Name: synthesis of thermogelling poly(n-isopropylacrylamide)-graft-chondroitin sulfate composites with alginate microparticles for tissue engineering
Uploaded: 2016-10-26T12:30:00
Description: Watch this Scientific Journal Video about Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering at JoVE.com

The authors would like to gratefully acknowledge the assistance of Dr. Jennifer Kadlowec in the development of the adhesive tensile testing protocol.
Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number 1R15 AR 063920-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Poly(N-isopropylacrylamide)-g-chondroitin Sulfate Synthesis
<ol>
	<li>Prior to the synthesis of the bioadhesive hydrogel, purify N-isopropylacrylamide (NIPAAm) monomer and methacrylate chondroitin sulfate (CS).
	<ol>
		<li>Weigh out at least 10 g of NIPAAm and dissolve the monomer in 400 ml&#160;of n-hexane at 60 &#176;C. Stir the container periodically until complete dissolution. Recrystallize the solution in a -20 &#176;C freezer for 24 hr.</li>
		<li>Remove the crystallized monomer from the container and vacuum f...

There are several critical steps in synthesizing the hydrogel-microparticle composite and evaluating its adhesive strength, swelling ability, and cellular biocompatibility. Free radical polymerization of PNIPAAm-g-CS requires successful methacrylation of chondroitin sulfate, complete dissolution of monomer components, and oxygen-free reaction conditions. The ratio of NIPAAm monomer to methacrylated chondroitin sulfate in the reaction mixture was chosen because it has been demonstrated, in our previous work, to generate c...

Injectable biomaterials are those that can be conveniently delivered into the body as a liquid and solidify in situ. Such materials have been applied extensively in regenerative medicine, where they are used to deliver encapsulated cells to the affected site 1-4 and act as a three-dimensional temporary extracellular matrix for the cells 5. For the patient, injectable biomaterials are advantageous because the surgical procedures for implantation are minimally invasive and the solid phase can fill irregularly shaped tissue defects, eliminating the need for custom-size implants.
Injectability can be achieved through a variety of mechanisms. External factors, like pH, have been investigated as a trigger for the formation of gels that encapsulate cells and bioactive molecules6-8. However, pH may not be the most convenient trigger to use in all physiological environments. Another traditional alternative for achieving injectability is using in situ chemical polymerization or crosslinking. A group developed a water-soluble redox system composed of ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine and used it for reacting macromers composed of polyethylene glycol and poly(propylene) glycol 9,10. Zan et al. 11 developed injectable chitosan polyvinylalcohol networks crosslinked with glutaraldehyde. In such systems, the cytotoxicity of reactive components must be considered, especially for applications involving cell encapsulation. Also, exothermic polymerization could produce high enough temperatures to compromise surrounding tissue, which has been reported for polymeric bone cements 12,13.
Still other injectable polymer systems have been developed that exhibit a change from the liquid to solid state with temperature as the trigger. Known as thermogelling systems, these are aqueous polymer solutions that do not require chemical stimulus, monomers, or crosslinkers to achieve in situ formation 14. Rather, a phase transition usually occurring near physiological temperature induces the formation of a physically crosslinked three-dimensional network. Poloxamers such as Pluronic F127 are among the most widely studied polymers for thermogelling drug delivery 15-17 and cell encapsulation 18,19. However, it is well accepted that these gels lack stability at physiological conditions. Studies have demonstrated increased stability using chain extenders 20 or chemical crosslinkers 21,22. Nevertheless, the use of these reagents may limit the potential of the materials for cell encapsulation.
Poly(N-isopropylacrylamide) is a synthetic thermogelling polymer that has received significant attention in tissue engineering and drug delivery 14. Aqueous solutions of poly(N-isopropylacrylamide) (PNIPAAm) exhibit a lower critical solution temperature (LCST), typically occurring around 32 - 34 &#176;C 23,24. Below the LCST, water hydrates PNIPAAm chains. Above the transition temperature, the polymer becomes hydrophobic, resulting in a dramatic phase separation 25-27 and formation of a solid gel without the use of toxic monomers or crosslinkers. However, PNIPAAm homopolymers exhibit poor elastic properties and hold little water at physiological temperature due to hydrophobicity 28. In this work, we choose to incorporate chondroitin sulfate covalently into the PNIPAAm network, which offers the potential for enzymatic degradability 29, anti-inflammatory activity 30,31, and increased water and nutrient absorption 32. PNIPAAm copolymers with CS were prepared in our laboratory by polymerizing the monomer NIPAAm in the presence of methacrylate-functionalized CS to form grafted copolymer (PNIPAAm-g-CS). Because of the low crosslinking density of the copolymer, PNIPAAm-g-CS forms a viscous solution in water at RT&#160;and an elastic gel at physiological temperature due to the LCST 29. The polymer solutions become flowable again upon cooling below the LCST due to the reversibility of the transition.
We have demonstrated that PNIPAAm-g-CS has the potential to function as a tissue engineering scaffold, due to mechanical properties that can be tailored, degradability, and cytocompatibility with human embryonic kidney (HEK) 293 cells 29. However, in certain load bearing areas, such as the intervertebral disc, tissue engineering scaffolds should have the ability to form a substantial interface with surrounding disc tissue to eliminate the risk of dislocation 33. This interface is also necessary for the adequate transmission of force across the interface between the implant and the tissue 33. In our work, we have suspended alginate microparticles in aqueous solutions of PNIPAAm-g-CS and found that gelation localizes the microparticles, which provide adhesion with surrounding tissue 34. In this paper, we outline the steps for preparation of the thermogelling, adhesive polymer. Standard techniques for biomaterials characterization, cell imaging, and assays for viability were adapted to take into account the temperature sensitivity of the polymer and the reversibility of the phase transition. The injectable polymer described in this paper has wide potential for drug delivery and tissue engineering applications outside of those described in this paper. Moreover, the characterization methods described here may be applicable to other thermogelling systems.

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">N-isopropylacrylamide, 99%, pure, stabilized</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">2210-25-5</td>
			<td style="width:347px;">Refrigerate and remove stabilier with hexane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Chondroitin sulfate A sodium salt (from bovine trachea)</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">39455-18-0</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Hexanes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">H302-4</td>
			<td style="width:347px;">Store in a flammable cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">50% (w/w) sodium hydroxide</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">SS254-1</td>
			<td style="width:347px;">Caustic in nature</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Methacrylic anhydride</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">276685</td>
			<td style="width:347px;">Strong fumes; use in a fume hood</td>
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			<td height="40" style="height:40px;width:255px;">Acetone</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">A18-4</td>
			<td style="width:347px;">Chill in a refrigerator prior to use</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Nitrogen Gas</td>
			<td style="width:164px;">Praxair</td>
			<td style="width:120px;">7727-37-9</td>
			<td style="width:347px;">Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tetramethylethylenediamine, 99% extra pure</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">110-18-9</td>
			<td style="width:347px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Ammonium persulfate</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">A3678</td>
			<td style="width:347px;">Hygroscopic and degrades in the presence of water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Phosphate buffered saline tablets</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">BP2944</td>
			<td style="width:347px;">Keep dry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Alginic acid, sodium salt</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">177775000</td>
			<td style="width:347px;">Use heat to aid in dissolving</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Calcium chloride dihydrate</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">C79</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Canola oil</td>
			<td style="width:164px;">Local store</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Obtain from a local store</td>
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			<td height="40" style="height:40px;width:255px;">Tween 20</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">93773</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">70% (v/v) Isopropoanol</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">A416-4</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Porcine ears</td>
			<td style="width:164px;">Haine&#39;s Pork Shop</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Obtain from a local butcher</td>
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			<td height="40" style="height:40px;width:255px;">Sodium Chloride</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">S271-3</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Human embryonic kidney 293 cells</td>
			<td style="width:164px;">ATCC</td>
			<td style="width:120px;">ATCC CRL-1573</td>
			<td style="width:347px;">Store in liquid nitrogen for long-term use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">DMEM: 1X, high glucose, no pyruvate</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">11965126</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Fetal bovine serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">10082-147</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Penn Strep: 10,000 U/ml</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">15140-122</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Trypsin-EDTA: 0.5%, 10X</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">15400-054</td>
			<td style="width:347px;">Refrigerate</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Methanol</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;">AAA44571-K7</td>
			<td style="width:347px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Live/Dead Cell viability kit</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">L3224</td>
			<td style="width:347px;">Light sensitive, keep frozen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">XTT cell viability kit</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">TOX2-1KT</td>
			<td style="width:347px;">Light sensitive, keep frozen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Clear DMEM: 1X, high glucose, no phenol</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">21063-029</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Dulbecco&#39;s PBS: 1X</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">14190136</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Sodium citrate</td>
			<td style="width:164px;">EMD</td>
			<td style="width:120px;">SX0445-1</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Positive displacement pipette</td>
			<td style="width:164px;">BrandTech Scientific, INC</td>
			<td style="width:120px;">2702904</td>
			<td style="width:347px;">Dispenses 100 - 500 &#181;L and comes with attachable tips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">No 3. Stainless Steel scalpel handle</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">S2896</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Miltex sterile surgical blades</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">12-460-440</td>
			<td style="width:347px;">Size 10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Power gem homogenizer</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">08-451-660</td>
			<td style="width:347px;">Model # 125</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Porcelain mortar and pestle</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">Z247464</td>
			<td style="width:347px;">Holds 50 mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">FreeZone 1 L benchtop freeze dry system</td>
			<td style="width:164px;">Labconco</td>
			<td style="width:120px;">7740020</td>
			<td style="width:347px;">Freeze samples prior to use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Oil sealed rotary vane pump</td>
			<td style="width:164px;">Edwards</td>
			<td style="width:120px;">A65301906</td>
			<td style="width:347px;">Model # RV5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Incubating orbital shaker</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;">12620-946</td>
			<td style="width:347px;">Model # 980153</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Benchtop refrigerated centrifuge</td>
			<td style="width:164px;">Forma Scientific, INC</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 5682</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Heated ovens</td>
			<td style="width:164px;">VWR</td>
			<td></td>
			<td style="width:347px;">Model # 1235PC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">2 N force gauge</td>
			<td style="width:164px;">Shimpo</td>
			<td style="width:120px;">FGV-0.5XY</td>
			<td style="width:347px;">Model # FGV-0.5XY</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">E-force test stand</td>
			<td style="width:164px;">Shimpo</td>
			<td style="width:120px;">FGS-200PV</td>
			<td style="width:347px;">Model # FGS-200PV</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tissue culture swinging bucket centrifuge</td>
			<td style="width:164px;">Beckman Coulter</td>
			<td style="width:120px;">366830</td>
			<td style="width:347px;">Model #6S-6KR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tissue culture microcentrifuge</td>
			<td style="width:164px;">Eppendorf</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model #5415C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Hemacytometer set</td>
			<td style="width:164px;">Hausser Scientific</td>
			<td style="width:120px;">3720</td>
			<td style="width:347px;">Requires replacement cover glass slips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Slide warmer</td>
			<td style="width:164px;">Lab Scientific</td>
			<td style="width:120px;">XH-2022</td>
			<td style="width:347px;">Model # XH-2002</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Portable heating lamp</td>
			<td style="width:164px;">Underwriters Laboratories</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Helps to maintain polymer temperature at 37&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Inverted fluorescent microscope</td>
			<td style="width:164px;">Zeiss</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model Axiovert 25 CFL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Heated water bath</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 1235PC</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Rocking platform</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Series 100</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Multiskan FC microtiter plate reader</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Type 357</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Cell culture incubator</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 2350T</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Purifier class II biosafety cabinet</td>
			<td style="width:164px;">Labconco</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Delta Series</td>
		</tr>
	</tbody>
</table>

synthesis of thermogelling poly(n-isopropylacrylamide)-graft-chondroitin sulfate composites with alginate microparticles for tissue engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

A thermally responsive grafted co-polymer was successfully synthesized and characterized for its bioadhesive strength, swelling properties, and in vitro cytocompatibility. We chose to investigate alginate due to its well-established mucoadhesive properties. Alginate microparticles, with an average diameter of 59.7 &#177; 14.9 &#181;m, were blended with 5% (w/v) PNIPAAm-g-CS at concentrations of 25, 50, and 75 mg/ml. These concentrations were based on one-half, equal to, and twice...

Watch this Scientific Journal Video about Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering at JoVE.com

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

The overall goal of this methodology is to assess both the adhesive properties and cellular biocompatibility of the proposed hydrogel composite for intervertebral disc tissue engineering of the nucleus pulposus. This method can help answer key questions in the field of tissue engineering, like how to design and prepare thermal reversible hydrogels with bioadhesive properties for cell encapsulation. The main advantage of this technique is that we are able to demonstrate in-vitro studies with an injectable, thermally-sensitive composite to evaluate its use as a potential replacement for the nucleus pulposus.In this current study, we evaluate a novel hydrogel scaffold by performing both tensile mechanical tests, and cellular viability assays. Demonstrating the biocompatibility assays will be Emily Schmidt, Mark Dittmer, Edward Goldschmidt, and Frantzeska Giginis, students from the departments of Biological Sciences and Biochemistry. Prior to the synthesis of the bioadhesive hydrogel, purify NIPAAm monomer and mCS, as described in the text protocol.Then, co-dissolve 10 grams of purified NIPAAm and 2.209 grams of mCS in 232 milliliters of deionized water. Purge the solution of oxygen by using inert nitrogen gas. Maintain a vigorous, yet low gas flow rate, to prevent the solution from bubbling over for 15 minutes.Continue purging the solution with nitrogen gas and add 0.976 milliliters of TEMED. Next, initiate the polymerization reaction by mixing 97.6 milligrams of ammonium persulfate. Mix the solution for approximately 20 seconds, and quickly seal the solution to prevent any air from entering the vessel.Allow the solution to polymerize under fluorescent light for 24 hours before processing the sample into a fine powder, as described in the text protocol. Create an oil and water mixture by combining 100 milliliters of canola oil, one milliliters TWEEN 20, and 20 milliliters of 2%of alginate. Emulsify the mixture for 10 minutes with a homogenizer at 15, 000 RPM.Additionally, mix with a magnetic stir bar at 200 RPM to create large microparticles, or 2, 000 RPM to create small microparticles. Aspirate the 2%calcium chloride solution using a syringe with an 18-gauge needle tip. Slowly add the calcium chloride drop-wise into the emulsion.After allowing the microparticles to cross-link for 10 minutes, evenly distribute the batch of microparticles into capped 50 milliliter conical tubes. Centrifuge at 1, 400 times G for two minutes to remove the initial layer of canola oil before washing and lyophilizing the microparticles as described in the text protocol. Use the resulting freeze-dried polymer powder to create a 5%weight volume PNIPPAm graft CS solution by first dissolving 50 milligrams of hydrogel powder in one milliliter of 1X phosphate buffered saline, or PBS.Mix the solution using a vortexer before chilling in a refrigerator at four degrees Celsius for 24 hours. Once the viscous solution forms, create a homogenous composite by adding 25 or 50 milligrams of freeze-dried alginate microparticles to the hydrogel solution and vortexing. Store the composite in a refrigerator at four degrees Celsius for later use.Prior to performing tensile tests, extract cartilage substrate from a porcine ear, as detailed in the text protocol. Then, attach the force gauge to the arm of the test stand. Place a hotplate on top of the force stand, and set the temperature to 50 degrees Celsius.Then, affix a one centimeter by one centimeter piece of cartilage to the stainless steel block with cyanoacrylate glue and a pair of tweezers. Glue an additional piece of cartilage to the Delrin cylinder, and attach the cylinder to the force gauge. It is very important that both cartilage surfaces face each other and contact the hydrogel.Place the plexiglass water bath on top of the hotplate, and insert the stainless steel block with the cartilage substrate inside the bath. Lower the arm of the test stand, and align both cartilage substrates with one another. Then, raise the arm, leaving enough room to apply the hydrogel.With a positive displacement pipette, dispense 200 microliters of the bioadhesive hydrogel to the bottom piece of cartilage. Lower the arm of the force stand at one milliliter per minute until the hydrogel contacts the upper piece of cartilage, and exerts a preload force of 0.001 newtons. Pour the 37-degrees Celsius saline solution into the bath until the volume reaches the designated water level.Check the temperature of the solution with a thermocouple. After allowing the hydrogel to set for a total of five minutes, raise the arm at a speed of two millimeters per minute. The test is completed once the bioadhesive detaches from the cartilage substrate, or when a significant drop in force occurs, signaling failure.Collect the recorded data, and raise the arm of the test stand. Remove the Delrin cylinder from the force gauge. Pour the saline solution into its previous container, and reheat to 37 degrees Celsius.Calculate the stresses, as described in the text protocol. Before performing the Live/Dead assay with PNIPAAm graft CS, prepare human embryonic kidney, or HEK 293 cells, as detailed in the text protocol. Create positive control monolayers by pipetting multiple 300-microliter volumes of the cell mixture into a 24-well plate to generate four to six replicate samples.Maintain the same cell concentration by re-suspending the cells in approximately two milliliters of PNIPAAm graft CS for each replicate sample. Pipette the polymer cell mixture up and down using a serological pipette until homogenous. If seeding with alginate microparticles, transfer two milliliters of the PNIPAAm graft CS cell suspension into a 35-millimeter culture dish, and introduce 200 milligrams of particles into the mixture.Pipette the cell mixture up and down using a serological pipette until homogenous. Then, with a serological pipette, dispense multiple 300-microliters volumes of the polymer cell mixture into each of the wells for killed control, PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles to generate four to six replicate samples. Incubate the plate at 37 degrees Celsius for 10 to 15 minutes, and allow the PNIPAAm graft CS to transition from a liquid to a gel.The polymer forms an opaque white disc. Position a slide warmer in the hood, and set the temperature to 37 degrees Celsius. Use the slide warmer to maintain well plate temperature, and pipette 600 microliters of pre-warmed media into each well.To further prevent the transitioning of the polymer to a liquid state, position a lamp with a fluorescent bulb above the dish to warm up the air above it. Incubate the cells for five days at 37 degrees Celsius with 5%carbon dioxide. After five days have elapsed, remove the media from the wells of the monolayer, PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles.Wash all of the wells thoroughly, but gently, with PBS, and then, remove the PBS. Allow the hydrogel discs containing alginate microparticles to liquefy at room temperature, and add two milliliters of 50 millimolar sodium citrate to each of the wells. Remove the media from the killed cell wells.Allow the hydrogel discs to liquefy at room temperature, and add 300 microliters of 70%methanol to each well before incubating the plate for 45 minutes at 37 degrees Celsius. Remove the sodium citrate and methanol solutions from the wells, and pipette each of the hydrogel suspensions into individual microcentrifuge tubes. Centrifuge the tubes at 2, 000 times G for 10 minutes to separate the cells from the hydrogel suspension.Carefully remove the suspension by pipetting the solution from the conical tube, and leaving behind the cell pellet. Re-suspend the pellet in 300 microliters of PBS, and transfer to the original well plate. Next, add 300 microliters of the Live/Dead dye mix to each of the wells.Wrap the well plate in aluminum foil, and incubate for 45 minutes on a rocker at room temperature before imaging all of the samples under an inverted fluorescence microscope with a 10X objective. Representative tensile strength results for both PNIPAAm graft CS, and PNIPAAm graft CS with varying alginate microparticle formulations are shown here. The tensile strength of PNIPAAm graft CS quadrupled with the addition of 50 or 75 milligrams per milliliter of alginate microparticles.As expected, PNIPAAm graft CS contracts and shrinks in volume above its lower critical solution temperature. Introducing microparticles within the hydrogel imparts a swelling effect over a seven-day period in PBS. HEK 293 cells encapsulated in both PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles showed active cellular fluorescence using Live/Dead dye after a period of five days.These results can be compared to the positive and negative control wells. In parallel, a quantitative assay was used to confirm the Live/Dead imaging, which showed no significant differences in cellular viability between PNIPAAm graft CS, PNIPAAm graft CS with alginate microparticles, and the control monolayer. A hydrogel composite consisting of thermally-sensitive poly(N-isopropylacrylamide)grafted with chondroitin sulfate and alginate microparticles was created for nucleus pulposus replacement.While attempting this procedure, it's important to remember that the composite is temperature-sensitive, and will liquefy below its lower critical solution temperature. Once mastered, tensile mechanical tests can be performed with ease as long as careful precision is practiced. Swelling studies reveal a hydrogel's ability to retain water.However, these tests must mimic the native in-vivo environment. Cellular viability of the thermally-sensitive gels can be confirmed and assessed by a combination of fluorescence-marking and metabolic-producing reagents. After watching this video, you should have a good understanding of how thermally-sensitive injectables are applied in intervertebral discs, tissue engineering, and how composites may enhance certain material properties.

synthesis-thermogelling-polyn-isopropylacrylamide-graft-chondroitin

Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

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

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...