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

Summary

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.

Abstract

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 °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 alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p<0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p<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.

Introduction

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 thro....

Protocol

1. Poly(N-isopropylacrylamide)-g-chondroitin Sulfate Synthesis

1. Prior to the synthesis of the bioadhesive hydrogel, purify N-isopropylacrylamide (NIPAAm) monomer and methacrylate chondroitin sulfate (CS).
   1. Weigh out at least 10 g of NIPAAm and dissolve the monomer in 400 ml of n-hexane at 60 °C. Stir the container periodically until complete dissolution. Recrystallize the solution in a -20 °C freezer for 24 hr.
   2. Remove the crystallized monomer from the container and vacuum filter the n-hexane using a Buchner funnel. Place the monomer in a vacuum oven at RT for 24 hr to remove any remaining n-hexane. Store the monomer....

Results

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 ± 14.9 µm, 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 the equiv.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
N-isopropylacrylamide, 99%, pure, stabilized	Acros Organics	2210-25-5	Refrigerate and remove stabilier with hexane
Chondroitin sulfate A sodium salt (from bovine trachea)	Sigma-Aldrich	39455-18-0	Refrigerate
Hexanes	Fisher Scientific	H302-4	Store in a flammable cabinet
50% (w/w) sodium hydroxide	Fisher Scientific	SS254-1	Caustic in nature
Methacrylic anhydride	Sigma-Aldrich	276685	Strong fumes; use in a fume hood
Acetone	Fisher Scientific	A18-4	Chill in a refrigerator prior to use
Nitrogen Gas	Praxair	7727-37-9	Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas)
Tetramethylethylenediamine, 99% extra pure	Acros Organics	110-18-9
Ammonium persulfate	Sigma Aldrich	A3678	Hygroscopic and degrades in the presence of water
Phosphate buffered saline tablets	Fisher Scientific	BP2944	Keep dry
Alginic acid, sodium salt	Acros Organics	177775000	Use heat to aid in dissolving
Calcium chloride dihydrate	Fisher Scientific	C79
Canola oil	Local store		Obtain from a local store
Tween 20	Sigma-Aldrich	93773
70% (v/v) Isopropoanol	Fisher Scientific	A416-4
Porcine ears	Haine's Pork Shop		Obtain from a local butcher
Sodium Chloride	Fisher Scientific	S271-3
Human embryonic kidney 293 cells	ATCC	ATCC CRL-1573	Store in liquid nitrogen for long-term use
DMEM: 1x, high glucose, no pyruvate	Life Technologies	11965126	Refrigerate
Fetal bovine serum	Life Technologies	10082-147	Refrigerate
Penn Strep: 10,000 U/ml	Life Technologies	15140-122	Refrigerate
Trypsin-EDTA: 0.5%, 10x	Life Technologies	15400-054	Refrigerate
Methanol	VWR	AAA44571-K7
Live/Dead Cell viability kit	Life Technologies	L3224	Light sensitive, keep frozen
XTT cell viability kit	Sigma Aldrich	TOX2-1KT	Light sensitive, keep frozen
Clear DMEM: 1x, high glucose, no phenol	Life Technologies	21063-029	Refrigerate
Dulbecco's PBS: 1x	Life Technologies	14190136	Refrigerate
Sodium citrate	EMD	SX0445-1
Positive displacement pipette	BrandTech Scientific, INC	2702904	Dispenses 100 - 500 µl and comes with attachable tips
No 3. Stainless Steel scalpel handle	Sigma Aldrich	S2896
Miltex sterile surgical blades	Fisher Scientific	12-460-440	Size 10
Power gem homogenizer	Fisher Scientific	08-451-660	Model # 125
Porcelain mortar and pestle	Sigma Aldrich	Z247464	Holds 50 ml
FreeZone 1 L benchtop freeze dry system	Labconco	7740020	Freeze samples prior to use
Oil sealed rotary vane pump	Edwards	A65301906	Model # RV5
Incubating orbital shaker	VWR	12620-946	Model # 980153
Benchtop refrigerated centrifuge	Forma Scientific, INC		Model # 5682
Heated ovens	VWR		Model # 1235PC
2 N force gauge	Shimpo	FGV-0.5XY	Model # FGV-0.5XY
E-force test stand	Shimpo	FGS-200PV	Model # FGS-200PV
Tissue culture swinging bucket centrifuge	Beckman Coulter	366830	Model #6S-6KR
Tissue culture microcentrifuge	Eppendorf		Model #5415C
Hemacytometer set	Hausser Scientific	3720	Requires replacement cover glass slips
Slide warmer	Lab Scientific	XH-2022	Model # XH-2002
Portable heating lamp	Underwriters Laboratories		Helps to maintain polymer temperature at 37 °C
Inverted fluorescent microscope	Zeiss		Model Axiovert 25 CFL
Heated water bath	VWR		Model # 1235PC
Rocking platform	VWR		Series 100
Multiskan FC microtiter plate reader	Thermo Scientific		Type 357
Cell culture incubator	VWR		Model # 2350T
Purifier class II biosafety cabinet	Labconco		Delta Series