Chitosan-gelatin-polypyrrole cryogel matrix for stem cell differentiation

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Tissue Engineering and Regenerative Medicine

Chitosan-gelatin-polypyrrole cryogel matrix for stem cell differentiation into neural lineage and sciatic nerve regeneration in peripheral nerve injury model Tanushree Vishnoi, Anamika Singh, Arun Kumar Teotia, and Ashok Kumar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00242 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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ACS Biomaterials Science & Engineering

Chitosan-gelatin-polypyrrole

cryogel

matrix

for

stem

cell

differentiation into neural lineage and sciatic nerve regeneration in peripheral nerve injury model Tanushree Vishnoi,§ Anamika Singh,§ Arun K. Teotia,§ and Ashok Kumar*,§,† §Department

of Biological Sciences and Bioengineering, and †Centre for Environmental

Science and Engineering & Centre for Nanosciences, Indian Institute of Technology Kanpur, Kanpur-208016, India

*Address for correspondence: Department of Biological Sciences and Bioengineering Indian Institute of Technology Kanpur Kanpur-208016 Tel.: +91-512-2594051 Fax: +91-512-2594010 Email: [email protected]

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Abstract With the advances in tissue engineering and regenerative medicine, various approaches have been developed for peripheral nerve tissue repair and regeneration. In the current study, we have synthesized a cryogel matrix from chitosan and gelatin incorporated with polypyrrole for neural tissue regeneration. The three-dimensional (3-D) cryogel matrix fabricated to mimic the in vivo microenvironment and analyzed for stem cell differentiation. Isolated bone marrow stem cells (BMSCs) cultured on 3-D cryogel matrix differentiated into neural lineage on the basis of scaffold properties, in a co-culture system and by treatment with the spent media of Neuro 2a cells. To validate the cell-cell contact and BMSCs differentiation, scanning electron micrography and fluorescent imaging was done which revealed the differentiation of the BMSCs. Immunostaining and gene expression analysis showed the BMSCs differentiation in all the three cases studied. However, BMSCs in the co-culture system showed increased neurotransmitter levels of dopamine (34%) and acetylcholine (16%) with a respective concentration of 2.04±0.03 ng/ml and 15.06±0.19 pg/ml. Based on these properties in vivo study explored the potential of the synthesized cryogel in regeneration of a 1.5 cm nerve gap in sciatic nerve of rats for a period of 12 weeks. The biocompatibility analysis showed that the scaffold did not induce any adverse immune response. Moreover, the walking track analysis, electrophysiological and immunostaining analysis revealed enhanced sciatic nerve regeneration in comparison to negative control group. This study reveals the regenerative potential of cryogel matrix for peripheral nerve regeneration.

Keywords: Cryogels, Neural differentiation, Neuro-transmitters, Co-culture, Spent media, Nerve regeneration 2 ACS Paragon Plus Environment

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1. Introduction Injury to tissues can be cured broadly either by repair or regeneration mechanism. Wherein the repair mechanism restores a part of the original tissue, in the regeneration mechanism there is a restitution of the whole injured tissue.1 Depending on the proliferative ability of native cells, the injured tissues are formed from continuously dividing cells like skin cells, bone marrow cells, hematopoietic cells, etc. or by quiescent cells which can divide in response to appropriate stimuli. Neural regeneration after accident, trauma or disease, suffer from this limitation to proliferate after injury, leading to the improper recovery of the lost/damaged part. Moreover, isolation of neural autologous or allogenic cells for regeneration has its disadvantages like donor site morbidity, shortage of donors, apoptosis of the cells at the site of injection, etc.2 Thus, alternatives like stem cells (neural stem cells, adult and embryonic stem cells) have been of great focus. Mesenchymal stem cells are multipotent stem cells, tending to differentiate into mesenchymal cells such as osteoblasts, chondrocytes, and adipocytes.3 They can also differentiate into nonmesenchymal cells such as neurons, glial cells, hepatocytes, and cardiomyocytes depending upon the environmental conditions and stimuli provided.4–7 The inductive signals mainly comprise the autocrine/paracrine factors, extracellular matrix (ECM) and cell-cell interaction leading to the transcriptional and translational level changes and thus the cell functionality.8 It has been reported that in vitro differentiated neurons from embryonic stem cell follows the same maturation pattern as the neural cells in vivo.9 In order to replicate such an effect, a simple approach can be adopted, wherein the BMSCs are co-cultured with the cell type in which differentiation is desired. The growth factors and ECM secreted by these cells in their native form are directly taken up by the undifferentiated stem cells inducing differentiation cascade in



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these BMSCs. Co-culture studies with astrocytes, chondrocytes, cardiac cells, etc., have been reported to show such desired results.4,10,11 It has been estimated that the nerve repair and regeneration market would be worth $13.98 billion by the year 2023. However, the current status in this field is unsatisfactory wherein 2.9% of trauma patients have been estimated to suffer from approximately 5 million bedridden days. This not only leads to physical and mental stress of an individual but also retards the growth and economy of a country.12 Nerve guidance channels (NGCs) along with their recent modifications like incorporation of growth factors, cells, ECM components as well as microchannels, fibers, microgrooves provide guiding cues to the growing axon across the nerve gap.13,14 There has also been a transition from non-degradable NGCs to degradable NGCs as well as from impermeable conduits to porous, permeable conduits for proper vascularization.15,16 Although there are few FDA approved NGCs available in the market none can overcome the limitations and challenges of peripheral nerve regeneration.17 In this study, we used the approach of cell-cell contact in co-culture system and further used the spent media collected from the in vitro cultured neural cells for differentiation of BMSCs to overcome usage of exogenous growth factors, peptides, and bioactive molecules. Neural cells during their culture secrete growth factors and other signaling molecules in the culture media, which when treated to BMSCs, can provide the respective bioactive molecules required for their growth and differentiation.18–20

The collected spent media can induce the BMSCs to

differentiate to the neural lineage. However, the two-dimensional (2-D) cultures cannot mimic the physiological three-dimensional (3-D) environment present in-vivo.21 Therefore 3-D matrices were synthesized closely mimicking the native ECM enabling efficient cell proliferation and differentiation. It has already been well established that the physio-


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mechanical, as well as chemical properties of the scaffolds, induce the differentiation of seeded stem cells into different lineages like chondrocytes, osteocytes, myocytes, etc.22,23 Thus, in the present work, the 3-D chitosan-gelatin (CG) polymeric cryogel scaffold incorporated with polypyrrole (PPy) were synthesized and used to study the differentiation of BMSCs based on i) presence of physical, mechanical and chemical cues from the scaffold materials; ii) co-culture wherein the stem cells are co-cultured with neural cell line, Neuro 2a, and iii) stem cell culture in the presence of conditioned media from Neuro 2a cells. The differentiation was confirmed in all the three approaches by the presence of neurotransmitters in the media, immuno-staining, and PCR based real-time expression analysis of neuronal markers. Further, we used these cryogel scaffolds as a potential implant material for nerve regeneration and a nerve graft substitute. The synthesized cryogels were extensively analyzed for in vivo biocompatibility and toxicity. The cryogel polymeric scaffolds were implanted in the 1.5 cm clinically relevant critical size sciatic nerve defect created in the rat and regeneration was analyzed for 12 weeks.

2. Materials and Methods Materials. Chitosan (low viscosity), gelatin (from cold water fish skin), Dulbecco’s Modified Eagle’s medium (DMEM), penicillin-streptomycin antibiotic, Haematoxylin and eosin (H&E) stain and DAB were purchased from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS) was procured from HyClone (Utah, USA). Sodium dodecyl sulfate (SDS) and ammonium persulfate (APS) was procured from Merck Chemical Co. (Mumbai, India). Cetyltrimethylammonium bromide (CTAB) and glutaraldehyde were purchased from S.D fine-chemicals limited (Mumbai, India). Iron (III) chloride (FeCl3) was bought from Hi-Media (Mumbai, India). Pyrrole was procured from Spectrochem (Andhra Pradesh, India). Cell strainers were purchased from BD Biosciences (New Jersey, USA). Anti-S100 antibody (sc-58839) and anti5 ACS Paragon Plus Environment


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neurofilament (sc-56575) were purchased from Santa Cruz (Mumbai, India). Hydrogen peroxide was procured from Ranbaxy (Okhla, New-Delhi). Wistar rats weighing (200-250 g) and BALB/c mice weighing (20-30 g) were procured from Indian Institute of Toxicology Research, Lucknow, India. 2.1. Fabrication of three-dimensional matrix with polypyrrole (PPy) Pyrrole (0.13 M) was added to the beaker containing sodium dodecyl sulfate (SDS), and this mixture was stirred for 15 min at 2-8 °C. To this iron (III) chloride (FeCl3) was added as an oxidizing agent and the solution was further stirred for 12 h. The synthesized PPy was dialyzed for 48 h and then used for the synthesis of cryogels. Gelatin (1.6% w/v) and chitosan (0.3% w/v) were dissolved in acetic acid (2% v/v) solution. To this solution, the dialyzed polypyrrole was added, and the volume was made to 10 ml. The solution was mixed properly, and glutaraldehyde (0.2% v/v) was added as the cross-linker. The solution was vortexed properly and immediately poured into 5 ml syringes and frozen at -12 °C for 12 h. After polymerization, these gels were thawed at room temperature (RT) and washed thoroughly to remove unreacted monomers.24 For in vivo nerve regeneration experiment, the cryogels were fabricated in insulin syringes. Apart from this polyacrylamide cryogels (PAAM) were synthesized by the method described previously.25 2.2. Isolation and characterization of bone marrow stem cells (BMSCs) BMSCs were isolated from 6-8 weeks old BALB/c mice. For isolating the cells 6 mice were sacrificed by cervical dislocation their femurs and tibia were cleaned of the tissue. The toe region was removed and the marrow region was harvested. DMEM was used to flush the marrow by inserting a syringe needle at one end of the medullary canal till a clear canal was observed. The obtained cells were passed through a 70 µm mesh filter and centrifuged. The obtained cell pellet was re-suspended in DMEM containing 20% FBS and 1% penicillin6 ACS Paragon Plus Environment

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streptomycin antibiotic. The cells were cultured at 37 °C, 5% CO2 and humidified atmosphere containing 95% air. The isolated BMSCs were characterized for their differentiation potential into osteogenic lineage in the presence of osteogenic induction medium as described earlier.26 The cells were maintained upto passage 3 and then seeded at a density of 5 x 104 cells/ well in 6 well plates. For BMSCs differentiation into osteogenic lineage, the cells were cultured in osteogenic differentiation medium (DMEM supplemented with 10% FBS, 1 µM dexamethasone, 10 mM β-glycerophosphate, 50 µM ascorbate and 1% antibiotic-antimycotic solution) for 2 weeks. As a control, the cells were maintained in DMEM medium supplemented with 10% FBS. The media was changed after every 2-3 days. After 12 days of induction, the cells were fixed with 4% paraformaldehyde for Alizarin red staining. After fixation, the cells were washed with PBS (3X) for 5 minutes each and incubated in Alizarin red stain for 20 minutes at 37 °C. 2.3. Differentiation of BMSCs via spent media from in vitro cultured Neuro 2a Neuro 2a cells were cultured in vitro in the culture flask. For Neuro 2a differentiation to neural lineage, the cells were grown in serum-free DMEM, and the media was collected after 8 h. Before its use, the collected spent media was filter sterilized by 0.22 µm syringe filter and mixed with fresh complete DMEM media in the ratio of 1:1. The cryogel monoliths were cut into sections of 5 mm thickness and were sterilized by passing through gradient ethanol (20% and 40% for 30 min, 70% overnight (O/N) and 100% for 2 h). Further, the scaffolds were washed with phosphate buffer saline (1X PBS) three times for 15 min each and then were allowed to saturate with complete DMEM media for 5-6 h in 24 well plates. The isolated BMSCs were seeded on the scaffolds at a density of 0.5 x 105 cells/ 500 µl and allowed to proliferate in 20%



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FBS containing DMEM media for a period of 24 h. After that, the media was replaced by the combination of 50% re-suspended spent media and 50% of fresh complete DMEM media. 2.4. Differentiation of BMSCs via co-culture with Neuro 2a Neuro 2a cells were seeded on the sterilized scaffolds at a density of 0.5 x 105cells/ 500 µl. The cells were allowed to adhere on the scaffold for 6 h after which the harvested BMSCs were seeded on the same scaffold at a seeding density of 0.5 x 105cells/ 500 µl for 1 month. Neuro 2a and BMSCs were seeded separately on the scaffolds at a cell density of 0.5 x 105 cells /500 µl, respectively were used as the control. Before seeding, Neuro 2a cells were cultured for 24 h in 0.1% FBS containing media to allow differentiation. Further, they were cultured in DMEM media with 20% FBS along with the BMSCs for differentiation studies. 2.5. Differentiation of BMSCs on the cryogels To evaluate the differentiation of BMSCs based on the material properties, the cells were seeded on the sterilized scaffolds at a density of 0.5 x 105 cells/500 µl and allowed to grow in the presence of DMEM media with 20% FBS. Samples in triplicates were collected every week for gene expression analysis whereas their media was analyzed for the secretion of neurotransmitters and NGF. 2.6. Metabolic activity of BMSCs Every fifth day, the scaffolds in triplicates, were removed and checked for cell viability using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. Briefly, the media was removed from the wells and the scaffolds were washed with PBS. Then 500 µl of MTT reagent dissolved in incomplete DMEM was added to each well and the scaffolds were incubated for 4 h at 37 °C. Thereafter, the formazan crystals thus formed were dissolved in 1.5 ml DMSO reagent and the absorbance was measured at 570 nm. The scaffolds from both coculture experiment and BMSCs cultured in spent media were removed in triplicates for analysis. 8 ACS Paragon Plus Environment

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2.7. Gene expression analysis The BMSCs cultured on the scaffold, as well as with the spent media and co-culture were collected every week and analyzed for the gene expression by TRIzol method. Briefly, the samples were treated with 1 ml of TRIzol reagent for 5 min and then vortexed. Chloroform (0.2 ml) was then added, and the mixture was centrifuged at 12,000 xg for 15 min. Further 0.5 ml isopropanol was added, and the samples were centrifuged at 12,000 xg for 10 min. RNA invisible before centrifugation appears on the walls of the tube. Ethanol (75%) was then added to it, and the mixture was centrifuged at 7000 xg for 5 min. After centrifugation, the ethanol was discarded, and the pellet was dried and then finally dissolved in RNA free water for further cDNA synthesis. The neural markers used were nestin and neurofilament. GAPDH was used as the housekeeping gene. Agarose gels (1%) were made to observe the bands of the expressed gene. 2.8. The matrix influence on cells The scaffolds seeded with cells were analyzed by scanning electron microscope (SEM). The samples were fixed with 2.5% glutaraldehyde for 4 h and then treated with a gradient of ethanol (20%, 40%, 70%, and 100%) and dried in desiccator. The dried samples were gold sputter coated and analyzed using FEI Quanta 200 SEM. For immuno-staining, scaffolds were cut into 100 µm sections using cryotome after fixing with 2.5% glutaraldehyde. The sections were incubated with 1° antibody against neurofilament (NF-200) at 4 °C for 24 h after which they were tagged with TRITC labeled 2° antibody. The nucleus of the cells was stained with 4',6diamidino-2-phenylindole (DAPI). 2.9. Identifying the neurotransmitter analysis Dopamine and acetylcholine concentrations were measured in the spent media collected from each well at different time points. The samples for dopamine were filtered through 0.22 µm size 9 ACS Paragon Plus Environment


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filters and then analyzed by HPLC (Waters, U.S.A) using the C18 column.27 The mobile phase composed of methanol (70% v/v), KH2PO4 (0.02 M) and SDS (30 mM) at pH 3.0 adjusted with phosphoric acid. The flow rate of the mobile phase was 0.5 ml/min, maintaining the column temperature at 40 °C. Sample volume injected for analysis was 20 µl. The procedure for choline/acetylcholine quantification was as per the method described in the MAK056 kit purchased from Sigma Chemical Co. (St. Louis MO, USA). 2.10.

Real-time PCR

Real time PCR was used to study the expression of NeuN in all the three systems. In brief, the cDNA synthesis was done using reverse transcriptase and oligo d (T). Real time PCR was carried out using 20 µl reaction (10 µl SYBR fast green PCR mix and primer at 0.4 µM). The primers were tested for their specificity and amplification efficiency before running the final experiment. Comparative CT method was used to study the relative quantity of mRNA wherein β-actin had been used as the endogenous control for normalization. 2.11.

Growth factor analysis

Enzyme-linked immunosorbent assay (ELISA) was done for the analysis of nerve growth factor. The optimal dilutions for the antigen as well as the antibodies were detected before the final experiment was performed. Recombinant nerve growth factor (NGF-2.5S; from murine submaxillary gland 1:100) and filtered spent media (1:6) collected from the control as well as the three different set-ups were coated on the 96 well plates using bicarbonate buffer (pH 9.6). Positive, negative and blank controls were included in the study. The coated antigen was incubated O/N at 4 °C. After which the plates were washed and BSA (10% w/v) was used as the blocking buffer at 37 °C for 2 h. The 1° antibody (cell signaling) was incubated O/N at 4 °C in the ratio of 1:1000 to each well. After incubation, the wells were washed with Tris-buffered saline with Tween-20 (TBST) to remove unbound and non-specific antibody. The wells were 10 ACS Paragon Plus Environment

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then treated with 2° antibody (1:3000) for 2 h at RT. The plates were again washed with TBST buffer three times following which the substrate, o-phenylenediaminedihydrchloride (OPD), dissolved in citrate buffer, over was added to the wells and incubated for 30 min after which the absorbance was read at 450 nm using the plate reader. 2.12.

In vivo biocompatibility studies

All the experimental procedures were conducted under the Institute Animal Ethics Committee guidelines. A total of 30 mice were divided into 2 groups: control and experimental set. Before implantation, the cryogels were sterilized by ethanol gradient. Mice were anesthetized using ketamine and xylazine, as per their body weight. The site of implantation was shaved and cleaned with 70% ethanol. Further, an incision was made in the skin at the back of the mice making a subcutaneous pocket in which the cryogel (2mm x 8mm) was placed. After that, the skin incision was closed using resorbable sutures. Tramadol was given intraperitoneally as a prophylactic pain treatment. The animals were kept under close observation and had access to food and water ad libitum. At each time point, 5 animals were sacrificed from both the groups after 72 h, 10 days and 30 days post implantation for the biocompatibility analysis. 2.13.

Toxicity evaluation

Systemic toxicity of the implant was evaluated by blood cell counter, using blood sample of control as well as samples implanted with the cryogel, for the presence of WBC’s, neutrophils and lymphocytes. Further, the serum samples were analyzed for the presence of various inflammatory interleukins by ELISA. Local toxicity was assessed by histological analysis of the nearby tissues for the presence of mast cells. In brief, the samples were excised at an interval of 72 h, 10 days and 30 days post-implantation. The harvested tissue was fixed in 4% paraformaldehyde (PFA) solution, after which the samples were dehydrated using ethanol gradient treatment (25%, 50%, 70%, and 100%). The samples were further embedded in the 11 ACS Paragon Plus Environment


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paraffin wax and sectioned of 10 µm thickness using microtome. The sections were stained with fast green and then counterstained with safranin. Organ toxicity was also evaluated by analyzing cell infiltration and tissue morphology after H&E staining of liver, kidney, pancreas, lungs, and spleen tissue sections at the end of one month for both control and experimental set of animals. 2.14.

Morphology of tissues surrounding the cryogel implants

The excised samples were embedded and sectioned as mentioned above and then stained with H&E to detect infiltration of cells, tissue morphology, and integration. 2.15.

In vivo study in peripheral sciatic nerve model

Adult Wistar male rats weighing 200-250 g were taken for the in vivo implantation experiments. The animals were divided into two groups having 20 animals in each group. The experimental set of animals in which the nerve injury was created and the scaffolds were implanted. The second set of animals were taken as the negative control in which the injury was created but no scaffolds were implanted. In the presence of anesthesia (ketamine 80 mg/kg and xylazine 10 mg/kg), the left sciatic nerve was exposed through an incision in the skin followed by the opening of the muscle. Prior to incision, the left side of the rat was shaved and wiped with 70% ethanol and povidone-iodine solution. After which the nerve transection of 15 mm was performed using microsurgical scissors. The injury was made above the point of nerve ramification. The scaffolds were cut longitudinally to create an upper and a lower part, which were 2 mm in diameter and 20 mm in length. The proximal and distal end of the cut nerve was placed on the lower scaffold such that the ends were in the midline of the scaffold with both ends at a distance of approximately 15 mm. After which the upper half of the scaffold was placed and the whole setup was tied with resorbable sutures at both ends resulting in a sandwich model. Following this implantation, lidocaine was applied and the muscles and the skin were sutured using resorbable and non-resorbable sutures respectively. The normal contralateral right 12 ACS Paragon Plus Environment

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sciatic nerve was taken as the control. Ceftriaxone (100 mg/kg body weight) and tramadol (12.5 mg/kg body weight) were given as the antibiotic and analgesic, respectively for 3 days postsurgery. All the animals have kept in individually ventilated cages with a regulated environment and had access to water and food ad libitum both before and after surgery. 2.16.

Histological and immunohistochemical staining

For histological studies, the animals were euthanized using a higher dose of pentobarbital. The implant was excised at regular interval of 2, 4, 6 and 12 weeks and fixed in 4% PFA for 24 h. After dehydration, the specimens were paraffin embedded and thin sections of 10 µm, both longitudinal and transverse, were cut from the proximal, middle and distal segment using microtome. These sections were then deparaffinized, rehydrated and stained with H&E, to study the nerve anatomy. At the same time, these specimens were also observed in SEM. The sections were immune-stained by using antibodies against neurofilament (neurites) and S100 (Schwann cells). The proximal, distal as well as the middle portion (the regenerated nerve along with the scaffold) were incubated with the 1° antibodies at 4 °C for 24 h after which either FITC labeled or peroxidase-labeled 2° antibodies were used for fluorescent imaging or DABperoxidase staining respectively, for observing cells. 2.17.

Electrophysiology assessment

To assess the functionality improvement of the regenerated nerve, electrophysiology analysis of all the experimental rats was performed after 8 and 12 weeks of surgery for measuring nerve conduction velocity (NCV) and compound muscle action potential (CMAP). In brief, the electromyogram (EMG) machine (Nicolet Viking Quest) was used for evaluating NCV and CMAP wherein bipolar electrodes were used. The operated area was shaved, and the nerve was stimulated at the proximal and distal end of the implanted cryogel. The distance between both the stimulated areas was noted to calculate the conduction velocity. The recording electrode was 13 ACS Paragon Plus Environment


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placed over the muscle innervated by the sciatic nerve, and the ground electrode was placed on the tail of the animal. As a normal control, the electrophysiology parameters of the contralateral leg were also calculated. 2.18.

Gastrocnemius muscle ratio (GM ratio)

Weight of gastrocnemius muscle from the surgery site as well as the contralateral right side was measured. Briefly, gastrocnemius muscle was isolated, and its wet weight was measured. Similarly, the weight of the control (contralateral leg) was also measured, and the ratio was calculated using the formula.28 𝐺𝑀 𝑅𝑎𝑡𝑖𝑜 =

2.19.

𝑊𝑒𝑡 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐺𝑀 𝑓𝑟𝑜𝑚 𝐸𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑠𝑖𝑑𝑒 𝑊𝑒𝑡 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐺𝑀 𝑓𝑟𝑜𝑚 𝑁𝑜𝑟𝑚𝑎𝑙 𝑆𝑖𝑑𝑒

𝐸𝑞. 1

Sciatic nerve functional index (SFI) analysis

To study functional recovery in control as well as experimental animals, their SFI was calculated. In brief, the rat was made to run on a wooden chamber (1m x 8cm) with raised sidewalls. The runway was attached with a dark box at its one end. Its hind legs paws were dipped in methylene blue ink such that they leave their footprints on the paper sheet (placed on the wooden block.). SFI was calculated using their print length (PL), toe spread (TS) and intermediate toe spread.29,30 SFI = ― 38.3

(EPL ― NPL) (ETS ― NTS) (EIT ― NIT) + 109.5 + 13.3 NPL NTS NIT

𝐸𝑞. 2

PL = Distance from the mid-toe to the heel TS = Distance between the extreme toes, i.e., the first and the fifth toe ITS = Distance between the second and fourth toe N = Normal E = Experimental 14 ACS Paragon Plus Environment

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The normal function and total dysfunction are represented by SFI values 0 and -100, respectively. 2.20.

Statistical analysis

All the in vitro experiments were carried out in triplicate keeping the minimum sample size of n = 3, and the quantitative experiments are expressed as mean ± standard deviation. All the analysis was carried out using GraphPad Prism 5 using two-tailed Student's t-test. The p-value of less than 0.05 was considered statistically significant. 2.21.

Animal ethics statement

All the animal experiments were performed under the guidelines of the Institute Animal Ethics Committee (IAEC) using the approval numbers IITK/IAEC/2014/1023, IITK/IAEC/1024, and IITK/IAEC/1025. All the animals were housed in standard conditions with free access to feed and water ad libitum. The animals were housed in cages with regulated temperature, light, and humidity.

3. Results and Discussion 3.1. Synthesis of cryogel scaffold Three-dimensional cryogel was synthesized using chitosan, gelatin, and polypyrrole wherein each of the constituents was responsible for imparting diverse properties to the synthesized cryogel (Figure 1a). Chitosan being a member of glycosaminoglycan’s mimics the extracellular matrix of native tissue. Moreover, the positive charge of chitosan also contributed to the adherence of the cells on the scaffolds.31,32 However, to overcome the brittle nature of chitosan, gelatin was incorporated. Gelatin in addition to providing mechanical strength by enhancing crosslinking also provides flexibility to the scaffold making it less brittle. Additionally, RGD



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motifs of gelatin serve as receptors for cellular adherence and proliferation.33 The SEM micrograph of the cryogels shows the presence of the polypyrrole and interconnected porous architecture important for cellular infiltration, nutrients, and metabolites transportation (Figure 1b). Polypyrrole provided the conducting properties to the scaffold, and it has already been reported to support the growth and proliferation of neural cells.24 3.2. Characterization of BMSCs The isolated BMSCs were analyzed for their in-vitro differentiation potential into the osteogenic lineage. After 12 days of culture in osteogenic medium, the BMSCs showed calcium deposition in alizarin red staining which is an indicator of osteogenesis as represented in Figure 1c. The undifferentiated cells did not show any calcium deposition as shown in Figure 1d. 3.3. Metabolic activity of proliferative cells The cell functionality is a direct indication of the cell behavior and in artificially fabricated 3D matrix cells could alter its natural growth cycle phenomena; hence constant monitoring is necessary. As we have already shown the potential of the cryogel scaffolds in cartilage34, bone35, cardiac tissue engineering36, etc., herein we further tried to explore the combined effect of the 3-D scaffold, its properties and use of spent media from neural cells as well as neural-stem cell co-culture system leading to BMSCs differentiation. The macroporous and highly interconnected network of the cryogels allowed the convective flow of nutrients and metabolites thus allowing the cells to grow and proliferate over the period. Moreover, the polymers chosen for the synthesis enhanced the cell material interaction leading to an increase in the cell proliferation for all the three cell systems, i.e., Neuro 2a, BMSCs and co-culture of Neuro 2a with BMSCs. Although the cell number seeded in the co-culture system was higher, but the cell proliferation rate was found not to be elevated as observed in Figure 1e. The BMSCs cocultured with Neuro 2a showed a slightly less cell proliferation as compared to the neural cell 16 ACS Paragon Plus Environment

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line seeded alone which could be attributed to the inverse relationship between proliferation and differentiation.37,38 Thus we also speculate that the seeded BMSCs started to differentiate into the neural lineage as a result of which the cell proliferation did not increase continuously as for the neural cell line. Apart from this, the proliferation of the BMSCs cultured in the presence of the spent media increased for two weeks as shown in Figure 1f. Thus, showing that the cells were able to grow and proliferate in the spent media collected from the differentiated Neuro 2a cells. A decrement in the cell proliferation was observed in the last week which could be because higher cell number was obtained over the two weeks. 3.4. Differentiation of BMSCs- Morphological and Immunostaining analysis 3.4.1. Scanning electron microscopy The SEM image of the cell-seeded scaffolds clearly showed the distribution of cells all over the scaffold at the end of 3rd week. Both the BMSCs as well as the Neuro 2a cells adhered to the scaffolds and attained their morphology. SEM images validated the existence of cell-cell interaction between BMSCs and Neuro 2a cells in the co-culture system as observed in Figure 1 (g & h). These cell-cell interactions modulated the signaling in the BMSCs which helped them to differentiate into the specific lineage. Similarly, BMSCs differentiation into neural lineage was observed in the presence of spent media. SEM image showed the initiation of neurite-like structures from the cell body resembling neural cell morphology as shown in Figure 1 (i). We speculate the extracellular cues from the spent media such as secreted factors and bioactive molecules instructed the cell for phenotypic changes. 3.4.2. Immunostaining analysis Immuno-staining of the cell-seeded scaffolds in all the three set-ups was performed. The sections were stained for neurofilament performing secondary staining by TRITC labeled



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secondary antibody. The cells were then counterstained with DAPI as a nuclear stain (Figure 1 j-l). The expression of neurofilament maker indicates the differentiation of BMSCs into the neural lineage. 3.5. Differentiation of BMSCs- Gene expression analysis 3.5.1. Expression in the presence of spent media In the case of BMSCs cultured in the presence of Neuro 2a spent media, the cells differentiated into neuronal-like cells and showed the presence of neural marker, nestin, and neurofilament (Figure 2a). Neuro 2a is derived from mice neural crest and is widely used for the study of neuronal differentiation. Therefore, as expected, the differentiated cells were more prone to grow towards neuronal lineage compared to glial lineage, and therefore we did not get a band for glial fibrillary acidic protein (GFAP). Neurite outgrowth is one of the important parameters required for neural differentiation, and NGF has been explored vigorously for the same. Therefore, we analyzed the presence of NGF in the spent media by ELISA. Moreover, one of the two NGF receptors, TrkA, is present on mice bone marrow stem cells. CD271, a surface marker for BMSCs, is also a low-affinity NGF receptor. During in vitro culture, the marker disappears as there is no stimulation in the media.39,40 However, NGF at a concentration of 20 pg/mg of total protein was detected in the spent media and is thought to be a constant stimulating factor for CD271. 3.5.2. Expression in the co-culture system Bands representing nestin and neurofilament were observed at the end of the first week. Neuro 2a cells as mentioned above directed the BMSCs to neural lineage by both cell-cell contacts as well as the secreted factors in the media as observed in Figure 2b. Both the factors contributed to modulating the cell-cell signaling and secretion of various bioactive molecules leading to an alteration in the gene expression and thus differentiation. 18 ACS Paragon Plus Environment

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3.5.3. Expression based on physical and chemical cues Nestin is an early marker for neural differentiation and codes for a class of an intermediate filament protein. During later stages of development, this protein undergoes remodeling and results in the formation of neurofilament/glial fibrillary acidic protein (GFAP).41 NF and GFAP are the markers for properly differentiated adult neural cells.42 The agarose gel showed the presence of nestin, neurofilament as seen in Figure 2c. Nestin was observed in the first week whereas NF expression was observed in the second week of analysis as also reported by others.43 It has also been known that human embryonic stem cells and induced pluripotent stem cells began to differentiate into neural cells in the first week with the appearance of nestin and further differentiated to either neurons or glial cells.44 However, the band for GFAP was not observed even after one month which suggests the differentiation of BMSCs into neuronal cells rather than the glial cells. This directed differentiation of stem cells on 3-D matrices has already been reported and might be attributed to various factors like the microenvironment to which the cells are exposed, the integrin-focal adhesion interactions, mechano-transduction phenomena, the protein layer and the charges on the surface of the 3-D substrate to which the cells are exposed.45–47 It has also been reported that neuronal cells prefer soft gels whereas glial cells differentiation occur more frequently on comparatively stiffer gels.48 In addition, chitosan being a cationic polymer is also known to interact with the negatively charged cell membrane of the mammalian cell due to electrostatic interactions leading to alterations in cell adherence and directed differentiation.48,49 Therefore to counteract its effect we synthesized polyacrylamide (PAAM) gel, and seeded it with the same cell density of BMSCs to further analyze its gene expression patterns.50 These gels formed a cross-linked polymer network without the presence of net



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charge. The cells seeded on PAAM gels didn’t show any adult neural marker expression during the course of one month experiment. However, nestin band was observed on agarose gel as seen in Figure 2d. Thus, it can be postulated that the positive charge of the cryogel allowed adhesion of negatively charged biomolecules like cytokines and growth factors which further influences cell adhesion, signaling and differentiation apart from its optimum mechanical strength. Thus, we speculate that it is the combination of the mechanical properties and surface properties such as surface charge which is inducing the differentiation of BMSCs to neural lineage. 3.6. Neurotransmitter analysis To confirm the in-vitro differentiation of the BMSCs to the neural lineage, certain neurotransmitters were examined quantitatively. HPLC analysis of dopamine, a known neuronal neurotransmitter involved in cell-cell signaling, motor activities was performed for evaluation. The amount of dopamine obtained in the co-culture system after normalizing with the cell number of individually seeded Neuro 2a cells were found to be 2.04±0.03 ng/ml, 2.27±0.04 ng/ml and 2.29±0.04 ng/ml at 1, 2 and 4 weeks of culture as shown in Figure 3a. Dopamine levels obtained for only Neuro 2a cell seeded cultures were 1.52±0.05 ng/ml, 1.82±0.04 ng/ml and 1.81±0.03 ng/ml at 1, 2 and 4 weeks of culture. These levels were observed to be 34%, 25%, and 24% higher for co-culture set up as compared to only Neuro 2a cell seeded cultures for 1, 2 and 4 weeks respectively. Neuro 2a cell lines do tend to differentiate into dopaminergic neurons.51 This must have induced the BMSCs co-cultured with Neuro 2a to differentiate into such types. Stem cells differentiation to dopaminergic neurons has also been induced by adding exogenous factors involved in activating sonic hedgehog and WNT signaling. Neuro 2a differentiation is regulated by one of the processes including WNT signaling as endogenous levels of Wnt7A results in increased neurite outgrowth. This might


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also activate WNT signaling in stem cells through cell-cell contact and thus increased the release of neurotransmitters.52 Human MSCs have shown the presence of dopamine receptors and transcription factors involved in dopaminergic neuron functions when analyzed for their gene expression.53,54 However, BMSCs cultured on the scaffolds as well as in the presence of spent media did not show the presence of dopamine in their medium. Similarly, the acetylcholine level in the media was also assayed for all the set-ups. The normalized values showed the presence of acetylcholine in the co-culture set-up as well as in the BMSCs cultured on scaffolds. However, there was no detectable acetylcholine in the set-up where BMSCs were cultured in the presence of spent media. The amount of acetylcholine detected in the co-culture system was 15.06±0.19 pg/ml and 13.36±0.20 pg/ml after 2 and 4 weeks respectively as shown in Figure 3b. However, in case of only stem cell-seeded cultures, the levels were 13±0.20 pg/ml and 12.50±0.20 pg/ml after 2 and 4 weeks, respectively. It was found that there was a 16% increase in the acetylcholine levels after 2 weeks. Further, we speculate that as BMSCs in the co-culture were directed towards dopaminergic type, there was less acetylcholine detected in their media as observed at 4 weeks. As we had not provided the cells with conditions to specifically differentiate into a specific neuronal cell type, the presence of both the neurotransmitters was detected in the co-culture system. Moreover, it has been shown that dopaminergic and cholinergic markers could co-localize in NeuN positive differentiated stem cells.55 3.7. Real-Time PCR NeuN is a mature neuron-specific marker widely used to study the differentiation of stem cells into the neuronal lineage.56 NeuN expression was checked by real-time PCR to study the differentiation efficiency of each of the systems. The positive control was Neuro 2a cells in the serum-free media, and the cycle threshold (CT) value was calculated in direct proportion to it. The co-culture system showed the highest fold increase in NeuN levels compared to the others 21 ACS Paragon Plus Environment


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and continued to remain high for 4 weeks whereas the BMSCs cultured in the presence of spent media showed the least differentiation. In the system where BMSCs differentiated based on scaffold properties, the NeuN expression reached a maximum during the second week as observed in Figure 3c. The co-culture system due to its constant cell-cell interaction and secretory factors constantly induced the BMSCs to differentiate. In the presence of spent media, the BMSCs differentiation did occur; however, the fold increase was very low thus proving that cell-cell contact is necessary for the stem cell differentiation to neural lineage. This decreased differentiation in comparison to the only BMSCs cultured system might be due to the 10% serum factors present in the media (50% spent media + 50% complete media). The serum factors do not promote neural differentiation but allow suitable growth and proliferation of these cells which further differentiate to neural lineage. Thus, different combinations of spent media with fresh media could be tried to achieve a higher differentiation rate. 3.8. In vivo biocompatibility analysis The implanted cryogel observed at the end of 72 h, 10 days and one month clearly showed well integration with the surrounding and nearby host tissue. The animals did not show any adverse signs of necrosis or tumor formation as can be observed in Figure 4A (a-c). Moreover, the presence of blood vessels indicated that the implanted scaffold assisted angiogenesis at the implanted site without any inhibitory effect as represented in Figure 4A (d-f). 3.8.1. Hematoxylin and eosin staining The sections stained with H&E showed infiltration of cells at the site of implantation at the end of the time points. It also showed the integration of the implanted scaffold with the nearby tissue over one month. A clear demarcated boundary was observed after 72 h between the host tissue and the implant which diminished by the end of 10 days and finally disappeared at the end of


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one month as shown in Figure 4B (a-c). Moreover, the degradation of the implanted scaffold could also be observed by the end of one month as shown in Figure 4B (d). 3.8.2. Blood cell count and interleukin analysis The local and systemic toxicity was evaluated by staining as well as blood sample analysis. Blood cell count for WBCs, neutrophils, and lymphocytes was done as these cells are primarily involved in the foreign body and immune response. Neutrophils are the first cells to arrive at the site followed by WBCs and lymphocytes. All these cells are involved in acute and chronic inflammation. It has been reported that the inflammatory response is resolved in 2-3 weeks in case of biocompatible implants. However, if the inflammation persists for more than a period of three weeks, it usually indicates infection.57 In this study, the lymphocyte count increased at the end of 72 h and continued until the end of 10 days. However, the number declined and became equal to the control by the end of one month (Figure 4C). Further, the samples were also studied for the presence of various interleukins. Macrophages secrete an array of interleukins and cytokines when activated in response to foreign material. The interleukin profile varied depending on the polymer type and the surface receptors available for cell binding. It also depends on whether the particular scaffold allows the induction of macrophage fusion. In our analysis, IL-1β, IL-4, and IL-6 were absent in the serum whereas the others like IL-2, IL-10, IFN-γ, TNF-α, G-CSF were present in the same concentration as the control with not much variation (Figure 4D). 3.8.3. Organ toxicity analysis Organ toxicity was also analyzed by H&E staining, and there was no undesirable outgrowth or immune response elicited as shown in Figure 5A (a-j). Therefore, it can be concluded that the synthesized scaffold was biocompatible and did not show any adverse immune response. 3.8.4. Safranin and fast green staining 23 ACS Paragon Plus Environment


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Further, to study the presence of mast cells, safranin staining was done as it primarily stains mast cells. It is already known that mast cells are involved in acute inflammation and are the prime candidate for allergic reactions.58 Mast cells degranulation leads to histamine release, which is an important recruiter for macrophages, at the site of implantation. Apart from this, mast cells also release IL-4 and IL-13, which are involved in foreign body reaction.59 The mast cells were observed at the end of 72 h and 10 days. However, their number drastically reduced by the end of one month as shown in Figure 5B (a-c). 3.9. Peripheral sciatic nerve rat model- an in vivo study The nerve was cut above the bifurcation, and the sandwich model was created as shown in the schematic in Figure 6A and digital image in Figure 6B (a-c). The cut nerve ends were intact within the scaffold and did not get displaced. The natural polymers have a high degradation rate, therefore it is speculated that chitosan-gelatin matrix had degraded to a greater extent by the end of 12 weeks. However, the polypyrrole filler along with the regenerating tissue supported the growth of the regenerated nerve. The Figure 7A (a) shows that at the end of two weeks, a fibrous mass was present in the nerve gap in the control and experimental animals. By the end of 4 weeks, it was difficult to observe the proximal, and the distal end of the transected nerves in control group as the cut ends innervated the nearby tissues as shown in Figure 7A (b, c). However, in the experimental animals, both the ends were intact in the implanted scaffold which provided guidance to the axonal sprouts and directed the proximal end to the distal end as shown in Figure 7A (d-f). Moreover, it was observed that the distal end of the animals implanted with the conducting cryogel did not have a shrunken nerve skeleton as compared in control animals. The blood samples of both the experimental and control animals were analyzed for the presence of immune responsive cells. The quantified results did not show much variation between the experimental and the control animals at the end of 4 weeks. However, the 24 ACS Paragon Plus Environment

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lymphocyte count was slightly higher in the sample at the end of 4 weeks as shown in Figure 7B. The body weight analysis of the animals taken before and after surgery showed that there was no sudden loss in appetite or weight post-operation as observed in Figure 7C. 3.10.

Histological and immunohistochemical analysis

Histological analysis showed the intact nerve fibers in the proximal end by the end of 2 weeks. However, this alignment was disrupted in the distal segment where myelin ‘ovoids’ could be observed. With time cryogel implants supported the nerve regeneration, and the healthy regenerated nerve was observed at the distal end after 12 weeks as shown in Figure 8. Whereas, in the negative control group the nerve cut ends degenerated. The transverse sections in Figure 9 (a-b) showed the interface between the cut end of the proximal nerve to that of the middle part of the scaffold, and therefore the cell number observed in the interface was higher compared to the cell number observed in the middle segment at the end of 12 weeks. The scaffolds also showed the presence of blood vessels both on the surface as well as the interior of the scaffold (Figure 9c), which could be attributed to the presence of porous and interconnected cryogel properties. The middle segments of the excised samples showed cellular infiltration and adhesion on the implanted scaffold as observed in Figure 9 (d, e). The longitudinal, as well as transverse sections, showed the presence of neurofilaments running through the middle segment of the implant after 6 weeks as represented in Figure 9 (f, g). With time, nerve regeneration improved and more neurofilament-positive regenerated neurons were observed in the middle segment at the end of 12 weeks (Figure 9 h). Further migration of Schwann cells to the middle segment of the implant was also analyzed. The cross sections were stained positive for S100, a marker for Schwann cells as shown in Figure 9i.



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Schwann cells are known to play an important role in peripheral nerve regeneration by providing both physical and chemical cues. 3.11.

Electrophysiological analysis

Electrophysiological parameters like NCV and CMAP were analyzed to determine the functionality of the regenerated nerve through the cryogel scaffold. After 8 weeks, postoperative, the recorded NCV for cryogel implanted group was 19.8 ± 1.5 m/sec which further increased to 30.6 ± 1.1 m/sec after 12 weeks of surgery as shown in Figure 10a. For the normal nerve NCV value was found to be 58.0 ± 1.4 m/sec. Thus, the cryogel implanted group showed 52% recovery in nerve conduction velocity of the regenerated nerve after 12 weeks of surgery representing that the unblocked conduction of nerve impulses has occurred through the cryogel scaffold. The CMAP exhibited by cryogel implanted group was 5.10 ± 0.65 mV after 8 weeks of surgery which also increased to 8.98 ± 0.36 mV after 12 weeks of surgery as presented in Figure 10b. For the normal contralateral nerve, the average CMAP was 16.40 ± 0.96 mV. After 12 weeks, the cryogel implanted group showed an improvement of 54% in CMAP values. However, no peaks were obtained for negative control as there was no connection between the two cut nerve ends. These results suggest that the functional reinnervation has occurred through the implanted cryogel scaffold. 3.12.

Gastrocnemius muscle ratio (GM ratio)

The sciatic nerve innervates the gastrocnemius muscle. It is already known if this nerve-muscle interaction is disrupted; it leads to muscle atrophy, and the fibers degenerate leading to a loss in function. It was observed that within 2 weeks the GM ratio decreased to around 0.5 both in the experimental and the negative control group. This value further declined to 0.2 in the control


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group for over 12 weeks. However, in the experimental animals, significant improvement was observed, and the values stabilized around 0.4 till the end of 12 weeks as seen in Figure 10c. 3.13.

Sciatic nerve functional index (SFI) analysis

Functional recovery of both the control and the animals implanted with scaffold was calculated using sciatic nerve functional index. At the end of 4 weeks both the groups showed SFI value of approximately -100 showing total dysfunction. However, at the end of 6 and 12 weeks, there was an improvement in the SFI value of the animals with the implant as compared to the control. The SFI values of the experimental animal were -40 as compared to the SFI value of -83 of the control as shown in Figure 10 (d). Improvement in the toe spread was observed by the end of 12 weeks in the animals with the implanted scaffold compared to their control as seen in Figure 10 (e-g).

4. Conclusion In this study, we tried to differentiate the BMSCs to neural lineage in three ways. Cell to cell interaction activates/inhibits certain pathways in the BMSCs so that they are forced to differentiate into specific cell lineage. In the co-culture system, Neuro 2a cells provided a niche which induces the BMSCs to differentiate to neural lineage both by cell-cell interaction as well as the secreted factors in the media allowing their differentiation. This approach can be applied in case of peripheral nerve injuries where there is a reduction in the number of native cells and thus optimum regeneration. Scaffolds seeded with BMSCs can be implanted at the injured site leading to cell-cell contact with the native cells and secretion of signaling molecules from the differentiated BMSCs which will assist in vivo regeneration mechanism. The cocktail of the bioactive molecules provided by the spent media of differentiating Neuro 2a cells also induced the BMSCs to differentiate into the neural lineage. It would be interesting to analyze these 27 ACS Paragon Plus Environment


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secreted molecules responsible for differentiation of BMSCs further and thus pave new pathways in the field of regenerative medicine. It is also motivating to compare the spent media from the two systems to study the difference in the type of factors present and thus the difference in the functionality of the differentiated cells. Similarly, this approach can prove beneficial in case of delayed peripheral nerve surgeries, wherein most of the native cells are dead, and the optimum ratio required for co-culturing is not adequate. Lastly, the synthesized cryogel had optimum mechanical and chemical properties which enabled the BMSCs to differentiate into the neural lineage. Further, this directed differentiation would also minimize the use of costly bioactive molecules. The synthesized conducting cryogel because of their inherent properties like interconnected channels, porosity, and biodegradable nature directed the growth of the proximal sprouts to their distal targets. Also, the lack of the suturing of the nerve ends during the implantation process is also advantageous as it avoids scar formation. The animals implanted with these scaffolds showed improved functional recovery as compared to their control. Further combining the synthesized conducting cryogel with other strategies like drug delivery, growth factors, bioactive molecules, cell therapy, etc., would lead to an enhancement in the nerve regeneration process. Thus, the technology mentioned here offers a rapid, simple, biocompatible and useful approach for synthesizing aligned cryogel scaffolds that could serve as a potential biomaterial, for the synthesis of nerve guidance conduits in the field of nerve regeneration.

 Author Information Corresponding Author *Email: [email protected], Phone: +91 512 2594051 (A.K.)

 Author Contributions 28 ACS Paragon Plus Environment

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A.K. and T.V. designed the whole study and experimental plan. T.V, A.S., and A.K.T carried out the experimental work and analysis. The manuscript was written by T.V. with inputs from A.S. and A.K. and further reviewed by all the authors.

 Conflict of interest The authors declare that they have no conflict of interest.

 Acknowledgments The authors would like to acknowledge the Department of Biotechnology (DBT) Ministry of Science and Technology, Govt. of India for funding this research project (Project No. BT/PR13561/MED/32/392/2016). T.V. would like to acknowledge the Council for Scientific and Industrial Research, India for providing research fellowship. A.S. and A.K.T. would like to acknowledge IIT Kanpur for providing research fellowship. A.K. would like to acknowledge TATA innovation fellowship from the Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India.

 Abbreviations NGCs: Nerve guidance channels DMEM: Dulbecco’s Modified Eagle’s medium H&E: Haematoxylin and eosin DAB: 3,3′-Diaminobenzidine FBS: Foetal bovine serum SDS: Sodium dodecyl sulfate CTAB: Cetyl trimethylammonium bromide BMSCs: Bone marrow stem cells PBS: Phosphate buffer saline MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 29 ACS Paragon Plus Environment


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DMSO: Dimethyl sulfoxide SEM: Scanning electron microscope DAPI: 4',6-diamidino-2-phenylindole ELISA: Enzyme-linked immunosorbent assay NGF: Nerve growth factor BSA: Bovine serum albumin NCV: Nerve conduction velocity CMAP: Compound muscle action potential SFI: Sciatic nerve functional index GFAP: Glial fibrillary acidic protein NF: Neurofilament

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Figures and Figure legends:

Figure 1: (a) Representative digital image of the chitosan-gelatin-polypyrrole cryogels; (b) SEM image of the synthesized cryogels showing interconnected porous architecture and the presence of incorporated polypyrrole (black arrows). Microscopic image of alizarin stained cells, (c) differentiated BMSCs into osteogenic lineage and, (d) undifferentiated BMSCs. Cell proliferation analysis of (e) Neuro 2a, BMSCs and Neuro 2a co-cultured with the BMSCs and; (f) BMSCs cultured in the presence of the spent media. SEM analysis representing; (g) homogenous distribution of cells in the co-culture system; (h) the magnified image shows the presence of Neuro 2a and BMSCs contact in the co-culture system; (i) BMSCs with neural like morphology in the presence of the spent media and inset shows the magnified image of the same. Immuno-staining by neurofilament-TRITC and DAPI for; (j) spent media; (k) co-culture; and (l) BMSCs seeded on synthesized scaffold.



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Figure 2: (a) Gene expression analysis by RT-PCR of the BMSCs cultured in the presence of the spent medium; and (b) co-cultured Neuro 2a and BMSCs; (c) BMSCs seeded on chitosangelatin-polypyrrole cryogel; and d) BMSCs seeded on polyacrylamide (PAAM) cryogel, L=Ladder, N=Nestin, NF=Neurofilament, GF= Glial fibrillary acidic protein, G= GAPDH.


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Figure 3: Neurotransmitter analysis in co-culture, (a) Dopamine analysis by HPLC; (b) Acetylcholine analysis by kit. NeuN gene expression analysis by, (c) Real-time PCR in coculture, spent media and based on chemical and physical cues of the scaffold.** p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001.



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Figure 4: A) Scaffold implanted subcutaneously in the back of mice was excised at the end of (a) 72h; (b) 10 days; and (c) 1 month; (d) scaffold integrated with the host skin; (e) scaffold with blood vessels underneath; (f) magnified image showing the scaffold assisted angiogenesis. B) Hematoxylin & eosin (H & E) staining showing scaffold integration at the end of; (a) 72 h; (b) 10 days; and (c) one month; (d) magnified image showing implanted scaffold degradation after one month. C) Blood cell count of the control as well as experimental samples for a period of one month; D) analysis of interleukins in the serum by ELISA at the end of one month.


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Figure 5: A) H&E staining of the organs (a-e) in control and (f-j) experimental animals to study systemic toxicity. The scale bar is 200 µm. B) Fast green and safranin staining for mast cells at the end of; (a) 72h; (b) 10 days; and (c) one month. The scale bar is 200 µm.



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Figure 6: (A) Schematic representation of the sandwich model. (B) a) Image showing the isolated sciatic nerve; b) Image representing the implanted cryogel of length 2 cm after removing nerve tissue of 1.5 cm above the ramification point (inset image), c) The proximal and distal nerve ends were placed in between the synthesized scaffolds and tied with resorbable sutures on both ends such that the cut ends are intact in between creating a sandwich model.


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Figure 7: (A) a) Fibrous tissue formation at the nerve gap generated in the control; b) proximal; and c) distal end of the cut nerve innervated in the nearby host tissue after 4 weeks. The cut end of the nerve was intact in the scaffold after d) 2 weeks; e) 4 weeks; and f) 12 weeks. (B) Blood cell count in the control and experimental samples. (C) Body weight before and post-surgery after 2, 4, 6 & 12 weeks in the control and experimental samples.



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Figure 8: H&E staining showing proximal and distal end after the nerve transection after 2 weeks in the control (a, b) and in the experimental samples after; 2 weeks (c, d); and 12 weeks (e).


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Figure 9: a) Excised implant was cut in proximal, middle and distal part; b) H&E staining of the interface between the proximal and middle part; c) H&E staining of blood vessels in the middle part; d) SEM image showing infiltrated cells and their adherence on synthesized scaffold; e) magnified image of infiltrated cells. DAB-peroxidase staining for neurofilament in, (f) transverse sections; and (g) longitudinal sections at the end of 6 weeks; (h) FITC staining of the excised middle segment after 12 weeks; (i) FITC staining for S-100 marker of Schwann cells in the excised middle segment.



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Figure 10: Functional analysis of regenerated nerve. a) Nerve conduction velocity calculated after 8 and 12 weeks of surgery. b) Compound muscle action potential calculated after 8 and 12 weeks of surgery. c) Gastrocnemius muscle weight ratio of the negative control and cryogel implanted groups post-surgery. (d) Sciatic functional index calculated by walking track analysis for 2, 4, 6 and 12 weeks. Digital image showing improvement in the toe spread of the experimental animal’s post-surgery after; (e) 2 weeks, (f) 6 weeks, and (g) 12 weeks, ** p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001.


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Table of Contents (TOC)


Chitosan-gelatin-polypyrrole cryogel matrix for stem cell differentiation

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