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Cytocompatibility studies of mouse pancreatic islets on gelatin - PVP semi IPN scaffolds in vitro: Potential implication towards pancreatic tissue engineering Sudhakar Muthyala, Ramesh R. Bhonde & Prabha D. Nair Published online: 01 Nov 2010.

To cite this article: Sudhakar Muthyala, Ramesh R. Bhonde & Prabha D. Nair (2010) Cytocompatibility studies of mouse pancreatic islets on gelatin - PVP semi IPN scaffolds in vitro: Potential implication towards pancreatic tissue engineering , Islets, 2:6, 357-366, DOI: 10.4161/isl.2.6.13765 To link to this article: http://dx.doi.org/10.4161/isl.2.6.13765

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research paper

Research paper

Islets 2:6, 357-366; November/December 2010; ©2010 Landes Bioscience

Cytocompatibility studies of mouse pancreatic islets on gelatin–PVP semi IPN scaffolds in vitro Potential implication towards pancreatic tissue engineering Sudhakar Muthyala,1 Ramesh R. Bhonde2 and Prabha D. Nair1,* Division for Tissue Engineering & Regeneration Technologies; Biomedical Technology Wing; Sree Chitra Tirunal Institute for Medical Sciences and Technology; Trivandrum, Kerala India; 2Stempeutics Research Pvt. Ltd.; Manipal Hospital; Bangalore, Karnataka India

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Key words: diabetes, islet culture, in vitro, gelatin scaffolds, inter penetrating network

Type 1 diabetes is a chronic disorder that results due to auto immune destruction of insulin producing cells, which leads to hyperglycemia in the blood. The development of an ideal scaffold for maintaining the structure and function of islets is a challenge in the field of pancreatic tissue engineering. In this study, gelatin (G) as well as gelatin/PVP (GP) semi interpenetrating polymer network scaffolds have been fabricated by freeze drying technique and cross linked with gluteraldehyde (GTA) and 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), which was abbreviated as GG, GPG (cross linked with GTA) GE and GPE (cross linked with EDC). The presence of gelatin and PVP in GPE and GPG scaffolds was confirmed through FTIR and TGA. The medium uptake ability of GPE and GPG scaffolds were higher than GG and GE scaffolds. The scaffolds were then analyzed for its ability to maintain the viability and function of mouse pancreatic islet cells in vitro. The results showed that the islets can adhere, but they tend to lose the structure and function on all the scaffolds after day 7, except on GPE where they remained intact up to day 30. Thus the present study clearly demonstrates that gelatin incorporated with PVP and cross linked with EDC scaffolds could support and maintain islet cells for prolonged period.

Introduction Type 1 diabetes is a chronic disorder that occurs due to auto immune destruction of insulin producing β cells and leads to hyperglycemia in the blood,1 which currently affects 200 million people all over the world.2 Intensive monitoring of blood glucose level and insulin injection is the current therapy to treat diabetes, but it is not a permanent cure because over or under treatment can lead to life threatening complications such as coma, diabetic retinopathy, neuropathy, nephropathy, vasculopathy etc. Normal blood glucose level may be obtained by replacing the damaged pancreatic islets with the whole pancreas, pancreatic islets, insulin secreting cell lines and stem cell-derived insulin producing cells. Shapiro et al.3 could achieve euglycemia in seven patients with type 1 diabetes up to one year by following the Edmonton protocol with human pancreatic islets. Since 2000, several hundred people have received islet transplants, of which 50–68% of patients did not require additional insulin a year subsequent to transplantation.4 Despite these promising results, insulin dependence is not sustainable, which can attributed to the inability to maintain the functionality of islets in vitro and in vivo for prolonged periods.

Transplanted islets are expected to survive longer by growing the cells on a three dimensional (3D) biodegradable interconnected porous scaffold, by applying the principle of tissue engineering. The scaffold serves as a substitute for extracellular matrix (ECM) and facilitate the cell-cell and cell-ECM interactions, which may help to achieve euglycemia.5 For that, the scaffolds are expected to have adequate (a) surface chemistry to facilitate cell attachment and proliferation, (b) interconnected porosity with open pores to favor the transport of nutrients and excretory products and (c) mechanical properties to match the intended site of implantation and handling.6 Based on the concept of generating tissue-engineered biohybrid pancreas, some natural as well as synthetic polymeric materials have been exploited in this field. Nevertheless, many of these scaffolds could not maintain the structure and function of islet for long period in vitro. Gelatin is a protein produced by partial hydrolysis of collagen, which is a major ECM component of connective tissue, skin, bone, cartilage and ligaments.7 Gelatin can form 3D gels in water and hydrophilic nature of these gels allows easy and rapid cell seeding as well as the maintenance of viability and functionality of cells in vitro.8 Even though it shows better cytocompatibility

*Correspondence to: Prabha D. Nair; Email: [email protected] Submitted: 08/18/10; Revised: 09/24/10; Accepted: 09/25/10 Previously published online: www.landesbioscience.com/journals/islets/article/13765 DOI: 10.4161/isl.2.6.13765 www.landesbioscience.com Islets

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and induces vasculature under in vivo conditions, the weak mechanical strength limits its use in pancreatic tissue engineering applications. The mechanical strength of hydrogels can be improved by preparing it as interpenetrating polymer network (IPN), which is defined as a physical mixture of at least two polymers that have been synthesized or cross linked in the presence of each other with no significant degree of covalent bonds between them.9 IPN made of a synthetic and a natural polymer combine the mechanical properties of the synthetic component with the biological properties of the natural one. For the present study, IPN of poly (vinylpyrrolidone) (PVP) and gelatin were prepared, in which gelatin was cross linked either with gluteraldehyde (GTA) or 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) in order to stabilise them in cell culture media for tissue engineering applications. The synthesis and characterization of IPN systems based on gelatin/synthetic polymers such as poly (2-hydroxyethylmethacrylate),10 PVP11 and PVP/chitosan12 have been reported. PVP has been widely used to coat the medical devices surface in order to prevent the protein and cell adhesion under in vivo applications13-15 and has been selected in order to improvise the hydrophilicity of gelatin scaffolds. Thus the objective of the study is to characterize four types of scaffolds (a) Gelatin: PVP IPN scaffolds cross linked with EDC (GPE) (b) Gelatin: PVP IPN scaffolds cross linked with GTA (GPG) (c) Gelatin scaffolds cross linked with EDC (GE) (d) Gelatin scaffolds cross linked with GTA (GG), for its ability to maintain the viability and functionality of islets in vitro. Results Physicochemical characterization of 3D porous scaffolds. FTIR analysis was done in order to confirm the presence of gelatin and PVP in Gelatin: PVP IPN scaffolds. Parental gelatin showed the characteristic peaks of NH2 and carbonyl stretching at 1,543 cm-1 and 1,627 cm-1 respectively. Parental PVP showed the characteristic peak of carbonyl group at 1,648 cm-1. Thus the peaks at 1,534 cm-1 and 1,630 cm-1 confirmed the presence of gelatin and PVP in GPE and GPG scaffolds. The peak of aldehyde groups (GTA) at 1,711 cm-1 and of N=C=N (EDC) at 2,861 cm-1 were absent in the spectra of four scaffolds, which indicates the absence of remnant GTA or EDC (Fig. 1). TGA curve showed the decomposition of gelatin and PVP at 261°C and 393°C respectively, where as GPE and GPG semi IPN decomposed at 278°C. In DTA spectra, gelatin showed the characteristic peak at 322.79°C and PVP at 430.54°C, with single step decomposition of these materials. However, semi IPN showed 2 stage decomposition, one of gelatin (316.63°C) and another of PVP (441.21°C), which indicates the presence of two components in the semi IPN scaffold (Fig. 2). SEM showed the interconnected porous structure with the pore size ranging from 60–100 μm. GPE and GPG have shown small pores on the walls of scaffolds, while non porous smooth walls was noted on GE and GG (Fig. 3). Micro-CT depicted average pore size and pore size distribution, which is represented in Table 1. In dry state, the scaffolds have shown pore size in the range of 60–80 μm, whereas in wet state, the pore size increased to 100–150 μm.

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GPE showed statistically significant high swelling percentage (p ≤ 0.05) within 5 min followed by GPG, GE and GG (GPE > GPG > GE > GG) (Fig. 4). This data was supported by contact angle studies where GPE and GPG showed a measurement of (27 ± 3)° and (47 ± 2)° respectively. However, GG and GE showed a measurement of (83 ± 3)° and (58.5 ± 3)° respectively. All the four scaffolds started degradation on day 10, and was in the order of GPE > GPG = GE > GG on day 30 (Fig. 5). The compressive modulus of GPE and GPG was lower when compared to GG and GE in dry state and wet state (Table 2). Cytocompatibility studies with mouse pancreatic islet cells in vitro. The isolated mouse islets were viable after 48 h (Fig. 6) and stained positive for DTZ staining as well as C-peptide/glucagon expression, which confirmed their endocrine origin (Fig. 6). As a preliminary cytotoxicity assay, the islets were cultured in presence of four types of scaffolds for 48 h and the results showed that the cells retained their morphology and viability (Fig. 7) when compared to control mouse islets (CMI). The islets were then cultured on/within the scaffolds and viability was determined by confocal microscopy. The cells were viable (Fig. 8) and could secrete insulin up to day 4, which was similar to CMI (Fig. 9). There was no statistically significant difference between all the scaffolds. However, on day 7, the cells lost their spherical morphology together with low level of insulin secretion on all scaffolds, except GPE (data not shown). The viability (Fig. 10) together with insulin secretion (Fig. 11) on GPE scaffolds were maintained up to day 30, whose performance were almost similar to freshly isolated islet cells (since CMI lost their morphology and functionality after day 7, fresh islets (300) was taken for comparison for glucose challenging studies). The islets on GPE scaffolds have also shown positivity for C-peptide and glucagon markers on day 30 (Fig. 12). Discussion In Edmonton protocol, cadaver pancreatic islets are infused into the portal vein of patients, as this site can aid the vascularization of islets graft and have the ability to secrete insulin in response to ambient blood glucose levels. But in long treatment regimes, failures occur and the exact reasons are undetermined.3 Loss of ECM proteins in the primary isolation of pancreatic islets, loss of cell-cell and cell-ECM interactions and disruption of microvasculature of the islets may cause islets to undergo apoptosis, and may result in the failure of the graft in the patient.16,17 Therefore, the provision of a matrix that mediate cell adhesion and activate intracellular signaling pathways may be an important requirement for maintaining the function and viability of transplanted islets. In this study, 3D porous gelatin–PVP semi IPN scaffolds have been fabricated to culture pancreatic islets under in vitro conditions. Gelatin has been selected because it is a partially denatured product of collagen (basement membrane of ECM of adult pancreatic islets) and contains Arg-Gly-Asp (R-G-D) sequence that help in binding of the integrins such as α5β1 and α5β3 of adult pancreatic islets and ensure its intact structure.18 Gelatin has also known to be less antigenic when compared to collagen19

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Figure 1. FTIR spectra of hybrid scaffold (A) GPE (B) GPG. The spectra were compared to parent materials gelatin and PVP.

and can be completely resorbable under in vivo conditions. Since gelatin can be easily dissolved in aqueous solution, it has to cross link, which was done either by GTA or by EDC. Cross linking with GTA involves reacting the free amino groups of the polypeptide chains (lysine or hydroxylysine) with aldehyde groups of the GTA.20 However during in vivo transplantation, GTA can be toxic due to the degradation of polymer.20,21 Hence biocompatible water soluble carbodiimide class cross linker EDC has also been tried, which is widely used for cross linking collagen in dermal tissue engineering.22 EDC crosslink the amino groups of

one polypeptide chain with the carboxylic groups of the adjacent polypeptide chain by forming the extra amide bond without its incorporation.23,24 The presence of gelatin and PVP in GPE scaffolds was analyzed by FTIR spectrum. The results suggest that neither the fabrication technique, cross linking mechanism nor the washing procedures altered the chemical composition. The carbonyl shift in the peak of PVP from 1,648 to 1,630 cm-1 was due to the formation of hydrogen bonding between C=O group of PVP and OH group of gelatin. This was supported by a shift in the peak of

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Figure 2. (A) TGA and (B) DTA of gelatin-PVP semi IPN, which was compared to parent materials gelatin and PVP. GPE is only represented here.

Figure 3. Scanning electron micrographs of the scaffolds showing interconnected porous structure (pore size ranging from 60–100 μM).

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hydroxyl group of gelatin from 3,282 to 3,273 cm-1. The presence of PVP/gelatin in GPE scaffolds were once again confirmed by TGA/ DTA, which showed two step decomposition. These results also confirmed the stability of material at 37°C. The interconnected open porous structure is an important parameter in promoting cellular ingrowth and angiogenesis. In this study, the scaffolds were fabricated by freeze drying method, and used water as porogen that could generate an interconnected open porous structure as confirmed by SEM and micro-CT. The result is likely due to highly parallel ice crystal growth, which in turn is caused by strong one dimensional nature of the thermal gradient established during the freezing.25,26 The average pore size and pore size distribution was determined by micro-CT, both in dry and wet state. The values were higher for GG and GE scaffolds than GPE and GPG scaffolds in dry state, which may be because of gelatin-water binary system. When the temperature of this system is altered (viz; freezing and subsequently drying), the system undergoes solidliquid phase separation, favoring a binodal form of decomposition. The pores of the resultant scaffold thus formed will be similar to that of the solvent crystallites (i.e., ice crystals). In GPE and GPG, the average pore size was low because the introduction of PVP into gelatin

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Table 1. Porosity parameters of scaffolds in dry and wet (W) state Parameters

GPE

GPEW

GE

GEW

GG

GGW

GPG

GPGW

Average pore size (µm)

60 ± 15

150 ± 20

80 ± 15

120 ± 15

80 ± 14

100 ± 15

60 ± 15

150 ± 23

Average pore size % distribution

45

25

48.5

20

50

25

50

30

Figure 4. Medium uptake ability of the scaffolds as a function of time (n = 6). The performance among the groups was in the order of GPE > GPG > GE > GG. The percentage of swelling of GPE was statistically significant (*) (p ≤ 0.05) when compared to all other scaffolds.

solution may creates a PVP/gelatin/water ternary system, which favors more of liquid-liquid phase separation and subsequent spinodal decomposition. Furthermore, PVP may interferes with the crystallization of water to ice, thus creating smaller ice crystallites that in turn give rise to scaffolds with smaller pore size. The morphology of the resulting scaffold was also different (lacy structure on scaffold walls—SEM image) as the final structure arises from the spinodal decomposition of PVP/gelatin/water system.25 Since the scaffolds are useful exclusively in contact with culture medium in vitro or body fluid in vivo, the porosity and pore size distribution of wet state was given more emphasis. In wet state, all the scaffolds could show the average pore size ranging from 80–150 μm, which is suitable for islet cell culture. Though gelatin is hydrophilic in nature, high water contact angles were observed on GG (83 ± 3)° and GE (58.5 ± 3)° scaffolds. Holly and Refojo27 connected this phenomenon with a preferred orientation of hydrophobic moieties at the hydrogel-air interface. Additionally, the swelling studies and contact angle studies showed that GTA cross linked scaffolds had lesser percentage of swelling (swelling studies-GPE > GPG > GE > GG and contact angle studies-GPE > GPG > GE > GG), which may be due to the introduction of extra aliphatic carbon chains in the GTA based scaffolds. In contrast, the presence of PVP has higher hydrophilicity and the water absorbing capacity, both in GTA and EDC cross linked materials. One of the desired properties of pancreatic tissue-engineered scaffolds is its mechanical strength, which is important to support extensive vasculature, the lymphatic system and nerve bundles during its implantation into intra peritoneal site. GPE

Figure 5. In vitro degradation of scaffolds in PBS (n = 6). The degradation pattern of four scaffolds was in the order of GPE > GPG = GE > GG, on day 30. The percentage of degradation of GPE was statistically significant (*) (p ≤ 0.05) when compared to all other scaffolds and the percentage of degradation of GE and GPG was statistically significant (+) (p ≤ 0.05) when compared to GG (day 30). Table 2. The compressive strength of scaffolds in dry and wet state Material

Modulus (kPa) in dry state

Modulus (kPa) in wet state

GPE

2.3 ± 0.27

0.018 ± 0.004

GE

12.9 ± 0.9

0.121 ± 0.037

GG

9.0 ± 2.5

0.258 ± 0.86

GPG

2.3 ± 0.27

0.013 ± 0.010

and GPG showed less compression modulus when compared to GE and GG because of the presence of PVP, which acts as a plasticizer and may interfere with efficient cross linking of the gelatin chains. In wet state, all the scaffolds showed less compression modulus when compared to dry state, which may be due the additive effect of water as plasticizer.28 Still, all the scaffolds exhibited better stability and handling properties in cell culture media throughout the study. As tissue engineering aims at the regeneration of new tissues, scaffolds are expected to be degradable in parallel to new tissue formation. In this study gelatin/PVP IPN based scaffolds degrade faster than gelatin scaffolds (GPE > GPG = GE > GG). The fast degradation of EDC cross-linked sample could be due to the cross linking mechanism,22 where EDC cross links amino and carboxylic groups of adjacent fgelatin molecules by forming new amide bond that in turn has increased possibility of hydrogen bonding with the water and other constituents of the storage media, leading to hydrolytic degradation. This study confirmed that the materials are degradable, but the degradation rate of these materials (in par with formation of new tissues) should be investigated in future through implantation studies.


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Figure 6. (A) Light micrograph showing the viability of mouse pancreatic islets—Trypan blue staining (B) DTZ staining to show the specificity of islet cells. Fluorescence micrographs showing the expression of (C) C-peptide (FITC-labeled anti-C-peptide) (D) Glucagon (FITC labeled anti-Glucagon) in mouse islet cells. Nuclei were stained with Hoechst 33258 (Scale bar 100 μm).

Figure 7. Fluorescence micrographs showing the viability of mouse pancreatic islets in direct contact with scaffolds after 48 h (Scale bar 100 μm).

Figure 8. Confocal micrographs showing the viability of islets on/within the scaffolds on day 4 (Scale bar 100 μm).

There were reports of growing islets for 49 days on chitoson sponge,29 and 2 weeks on agarose cryogel.30 So the behavior of mouse islets on gelatin/PVP scaffolds were determined in vitro. The direct contact assay showed that the cells were viable on all the scaffolds up to 48 h. In order to view the in growth of cells towards interior portion of the scaffolds, z sectioning was done

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with confocal microscopy. The results showed that the cells lost their spherical morphology, leading to adherence of cells on scaffolds and losing their function after day 4. Interestingly, GPE could maintain even distribution of viable cells for 1 month. The functionality of these cells was confirmed by glucose challenging assay and C-peptide/glucagon expression. In the

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Figure 9. The relative comparison of insulin secretion (U/mL) from control mouse islets (CMI) and islets cultured on different scaffolds when challenged with low glucose (5.5 mM) and high glucose (25 mM) media on day 4. There was no statistically significant difference between all the scaffolds (n = 5).

present scenario, it is expected that the presence of PVP is an added benefit for maintaining structure and function of islet cells. PVP increased the hydrophilicity of materials that can improve the nutrition uptake capability of cells on the scaffolds. Still, the construct needs to be evaluated under in vivo transplantation which is currently under progress. Still, a question arises, why did GPG scaffolds perform poorly even if they contained PVP? In GPG scaffolds, GTA was used as the cross linking agent. The unstable GTA polymers that may have been retained during the cross linking procedure may have implicated in the toxicity of cells.31 Hence we presume that the concentration of GTA used in this study (1% GTA) may not be suitable for islet cell culture. It is also possible that trace quantities of GTA are released on degradation of GPG while immersed in media, thereby having implications on the viability of islet cells. Materials and Methods Materials. Gelatin porcine skin type A (~300 bloom), PVP (MW 1300000); EDC; Hoechst 33258, Dithiocarbazone (DTZ) and 6-diamidino-2-phenylindole (DAPI), soybean trypsin inhibitor, bovine serum albumin (BSA) (Sigma-Aldrich, USA); GTA (MW 100.12, Laboratory Rasayan, India); Cell culture media like RPMI, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotic/antimycotic solution (AB/AM), Trypan blue and N-(2-Hydroxy ethyl) Piperazine-N-(4-butane sulfonic acid) [HEPES] (Gibco, USA); Mouse anti-human C-Peptide IgG, mouse anti-human glucagon IgG, rabbit anti-mouse IgG FITC, rabbit anti-mouse IgG-PE (Santa Cruz Biotechnology, USA); Mouse insulin ELISA kit (Biosources, USA). Fabrication of scaffolds. Gelatin and Gelatin PVP IPN scaffolds were prepared by freeze drying method. Gelatin scaffolds. Gelatin solution (6 w/v %) was prepared at 40°C and mixed by mechanical stirrer at 3,000 rpm for 90

Figure 10. Confocal micrograph showing the penetration (3D projection depth code) of viable pancreatic islets towards GPE scaffolds after 1 month. (About five fields were randomly selected in x-y direction). The z- stack size was created up to 228 μm with a scaling of 19 μm. The signals were viewed through the Laser He 515 nm.

min in order to get stable uniform foam. The foam was poured into plastic vials and frozen at -80°C (Ultra low Sanyo, MDF44086S) for 2 days and freeze dried at -78°C for 2 days (Christ, Alpha 2–4D) to obtain open porous 3D scaffolds. Gelatin. PVP IPN scaffolds (2:1): Gelatin solution (8 w/v %) and PVP solution (4 w/v %) were prepared at 40°C and 27°C respectively followed by the mixing using a mechanical stirrer at 3,000 rpm for 90 min in order to get stable uniform foam. The foam was poured into plastic vials and frozen at -80°C for 2 days and freeze dried at -78°C for 2 days to obtain open porous 3D scaffolds. In order to increase the stability of scaffolds in cell culture media, gelatin was cross linked either with GTA or with EDC. For this, gelatin and gelatin: PVP scaffolds (1 mg/ml) were immersed in 90 v/v % acetone-water containing EDC (0.1 mg/ mL), N-Hydroxy Succinimide (0.02 mg/ml) and swirled at 100 rpm at 37°C for 12 h. To crosslink with GTA, gelatin and gelatin: PVP scaffolds (1 mg/mL) were immersed in 90 v/v % isopropanol-water containing 1 w/v % GTA as well as 0.1 N HCl and swirled at 100 rpm at 37°C for 12 h. EDC cross-linked scaffolds were washed in 50% acetone, while GTA cross-linked samples were washed in 90% isopropanol for 3 h with continuous shaking at 100 rpm followed by washing with double distilled water for three changes. The cross-linked scaffolds were frozen at -80°C for 24 h and lyophilized at -78°C for 2 days to obtain the complete dried samples.


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Figure 12. Confocal Laser micrographs showing pancreatic islets expressing (A) C-peptide (PE labeled anti-C-peptide antibody) and (B) Glucagon (PE-labeled anti-Glucagon) after culturing on GPE scaffolds for 1 month. The signals were viewed through the Laser Argon 488 nm.

Figure 11. The comparison of insulin secretion (U/mL) from islets (300) cultured for 1 month on GPE scaffold and freshly isolated mouse pancreatic islets (FMI). There was no statistically significant difference between two groups (n = 5).

Physico-chemical characterization of 3D scaffolds. Fourier transform infrared (FT-IR) spectroscopy. The chemical composition of the hybrid scaffolds was analyzed using FTIR spectroscopy (Thermo electron corporation, USA) and compared to the parent materials (Gelatin/PVP). The spectra of the 3D scaffolds were taken in Nicolet 5700 FTIR spectrophotometer with DTGS (Deutrated triglycine sulfate) detector using diamond Attenuated Total Reflectance (ATR) accessory in reflectance mode with a resolution 4. Fifty scans were recorded per sample in the range of 4,000400 cm-1 and the spectra were analyzed using Omnic software. Thermal analysis. Thermal stability of the scaffold was studied using SDT 2960 (Simultaneous Differential Scanning Calorimeter (DSC)-Thermogravimetric Analyzer (TGA), TA Instruments Inc., USA) as per American Society for Testing and Materials (ASTM) standards.32 For TGA analysis about 10–12 mg of samples were taken in a platinum cup and heated under nitrogen atmosphere at a rate of 10°C/min from 0°C to 600°C. The decomposition pattern of the hybrid scaffold was compared to parent materials (Gelatin and PVP). Swelling percentage studies. The dried samples (10 mm diameter x 5 mm thickness) (n = 6) were initially weighed and immersed in phosphate buffered saline (PBS) (pH 7.4) at 37°C. The samples were blotted with tissue paper at an interval of 5 min and the swollen gel was weighed. This was done until the equilibrium weight was reached. The degree of swelling of these samples was calculated by the formula:

Contact angle. The contact angle was measured using video contact analyzer (Data Physics, OCA 15 Plus, Germany) and imaged using SCA 20 software by sessile drop method on thin films. Deionized water (3 μL) was automatically dropped on the

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film using a gas tight Hamilton precision syringe and the images were captured within 5 seconds. The baseline and the tangent were drawn using the software and the contact angles from both sides were measured. Contact angle was reported as an average measured from four replicates and from seven different portions of the same film. In vitro degradation studies. The scaffolds (12 mm height x 2.5 mm diameter) (n = 6) were weighed and immersed in PBS (pH 7.4) at 37°C for 10, 20 and 30 days. At every time interval, scaffolds were washed with distilled water by keeping at orbital shaker at 100 rpm overnight. The samples were further frozen at -80°C for 12 h and freeze dried at -70°C for 1 day. The percentage of in vitro degradation was calculated by the formula below:

Mechanical properties. The compression modulus of the scaffolds (n = 6) in dry and wet state was tested using Instron (Series IX automated Material Testing system 8.30.00.) with a 100 N load cell at a speed of 1.3 mm/min. The sample size and the testing speed were formulated as per the ASTM standard.33 For wet state measurement, the materials were kept in RPMI media (pH 7.4) overnight and excess medium was blotted before analysis. Surface morphology. The specimens were mounted on aluminium stubs and coated with 300 Å ultra thin layer of gold and observed under the scanning electron microscope (SEM) (Hitachi-S2400 Japan). The images were analyzed using image analysis software (Optima’s TM 6.1, West Ford and MA). Porosity. The porous structure of the scaffolds in dry and wet state was imaged using a high-resolution micro computed tomography (μCT) (μCT 40 Scan Co., Medical, Bassersdorf, Switzerland). For wet state measurement, the materials were kept in RPMI media (pH 7.4) overnight and the excess medium was blotted before analysis. The cylindrical samples were irradiated using X-rays of power 45 KV and the data was analyzed using μCT evaluation program V 6.0. An optimized threshold was used to isolate the scaffolds from the background. About two

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hundred 2D slices (2,048 x 2,048 pixels) were scanned for every sample. The resulting gray-scale images were segmented and a threshold was applied to extract the polymer architecture and then inverted to extract the pore volume architecture. Cytocompatibility studies with mouse pancreatic islets cells in vitro. Isolation of mouse pancreatic islets. The usage of animals in this experiment was approved by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA, Chennai, India). Mouse islets were isolated from 6–8 week old BALB/C mice as per standard protocol.34 Briefly the pancreas of mice was collected in PBS containing 1% AB/AM under aseptic condition. Pancreas were then treated with DMEM containing 1% collagenase type V (Sigma), 2 mg/mL soybean trypsin inhibitor, 2% BSA for 10 min with a magnetic stirrer. The digestion was followed by the centrifugation at 1,000 rpm for 10 min at 4°C. The pellet was dispersed in RPMI 1640 with 10% FBS and 1% AB/AM; seeded in 25 cm2 cell culture flask (Nunc, Denmark) and incubated at 37°C in a 5% CO2 incubator with medium change once in three days. Trypan blue staining. The viability of islets was checked by trypan blue dye exclusion assay by adding 100 μL of 0.4% trypan blue to islets suspension (600 cells) and incubated for 5 min at 27°C ± 2. Blue stained islets were scored as nonviable and unstained scored as viable using inverted phase contrast microscope (Nikon Eclipse TS 100, USA). Dithiocarbazone staining. Dithiocarbazone staining was done to assess the specificity of islets. DTZ staining was carried out by adding 10 μL of DTZ stock (39 mM) to islets (60 islets) suspended in 1 mL of Krebs Ringer bicarbonate buffer (pH 7.4) with HEPES (10 mM) and incubated at 37°C in 5% CO2 incubator for 15 min. The cells were viewed and counted using inverted phase contrast microscope (Nikon Eclipse TS 100, USA). Immuno staining of C-peptide and glucagon. The islets cultured for 48 h were separated and centrifuged at 500 g for 5 min. The islets were then fixed with 4% paraformaldehyde (w/v) in PBS (pH 7.4) for 10 min at room temperature; permeabilized with Triton X-100 for 5 min at 4°C followed by blocking with 1% BSA in PBS for 30 min. Mouse anti-human C-peptide and mouse antihuman glucagon antibodies were used as primary antibodies and rabbit anti-mouse IgG FITC (495/505 nm) was used as secondary antibodies to stain C-peptide and glucagon hormones. C-peptide, glucagon primary antibodies (1:100 in 1% BSA) were added and incubated overnight at 4°C followed by secondary antibody (1:100 in 1% BSA) for 1 h at room temperature. Finally, 10 μL of nuclear stain Hoechst 33258 (350/460 nm) was added and mounted on a glass slide. The images were captured under Inverted fluorescence microscope (DMIL generic, Leica, Switerzerland). At every step cells were washed with PBS by centrifuging at 500 g for 5 min. Direct contact assay. As a preliminary cytotoxicity study, the scaffolds (5 mm diameter x 2 mm height) (n = 3) were kept in direct contact with islets and incubated in RPMI media containing 10% FBS and 1% AB/AM at 37°C, 5% CO2 for 48 h. These islets were checked for viability by staining with acridine orange (15 μg/mL of PBS and 502/526 nm) and ethidium bromide (10 μg/mL of PBS and 518/605 nm) and the images were captured using Inverted fluorescent microscope (DMIL generic, Leica, Switerzerland).

Culturing of pancreatic islets on scaffolds. Pancreatic islets were centrifuged in RPMI media at 500 g for 10 min at 4°C and the cell pellet was dispersed in RPMI media. Three hundred islets were seeded on each scaffold (5 mm diameter x 2 mm height), which were kept in 24 well plate; incubated for 1 h followed by the adding of 2 mL RPMI media containing 10% FBS and 1% AB/AM and cultured. Similarly, same number of islets were cultured on standard tissue culture plate (TCPS) (6 well plate) at 37°C in 5% CO2 incubator as control (CMI). Cell viability—confocal microscopy. In order to view the indepth migration of cells towards the centre of the scaffold, the cells on the scaffolds (Day 4, 7 and 30) was checked for viability by acridine orange and Ethidium bromide staining (as explained above) and imaged using confocal microscope (Carl Zeiss LSM 510 META, Germany). In z-axis the stack size was created up to 228 μm with a scaling of 19 μm. Cell functionality by glucose challenging assay. The cell seeded scaffolds (n = 5) and CMI (n = 5) after 4, 7 and 30 days were washed with serum-free media and challenged with 100 μL of low glucose challenging media (5.5 mM), which contains KRBH buffer (pH 7.4), 5.5 mM glucose and 1% BSA and incubated for 1 h at 37°C, 5% CO2. These islets-seeded scaffolds and CMI were washed with PBS and again challenged with high glucose (25 mM) challenging media and incubated for 1 h. After every hour of incubation, 100 μL of supernatant was collected and stored at -4°C for insulin analysis by mouse insulin ELISA kit. Cell identity by immuno staining C-peptide and glucagon hormones. The islets were cultured on scaffolds for 1 month with frequent medium change at every four days. The cells were then stained with C-peptide and glucagon as described above and viewed under confocal microscope (Carl Zeiss LSM 510META). Mouse anti-human C-peptide and glucagon were used as primary antibodies and rabbit anti-mouse IgG-PE (488/578 nm) was used as secondary antibody. The nucleus was stained with DAPI (345/458 nm). Statistics. The results were expressed as a mean ± SD (standard deviation). The differences between groups were tested by one way analysis of variance (ANOVA). Values less than 0.05 were considered significant. Computations were performed using Microcal Origin.6 (Origin Lab Corporation, Northampton USA). Conclusions The present study suggests that a gelatin–PVP semi IPN scaffold cross linked with EDC could support and maintain islet cells for one month. However, substitution of the crosslinker by GTA has caused loss of morphology and functionality of islet cells after four days. Acknowledgements

The authors acknowledge The Director, SCTIMST and The Head, BMT Wing for the facilities provided; DBT, Govt of India for financial support; ICMR for the Senior Research Fellowship for Mr. Sudhakar. Dr. Roy Joseph for UTM, Instron, Dr. Kalliyana Krishnan for micro-CT studies and Dr. Shiny Velayudhan for helpful discussions.


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Volume 2 Issue 6