Extracorporeal Shock Wave-Stimulated Periosteum for Bone Reconstruction

描述(由申请人提供)： 本研究有四个具体目标。具体目的1是通过进行剂量-反应实验，确定体外冲击波(ESWs)对同一动物的胫骨和无牙下颌骨骨膜中成骨细胞增殖的影响。将采用三种能量密度(0、0.15和0.45MJ/mm2；每种能量密度一次性施加3000次冲击)，并在2、4和8天后比较结果。结果变量将从免疫组织化学的组织形态计量学和成骨细胞培养实验中选择细胞计数和骨膜厚度测量。我们的目标是找到在骨形成之前产生最多成骨细胞的治疗剂量(0.15mJ/mm2比0.45mJ/mm2)和治疗后时间。其目标是能够提升或收获正在增殖的细胞形成层；骨小梁的存在将干扰细胞的收获。在这个方案中，我们最初的目标是获取成骨细胞；未来的研究可能指向获取在ESW刺激的骨膜中形成的松质骨。第二个特定目标是确定ESWs对相同的胫骨和下颌骨样本骨髓中成骨细胞的影响。我们已经在先前的工作中发现，ESWs会引起骨髓的变化。病理学家告诉我们，这些变化与预期可逆的变化是一致的，并且没有不良的临床后果。我们想要确定体外冲击波刺激的骨髓中是否含有更多比例的间充质干细胞(MSCs，即骨髓基质细胞或骨祖细胞)。我们将通过在细胞培养实验中分析骨髓的成骨能力来确定这一点。如果我们发现，事实上，ESWs确实刺激了骨髓中的成骨细胞活动，这将在未来的研究中进行研究。这一发现可能对开发一种简单、非侵入性的程序来增强骨重建程序的替代自体细胞来源(即骨髓)具有价值。具体目的#3是确定ESW刺激的骨膜对下颌骨缺损的多孔磷酸钙(CAP)块中骨再生的影响。在特定目标#1中产生最多成骨细胞的ESW剂量将应用于山羊无牙前磨牙区的骨膜。在ESW术后规定的时间后，骨膜将被抬高，一个包含2个螺纹钛种植体的多孔帽块将被放置到手术准备的部位。然后将升高的骨膜应用于块的表面。在另一组动物中，电波冲击波刺激的胫骨骨膜将作为游离的自体移植物应用于帽块。在特定目标#4中，重组人(Rh)PDGF-BB将被整合到帽块中，然后将其植入到缺损处并覆盖ESW刺激的骨膜，如特定目标#3中那样。

DESCRIPTION (provided by applicant): There are 4 specific aims for this study. Specific Aim #1 is to determine the effects of extracorporeal shock waves (ESWs) on the proliferation of osteogenic cells in the periosteum of the tibia and edentulous mandible of the same animals, by conducting a dose-response experiment. Three energy densities will be employed (0, 0.15, and 0.45 mJ/mm2; a one-time application of 3,000 shocks for each energy density), and the results compared after 2, 4, and 8 days. The outcome variables will be selected cell counts and periosteum thickness measurement from histomorphometry employing immunohistochemistry, and osteogenic cell culture assay. Our objective is to find the dose (0.15 vs 0.45 mJ/mm2) and time post-treatment that yields the greatest number of osteogenic cells prior to the formation of bone. The goal is to be able to elevate or harvest the proliferating cambium layer of cells; the presence of bony trabeculae would interfere with the harvest of the cells. Our initial goal, in this proposal, is to harvest the osteogenic cells; future studies may be directed toward harvesting the cancellous bone that forms in the ESW-stimulated periosteum. Specific Aim #2 is to determine the effects of ESWs on the osteogenic cells in the marrow of the same tibial and mandibular samples employed in Specific Aim #1. We have already found in prior work that ESWs induce changes in the marrow. We have been informed by a pathologist that the changes are consistent with those that would be expected to be reversible, and of no adverse clinical consequence. We would like to determine if the ESW-stimulated marrow contains a greater percentage of mesenchymal stem cells (MSCs; i.e., marrow stromal cells, or osteoprogenitor cells). We will make this determination by assaying the osteogenic potential of the marrow in the cell culture assay. If we find that ESWs do, in fact, stimulate osteogenic cellular activity in the marrow, this will be investigated in future studies. The findings could be of value in developing a simple, non-invasive procedure for enhancing an alternative autologous cell source (i.e., marrow) for bone reconstruction procedures. Specific Aim #3 is to determine the effects of ESW-stimulated periosteum on bone regeneration in porous calcium phosphate (CaP) blocks in defects in the mandible. The ESW dose that yields the greatest number of osteogenic cells in Specific Aim #1 will be applied to the periosteum of the edentulous premolar region of goats. After the prescribed period post-ESW, the periosteum will be elevated and a porous CaP block containing 2 threaded titanium implants placed into the surgically prepared site. The elevated periosteum will then be applied to the surface of the block. In another group of animals the ESW-stimulated periosteum of the tibia will be harvested as a free autologous graft to be applied to the CaP block. In Specific Aim #4, recombinant human (rh) PDGF-BB will be incorporated into the CaP block before it is implanted into the defect and covered with the ESW-stimulated periosteum as in Specific Aim #3.