Name: composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules
Uploaded: 2015-08-19T14:00:00
Description: Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

这项工作是由它的力学生物学研究所，新加坡研究卓越中心，一个管理的新加坡国家研究基金会的支持。 MFAC是1122703037. BKKT是支持的力学生物学研究所机构科学，技术和研究（新加坡）和国家研究机构（法国）项目下的多次联合计划的支持。我们感谢丹尼尔HC黄先生的校对书稿和黎明JH新女士协助视频制作。

1.准备聚电解质溶液的<ol><li>纯化脱乙酰壳多糖，如在Liao 等详细说明。简言之，将创建一个1％（重量/体积）壳聚糖在2％的溶液使用（体积/体积）乙酸和真空过滤级93过滤纸。中和使用5M的NaOH将滤液直到pH稳定至7。离心沉淀的脱乙酰壳多糖在1200×g离心10分钟。滗析出上清液，并添加去离子水以洗涤脱乙酰壳多糖。重复离心和洗涤步骤两次以上。冻结的沉淀壳聚糖在-80℃和冷冻干燥O...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+in+vitro+characterization+of+vascular+endothelial+growth+factor+(VEGF)-loaded+poly(DL-lactic-co-glycolic+acid)/poly(ethylene+glycol)+microspheres+using+a+solid+encapsulation/single+emulsion/solvent+extraction+technique.">Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique.</a> J. Biomed. Mater. Res. 51 (3), 383-390 (2000).</li><li>Cutiongco, M. F. A., Tan, M. H., Ng, M. Y. K., Le Visage, C., Yim, E. K. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated-PLGA+microparticles+containing+VEGF+for+long+term+drug+delivery.">PEGylated-PLGA microparticles containing VEGF for long term drug delivery.</a> Int. J. Pharm. 440 (1), 13-18 (2013).</li><li>Patel, Z. S., Ueda, H., Yamamoto, M., Tabata, Y., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+release+of+vascular+endothelial+growth+factor+from+gelatin+microparticles+and+biodegradable+composite+scaffolds.">In vitro and in vivo release of vascular endothelial growth factor from gelatin microparticles and biodegradable composite scaffolds.</a> Pharm. Res. 25 (10), 2370-2378 (2008).</li><li>Sun, H., Xu, F., Guo, D., Liu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+effects+of+various+additives+and+polymers+on+nerve+growth+factor+microspheres.">In vitro evaluation of the effects of various additives and polymers on nerve growth factor microspheres.</a> Drug Dev. Int. Pharm. 40 (4), 452-457 (2014).</li><li>Lee, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+drug+release+from+hydrogel+matrices.">Kinetics of drug release from hydrogel matrices.</a> J. Control. Release. 2, 277-288 (1985).</li><li>Cutiongco, M. F. A., Choo, R. K. T., Shen, N. J. X., Chua, B. M. X., Sju, E., Choo, A. W. L., Le Visage, C., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+scaffold+of+poly(vinyl+alcohol)+and+interfacial+polyelectrolyte+complexation+fibers+for+controlled+biomolecule+delivery.">Composite scaffold of poly(vinyl alcohol) and interfacial polyelectrolyte complexation fibers for controlled biomolecule delivery.</a> Front. Bioeng. Biotechnol. 3 (3), 1-12 (2015).</li><li>Yow, S. Z., Lim, T. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Electroactive+Polypyrrole-Collagen+Fibrous+Scaffold+for+Tissue+Engineering.">A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering.</a> Polymers. 3 (1), 527-544 (2011).</li><li>Leong, M. F., Toh, J. K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+prevascularised+tissue+constructs+by+assembly+of+polyelectrolyte+hydrogel+fibres.">Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres.</a> Nat. Commun. 4, 2353 (2013).</li><li>Di Benedetto, F., Biasco, A., Pisignano, D., Cingolani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+polyacrylamide+hydrogels+by+soft+lithography.">Patterning polyacrylamide hydrogels by soft lithography.</a> Nanotechnology. 16 (5), S165 (2005).</li><li>Revzin, A., Russell, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+poly(ethylene+glycol)+hydrogel+microstructures+using+photolithography.">Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography.</a> Langmuir. 17 (18), 5440-5447 (2001).</li><li>Yim, E. K. F., Liao, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+compatibility+of+interfacial+polyelectrolyte+complexation+fibrous+scaffold:+evaluation+of+blood+compatibility+and+biocompatibility.">Tissue compatibility of interfacial polyelectrolyte complexation fibrous scaffold: evaluation of blood compatibility and biocompatibility.</a> Tissue Eng. Part A. 13 (2), 423-433 (2007).</li><li>Chaouat, M., Le Visage, C., Autissier, A., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+a+small-diameter+polysaccharide-based+arterial+graft+in+rats.">The evaluation of a small-diameter polysaccharide-based arterial graft in rats.</a> Biomaterials. 27, 5546-5553 (2006).</li><li>Eshraghi, S., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+microstructural+properties+of+polycaprolactone+scaffolds+with+one-dimensional,+two+dimensional,+and+three-dimensional+orthogonally+oriented+porous+architectures+produced+by+selective+laser+sintering.">Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering.</a> Acta Biomater. 6, 2467-2476 (2010).</li></ol>

IPC纤维通过两个带相反电荷的聚电解质的相互作用形成。该方法使用从聚电解质的界面的复杂的提取，便利了一种用于纤维形成稳定的自组装过程。 IPC的纤维形成的机制可确保任何生物分子加入到类似电荷的聚电解质可以在络合过程中引入。10,11相反，除了生物分子的成带相反电荷的聚电解质将导致瞬间沉淀。用于IPC纤维的简单的制造方法借给多功能性在将各种生物材料如细胞，生长因?...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pullulan&#160;</td>
			<td>Hayashibara Inc Okayama Japan</td>
			<td></td>
			<td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma Aldrich</td>
			<td>D1037</td>
			<td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Trimetaphosphate</td>
			<td>Sigma Aldrich</td>
			<td>T5508</td>
			<td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td>
		</tr>
		<tr>
			<td>Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>448877</td>
			<td>MW 190-310 kDa. Acetylation degree of 75 to 85%. Purification of chitosan is required to create stable IPC fibers.</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td></td>
			<td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&#160;</td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt from brown algae, low viscosity</td>
			<td>Sigma Aldrich</td>
			<td>A2158</td>
			<td>Dissolve in water overnight. Filter through sterile 0.2&#181;m syringe filter before use. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td></td>
			<td>Dissolve in sterile PBS and filter using 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>This kit was used to measure BSA release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Human Recombinant Vascular Endothelial Growth Factor</td>
			<td>R&#38;D systems</td>
			<td>293-VE</td>
			<td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5 mg/ml.</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt From Porcine</td>
			<td>Sigma Aldrich</td>
			<td>H3393</td>
			<td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&#160;</td>
		</tr>
		<tr>
			<td>ScanVac Coolsafe Lyophilizer</td>
			<td>Labogene</td>
			<td>7.001.200.060</td>
			<td>This is a non-programmable freeze dryer that operates at -105 to -110 &#176;C, This can be replaced by other standard lab lyophilizers.</td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL)</td>
			<td>Sigma Aldrich</td>
			<td>181609</td>
			<td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>V800151</td>
			<td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td>
			<td>Dow Corning</td>
			<td>(240)4019862</td>
			<td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td>
		</tr>
		<tr>
			<td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td>
			<td>R&#38;D systems</td>
			<td>256-GF</td>
			<td>Reconstituted in sterile DI water to a final concentration of 100 &#181;g/ml. Aliquot and store in -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells (hMSC)</td>
			<td>Cambrex</td>
			<td></td>
			<td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td>
		</tr>
		<tr>
			<td>Rat PC12 Pheochromocytoma Cells&#160;</td>
			<td>ATCC</td>
			<td></td>
			<td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td>
		</tr>
		<tr>
			<td>Grade 93 filter paper</td>
			<td>Whatman</td>
			<td>Z699675</td>
			<td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH 7.</td>
		</tr>
		<tr>
			<td>Swing bucket centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>To be used during the purification of chitosan using 1200 x g speed.&#160;</td>
		</tr>
		<tr>
			<td>Motor with mandrel rotating at constant speed</td>
			<td>Rhymebus</td>
			<td>RM5E</td>
			<td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>FirstBase</td>
			<td></td>
			<td>Sterilize through filtration (0.2 &#181;m filter) and autoclave.&#160;</td>
		</tr>
		<tr>
			<td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td>
			<td>Greiner</td>
			<td></td>
			<td>The TCPS dish is used for casting of pullulan frame.&#160;</td>
		</tr>
		<tr>
			<td>Human VEGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DVE00</td>
			<td>The ELISA kit is used for detection of VEGF in the release medium.</td>
		</tr>
		<tr>
			<td>Human NGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY256</td>
			<td>&#160;The ELISA kit is used for detection of NGF in the release medium.</td>
		</tr>
		<tr>
			<td>Plastic Coated Adhesive Tape</td>
			<td>Bel-Art</td>
			<td>9040336</td>
			<td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td>
		</tr>
	</tbody>
</table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

在这篇文章中，我们试图建立复合支架与IPC纤维各种生物分子的持续释放。在此研究中使用的生物分子的特征见于表1。的IPC纤维第一嵌入到亲水的PD水凝胶来创建的PD-IPC复合支架（ 图1B）。模型分子BSA的第一测试，以确定使用复合支架用于控制生物分子释放的可行性。 BSA的掺入的PD-IPC支架为45±0.97％的效率。 BSA的从PD-瞳距释放呈接近线性的动力学具有初始减毒突释之后?...

Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

科研

生物工程

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...