FGF-18 incorporated Chitin-PLGA

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Injectable Shear Thinning CaSO4/FGF-18 incorporated Chitin-PLGA Hydrogels Enhances Bone Regeneration in Mice Cranial Bone Defect Model Sivashanmugam Amirthalingam, Pornkawee Charoenlarp, Sankar Deepthi, ArunKumar Rajendran, Shantikumar V Nair, Sachiko Iseki, and Rangasamy Jayakumar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15845 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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ACS Applied Materials & Interfaces

Injectable Shear Thinning CaSO4/FGF-18 incorporated Chitin-PLGA Hydrogels Enhances Bone Regeneration in Mice Cranial Bone Defect Model A. Sivashanmugam1, Pornkawee Charoenlarp2, S. Deepthi1, Arunkumar Rajendran1, Shantikumar V. Nair1, Sachiko Iseki2*, R. Jayakumar1* 1

Center for Nanosciences and Molecular Medicine, Amrita University, Kochi-682041,

India 2

Section of Molecular Craniofacial Embryology, Graduate School of Medical and

Dental Sciences, Tokyo Medical and Dental University, Tokyo-113 8510, Japan

---------------------------------------------------*Corresponding Author E-mail: [email protected] (Dr. R.Jayakumar) Tel: +91-484-2801234. Fax: +91-484-2802020 E-mail: [email protected] (Dr. Sachiko Iseki) Tel.: +81 3 5803 5579; fax: +81 3 5803 0213

1 ACS Paragon Plus Environment


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Abstract For craniofacial bone regeneration, shear thinning injectable hydrogels are favoured over conventional scaffolds due to their improved defect margin adaptability, easier handling and ability to be injected manually into deeper tissues. The most accepted method, after autografting, is the use of recombinant human BMP-2; however, complications such as inter-individual variations, edema, and poor cost-efficiency in supraphysiological doses have been reported. The endogenous synthesis of BMP-2 is desirable, and a molecule which induces this is FGF-18 since it can up-regulate the BMP-2 expression and positively modulate other osteogenic genes. We developed a Chitin-PLGA composite hydrogel by regeneration chemistry, and then incorporated, CaSO4 and FGF-18 for this purpose. Rheologically, a 7-fold increase in elastic modulus was observed in the CaSO4 incorporated Chitin-PLGA hydrogels as compared to the Chitin-PLGA hydrogel. Shear thinning HerschelBulkley fluid nature was observed for both hydrogels. Chitin-PLGA/CaSO4 gel showed sustained release of FGF-18. In vitro osteogenic differentiation showed an enhanced ALP expression in FGF-18 containing Chitin-PLGA/CaSO4 gel when compared to cells alone. Further, it was confirmed by studying the expression of osteogenic

genes

(RUNX2,

ALP,

BMP-2,

osteocalcin

and

osteopontin),

immunofluorescence staining of BMP-2, osteocalcin and osteopontin, and alizarin red S staining. Incorporation of FGF-18 in the hydrogel increased the endothelial cell migration. Further, regeneration potential of the prepared hydrogels was tested in vivo and µ-CT was performed at implantation, 1st, 3rd, 6th and 8th weeks. FGF-18 loaded Chitin-PLGA/CaSO4 showed early and almost complete, bone healing in comparison with Chitin-PLGA/CaSO4 and Chitin-PLGA/FGF-18, Chitin-PLGA and sham control systems, as confirmed by H&E and Osteoid tetrachrome staining. This


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shows that the CaSO4 and FGF-18 incorporated hydrogel is a potential candidate in craniofacial bone defect regeneration. Keywords: Shear thinning hydrogels, FGF-18, Chitin-PLGA, bone regeneration, craniofacial bone regeneration, Calcium Sulfate.



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1. INTRODUCTION Bone regeneration in the craniofacial complex (CFC), following tumour removal, trauma, and congenital deformities is a formidable obstacle in clinical practices1,2 which may lead to serious functional, aesthetic and psychological compromises. The current treatments for CFC reconstruction include usage of autografts, allografts and synthetic bone grafts. Although, bone grafts have features required for osteogenesis, like molecular cues, microenvironments and committed cells for osteogenesis, the limited availability, patient compliance, and regulatory body restrictions are the major limitations3,4 for their continued use. In this aspect, synthetic bone grafts are the next viable option for clinicians2. Injectable hydrogels are currently being explored for bone regeneration as they can possess similar properties to native tissues, have good bioadhesion at the defect site and provides a favourable niche for the cells to grow5,6. Injectability provides an additional benefit as they can adapt well to the defect margins, are easily handled, and can be injected manually even into deeper tissues through a minimally invasive procedure2,7,8. In situ hydrogels, a class of injectable hydrogel systems, requires either a cross-linking agent, or it may utilize physical properties (such as temperature, etc), to transit from sol to gel9 when injected at liquid state into the defect site, Other classes of injectable hydrogels include shear thinning hydrogels, in which viscosity drops down drastically at high shear rates allowing them to be injectable. The faster the reversion of the hydrogel into its initial viscosity, the better will be the control over the placement of the material at the defect site8,10. Chitin is natural amino-polysaccharides, possess several unique and suitable properties

for

biomedical

applications11.

Further,

chitin

would

elicit

mild

inflammation12 in comparison to its derivative chitosan, which is known to promote


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neutrophil chemotaxis13,14. Hence, chitin was preferred in the work over chitosan, even though; chitosan could also be prepared for injectable shear thinning hydrogel. Injectable shear thinning chitin hydrogels can be prepared using a chemical crosslinker free method which, provides better cyto-compatibility15. Blending chitin with synthetic polymers can improve the mechanical and degradation properties16. Poly(lactide-co-glycolide) (PLGA) an FDA approved polymer is often blended with other natural polymers to obtain favourable mechanical properties, and a degradation time suitable for bone regeneration17-15. The degradation property of PLGA can easily be fine-tuned by adjusting the molecular weight and the ratio of lactic acid to glycolic acid in the polymer. All these mutually exclusive properties can be used to our advantage for preparing composite hydrogels with improved properties7. α-Calcium sulfate hemihydrate (CaSO4.½H2O) has a long history of use in clinics for ridge and sinus augmentations, and guided bone regeneration20. It is well tolerated in the body, and evokes very minimal inflammatory response and rapidly dissolutes in comparison with calcium phosphate. The dissolute is shown to support osteogenic differentiation20. CaSO4 injectable pastes are available in the market for clinical use, but they suffer from shrinkage during setting and brittleness etc2,21. The aforementioned problems can be overcome by using a polymeric hydrogel/ceramic composite system for better bone regeneration in small and non-load bearing bone defects22–24 To improve the bone regeneration potential, growth factors such as the members of the bone morphogenetic protein family, especially BMP-2 and BMP-7 are clinically

25

. However, they are clinically used at supraphysiological

concentrations in humans (1.5mg for BMP-2 and 3.3mg for BMP-7)26, due to which complications such as oedema, inter-individual variations, poor cost-efficiency



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etc27,28. have been reported and side effects such as bone cysts, osteolysis, hematoma formation

have

also

been documented clinically27,29–31.

Hence,

researchers have shown interest in molecules which help in inducing the endogenous synthesis of BMP-2. One such candidate is fibroblast growth factor-18 (FGF-18), which increases BMP-2 expression by suppressing noggin28,32,33. FGF-18 is also known to regulate fibroblast growth factor receptors (FGFR-1, -2, -3, & -4)33. In addition to this, FGF-18 helps in the migration of endothelial cells34, which could aid in angiogenesis and thereby results better bone regeneration. In our study, we developed injectable shear thinning chitin-PLGA hydrogels containing CaSO4 and FGF-18 and tested their efficacy in cranial defects in mice. To the best of our knowledge, this is the first manuscript to study the combined effect of ceramic and FGF-18 under in vivo conditions.

2. Materials & Methods 2.1 Materials PLGA with molecular weight of 20kDa and lactic acid to glycolic acid ratio of 50:50 was purchased from Wako Chemicals, Japan. α-Chitin of average molecular weight ~100kDa and degree of acetylation >90% was purchased from Koyo Chemical Co., Japan.

FGF-18 was purchased from PeproTech Inc., Rocky Hill, USA. α -Calcium

Sulfate hemihydrate was purchased from Fischer Scientific, USA. Calcium Chloride dihydrate and Methanol were purchased from Merck Chemicals, USA. Alamar Blue, Collagenase Type I, Fetal bovine serum, Minimum essential media–alpha modification (α-MEM), Iscove’s modified Dulbecco’s media (IMDM), Large Vessel Endothelial Supplement (LVES), and Trypsin-EDTA were purchased from Gibco, Thermo Fisher Scientific, USA. All the chemicals were used without any modification


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and purification. For all the preparation, wherever required, milliQ water (18.2 MΩcm) was used.

2.2 Methods 2.2.1 FGF-18 loaded Chitin-PLGA/CaSO4 hydrogel preparation: Chitin-PLGA hydrogel was prepared by chitin regeneration technique with modification of a reported method35. Briefly, 0.5 w/v% α-chitin solution was prepared in methanol saturated with CaCl2.2H2O at room temperature (RT). PLGA was also dissolved in methanol saturated with CaCl2.2H2O at 65°C. The w/w ratio of chitin and PLGA was maintained at 4:1. α-Chitin and PLGA solution was mixed together and stirred for 2 hours at 65°C. Further, the temperature was reduced to RT and stirred for 8 hours. In order to regenerate α-chitin-PLGA anhydrous gel, to the α-chitin and PLGA solution, an equal volume of methanol was added. The regeneration was performed for 24 hours and then the gel was washed and rehydrated with water (Fig. 1). The gel was washed with water 6 times to completely remove methanol and CaCl2 by centrifuging it at 20,000 rcf. Prior to each wash, gel was incubated in water for 30 min and centrifugation was performed. The prepared chitin-PLGA hydrogel (CP Gel) was stored at RT until further use. For preparing CaSO4 incorporated hydrogel, 20 w/w% to chitin-PLGA hydrogel was mixed using agate spatula in Petri plate and will be referred as CP CS Gel. FGF-18 incorporated gel was prepared by adding FGF-18 solution (of 100 and 500ng) to the CP Gel (20mg). The hydrogel was then incubated at 37°C for 30 minutes. These hydrogels will be represented as CP F100 Gel and CP F500 Gel, with CP Gel containing 100 and 500ng of FGF-18, respectively. To prepare, CaSO4 and FGF-18 incorporated hydrogel, they were added sequentially as per the



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procedure mentioned above (Fig. 1). For in vitro and in vivo studies, the CP Gel and CaSO4 was UV sterilized before use.

Figure 1: Schematic representation of preparing Chitin-PLGA Hydrogel and incorporation of CaSO4 and FGF-18 to it. 2.2.2 Physio-Chemical Characterization: The prepared hydrogel was analysed using scanning electron microscope (JEOL JSM-6490LA SEM). Briefly, 10mg of the hydrogel was placed over the specimen stub, allowed to air dry at RT (overnight), gold sputtered and imaged. To observe the size and the shape of the individual chitin-PLGA microgels, 1mg of the sample was dispersed in water at a ratio of 1:100, probe sonicated for a minute at 30% amplitude and then drop casted, gold sputtered and imaged. Fourier transformed infrared spectroscopy (FTIR) was carried out using the lyophilized samples in Shimadzu IRAffinity-1S. The transmitted IR spectra were recorded from a range of 4000 to 500 cm-1. 2.2.3 Rheological Studies: The prepared hydrogels were subjected to rheological characterization using Malvern Kinexus pro (Malvern Instruments, UK) as per the


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protocols published earlier36. Briefly, dynamic shear strain sweep was performed to determine the linear viscoelastic region (LVER) for the hydrogel at constant frequency (1Hz) from 10-2 %. All the rheological studies were performed in LVER. Dynamic frequency sweep studies were carried out from 10 to 0.1 Hz to understand the structural response to deformation. The flow behaviour of prepared hydrogel was performed under steady shear. The three structural rheological model fits were studied to calculate the yield stress, which corresponds to minimum stress necessary for initiating the flow of the hydrogel. The model fit was studied for Bingham, Casson and Herschel–Bulkley models. The change in the viscosity during the injection and the recovery after ejection of gel has to be studied, as these hydrogels would be injected in the defect site. The shear rate during the injection was determined using the following expression:

ߛሶ =

ொ గ௥ య

ଵ

[3 + ] ௡

Where, ‘Q’ is volumetric flow rate, ‘r’ inner radius of the needle through which gel will be injected (in this case 18G needle was used), ‘n’ is the flow behavior index and ’ߛሶ ‘ is calculated application shear rate. The calculated shear rate was used to study the change in viscosity during the injection and to the ejected hydrogel. The shear rate loop test was ramped up from 0.1 s-1 to the calculated application shear rate and then to initial value of 0.1 s-1. This study will help to calculate the shear rate required to extrude the hydrogel and the extent of hydrogel recovery in terms of viscosity following this process. 2.2.4 Injection Force and Inversion Test: Injectability test was performed to calculate the injection force required for extruding the hydrogel according the protocol

published8.

Experiment

was

performed

using

mechanical

tester

(TiniusOslen, USA). Prepared hydrogel was loaded in 2ml syringes (Hindustan 9 ACS Paragon Plus Environment


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Syringes & Medical Devices Ltd) and injected through 18G needle (BD Biosciences). The upper compression platen lowered down so as to achieve the flow speed of 1ml/min and the needle was clamped onto a tensile grip, to avoid the movement during the injection. The injection force was measured using a 250N load cell. To check the Injectability and smoothness of the hydrogel visually, the prepared hydrogel was loaded onto 2ml syringe fitted with 18 G needle and manually injected. Inversion test was carried to study whether the prepared hydrogel showed time dependent shear thinning property according to the published protocols37. It was carried out in a flat bottom 5ml storage vial (Tarsons, India) with an internal diameter of 20mm. The prepared hydrogel of volume 2ml was injected in each tube and inverted vials were placed on flat surface, undisturbed. 2.2.5 In Vitro FGF-18 Release Profile: The prepared CP F500 Gel and CP CSF500 Gel were used to study the FGF-18 release profile. To 80mg of FGF-18 loaded hydrogel, 500µl of PBS was added on top of the hydrogel and incubated at 37°C for 10 days. At each pre-determined point, 50µl of supernatant was sampled for analysis and replenished with equal volume of fresh PBS. Release of FGF-18 from these two hydrogel was analysed using ELISA kit, according to the manufacture’s protocol (Cusabio, Hubei, China). 2.2.6 Cell Proliferation Assay: Cell proliferation assay was carried out in rat adipose derived stem cells (rADSCs). rADSCs were isolated from the adipose tissue of Sprague Dawley rats after the ethical consent from Institutional Animal Ethical Committee, Amrita Institute of Medical Sciences. The isolation procedure was followed as per the protocol published38. For the cell culture experiments, passage no 3-6 was used. rADSCs were used for both cell proliferation and osteogenic differentiation studies to understand the viability and proliferation of stem cells


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towards the prepared hydrogel systems and later to understand the osteogenic differentiation. For the cell proliferation assay, 1*104 rADSCs were seeded on 24 well plate containing 20mg of hydrogel (n=3). The cell proliferation was studied for 24 and 48 hours of time period. After the respective time points, the media was removed and washed twice using PBS. Cells were incubated with 10% Alamar blue reagent in media and optical density values were recorded at 570nm, with 600nm as reference using spectrophotometer after 4 hours of incubation39 (Biotek Power 223 Wave XS). A graph of cell number standard plot (cell number versus alamar O.D values) was plotted to study the cell proliferation in the presence of the hydrogels tested. 2.2.7 Cell Migration Assay: Two different kinds of cell migration assay were performed to study cell migration through the CP and CP CS hydrogel, and migration of cells towards hydrogels, tested for all the prepared samples. Cell migration through the hydrogel was studied according the protocol published earlier with modifications40. Briefly, 20mg of the hydrogel was added into the transwell inserts (Corning, USA) with 8µm pore size and 6.5mm diameter inserts. Human umbilical cord derived endothelial cells (HUVEC) were isolated from umbilical cord using already published protocol41 with institutional ethical clearance from the Amrita Institute of Medical Sciences. HUVECs were used to study the migration of endothelial cells, as FGF-18 positively affects it, and migration of endothelial cells in the defect area would be one of the important steps in bone regeneration. 2*104 HUVEC were seeded on top of the hydrogel and 60µl of the IMDM media was added on the hydrogel. To induce chemotaxis, 600µl of IMDM with LVES was added. The plate was incubated for 20 hrs to allow the cells to migrate. After the end point, hydrogel was removed, washed 3 times in PBS and mopped using cotton tip applicator to remove cells in the top layer. The migrated cells which are present in



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the underside of the membrane were fixed stained using DAPI and imaged. The micrographs were captured using fluorescence microscope (Leica, DMI3000 B with fluorescent bulb attachment, USA). The number of cells per microscopic field was counted using ImageJ 1.6.0V(NIH, USA). Cell migration in the presence or towards the prepared hydrogel was performed in accordance with the protocol published42. Briefly, 2*104 HUVEC were seeded on the inserts and 60µl of IMDM was added. 20 mg of the hydrogel was injected in the well plate and 600 µl of IMDM was added. The migration of the cells on the underside of the membrane was analysed after 20 hours of incubation. 2.2.8 In vitro Osteogenic Differentiation: Osteogenic differentiation was carried out for the prepared hydrogels. Briefly, 3*104 rADSCs were seeded on 24 well plate. After 24 hours, 20 mg of prepared hydrogel were added in wells. Cells were cultured in

osteogenic

media

(50µg/ml

ascorbic

acid-2

phosphate,

10mM

β-

glycerophosphate, and 10nM dexamethasone in α-MEM containing 10% FBS and 1% antibiotic-antimycotic). Cells were replenished with media every 3 days. ALP was analysed at end of 7th, 14th, 21st& 28th day time point. The cell lysates were obtained by treating the cells with 1% TritonX-100 for 1 hour. To 50µl of p-nitrophenyl phosphate (PNPP) substrate solution, 80µl glycine buffer and 50µl cell lysate were added in 96 well plate. After 30 min of incubation, the reaction was stopped by adding 20µl of 3N NaOH. The absorbance was measured at 405nm in spectrophotometer (Biotek Power 223 Wave XS, USA). The ALP concentration was obtained from the standard ALP graph and they were normalized against the protein concentration. The protein concentration was assayed using bicinchoninic acid (BCA) assay. Mineralization associated with osteogenesis was studied by alizarin red S staining. On 21st day of osteogenic differentiation, the cells were washed with


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PBS and fixed with 4% PFA solution for 20 minutes. Upon washing with distilled water, cells were stained with 2% Alizarin red S solution (pH 4.2) for 30 minutes. Cells were viewed under microscope after rinsing with distilled water16. 2.2.9 Gene Expression Studies: Total RNA was isolated from cells at experimental time points (7th Day, 14th Day and 21st Day) using TRIzol Reagent (Invitrogen, USA). cDNA was synthesized according to manufacturer’s instruction (Verso cDNA synthesis kit, Thermo Scientific, USA). Early Osteogenic gene markers (RUNX-2, ALP, & BMP-2) were analysed on day 7 and day 14. Late Osteogenic gene markers (Osteocalcin and Osteopontin) were analysed on day 14 and day 21. Quantitative RT-PCR was performed using DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific, USA) in ABI PRISM 7900 HT system (Applied Biosystems). The obtained Ct values for target genes were normalized using β-actin as housekeeping gene, to finally obtain 2-∆∆Ct value expressing as relative fold expression. RUNX-2 FP: 5’-CGG TCTTCACAAATCCTCCC-3’, RP:5’-GGACACCTACTCTCATACTGGG-3’; ALP FP: 5’-ACAATGAGATGCCGCCAGA 3’, RP:5’-CAAATGCTGATGAGGTCCA GG- 3’; BMP-2 FP: 5’-TCCATCACGAAGAAGCCATC-3’, RP: 5’-CTTCCTGCATTTGTTCCC G A-3’; OCN FP: 5’-CAAAGCCTTCATGTCCAAGCA-3’, RP: 5’-AGCTCGTCACAAT TGGGGTT-3’; OPN FP: 5’-GAAACTCTTCCAAGCAACTCCA-3’, RP: 5’-AGTTCACA GAATCCTCGCTCT-3’; and β-actin FP: 5’-TTCAACACCCCAGCCATGT-3’, RP: 5’CAGTGGTACGACCAGAGGCATAC-3’. 2.2.10

Immunocytochemical

Staining

Studies:

Immunocytochemistry

was

performed for studying bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). The protein expressions were studied after performing osteogenic differentiation in the presence of the prepared hydrogels. BMP-2 expression was studied by tagging it with rabbit anti-BMP2 antibody (Novus



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Biologicals, USA) at the end of 7th and 14th day, OCN and OPN expressions were studied at 28th day using mouse anti- OCN antibody (Pierce Antibodies, USA) and mouse anti-OPN antibody (OriGene Technologies, USA), respectively. The cells after the specific time points were fixed in 4% PFA, permeabilized with 0.5 v/v% triton X-100 in PBS and blocked with 3% bovine serum albumin in PBS. The cells were incubated with primary antibodies of anti-BMP-2, anti-OCN, and anti-OPN at a dilution of 1:100, 1:200 and 1:100, respectively, for overnight at 4°C. Goat anti-rabbit IgG (Life Technologies, USA) and goat anti-mouse IgG (Life Technologies, USA) conjugated to FITC was added at a dilution of 1:200 and incubated for 5 hrs. TRITC conjugated phalloidin (Sigma-Aldrich, USA) was used to counter stain actin filaments and analyzed using confocal laser microscopy (Leica TCS SP5 II, USA). 2.2.11 In Vivo Mouse Cranial Defect Studies: Four week old ICR mice were used for the study. Animal experiments were approved by institute animal care and use ethical committee, Tokyo Medical & Dental University (Approval No: 82460). Full through cranial defect was created using 3mm biopsy punch and 20mg of the hydrogel was injected in the defect area (Fig. S1). Sham Control (n=4), CP Gel (n=6), CP CS Gel (n=6), CP F500 Gel (n=6) & CP CSF500 Gel (n=6) were the groups studied. The dissected calvarial vault was histologically analysed. Refer the Supporting Information for the detailed steps involved in creating cranial defect model. 2.2.12 In vivo Live Animal µ-CT imaging: µ-CT was performed using inspeXio SMX100CT (Shimadzu, Japan). Each operated mouse was radiologically imaged on 0th, 1st, 3rd, 6th, and 8th week time points. Volume rendering software was used to reconstruct and visualize the images in three-dimensionally. ImageJ Software (1.6.0v, NIH, USA) was used to analyse the bone formation and coverage %. BoneJ


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plugin was used to measure the bone volume (BV) and bone volume to total volume ratio (BV/TV)43. 2.2.13 Histological Imaging: The upper cranial vault was fixed in normalized formalin buffer and then decalcified in Morse’s Solution for 24 hours. The samples were dehydrated in graded ethanol series, paraffin embedded, and sectioned at 5 µm thickness for staining. The sections were stained by Hematoxylin-Eosin (H&E) and by osteoid tetrachrome staining with the protocol as published earlier44,45. 2.2.14 Statistical Analysis: The results were represented as mean ± S.D, n>3. Statistical analysis was performed using ANOVA and post modification for multiple comparisons was performed using Tukey’s test. p value less than 0.05 was considered to be statistically significant.

3. Results and Discussion 3.1 Preparation of FGF-18 loaded Chitin-PLGA/CaSO4 Hydrogel: Chitin and PLGA solution were blended together to have physical interlocking in the polymeric chains. Methanol was added to regenerate chitin-PLGA in hydrogel form by simple regeneration chemistry. The probable chemical interaction between chitin and PLGA polymeric chains were through hydrogen bonding, which were between the carbonyl group of PLGA with hydroxyl and acetamide groups of chitin46. Repeated gel washings were performed to ensure removal of CaCl2 and methanol. SEM images revealed that individual chitin-PLGA microgels are of size 3±.5µm (Fig. 2A-Insert) They are further self-assembled to form bulk hydrogel, which is consistent with published data35,46. Incorporation of CaSO4 in CP Gel resulted in consistent and moldable hydrogel. Different concentration of FGF-18 solution in the hydrogel



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resulted in 100ng and 500ng incorporated CP and CP CS hydrogel. Refer Table 1 for the hydrogel composition and the abbreviation which are used in the manuscript.

Material Name

CaSO4 (% W/W)

FGF-18 (ng)

CP Gel

--

--

CP CS Gel

20

--

CP F100 Gel

--

100

CP F500 Gel

--

500

CP CSF100 Gel

20

100

CP CSF500 Gel

20

500

Table 1: The Composition of the hydrogel prepared. 3.2 Physico-Chemical Characterization of Chitin-PLGA Hydrogel: SEM analysis of the hydrogel revealed that CP Gel (Fig. 2A) has a smooth morphology and, CaSO4 incorporated CP Gel (Fig. 2B) shows needle shaped crystals of CaSO4.2H2O due to the setting morphology of calcium sulfate hemihydrate47. FTIR analysis showed the characteristic peaks of chitin’s35,46 amide I (1645cm-1), amide II (1550cm1

), asymmetric deformation (1370cm-1) and β-1,4 glycosidic (1068cm-1) bonds, and

characteristic peak of PLGA46 of carbonyl (1735cm-1) and -C-O stretching (1035cm-1) bonds were present in CP Gel (Fig. 2C). The characteristic peaks of CaSO415 are -SO4 stretching vibrations (596 & 665 cm-1), and -SO4 stretching (1120cm-1). The characteristic peaks of chitin, PLGA and CaSO4 were present in the CP CS Gel, (Fig. 3C) indicating the blending of the parent materials.


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Figure 2:

Physio-Chemical Characterization of prepared hydrogels. Scanning

electron micrograph of CP Gel/insert image shows the SEM image of individual microgel (A) and CP CS Gel (B), and (C) FT-IR spectra of chitin, CaSO4.2H2O, PLGA, CP Gel and CP CS Gel, and their corresponding peaks were marked. 3.3 Rheological Studies: The storage modulus (G’), loss modulus (G”), phase angle (δ) and viscosity (η) were the parameters for studying the hydrogel strength. For all the prepared hydrogels, the G’ (elastic component) was higher than the G” (viscous component) attribute of the hydrogel. The rheological properties were studied for only CP Gel and CP CS Gel, as the addition of 100 and 500ng of FGF-18 didn’t change any rheological properties of the parent CP and CP CS Gel. The storage modulus of the CP Gel was found to be 10.5±1.2 kPa and that of the CP CS Gel was 165±3 kPa. Thus a 16-fold increase in the storage modulus was observed upon to the incorporation of CaSO4 (Fig. 3A). The phase angles (δ) of CP and CP CS Gels were found to be less than 10°, indicating that the hydrogel is solid dominant. There were no variations in the storage modulus for both the hydrogels over the studied



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frequency range. So, the hydrogel remained stable over a period of time. This is one of the parameters required for injectable hydrogels36. The flow behaviour of the CP Gel and CP CS Gels was studied under steady shear sweeps and the model fitting of shear rate vs shear stress for the 3 different models were analyzed, viz Bingham, Casson and Herschel-Bulkley fluid models. The yield stress (σ), flow behaviour index (n) and the correlation coefficient (r2) were analysed in these model fits. Both gels behave as Herschel-Bulkley fluids with r2 being 0.9803 and 0.9665, respectively. The other two models had lower r2 values as compared to the Herschel-Bulkley fluid model fit (Table 2). The n values for the CP Gel and CP CS Gel were found to be 0.5345 and 0.4816, respectively (Fig. 3B). A flow behaviour index less than 1 indicates the prepared hydrogel48 is not Newtonian. The yield stress values are 959.7 Pa for the CP Gel and 1,419 Pa for the CP CS Gel (Table 2). These studies were performed before the setting time of the hydrogel, so that it doesn’t affect the rheological properties during the study. The Herschel– Bulkley fluid model is an extension of the Ostwald-de Waale (Power law model) with the yield stress10,48,49. The yield stress is defined as the threshold stress value beyond which the hydrogel will begin flowing, below which, the hydrogel behaves as the solid, with strong interactions between polymeric chains. Beyond the yield stress value, there can be a rearrangement in polymeric chain interactions which allows the hydrogel to flow in the direction of stress applied. The k, σ and r2 values under the Casson and the Bingham model are showed in Table 2. These models failed, probably since they consider the material to be more viscous and lacking in an exponential stress growth term48,49.


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Model fit type

Yield Stress (Pa) CP CP CS Gel Gel

K CP Gel

Chi Square

CP CS Gel

CP Gel

CP CS Gel

HerschelBulkley 703 3040 37.62 1141 3.108*104 6.596*106 Model Casson 719.3 3501 0.967 233.8 2.395*104 8.156*107 Model Bingham 654.1 3001 4.545 199.3 7.219*104 6.825*108 Model Table 2: Model fit analysis of CP Gel and CP CS Gel

Correlation Coefficient CP Gel

CP CS Gel

0.9813

0.9665

0.9671

0.8802

0.9208

0.8710

Ideally, from an application point of view, shear thinning hydrogels should have the same viscosity before and after injection. The shear rate required to inject the hydrogel into the defect site was calculated through the Eq. 1. The flow curve experiment was performed from 0.1 s-1 (stress which doesn’t affect the hydrogel property) to the calculated shear rate and ramped up again to study the changes in the viscosity15,50,51. The value of Q and the needle gauge were unchanged, so as to easily compare the properties of CP Gel and CP CS Gel. The parameters taken into the study are mentioned in the Table 3. The calculated shear rate for CP Gel and CP CS Gel were found to be 346.3 s-1 and 385.3 s-1, respectively (Table 3). The viscosity of the CP Gel reduced from 5178 Pa.s to 2.713 Pa.s after the application of the 346.3 s-1 shear rate. In the case of CP CS Gel, the viscosity was found to reduce from 41,430 Pa.s to 10.75 Pa.s after the application of 385.3s-1 shear rate. The area under the curve (AUC) between the shear rate in the ‘up’ and ‘down’ cycles was calculated to show the shift in the viscosity during the application.



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Inner n Shear Volumetric Diameter Shear Material (power Viscosity Flow of the Rate Viscosity(Pa Name law (Pa s) -1 Rate (µl/s) Needle (s ) s) index) (mm) CP Gel 10 0.84 0.5315 346.7 1849 2.713 CP CS Gel 10 0.84 0.4816 385.3 1104 10.75 Table 3: Parameters and results of steady shear rate sweep analysis Material The AUC for the CP Gel was found to be 3157, indicating that there is a change in the viscosity of injected hydrogel, after recovery. In the case of CP CS Gel, the AUC was found to be 346.7, which is a relatively small change in the viscosity during recovery. The viscosity of the CP hydrogel after applying the calculated shear rate was found to be 3190 Pa.s after reverting back to 0.1s-1, whereas in the case of CP CS hydrogel, it was found to be 38500 Pa.s (Fig. 3C). The alteration in the viscosity before and after the application of calculated shear rate and the AUC in the case of CP Gel indicates a inadequacy in the complete recovery and rearrangement of the polymeric chains, so, the CP CS Gel, CaSO4 could act as reinforcing agent to achieve greater than 95% recovery in the viscosity. In specific, the charge interactions between CP polymeric chains and CaSO4 could help in recovery of the hydrogel, after injection.


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Figure 3: Rheological characterization of the prepared hydrogels. (A) Frequency sweep analysis of CP Gel (triangles) and CP CS Gel (squares) showing the G’ (Storage Modulus) (in red), G’’ (Loss Modulus) (in blue) and the δ (phase angle) (in green); (B) Herschel-Bulkley model fit curves for CP Gel (in blue) and CP CS Gel (in red), wherein original data (in squares) and the expected model fit (in triangles); (C) Steady shear rate ramp ‘down’ and ‘up’ for the CP Gel (in blue) and CP CS Gel (in red); 3.4 Injection Force and Inversion Test: To imitate the injection process in a clinical scenario, and to identify the injection force required to extrude the hydrogel from a needle, an experiment was carried out where, the hydrogel was loaded in a 2ml syringe at a flow rate of 1ml/min (same as Q tested in rheological studies) with a 18G needle in a mechanical tester set up as shown in the Fig. 4A. The injection force 21 ACS Paragon Plus Environment


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was found to be 8.1±2.5 N and 28±1 N for CP Gel and CP CS Gel, respectively (Fig. 4B & C). The obtained injection forces are well within the range for manual injection, without the requirement of additional instruments8. For visualization, the hydrogel was injected through 18G fitted syringes. Both CP Gel and CP CS Gel were injectable with smooth edges and continuously flowed without any breakage (Fig. 4E). The inversion test can give a visual cue of time dependent shear thinning property of the visco-elastic material. The inversion study revealed that the prepared hydrogel doesn’t flow under gravitational force even over a period of 24 hrs (Fig. 4D). This could be because, the yield stress of these prepared hydrogels (Table 2) were larger than the gravitational stress because of which the hydrogel matrix remained undisturbed37. The result of the inversion test can also be attribution to the adhesion property of the hydrogel towards the surface of the container, which is proportional to the water content in the hydrogel. This is an important property for shear thinning hydrogels since they should be injectable only when the yield stress is applied and should retain their shape at the defect site.


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Figure 4: Injectability of the hydrogels and FGF-18 release profile. (A) Schematic representation of injection force measurement using mechanical tester; (B) Injection force required for the hydrogel to be injected through a 18G needle and the (C) Injection force graph for CP Gel and CP CS Gel; (D) Inversion test of CP Gel and CP CS Gel for 0th and 24th hour time periods, (E) Manual injectability test of CP Gel and CP CS Gel and (F) In Vitro FGF-18 release profiles of CP F500 Gel and CP CSF500 Gel in PBS. 3.5 In Vitro FGF-18 Release Profile: CP F500 gel showed a burst release of 26.74±3.27% of FGF-18 within 6 hours and 60.07±2.01% of FGF-18 by 48 hours. Nearly 100% of the FGF-18 loaded was released by 10th Day. In case of CP CSF500 gel, only 9.15±0.98% of FGF-18 was released by 6 hours and 16.99±2.98% of FGF18 was released by 48 hours. By 10th day, 53.3±3.78% of FGF-18 loaded was released in PBS (Fig. 4F). FGF-18 would have been adsorbed on to CP Gel matrix and in the presence of PBS, the physical interaction between FGF-18 and CP Gel 23 ACS Paragon Plus Environment


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matrix, being weak, 60% of loaded FGF-18 was released within 48 hours, whereas, it will take more than 10 days for FGF-18 to be released from the CP CSF500 Gel. The release rate of FGF-18 would be regulated by CaSO4 as FGF-18 would have been bound or entrapped in CaSO4-CP gel matrix20,26,52. 3.6 Cell Proliferation Assay: Cell proliferation assay was studied with rADSCs for 24 and 48 hours using Alamar Blue assay. The results indicate that the prepared CP Gel is cytocompatible and the incorporation of FGF-18 in the hydrogel increases the proliferation of the cells (Fig. 5A). There was a statistical significance in the cell proliferation for CP F100 and CP F500 Gel as compared to all the other samples. This could be because of the presence of FGF-18, a member of FGF family, which increases the cell proliferation53,54 by a dual receptor system, namely by FGF receptors and heparin sulfate proteoglycan55–57. The release rate of FGF-18 was regulated in presence of CaSO4 which then results in a lower level of FGF-18 release from the hydrogel system (Fig. 4F) and thereby a comparatively lesser proliferation rate as compared to CP F100 and CP F500 Gel. Yet the cell proliferation rate of CP CSF100 and CP CSF500 was statistically higher at 48-hour time point, when compared to cells alone, CP Gel and, CP CS Gel. 3.7 Cell Migration Assay: Cell Migration through the hydrogel was performed in hydrogel injected transwell inserts, so as to better understand the migration of cells in vivo, and the schematic representation of the experiment is depicted in Fig. 5B. The Fig. 5B represents the cell migration through the hydrogel system, wherein, gel systems were added on top of the transmembrane, upon which the cells were added, to study the migration of cells through the hydrogel layer. The cells which migrated through the hydrogel and the insert membrane were taken into consideration. The number of cells which migrated were counted and recorded per


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field of view (Fig. 5D) and the representative fluorescent images of the DAPI stained nuclei are shown in the Fig. 5C. The number of cells which migrated in CP Gel and CP CS Gel had no statistical significance, which shows that the cells were able to migrate through the calcium sulfate incorporated gel. The addition of CaSO4 to the CP Gel (at 20 w/w%) did not hamper the migration of cells through the hydrogel system. The migration of the cells could be possible because the prepared chitinPLGA hydrogel were microgels as described earlier, and the cells would have migrated through the gaps between the individual microgel particles. Li et al58 have showed that pockets of cell migration were seen in the gelatin microgel incorporated PEG hydrogels and cell migration was not seen in the control PEG hydrogel. This was because of the incorporation of the microgels in the PEG hydrogel, as the cells were able to migrate through the space between the gelatin microgel particles. After studying the cell migration through the hydrogel, experiment was performed to analyze the migration of cells towards to the prepared hydrogel system (Fig. 5E). The Fig. 5E represents the migration of cells towards the hydrogel system, wherein the gel is being added at the well plate and cells were added on top of transmembrane. The transwell inserted is placed in the gel containing well plate to understand the components of hydrogel is acting as molecular cues for attracting the cells towards them. For this study, the cells were seeded directly on to the membrane42. In the well plate, 20mg of the prepared hydrogel was injected and culture media without any supplements were added. The cells were allowed to migrate for 20 hours to understand the chemo-attraction of the cells towards all the prepared hydrogel systems (Fig. 5G). The results show that the incorporation of FGF-18 in the hydrogel increased the HUVEC cell migration. CPF100 Gel and CPF500 Gel showed a similar cell migration after the incubation time and, the



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number of cells migrated/field were higher than CP CSF100 Gel and CP CSF500 Gel (Fig. 5F). This phenomenon could be because of higher quantity of FGF-18 that have been released from CP F100 and CP F500 Gel when compared to calcium sulfate incorporated hydrogel. It could also be because the FGF-18 was bound to the CaSO4 crystals and the setting of the CaSO4 crystals prolonged the release of FGF-1820,52,59. This might have led to a lower FGF-18 release into the culture media, and thereby reduced cell migration in comparison to FGF-18 incorporated CP Gel. Even though the number of cells which migrated was lower at the studied time point for the FGF-18 incorporated CP CS Gel, the prolonged release of FGF-18 would be favourable for in vivo bone regeneration60–62.


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Figure 5: Effect in Cell proliferation and Cell migration of FGF-18 loaded CP and CP CS Gel. (A) Cell proliferation of rADSCs in the presence of prepared hydrogel at 24 and 48 hours. (B-D) Results of cell migration through CP Gel and CP CS Gel, (B) Schematic representation of the cell migration experiment through hydrogel, (C) Representative image of endothelial cells migrated through CP Gel and CP CS Gel, (D) Quantification of cells migrated through the hydrogel and it is represented as no. of cells migrated per microscopic field. The images are taken at 100x magnification and the cells are counted using ImageJ software and (E-G) Cell migration towards the FGF-18 loaded CP and CP CS Gel. (E) Schematic showing the for cell migration towards the hydrogel. (F) Quantification of cells migrated towards the hydrogel and the representative images of cell migrated towards hydrogel is shown in (G). Scale bar represents 100µm. 3.8 In Vitro Osteogenic Differentiation, Gene Expression and Immunocytochemical Staining Studies: ALP enzyme was quantified on 7, 14, 21 & 28 days of rADSCs osteogenic culture in the presence of the prepared hydrogels. The cells exposed to every hydrogel prepared showed a rise in ALP concentration on 14th day, and declined after that time point, a phenomenon which is typically seen for osteogenic differentiation63. There was a 1.9-time increase in the ALP release from the cells which were exposed to FGF-18 loaded CP CS Gel when compared to cells alone. There was a 1.3-fold increase in the ALP expression for cells exposed to CP CS Gel when compared to the control cells. The cells exposed to CP F500 Gel showed a 1.4-fold increase in the ALP expression when compared to the control cells (Fig. 6A). Mineralization associated with osteogenic differentiation was studied by staining the cells with Alizarin Red S staining. A dense and increased number of foci of calcium deposition was seen by alizarin red S staining for cells differentiated


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in the presence of CP CSF500 Gel, compared to all other samples. Mineral deposition for CP CS Gel and CP F500 Gel group showed higher positive region for mineral deposition in comparison to cells alone (Fig. 6B). Hence, individually, CaSO4 and FGF-18 were able to positively affect the osteogenic differentiation. Hence, in combination, CP CSF500 Gel sample showed denser, wider and highest number of foci for mineral deposition. Jeon et al53

and Hamidouche et al64 has previously

showed that FGF-18 exposure to mesenchymal stem cells during osteogenic differentiation increased ALP activity and calcium deposition.

Figure 6: Osteogenic differentiation of rADSCs in the presence of prepared hydrogels. (A) ALP expression of rADSCs during osteogenic differentiation on 7th, 29 ACS Paragon Plus Environment


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14th, 21st, & 28th day in the presence of prepared hydrogels. (B) Alizarin Red S staining of rADSCs on 28th day of osteogenic differentiation in the presence of prepared hydrogel. Scale Bar Represents 50µm. p-value

FGF-18 incorporated Chitin-PLGA

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