Molecular Heparan Sulfate Delivery in Guided Tissue Regeneration

描述（由申请人提供）：蛋白聚糖和碳水化合物聚合物（如硫酸乙酰肝素）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领域。这项拟议中的研究完成后，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。串珠素，最初命名为硫酸乙酰肝素蛋白聚糖-2，现在已知是所有基底膜的重要组分（沿着IV型胶原蛋白和层粘连蛋白）。硫酸乙酰肝素修饰的串珠素在血管生成或新血管形成中的作用有强有力的科学支持和理论基础。据报道，串珠素的硫酸乙酰肝素促进细胞粘附，促进增殖和/或分化，并结合和递送生长因子。硫酸乙酰肝素修饰的重组串珠素D1（rD 1）在多种细胞类型中的表达已经得到验证。rD 1被证明可以结合成纤维细胞生长因子，并在体外促进细胞粘附。在牙周再生中，屏障膜是旨在恢复口腔形式和功能的程序的重要辅助手段。在这些引导组织再生（GTR）手术期间，屏障膜位于手术皮瓣和下方再生部位之间。然而，在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上的初次闭合愈合失败，通常导致再生部位暴露于口腔环境。该I期申请的目的是1）确定在临床上可接受的参数内用串珠素结构域1表达构建体加载常用屏障膜的有效方式，2）比较由加载有串珠素D1转基因递送系统的膜与假加载的膜产生的串珠素结构域1构建体的体外细胞表达和细胞应答，和3）比较在动物模型中放置负载的与假负载的手术膜以通过2 μ意图愈合所产生的体内反应。将通过体外建立的参数对膜进行预处理，然后将其固定在牙龈瓣下，并确定膜暴露区域。愈合将在临床和组织学上进行测量，其中上皮细胞对暴露膜的覆盖率将是主要结局。硫酸乙酰肝素和珍珠层聚糖D1输送支持组织再生的充分支持的理由，加上这些初步数据和Alfreta的相关专利，使Alfreta能够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.