulfate in biological processes are becoming better understood, but this new frontier has only begun to be explored. Tissue regeneration is also an emerging frontier. This proposed research, when completed, will make an important contribution to both the proteoglycan and tissue regenerative fields, with a specific, clinical impact on periodontal regeneration. Perlecan, originally named heparan sulfate proteoglycan-2, is now known to be an important component of all basement membranes (along with collagen type IV and laminin). There is strong scientific support and rationale for a role of heparan sulfate-decorated Perlecan in angiogenesis or neovascularization. The heparan sulfate of Perlecan is reported to promote cell adhesion, to promote proliferation and/or differentiation, and to bind and deliver growth factors. Expression of a heparan sulfate-decorated recombinant Perlecan D1 (rD1) in a variety of cell types has since been validated. The rD1 was shown to bind fibroblast growth factor and promoted cell adherence in vitro. In periodontal regeneration, barrier membranes are an important adjunct to procedures aimed at restoring the form and function of the mouth. During these guided tissue regenerative (GTR) procedures, barrier membranes are positioned between the surgical flap and the underlying regenerative site. Nevertheless, an unnecessarily high failure rate exists in GTR procedures due to failure of the flaps to heal with primary closure over the membranes and regenerative site, typically leading to exposure of the regenerative site to the oral environment. The objectives of this Phase I application are 1) to determine an effective manner of loading commonly used barrier membranes with Perlecan domain 1 expression constructs within clinically acceptable parameters, 2) to compare in vitro cellular expression of, and cellular responses to, the Perlecan domain 1 construct generated by membranes loaded with the Perlecan D1 transgene delivery system vs. the sham-loaded membranes, and 3) to compare in vivo responses generated by placement of the loaded vs. the sham-loaded surgical membranes in an animal model for healing by 2¿ intention. Membranes will be pre-treated by parameters established in vitro then secured beneath a gingival flap with a defined area of membrane exposure. Healing will be measured clinically and histologically where rate of exposed membrane coverage by epithelium will be the primary outcome. The well-supported rationale for heparan sulfate and Perlecan D1 delivery to support tissue regeneration, coupled with these preliminary data and Agenta's relevant patent, uniquely positions Agenta to fulfill the objectives of this proposed project.