Journal of Materials Science: Materials in Medicine

, 20:173

Macroporous interpenetrating cryogel network of poly(acrylonitrile) and gelatin for biomedical applications

Authors

  • Era Jain
    • Department of Biological Sciences and BioengineeringIndian Institute of Technology Kanpur
  • Akshay Srivastava
    • Department of Biological Sciences and BioengineeringIndian Institute of Technology Kanpur
    • Department of Biological Sciences and BioengineeringIndian Institute of Technology Kanpur
Article

DOI: 10.1007/s10856-008-3504-4

Cite this article as:
Jain, E., Srivastava, A. & Kumar, A. J Mater Sci: Mater Med (2009) 20: 173. doi:10.1007/s10856-008-3504-4

Abstract

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.

1 Introduction

“Cryogels” are supermacroporous hydrogels formed at subzero temperature which provides unique properties for it application in bioengineering. Synthesis of these materials at subzero temperatures provide the cryogels with large pores (upto 200 μm) having spongy and elastic morphology [13]. The monomer or polymer precursors are dissolved in aqueous media and polymerized at subzero temperature for desired time period. At such low temperature water forms ice crystals which grow and connect to each other. After complete polymerization and thawing of gels at room temperature the ice crystal melts, leaving behind large interconnected pores. The cryogel materials can be produced from both hydrophilic and hydrophobic monomers and polymeric precursors in different sizes and formats (monoliths, sheets, discs, micro-titer plate formats, etc.) depending upon the application and scale of operation. These characteristics in combination with osmotic, chemical and mechanical stability and good mass transport and convective flow properties can be well utilized in cell separations, cell culture and cell-biomaterial applications [35]. The cryogel scaffold has shown interesting applications in the area of cell separation like release of particular cell type after they are affinity bound [6], by squeezing method which is possible due to highly elastic and spongy nature of cryogels [7]. The cryogels have also shown application as scaffolds for culturing of the cells for therapeutic protein production such as urokinase [8] and monoclonal antibodies [9]. The porous structure of the cryogels has been studied by various microscopic techniques like using optical microscopy [10], scanning electron microscopy (SEM) [11], environmental scanning electron microscopy (ESEM) [12], confocal microscopy [13] and also microcomputed tomography [13] (Fig. 1). The microcomputed tomography was used for the analysis of porous structure of the gelatin macroporous gel, prepared through combination of cryogenic treatment of a chemically cross-linked gelatin gels, followed by removal of the ice crystals formed through lyophilization (freeze-drying technique) [14]. The two-dimensional (2-D) cross-sections of gelatin gels were used to segment the images and determine their 3-D porosity and pore size distribution using the software (μCT analysis).
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-008-3504-4/MediaObjects/10856_2008_3504_Fig1_HTML.jpg
Fig. 1

Macroporous cryogel studied through various microscopy techniques. (a) Environmental Scanning Electron Microscope (ESEM), (b) Optical Microscope (OM), (c) Confocal Microscope (CM), (d) Scanning Electron Microscope (SEM) and (e) Micro Computer Tomography (μCT analysis). The ESEM (a) is presented for poly(vinyl alcohol) (PVA) cryogel and reproduced from [10] with permission. OM (b) is presented for PVA cryogel and reproduced from [10] with permission. CM (c) is presented for dextran-methyl methacrylate cryogel and reproduced from [23] with permission. SEM (d) is presented for PNIPAAm cryogel and reproduced from [7] with permission. Micro-CT images of the hydroxy ethylmethacrylate cryogel 3-D reconstruction (e) is reproduced from [17] with permission

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 [3]. Similar results were obtained when studying the binding of CD34+ cell to protein A-cryogel matrix [15]. 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 [6]. 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 [8] and monoclonal antibody [9]. 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 [8]. Similarly monoclonal antibodies were secreted continuously after the hybridoma cell line M1239 were grown on the polymeric cryogel support [9]. 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 [16] 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 [17].

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

2.1 Materials

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 Methods

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

The PAN-gelatin cryogels were dried under vacuum for swelling studies. The dried samples were weighed and then immersed in buffer to determine the equilibrium swelling ratio. PAN-gelatin cryogels were immersed in phosphate buffer (pH 7.5) and samples were removed at regular time intervals and weighed after wiping of the excess water with filter paper. This was done till gels attained a constant weight. The equilibrium degree of swelling is expressed as:
$$ \alpha _{{{\text{eq}}}} = \left[ {\left( {W_{{{\text{eq}}}} - W_{{\text{d}}} } \right)/W_{{\text{d}}} } \right] \times 100 $$
where Weq is the weight of the swollen gel at equilibrium and Wd is the dried weight of the gel. The results were taken as the mean values of five measurements.

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. [18].

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

The compressive modulus of PAN-gelatin cryogels was determined using dynamic mechanical analyzer (DMA) Zwick/Roell Z010. Water swollen samples were taken of the dimensions, 10 mm height and 13 mm diameter, and subjected to compression analysis. The swollen samples were mounted on DMA. The samples were placed between two arms of load frame and then compressed up to 80% of the total length, from where the compressed cryogel can regain its original shape on addition of liquid. The applied force was recorded and change in column length due to compression was measured. A test speed of 1 mm/min was selected for each sample. The compressive modulus was calculated at 10% strain using the following equation:
$$ E = \left( {F/A} \right)/\left( {{{\Updelta}}l/l} \right) $$
where E is the elastic modulus, F is the applied force, A is the cross-sectional area of the test sample, l is the initial length of the test sample and Δl is the change in length under the compressive force. For each type of cryogel, five samples were analyzed and the average modulus was determined.

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

Interpenetrating networks of polyacrylonitrile-gelatin (PAN-gelatin) was prepared at sub-zero temperature by a combination of free radical crosslinking polymerization of acrylonitrile and MBAAm and covalent crosslinking of gelatin chains using glutaraldehyde. The interpenetrating cryogel was formed by single step freezing of both the precursors simultaneously. The ratio of PAN:gelatin was varied and two different cryogels with ratios of 5:1 and 2:1 were synthesized. The total concentration of polymers in PAN-gelatin (2:1) and (5:1) was 12% and 9.6%, respectively. The interpenetrating network obtained by polymerization at low temperature resulted in a supermacroporous gel which was very rigid in nature (Fig. 2). This supermacroporous nature was generated by the mechanism of cryogelation which plays an important role at such low temperatures [19]. The scaffolds generated using this mechanism had high porosity along with high mechanical stability. This high mechanical strength is generated due to dense pore walls formed by cryoconcentration of polymers. One added advantage of combining synthetic polymer with a natural polymer like gelatin was that, highly rigid yet porous and mechanically stable cryogels were obtained which was not possible when any of the polymers were used individually. Thus an interpenetrating network of gelatin with acrylonitrile is beneficial in this respect.
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-008-3504-4/MediaObjects/10856_2008_3504_Fig2_HTML.jpg
Fig. 2

Digital photographs of PAN-gelatin cryogel interpenetrating network

3.2 Swelling kinetics

The equilibrium degree of swelling (αeq %) for both the cryogels showed that both of them swelled very fast and attained equilibrium within 2 min (Fig. 3). PAN-gelatin (2:1) cryogels interpenetrating network had αeq % value of 862 while PAN-gelatin (5:1) had a value of 1299. This can be explained on the basis of the total polymer concentration which increases as the PAN:gelatin ratio is varied. In case of PAN:gelatin (5:1) the total concentration is 9.6% while in PAN:gelatin (2:1) cryogel it is 12%. Thus as the polymer concentration increases the walls become thicker and could not swell upto the same extent in the case of a lower concentration gel. This fast swelling behavior is a characteristic response shown by such large macroporous polymeric cryogels. Cryogels are made up of large interconnected pores which allow fast transport of solvent molecules within thin walls over short distances across the macroporous structure. The rapidity of response in such supermacroporous structure depends upon total monomer concentration, cross-linking density, pore wall thickness, temperature at which the gels are prepared, etc. The response time is also affected by the thickness of the gel i.e., the distance between the outer boundaries to central parts of cryogel, larger the distance slower the rate of swelling and shrinkage due to slow rate of mass and heat exchange due to increased distance [12, 19]. Interconnectivity of pores plays a crucial role in fast swelling of cryogels as solvent molecule could move by convection across this network, while in conventional hydrogels this process is diffusion dependent and thus slower [7, 12, 1921].
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-008-3504-4/MediaObjects/10856_2008_3504_Fig3_HTML.gif
Fig. 3

Swelling kinetics of PAN-gelatin cryogel interpenetrating network: Plot of αeq % versus swelling time. The swelling behavior was estimated for PAN-gelatin 2:1 (■); 5: 1 (▲)

3.3 Scanning electron microscope analysis

Surface morphology of PAN-gelatin cryogel interpenetrating network, showed some unique feature which were evident in the scanning electron microscope (SEM) images (Fig. 4). The pore size of the two cryogels PAN-gelatin interpenetrating network (2:1) and PAN-gelatin interpenetrating network (5:1) was almost similar and lied in the range of 50–100 μm. As seen in the SEM pictures the surface of pore walls of PAN-gelatin cryogel interpenetrating network was smooth and walls had pores too. The size of the pores in the walls was found to vary between 5 and 20 μm. There was some difference in the surface smoothness and the structure of pores present in the pore walls as the concentration of gelatin was varied. The presence of pores in the walls is an advantageous feature since it increases the total surface area available for cell growth and proliferation and it may also help in better nutrient transport.
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-008-3504-4/MediaObjects/10856_2008_3504_Fig4_HTML.jpg
Fig. 4

SEM images of PAN-gealtin cryogel interpentrating network (a) PAN gelatin (2:1); (b) PAN-gelatin (5:1)

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 [22]. Thus these cryogel scaffolds can be a potential cell scaffold material and may be used for various tissue engineering applications.

3.5 Mechanical analysis

The compression properties of cryogels was determined by exerting uniaxial stress on the cryogel which gives a relationship between the stress exerted and proportional strain experienced by the gel. The slope of the curve between stress versus strain gives the Young’s modulus of the gel which is an estimate of the elasticity and mechanical strength of the cryogel. The Young’s modulus for PAN-gelatin cryogel interpenetrating network varied with the increasing concentration of gelatin. For a ratio of PAN to gelatin of 5:1 it was 819 kPa while for a ratio of 2:1 the value is 123 kPa (Fig. 5). This shows that increasing concentration of gelatin makes the cryogel more spongy and elastic. This is due to hydrophilic nature of gelatin as its concentration increases the amount of polymer bound water increases making the polymer chains more flexible and elastic such that they can move freely thus making cryogels more elastic. In general the gels were quite rigid yet elastic and could be compressed upto 80% of their original length without loosing shape. The cryogels took 15–20 min to regain their original shape after stress was applied. This shows that though the cryogels are spongy, the polymer chains are not free enough to regain shape immediately.
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-008-3504-4/MediaObjects/10856_2008_3504_Fig5_HTML.gif
Fig. 5

Compression analysis of PAN-gelatin cryogels: Stress versus Strain curve were plotted for different ratio of PAN:gelatin

3.6 In vitro cell culture studies

The CHO cells were allowed to grow for a period of 7 days over the PAN-gelatin cryogel scaffold. PAN-gelatin scaffolds were found to be biocompatible and a potential material for cell growth and proliferation as is evident by SEM images demonstrating uniform growth of CHO cells over the material surface (Fig. 6a). CHO cells adhered well over the surface of the cryogel scaffold and secreted extracellular matrix which was spread all over the matrix (Fig. 6b). Since polyacrylonitrile is a moderately hydrophobic polymer and gelatin being a naturally occurring denatured form of collagen, thus combination of the two provide a good scaffold material for cell adherence and proliferation.
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-008-3504-4/MediaObjects/10856_2008_3504_Fig6_HTML.jpg
Fig. 6

Scanning electron microscope images of CHO fibroblast cells adhering over the surface of PAN-gelatin cryogel (a) at 500× (200 μm scale); (b) 5000× (20 μm scale) showing the presence of extracellular matrix all over the scaffold as rough white matrix. The images were taken after 7 days of culture

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.

Acknowledgements

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.

Copyright information

© Springer Science+Business Media, LLC 2008