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.
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.
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.
IPC fibras se forman por la interacción de dos polielectrolitos con cargas opuestas. El proceso utiliza la extracción del complejo desde la interfaz de los polielectrolitos, facilitando un proceso de autoensamblaje para la formación de fibras estable. El mecanismo de formación de la fibra IPC asegura que cualquier biomolécula añadió en un polielectrolito cargado de manera similar se puede incorporar durante el proceso de complejación. 10,11 A la inversa, la adición de una biomolécula en el polielect…
The authors have nothing to disclose.
Este trabajo fue apoyado por la Fundación Nacional de Investigación de Singapur administrada por uno de sus Centros de Investigación de Excelencia, el Instituto Mecanobiología, Singapur. MFAC con el apoyo de la Agencia para la Ciencia, Tecnología e Investigación (Singapur) y la Agencia Nacional de Investigación (Francia) el programa conjunto con el número 1122703037. BKKT proyecto es apoyado por el Instituto Mecanobiología. Agradecemos al Sr. Daniel HC Wong para la prueba de lectura del manuscrito y la Sra Amanecer JH Neo para ayudar en la producción de vídeo.
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 |