生体分子の時間的に制御された放出のための界面高分子電解質繊維の複合足場

Summary

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.

Abstract

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.

Introduction

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 factors^2,7 even cells^8,9 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.

Protocol

高分子電解質溶液の調製簡単に言えば。リャオらに詳述されるように、キトサンを精製し、グレード93濾紙を使用して、2％キトサンの1％（w / v）の溶液（v / v）の酢酸、真空フィルタを作成します。 pHは10分間1200×gで7遠心沈殿したキトサンに安定するまで5MのNaOHを使用して、ろ液を中和します。上清を除去し、キトサンを洗浄し、脱イオン水を加えます。遠心分離および…

Representative Results

本稿では、様々な生体分子の持続放出のためのIPC繊維との複合足場を作成しようとしました。本研究で用いた生体分子の特性を表1に見出される。IPC繊維が最初PD-IPC複合足場（ 図1B）を作成するために、親水性PDヒドロゲル中に包埋しました。モデル分子BSAは、第1の被制御生体分子の放出のための複合足場を使用することの実現可能性を決定するために試験しました?…

Discussion

IPC繊維は、2つの反対に帯電した高分子電解質との相互作用によって形成されます。プロセスが安定した繊維形成のための自己組織化プロセスを容易にする、高分子電解質の界面からの複合体の抽出を利用します。 IPCの繊維形成の機構は同様に荷電した高分子電解質に添加される任意の生体分子は、錯体形成プロセス中に組み込むことができることを保証する^。10,11逆に、反対に荷電…

Materials

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