Injectable in Situ Shape-Forming Osteogenic Nanocomposite

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Injectable in Situ Shape Forming Osteogenic Nanocomposite Hydrogel for Regenerating Irregular Bone Defects Sivashanmugam Amirthalingam, Ashvin Ramesh, Seunghun S. Lee, Nathaniel S. Hwang, and Rangasamy Jayakumar ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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ACS Applied Bio Materials

Injectable in Situ Shape Forming Osteogenic Nanocomposite Hydrogel for Regenerating Irregular Bone Defects Sivashanmugam Amirthalingam1#, Ashvin Ramesh1#, Seunghun S. Lee2#, Nathaniel S. Hwang2* and Rangasamy Jayakumar1* 1

Center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Center, Amrita Vishwa Vidyapeetham, Kochi-682041, India 2

School of Chemical and Biological Engineering, NBio Institute, Institute of Chemical Processes, Seoul National University, Seoul, 151-744, Republic of Korea

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These authors contributed equally.

* Corresponding Author email Id: [email protected], [email protected](Dr. R. Jayakumar) Tel: +91-484-280-1234 email Id: [email protected] (Dr. Nathaniel S. Hwang) Tel: +82-02-880-1635

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ABSTRACT The in situ forming injectable hydrogels are appealing for irregular bone defects due to the ease of administration and the addition of ceramics, molecules and proteins into the hydrogel. We have developed in situ shape forming hydrogel using oxidized alginate and gelatin as the hydrogel matrix. Whitlockite bioceramic nanoparticles (WH NPs) were incorporated as they provide enhanced osteogenic stimulation compared to hydroxyapatite via providing higher local ion concentration. The drug simvastatin was also incorporated into the hydrogel system as it increases the expression of BMP-2 thereby provide environment for bone regeneration. The presence of both WH nanoparticles and simvastatin would enhance bone regeneration potential. The whitlockite nanoparticles (80±8nm) were synthesized by precipitation method and were characterized. The nanocomposite hydrogel system was characterized by SEM, FTIR and rheologically. The gelation time of the in situ nanocomposite hydrogel was determined by rheological analysis as 28s, whereas hydrogel alone showed 132s. On addition of WH NPs not only shortened the gelation time but also increased the gel strength. The in vitro release of simvastatin from the nanocomposite hydrogel showed a release over a period of 28 days. The Alkaline Phosphatase (ALP) level also showed a significant increase. RUNX2 and BMP2 expressions showed that nanocomposite hydrogel enhanced the osteogenic differentiation. In vivo bone regeneration studies in mice cranial defect studies showed nanocomposite hydrogel was effective in regenerating the bone compared to controls. Thus, the simvastatin incorporated oxidized alginate-gelatin/WH NPs hydrogel system has potential to be used as repair and regenerative system in cranial bone defects. KEYWORD: injectable hydrogel, cranial defect, whitlockite nanoparticles, simvastatin, osteogenesis.


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1. Introduction Defects in craniofacial complex are due to trauma, infections or tumor removal requires closure for structural, functional and aesthetic reasons1. Craniofacial bone has complex shapes and structurally varies from person to person. In this regard, injectable in situ forming hydrogel could adapt well to the irregular defect margin and as it forms a 3D scaffold within the defect is of great interest. Further, injectable hydrogels could reduce the surgical procedure, post surgical pain (as only few muscles could be retracted during the procedure) and recovery time2. Consideration of the ideal scaffold other than biocompatibility, biodegradability and mass transport of nutrients it also includes biomimetic nature, surface featuring for cell attachment and migration3–6.Among the wide range of available biocompatible materials, alginate and gelatin based hydrogels have been widely used for the tissue engineering7–9. To prepare injectable hydrogel, oxidized alginate and gelatin have been used for in situ forming hydrogel by utilizing the Schiff base reaction10,11. The gelatin provides the collagen structural framework and sites for cell attachment and oxidized alginate controls the degradability of the hydrogel. Schiff base reaction utilized the aldehyde group present in the partially oxidized alginate and the free amide group of the amino acid, lysine or hydroxyl lysine11,12. The hydrogel matrix formed could also encompass drugs, ceramics, proteins etc. therefore acts a delivery matrix. Whitlockite (Ca18Mg2(HPO4)2(PO4)12), a biomimetic ceramic which accounts for 20 wt% mineral present in human bone13. It is second most abundant ceramic material naturally seen in the bones14,15. Synthesized Whitlockite nanoparticles (WH NPs) have shown to have faster ion release and better protein adsorption, compared to hydroxyapatite16. It is reported to inhibit osteoclast differentiation from monocyte attributed to higher release of Mg2+ and PO43- which reduces the



number of active osteoclasts. Its role in early bone formation could be established by the increase in the alkaline phosphatase activity and osteogenic gene expression16,17. Simvastatin which is 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors which is a cholesterol lowering agent has some pleiotropic effect towards bone remodeling process at the molecular aspect18. The prenylation inhibition by statin in the mevalonate pathway is the major factor responsible for pleiotropic effect. Simvastatin has a direct effect on the osteoblast activation leading to the bone regeneration by up regulating BMP2 expression, through antagonizing TNF-α to Ras/Rho/mitogen activated protein kinase and augment BMP2-Smad pathway19.This aids us in preventing the direct use of BMP2 at supraphysiological dose owing to their faster degradability. Studies have revealed the positive effect on bone formation and bone mineral density in vivo20. These findings show that the system with incorporation of WH nanoparticles and simvastatin in an oxidized alginate-gelatin hydrogel system is promising biomimetic scaffold for bone tissue engineering. In this study, the physico-chemical characteristics of this nanocomposite material were determined and osteogenic potential was evaluated in vitro and in vivo mice cranial defect model.

2. MATERIALS AND METHODS 2.1. Materials Sodium alginate from brown algae (2750cps at 2% solution at 25⁰C), sodium metaperiodate, Calcium hydroxide, Magnesium hydroxide and Dialysis membrane (MWCO 14000) were purchased from Sigma Aldrich, USA. Gelatin type A was purchased from HiMedia, India. Simvastatin was purchased from AK Scientific, USA. α-MEM, Trypsin-EDTA, and fetal bovine


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serum (South American origin) were purchased from Gibco, Thermo Scientific, USA. For all the experiments, milliQ water (18.2MΩ.cm) was used. 2.2 Methods 2.2.1 Preparation of Whitlockite NPs Whitlockite NPs were synthesized using a precipitation method using calcium hydroxide, magnesium hydroxide and phosphoric acid in a water-based ternary system according to the method used by Jang et al13. Briefly, under vigorous stirring condition both calcium hydroxide (0.74 M) and magnesium hydroxide (0.26 M) were added to100 mL water and heated at 70 °C for 1 hour. Further, an aqueous solution of H3PO4 (1 M, 100 mL) was dropwise added to Ca(OH)2 – Mg(OH)2 solutions. After aging the precipitate for 18h, it was washed and freeze dried to obtain the whitlockite nanoparticles. 2.2.2 Periodate Oxidation of Sodium Alginate Oxidized sodium alginate (OA) was synthesized with the oxidization degree of 57%, by a modified procedure described11. Briefly, sodium metaperiodate (NaIO4) in water (25mL) was added to sodium alginate-ethanol suspension mixture (20w/v %). The reaction was carried out in dark for 6h, further ethylene glycol (equimolar to sodium metaperiodate) was added to stop the reaction. The reaction solution was dialyzed (MWCO 14kDa) against water for 3 days with change of double distilled water for every 12 hours. The dialysate was lyophilized to give OA and stored in an air tight container until further use. Iodometric reaction was carried out to analyze the degree of oxidization11. 2.2.3 Preparation Oxidized Alginate-Gelatin Composite in situ Gel The preparation of oxidized alginate-Gelatin composite hydrogel was prepared based on the protocol published earlier with modifications12. Briefly, 15% w/v gelatin was solubilized in



PBS buffer (pH 7.4) at 37oC and WH NPs powder was added (10 wt %) to the solution. The gelatin-whitlockite nanoparticles mixture was loaded into a syringe. Similarly, the 20% w/v oxidized alginate with SIM drug (0.1µM) is loaded into another syringe. Both of the syringe is loaded into a syringe mixer and fitted with an 18-gauge needle. Both the syringe plungers were simultaneously pressed in order to dispense equal volume of the solution through the needle were in the cross-linking of the gel occur (Figure 1). Hereafter, oxidized alginate gelatin hydrogel would be represented as OA-G, WH NPs incorporated OA-G as OA-G/W, SIM added OA-G as OA-G/S and both WH NPs and SIM incorporated OA-G as OA-G/W/S. Concentration of WH NPs16 and SIM21 were selected from the previous literature.

Figure 1: Schematic representation showing the preparation of whitlockite NPs and simvastatin drug incorporated in situ oxidized alginate-gelatin hydrogel. 6 ACS Paragon Plus Environment

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2.2.4 Physicochemical Characterization of the prepared WH NPs The synthesized nanoparticles were analyzed for the morphology and size with Scanning Electron microscope (JEOL JSM-6490LA Analytical SEM) by spotting an appropriate dilution of WH NPs particles on a stub and left overnight for drying. EDAX was performed for the elemental analysis.HR TEM (FEI Tecnai F20) was also performed to study the size,shape and d-spacing values of the nanoparticles prepared. 5µl of the suspended particle solution were drop casted on a copper grid and left overnight to dry and later analyzed. FTIR spectroscopy (Shimadzu IRAffinity-1S Fourier Transform Infrared Spectrophotometer) was conducted to confirm the functional group of the obtained particle. The WH NPs, were lyophilized and were analyzed using a transmission type spectroscope using KBr pellet. XRD analysis was done to confirm the major peak obtained of the prepared nanoparticles with the WH from database (JCPDS 70-2064). 2.2.4Physico-chemical Characterization of prepared Hydrogel The prepared hydrogels were freeze dried and observed using SEM for the porosity. FTIR spectroscopy was done for different gels using KBr pellet to confirm the presence of the different compounds in the gel matrix. 2.2.5 Rheological studies The different gel systems were evaluated using Malvern Kinexus Pro rheometer. For all measurement parallel plates were used with a constant gap between the plates which is 1 mm and a constant temperature of 37°C. During a cross-linking reaction, the gelation point is measure as a function of time was studied using oscillating measurements. The elastic modulus is the measurement of the resistance of the substance against deformation under shear. Loss modulus reflects the viscous properties of the gel. Gelation time is defined as a point where the transition of phase angle from >45° to

Injectable in Situ Shape-Forming Osteogenic Nanocomposite

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