Macroporous interpenetrating cryogel network of poly(acrylonitrile) and gelatin for biomedical applications
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- Jain, E., Srivastava, A. & Kumar, A. J Mater Sci: Mater Med (2009) 20: 173. doi:10.1007/s10856-008-3504-4
Cryogels are supermacroporous gel network formed by cryogelation of appropriate monomers or polymeric precursors at subzero temperature. The beneficial feature of this system is a unique combination of high porosity with adequate mechanical strength and osmotic stability, due to which they are being envisaged as potential scaffold material for various biomedical applications. One of the important aspect of cryogel is simple approach by which they can be synthesized and use of aqueous solvent for their synthesis which make them suitable for different biological applications. Various modifications of the cryogels have been sought which involves coupling of various ligands to its surfaces, grafting of polymer chain to cryogel surface or interpenetrating networks of two or more polymers to form a cryogel which provides diversity of its applications. In the following work we have synthesized full interpenetrating network of polyacrylonitrile (PAN)-gelatin with varied gelatin concentration. The PAN-gelatin cryogel interpenetrating network is macroporous in nature and has high percentage swelling equlibirium in the range of 862–1,200 with a flow rate greater than 10 ml/min, which characterizes the interconnectivity of pores and convective flow within the network. PAN-gelatin interpenetrating cryogel network has good mechanical stability as determined by Young’s modulus which varies from 123 kPa to 819 kPa depending upon the polymer concentration. Moreover they are shown to be biocompatible and support cell growth within the scaffolds.
The application of cryogels matrices was successfully demonstrated where the affinity supermacroporous adsorbent can be used to design a novel cell separation strategy. Protein A when covalently coupled to supermacroporous cryogel matrix could specifically bound more than 90% of IgG-labeled B-lymphocytes, while non-labeled T-lymphocytes passed through the column. The bound lymphocytes were eluted with 60–70% recoveries without significantly impairing the cell viability . Similar results were obtained when studying the binding of CD34+ cell to protein A-cryogel matrix . A breakthrough study showed that these cryogel matrices can be used for releasing the cells by squeezing the gels when the cells are affinity bound on such matrices . These results have shown tremendous potential of such cell separation strategy to be applicable to different types of cell populations and particularly targeting stem cells and other medically relevant cell types. Also this provided a very convenient and elegant way to release the bound cells from the matrices which otherwise can be a major bottleneck in positive cell selection and separation.
In a different application, supermacroporous cryogel matrices of poly(acrylamide) modified with gelatin was used as a polymeric support for the cultivation of mammalian cells and for the design of the continuous bioreactor for the production of therapeutic proteins like urokinase  and monoclonal antibody . A polymeric cryogel support with covalently coupled gelatin has been used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. Two different cell lines Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor . Similarly monoclonal antibodies were secreted continuously after the hybridoma cell line M1239 were grown on the polymeric cryogel support . The interconnected pore morphology in cryogel bioreactor provide a network of capillary through which the media is circulated and thus consumed by the cells which are being cultured on the porous surfaces. This unique pore morphology and flexibility of using different polymeric precursors for development of cryogel makes them potential materials for cell cultivation matrices and thus as bioreactors. Essentially the continuous pore structure in cryogel helps to mimic currently most widely used bioreactors for mammalian cell culture i.e. hollow fiber bioreactor. This provides cell growth conditions that are very similar to in vivo conditions. Thus a cryogel holds considerable potential to be developed into disposable bioreactor for mammalian cell culture.
Recently grafting of suitable polymer inside porous surface of cryogel has been reported  for binding and elution of positively charged proteins. The large surface area and suitable chemistry of poly(acrylamide) cryogel provide suitable surface for grafting of acrylic acid for selective removal of oppositely charged biomolecules. The grating of PNIPAAm onto poly(hydroxyethyl methacrylate) (PHEMA) cryogel using atom transfer radical polymerization (ATRP) has also been reported which present a temperature responsive matrix for fine tuning of cell adhesion properties .
Macroporous interpenetrating networks (IPNs) of cryogels have also been prepared which provide new properties to cryogel materials with respect to mechanical strength and porous architecture. These interpenetrating cryogel networks can be synthesized in two ways either as single freezing or double freezing methods. In single freezing method more than one monomer/polymer precursors are dissolved in aqueous system and allowed to polymerize and/or gelate at low temperature. After certain incubation time the interpenetrating cryogel network can be obtained after thawing at room temperature. In another approach the recurrent synthesis of the new cryogel network inside the interconnected macropores of another pre-synthesized cryogel network is being done. The widely distributed and open porous structure of the interpenetrating cryogel network with soft tissue like elasticity, mechanically stable and pore volume upto 80–91% make them attractive in many biotechnological applications, especially as potential cell scaffolds in tissue engineering, bioreactor development, chromatography medium for processing particulate containing matter, cell chromatography and for the preparation of robust immobilized cell systems. These can also be designed or tailored while incorporating appropriate gel surface chemistry with required mechanical strength and formats (rods, slabs, sheets).
As an example here we are presenting the synthesis of interpenetrating cryogel network of gelatin with poly(acrylonitrile) by single freezing technique. Gelatin is known to be biocompatible and biodegradable in nature. As is the case with some of the other synthetic polymers, poly(acrylonitrile) (PAN) is biocompatible, moderately hydrophobic and is known to be supporting cell growth. The interpenetrating network of PAN-gelatin was synthesized to establish cell matrix interaction for its application in bioreactor development and tissue engineering. These IPN were further characterized with respect to porosity, mechanical strength and biocompatibility.
2 Materials and methods
Acrylonitrile (AN) monomer was purchased from Lancaster (Morecambe, England). Gelatin (from cold fish skin), Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Sigma Chemical Company (St. Louis, USA). N,N′-methylene bis(acrylamide) (MBAAm), ammonium persulphate (APS), N,N,N′,N′ tetramethylethylenediamine (TEMED) were bought from Sisco Research Laboratories (Mumbai, India). Glutaraldehyde was obtained from S.D. fine chemicals (Mumbai, India). Fetal Bovine Serum (FBS), antibiotic Penicillin/Streptomycin solution were obtained from Hyclone (Utah, US). All other chemicals used were of analytical grade.
2.2.1 Preparation of PAN-gelatin cryogel
Polyacrylonitrile gelatin cryogel interpenetrating network was prepared by free radical polymerization of acrylonitrile (AN) and simultaneous gelation with MBAAm and glutaraldehyde as crosslinkers for polyacrylonitrile and gelatin polymers, respectively. The cross-linker concentration for AN: MBAAm was kept constant at (5:1), and while the monomer concentration for acrylonitrile was 8%. The ratio of acrylonitrile to gelatin was varied and two ratios of AN: gelatin was used that is 2:1 and 5:1 glutraldehyde was used as a crosslinker for gelatin at a ratio of 20:1 (gelatin: glutaraldehyde). Briefly 8 ml of AN (8% v/v) was mixed with MBAAm (1.6 g) [AN: MBAAm; 5:1]; in degassed deionized water. To this solution gelatin was added and the ratio of gelatin to acrylonitrile was varied as 2:1 and 5:1 thus 4 g and 1.6 g of gelatin was added, respectively and volume was made upto 100 ml. The solution was degassed again and APS (500 mg), TEMED (500 μl) and glutaraldehyde (800 μl for 2:1 AN-gelatin, 320 μl for 5:1 AN-gelatin of 25% solution, respectively) was added and reaction mixture was stirred. The solution was then poured in 2.5 ml syringes and frozen immediately at −12°C. The polymerization was allowed to proceed for 16 h after which the gels were thawed at room temperature. The gels were washed immediately with water to remove any unreacted monomer. The gels were then vacuum dried and stored at room temperature. Further the gels were used for characterization studies.
2.2.2 Swelling studies
2.2.3 Measurement of flow resistance of cryogel column
The flow resistance of the cryogel columns (2.5 ml) was evaluated at flow rates of 1–10 ml/min using peristaltic pump, registering the flow rate at given pump settings. In a separate experiment, the pump settings were calibrated at flow rate with no column connected according to Adrados et al. .
2.2.4 Scanning electron microscopic (SEM) analysis
PAN-gelatin cryogel were subjected to SEM analysis. All the samples were ethanol dried. The samples were put consecutively in increasing concentration of ethanol that is 20% v/v, 40% v/v, 60% v/v, 80% v/v and finally in 100% v/v ethanol. The samples were then vacuum dried overnight before gold coating. The SEM pictures were taken using FEI Quanta 200 and the pore diameters of cryogel column were measured arbitrarily.
2.2.5 Mechanical analysis
2.3 In vitro cell culture studies
The fibroblast cell line i.e. CHO were grown over PAN-gelatin cryogel to check the adherence of cells over the material. The cryogel samples were sterilized by ethanol and then autoclaved. The scaffolds of 1 cm diameter and 4–5 mm thickness were placed in 12-well polystyrene tissue culture dish. The dried scaffolds were equilibrated with DMEM supplemented with 10% FCS and 1% P/S. The medium were then removed from the scaffolds and 2 × 105 cells/ml were seeded in a total volume of 1 ml. The cells were allowed to culture in 5% CO2 and humidified environment at 37°C. The medium was changed after every 2 days and the cell attachment was observed on SEM after 10 days of culture.
3 Results and discussion
3.1 Synthesis of polyacrylonitrile-gelatin interpenetrating cryogel network
3.2 Swelling kinetics
3.3 Scanning electron microscope analysis
3.4 Flow rate analysis
PAN-gelatin cryogel interpenetrating network showed low resistance to flow of water and water could easily pass through them at a flow rate greater than 10 ml/min, respectively. Flow rate measurement is simple and indirect estimation of the interconnectivity and porosity of macroporous scaffolds. Such flow rate through the cryogel scaffold is high enough for efficient cell seeding and media transport to immobilized cells . Thus these cryogel scaffolds can be a potential cell scaffold material and may be used for various tissue engineering applications.
3.5 Mechanical analysis
3.6 In vitro cell culture studies
In conclusion the supermacroporous cryogels provide useful scaffold material properties for biomedical applications. High porosity, interconnected pore morphology and high surface area are the typical characteristics of these cryogel scaffolds. All these parameters have been found to play an important role in cell seeding, growth, and migration, mass transport, gene expression, and new tissue formation in three dimensions. In this regard polyacrylonitrile-gelatin interpenetrating cryogel network with their suitable properties like high mechanical stability and biocompatibility can be a potential material for tissue engineering and as scaffold for cell immobilization.
The work was financially supported from Department of Biotechnology (DBT) and Department of Science and Technology (DST), Govt. of Indian organizations. EJ would like to thank IITK for granting fellowship during the Ph.D. programme. AS gratefully acknowledges the fellowship received from University grants commission, India.