Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds.
Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 µg/g, and RNA by 61% down to less than 10 µg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria.
Department of Surgery, Columbia University Medical Center, USA Department of Physiology, Maastricht University Medical Center, The Netherlands hugo@sondermeijer.net.
Hugo P
HP
Sondermeijer
Department of Surgery, Columbia University Medical Center, USA Department of Surgery, Section of Transplantation, University of Chicago Medicine, USA.
Piotr
P
Witkowski
Department of Surgery, Columbia University Medical Center, USA.
David
D
Woodland
Department of Surgery, Columbia University Medical Center, USA.
Tetsunori
T
Seki
Department of Mechanical Engineering, Eindhoven University of Technology, The Netherlands.
Frank J
FJ
Aangenendt
Departments of Cardiology and Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, The Netherlands.
Arnoud
A
van der Laarse
Department of Surgery, Columbia University Medical Center, USA Mesoblast Limited, Melbourne, Australia.
Silviu
S
Itescu
Department of Surgery, Columbia University Medical Center, USA.
Mark A
MA
Hardy
D065966:Absorption, Physicochemical; D017607:Aluminum Compounds / Q000009:adverse effects* / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D001672:Biocompatible Materials / Q000009:adverse effects / Q000737:chemistry / Q000302:isolation & purification; D002606:Charcoal / Q000737:chemistry; D011232:Chemical Precipitation; D000431:Ethanol / Q000737:chemistry; D005549:Foreign-Body Reaction / Q000276:immunology* / Q000517:prevention & control; D007113:Immunity, Innate / Q000187:drug effects / Q000276:immunology; D008297:Male; D008422:Materials Testing; D008567:Membranes, Artificial; D011145:Polyvinyls / Q000737:chemistry*; D051381:Rats; D011917:Rats, Inbred Lew; D017670:Sodium Compounds / Q000009:adverse effects* / Q000737:chemistry / Q000302:isolation & purification*; D054457:Tissue Scaffolds / Q000009:adverse effects*; D014462:Ultrafiltration / Q000379:methods*; D005465:Fluorocarbon Polymers
D017607:Aluminum Compounds; D001672:Biocompatible Materials; D002606:Charcoal; D000431:Ethanol; D008567:Membranes, Artificial; D011145:Polyvinyls; D017670:Sodium Compounds; C513667:sodium aluminum hydride; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers
Alginate; bioengineering; purification; scaffolds; transplantation