Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan, ROC.
Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.
J Chin Med Assoc. 2018 Aug;81(8):699-707. doi: 10.1016/j.jcma.2017.12.004. Epub 2018 Mar 15.
Gene-activated matrix (GAM) induces sustained local production of growth factors to promote tissue regeneration. GAM contains a plasmid DNA (pDNA) encoding target proteins that is physically entrapped within a biodegradable matrix carrier. GAM with a pDNA encoding the first 34 amino acids of parathyroid hormone (PTH 1-34) and a collagen matrix enhances bone regeneration in long bone defects. Demineralized freeze-dried bone allograft (DFDBA) is a widely used osteoinductive bone graft. The present study determined the osteogenic effects of PTH-GAM with a collagen or DFDBA/collagen composite (D/C) matrix for treating craniofacial bone defects.
We constructed a pDNA encoding human PTH 1-34 and performed cyclic AMP ELISA to verify the bioactivity of PTH 1-34. Next, we generated a D/C matrix and PTH-GAMs containing a collagen matrix (PTH-C-GAM) or D/C matrix (PTH-D/C-GAM). Rats with critical-sized calvarial bone defects were divided into four groups, namely, untreated rats (sham group) and rats grafted with D/C matrix, PTH-C-GAM, or PTH-D/C-GAM (D/C, PTH-C-GAM, or PTH-D/C-GAM groups, respectively). PTH expression was determined by performing immunohistochemical staining after 4 and 8 weeks. New bone formation was evaluated by performing radiography, dual-energy X-ray absorptiometry, microcomputed tomography, and histological examination.
PTH pDNA-transfected cells secreted bioactive PTH 1-34. Moreover, PTH was expressed at 4 and 8 weeks after the surgery in rats in the PTH-C-GAM group but not in rats in the D/C group. New bone formation in the calvarial bone defects, from more to less, was in the order of PTH-D/C-GAM, PTH-C-GAM, D/C, and sham groups.
Our results indicate that PTH-GAM with a collagen matrix promotes local PTH production for at least 8 weeks and bone regeneration in craniofacial bone defect. Moreover, our results indicate that replacement of the collagen matrix with the D/C matrix improves the osteogenic effects of PTH-GAM.
基因激活基质（GAM）诱导生长因子的持续局部产生，以促进组织再生。GAM 包含一个质粒 DNA（pDNA），该质粒 DNA 编码目标蛋白，这些目标蛋白被物理包埋在可生物降解的基质载体中。含有编码甲状旁腺激素（PTH 1-34）的前 34 个氨基酸的 pDNA 和胶原基质的 GAM 可增强长骨缺损中的骨再生。脱矿冻干骨同种异体移植物（DFDBA）是一种广泛使用的成骨诱导骨移植物。本研究确定了含有胶原或 DFDBA/胶原复合（D/C）基质的 PTH-GAM 治疗颅面骨缺损的成骨效果。
我们构建了一个编码人 PTH 1-34 的 pDNA，并进行环磷酸腺苷 ELISA 验证 PTH 1-34 的生物活性。接下来，我们生成了 D/C 基质和含有胶原基质的 PTH-GAM（PTH-C-GAM）或 D/C 基质（PTH-D/C-GAM）。将具有临界尺寸颅骨骨缺损的大鼠分为四组，即未处理大鼠（假手术组）和用 D/C 基质、PTH-C-GAM 或 PTH-D/C-GAM 移植的大鼠（D/C、PTH-C-GAM 或 PTH-D/C-GAM 组，分别）。术后 4 周和 8 周，通过免疫组织化学染色确定 PTH 表达。通过进行放射照相、双能 X 射线吸收测定法、微计算机断层扫描和组织学检查评估新骨形成。
转染 PTH pDNA 的细胞分泌有生物活性的 PTH 1-34。此外，在 PTH-C-GAM 组大鼠中，术后 4 周和 8 周时均可检测到 PTH 表达，但在 D/C 组大鼠中则不能检测到。颅骨骨缺损中的新骨形成，从多到少，依次为 PTH-D/C-GAM、PTH-C-GAM、D/C 和假手术组。
我们的结果表明，含有胶原基质的 PTH-GAM 可至少促进局部 PTH 产生 8 周，并促进颅面骨缺损中的骨再生。此外，我们的结果表明，用 D/C 基质替代胶原基质可提高 PTH-GAM 的成骨效果。
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