聚电解质复合物肝素结合域成骨生长因子交货
1Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine,National University of Singapore, 2Centre for Research in Medical Devices (CÚRAM),National University of Ireland Galway, 3Department of Bioengineering, Faculty of Engineering,National University of Singapore, 4Tissue Engineering Program,National University of Singapore, 5Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center,University of California, Los Angeles
Summary
Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.
Abstract
在骨重建手术，生长因子超生理金额经验装上支架，以促进成功的骨融合。大剂量的高度有效的生物制剂的需要，由于生长因子的不稳定性如快速酶降解的结果，以及在植入物位点定位足够量的生长因子的载体的低效率。因此，该延长的生长因子的稳定性，如BMP-2 / NELL-1，并控制其释放策略实际上可以降低其有效剂量，从而减少了需要更大的剂量期间未来骨再生手术。反过来，这将减少副作用和生长因子的成本。自组装胸肌已经制造通过增强的体内稳定性通过肝素结合，并进一步使可能生长因子的生物活性提供的BMP-2 / NELL-1递送的更好的控制。在这里，我们说明了PEC制造这有助于在国内配送简单的过程中重建骨手术的各种生长因子RY。
Introduction
假的发病率已经报道高达10〜45％，在退化性脊柱融合和修订脊柱手术1。以减少脊柱融合和其他重建骨手术，骨生长因子如BMP-2，内尔-1 1和血小板衍生的生长因子（PDGF）已被引入，以促进从头成骨过程中假关节的速度。其中，BMP-2是脊柱融合2的热门选择。虽然BMP-2的诱导和促进新骨形成的效力已被很好地建立3;临床显著并发症，如异位骨形成，血清肿和血肿形成，炎症反应，神经根，椎体骨溶解和逆行射精继续关注的问题，由于使用的4,5超生理金额。
因此，降低的BMP-2的剂量保持在一个相关的策略引诱最小化的副作用。此外，需要有效的载体系统来抑制BMP-2在当代胶原海绵载波系统观察到的突释，并进一步加强这种有效的细胞因子的长期和本地化交货。交替阳离子和阴离子聚电解质的层-层自组装，可以用作建立脚手架基质或植入材料6的表面上的聚电解质复合物的可调谐方法。在这方面，肝素（对于具有所有生物剂的最高负电荷密度已知）已被识别为贪婪地通过静电和肝素结合结构域的多种生长因子结合。的确，肝素已经显示出延长的半衰期，从而使可能的多种生长因子的生物活性。
在此基础上，我们的组适于层 – 层自组装协议来制造基于肝素的聚电解质复合物（PEC），该负载和固定7,8中保留了成骨生长因子的生物活性。藻酸盐微球芯由交联的α-L-古洛糖醛酸盐（G），用二价阳离子钙或锶离子藻酸盐的残基制成。藻酸盐核心是可生物降解的支架基质;该植入后，它是在融合床提供余地骨向内生长吸收。聚-L-赖氨酸（PLL）或鱼精蛋白被用作阳离子层与两个支架矩阵（在这种情况下，藻微珠载体芯）和带负电的肝素隔行;而阴离子肝素层的功能，以稳定和定位加载生长因子。三重层的PEC已经显示出增加的生长因子负载能力在猪模型9。最近，PEC载体已显示出至少20倍的成功减少在大鼠10和脊骨融合8的猪模型的BMP-2的有效剂量。
ntent“>在这里，用BMP-2作为模型骨生长因子，我们报告脊柱融合增强生长因子交付和其他骨重建手术制造佩奇方法。Protocol
1.海藻酸钠溶液制备溶解200毫克藻酸钠（未照射），或者400毫克8兆拉德的在10ml双蒸水照射海藻酸钠振摇1小时的非辐射藻酸盐和辐照藻15分钟。过夜储存在4℃的藻酸盐溶液。滤波器海藻酸钠微珠制作之前无菌0.2微米的注射器过滤的藻酸盐溶液。 2.海藻酸钠微珠制作消毒静电珠发生器和注射器泵用70％乙醇，并放置在一类二生物安全柜（ 图1）。…
Representative Results
在我们的载体，鱼精蛋白被选为聚-L-赖氨酸的替代，因为它有类似的化学性质，它是FDA批准作为肝素的解毒剂。光学显微镜结果表明，非照射微珠呈球状的形状，直径为267±14微米。 （0.35毫米喷嘴，流量为5ml /小时和5.8千伏率）。大部分照射微珠是泪滴形状。上照射微珠的轮部测得的直径为212±30微米（0.35毫米喷嘴，流速为4毫升/小时和6千伏率）。 （ 图4）。 …
Discussion
该协议提出了通过层 – 层自组装制备的PEC的方法。的层 – 层结构，使用鱼精蛋白，肝素，BMP-2和NELL-1和共聚焦显微镜的荧光类似物可视化。吸收和释放试验表明，肝素对PEC介导成骨生长因子的吸收和释放。在PEC方法的摄取效率是：NELL-1：86.7±2.7％，BMP-2：70.5±3.1％。在PEC载体具有NELL-1（20％）释放的更好的调制相比，纯表面吸附载体如钙磷灰石颗粒（40-80％）11。
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Disclosures
The authors have nothing to disclose.
Acknowledgements
These studies were funded by National Medical Research Council Clinician Scientist – Individual Research Grant (CS-IRG) NMRC/CIRG/1372/2013 and NMRC EDG/0022/2008.
Materials
| Life Science Acrodisc 25mm Syring Filter w/0.2 µm Supor Membrane | PALL | PN4612 | Sterile protamine, heparin solution by ultrafiltration |
| 24 well plate | Cell Star | 662160 | |
| 96 well plate Nuclon Delta Surface | Thermo Fisher Scientific | 167008 | |
| (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), MTT | Sigma Aldrich | M5655 | Measure cytotoxicity of PEC-NELL-1 |
| Acetone | Fisher Scientific | A/0600/17 | Precipitate CF-405 Labelled protamine |
| Alamar Blue | Invitrogen, Life Technologies | DAL 1025 | Measure cytotoxicity of PEC-BMP-2 |
| Alkaline Phosphatase Assay (ALP) assay kit | Anaspec | AS-72146 | |
| Ammonium Chloride | Merck | Art 1145 | Stop reagent in FITC labelling |
| Anhydrous Dimethyl Sulfoxide (DMSO) | Invitrogen, Life Technologies | D12345 | Solvent for fluorescent isothiocyanate I |
| Dimethyl Sulfoxide (DMSO) | Sigma Aldrich | Dissolve formazan | |
| Autoclave | Hirayama | HU-110 | Sterilize alginate beads by steam |
| Beta-glycerophosphate | Sigma Aldrich | G9422 | |
| BMP-2 (Infuse Bone Graft Large II Kit) | Medtronic Sofarmor Danek, Memphis TN, USA | 7510800 | Osteogenic Growth Factor, dialysis is needed to remove stabilizer component that interferes with FITC coupling |
| Carboxybenzoyl quinoline-2-Carboxaldehyde (CBQCA) | Thermo Fisher Scientific | A-6222 | To quantify NELL-1 protein |
| Cell Strainer (100µm) | BD Science | 352360 | Hold PEC for ALP assay |
| Cell Scraper 290mm Bladewide 20mm | SPL Life Science | 90030 | Detach the cell from 24 well plate |
| CF 405S, Succinimidyl Ester | Sigma Aldrich | SCJ4600013 | Blue fluorescent dye for protamine labelling |
| CF 594, Hydrazide | Sigma Aldrich | SCJ4600031 | Deep red fluorescent dye for heparin labelling |
| Centrifuge | Beckman Coulter | Microfuge 22R | |
| Confocal Microscope | Olympus | FV1000 | |
| Dexamethasone | Sigma Aldrich | D4902 | Component of osteogenic growth medium |
| Dextran Desalting Columns | Pierce (Thermo Scientific) | 43230 | |
| DMEM | Gibco | 12320 | |
| BMP-2 Quantikine ELISA Kit | R&D System | DBP200 | Determine BMP-2 release |
| Fetal Bovine Serum FBS | Hyclone | SV30160.03 | |
| Fluoescein Isothiocyananate, Isomer I | Sigma Aldrich | F7250 | Green fluorescent dye for NELL-1 and BMP-2 labelling |
| ThinCert Cell Culture Inserts, For 24 Well plates, Sterile |
Greiner | 662630 | Prevents PEC wash out when changing osteogenic medium |
| Havard Appartus Syringe Pump (11 plus) | Havard Apparatus | 70-2208 | |
| n-Hexane (>99%) | Sigma Aldrich | 139386 | |
| Heparin | Sigma Aldrich | H3149 | Binds with osteogenic growth factor with heparin binding domain |
| Hydrochloric acid (37%) | Merck | 100317 | Highly Corrosive |
| Incubator | Binder | C8150 | |
| MicroBCA Protein Assay kit | Thermoscientific | 23235 | |
| Microplate Reader | Tecan | Infinite M200 | For ALP and microBCA assays |
| NELL-1 | Aragen Bioscience Morgan Hill, CA, USA | N/A | Osteogenic growth factor, keep at -80˚C |
| Nisco cell encapsulator | Nisco Engineering Inc | Encapsulation unit VAR V1 | |
| Fluorescent Microscope | Olympus | IX71 | |
| mPCL-TCP Scaffold (Pore size is 1.3mm) | Osteopore | PCL-TCP 0/90 | Hold PEC for in vivo study |
| Penicillin-Streptomycin 10,000 unit/ml, 100ml | Hyclone Cell Culture | SV30010 | Antibiotic |
| 10X Phosphate Buffered Saline (PBS) | Vivantis | PB0344-1L | 10x Solution, Ultra Pure Grade |
| Poly-L-Lysine MW 15,000-30,000 | Sigma Aldrich | P2568 | Polycation |
| Protamine Sulfate salt, from Salmon | Sigma Aldrich | P4020 | Polycation |
| Shaker | Labnet | S2025 | |
| Snakeskin Dialysis Tubing 3,500 MWCO 22mm x 35 feet | Thermo Fisher Scientific | 68035 | Remove unreacted FITC by dialysis |
| Sodium Chloride | Merck | 1.06404.1000 | |
| Sodium Hydroxide | Qrec | S5158 | |
| Sodium Bicarbonate | US Biological | S4000 | Buffer |
| Sodium carbonate | Sigma Aldrich | S7795-500G | Buffer |
| Strontium Chloride Hexahydrate | Sigma Aldrich | 255521 | Crosslinker for alginate |
| Spatula | 3dia | ||
| 5ml syringe | Terumo | 140425R | Diameter of syringe affects the flow rate |
| 75cm2 Cell Culture Flask Canted Neck | Corning | 730720 | |
| Toluidine Blue | Sigma Aldrich | 52040 | Heparin assay |
| Trypsin 1X | Hyclone Cell Culture | SH30042.01 | |
| Sodium alginate | Novamatrix (FMC Biopolymer, Princeton, NJ) | Pronova UPMVG | Core material of microbeads |
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Cite This Article
Wing Moon Lam, R., Abbah, S. A., Ming, W., Naidu, M., Ng, F., Tao, H., Goh Cho Hong, J., Ting, K., Hee Kit, W. Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery. J. Vis. Exp. (114), e54202, doi:10.3791/54202 (2016).