Gunnella Francesca, Kunisch Elke, Horbert Victoria, Maenz Stefan, Bossert Jörg, Jandt Klaus D, Plöger Frank, Kinne Raimund W
Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital, Waldkrankenhaus "Rudolf Elle", Klosterlausnitzer Str. 81, 07607 Eisenberg, Germany.
Chair of Materials Science, Otto Schott Institute of Materials Research, Friedrich Schiller University Jena, 07743 Jena, Germany.
Pharmaceutics. 2019 Sep 3;11(9):455. doi: 10.3390/pharmaceutics11090455.
Bone regeneration of sheep lumbar osteopenia is promoted by targeted delivery of bone morphogenetic proteins (BMPs) via a biodegradable, brushite-forming calcium-phosphate-cement (CPC) with stabilizing poly(l-lactide--glycolide) acid (PLGA) fibers. The present study sought to quantify the release and bioactivity of BMPs from a specific own CPC formulation successfully used in previous in vivo studies. CPC solid bodies with PLGA fibers (0%, 5%, 10%) containing increasing dosages of GDF5, BB-1, and BMP-2 (2 to 1000 µg/mL) were ground and extracted in phosphate-buffered saline (PBS) or pure sheep serum/cell culture medium containing 10% fetal calf serum (FCS; up to 30/31 days). Released BMPs were quantified by ELISA, bioactivity was determined via alkaline phosphatase (ALP) activity after 3-day exposure of different osteogenic cell lines (C2C12; C2C12BRlb with overexpressed BMP-receptor-1b; MCHT-1/26; ATDC-5) and via the influence of the extracts on the expression of osteogenic/chondrogenic genes and proteins in human adipose tissue-derived mesenchymal stem cells (hASCs). There was hardly any BMP release in PBS, whereas in medium + FCS or sheep serum the cumulative release over 30/31 days was 11-34% for GDF5 and 6-17% for BB-1; the release of BMP-2 over 14 days was 25.7%. Addition of 10% PLGA fibers significantly augmented the 14-day release of GDF5 and BMP-2 (to 22.6% and 43.7%, respectively), but not of BB-1 (13.2%). All BMPs proved to be bioactive, as demonstrated by increased ALP activity in several cell lines, with partial enhancement by 10% PLGA fibers, and by a specific, early regulation of osteogenic/chondrogenic genes and proteins in hASCs. Between 10% and 45% of bioactive BMPs were released in vitro from CPC + PLGA fibers over a time period of 14 days, providing a basis for estimating and tailoring therapeutically effective doses for experimental and human in vivo studies.
通过具有稳定聚（L-丙交酯-乙交酯）酸（PLGA）纤维的可生物降解、形成透钙磷石的磷酸钙骨水泥（CPC）靶向递送骨形态发生蛋白（BMP），可促进绵羊腰椎骨质减少的骨再生。本研究旨在量化BMP从先前体内研究中成功使用的特定自制CPC制剂中的释放量和生物活性。将含有不同剂量GDF5、BB-1和BMP-2（2至1000μg/mL）的PLGA纤维（0%、5%、10%）的CPC固体研磨，并在磷酸盐缓冲盐水（PBS）或含有10%胎牛血清（FCS）的纯绵羊血清/细胞培养基中提取（长达30/31天）。通过酶联免疫吸附测定（ELISA）对释放的BMP进行定量，在不同成骨细胞系（C2C12；过表达BMP受体-1b的C2C12BRlb；MCHT-1/26；ATDC-5）暴露3天后，通过碱性磷酸酶（ALP）活性测定生物活性，并通过提取物对人脂肪组织来源的间充质干细胞（hASC）中成骨/软骨生成基因和蛋白质表达的影响来测定生物活性。在PBS中几乎没有BMP释放，而在培养基+FCS或绵羊血清中，GDF5在30/31天的累积释放率为11-34%，BB-1为6-17%；BMP-2在14天内的释放率为25.7%。添加10%的PLGA纤维显著增加了GDF5和BMP-2的14天释放量（分别增至22.6%和43.7%），但BB-1的释放量未增加（13.2%）。所有BMP均被证明具有生物活性，这在几种细胞系中通过增加的ALP活性得到证明，10%的PLGA纤维有部分增强作用，并且在hASC中通过对成骨/软骨生成基因和蛋白质的特异性早期调节得到证明。在14天的时间段内，10%至45%的生物活性BMP从CPC+PLGA纤维中体外释放，为估计和调整实验和人体体内研究的治疗有效剂量提供了依据。
