Enhanced Skull Bone Regeneration by Sustained Release of BMP-2

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Enhanced Skull Bone Regeneration by Sustained Release of BMP-2 in Interpenetrating Composite Hydrogels Sungjun Kim, Junhyung Kim, Gajendiran Mani, Minhyuk Yoon, Mintai P. Hwang, Yadong Wang, Byung-Jae Kang, and Kyobum Kim Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01013 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 20, 2018

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Enhanced Skull Bone Regeneration by Sustained Release of BMP-2 in Interpenetrating Composite Hydrogels Sungjun Kim1,#, Junhyung Kim2,#, Gajendiran Mani1, Mintai P. Hwang3, Yadong Wang 3 , Byung-Jae Kang2,*, and Kyobum Kim1,*

1

Division of Bioengineering, College of Life Sciences and Bioengineering, Incheon National

University, Incheon, 22012 Korea 2

Department of Veterinary Surgery, College of Veterinary Medicine and Institute of

Veterinary Science, Kangwon National University, Chuncheon 24341, Korea 3

Meinig School of Biomedical Engineering, Cornell University, Ithaca, USA

# These authors equally contributed to the manuscript. *Corresponding Author Byug-Jae kang, D.V.M., Ph.D. : [email protected]

Kyobum Kim, Ph.D. : [email protected]

Keywords: bone morphogenetic protein-2, coacervate, gelatin microparticle, sustained release, interpenetrating hydrogel, calvarial bone regeneration

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Abstract Direct administration of bone morphogenetic protein-2 (BMP-2) for bone regeneration could cause various clinical side effects such as osteoclast activation, inflammation,

adipogenesis,

and

bone

cyst

formation.

In

this

study,

thiolated

gelatin/poly(ethylene glycol) diacrylate (PEGDA) interpenetrating (IPN) composite hydrogels were developed for guided skull bone regeneration. To promote bone regeneration, either polycation-based coacervates (Coa) or gelatin microparticles (GMPs) were incorporated within IPN gels as BMP-2 carriers. Both BMP-2 loaded Coa and BMP-2 loaded GMPs showed significantly enhanced in-vitro alkaline phosphate (ALP) activity of human mesenchymal stem cells (hMSCs) than non-BMP-2 treated control. Moreover, BMP-2 loaded GMPs group exhibited statistically increased ALP activity than both bolus BMP-2 administration and BMP-2 loaded Coa group, indicating that our carriers could protect and maintain biological activity of cargo BMP-2. Sustained release kinetics of BMP-2 from IPN composite hydrogels could be controlled by different formulations. For in-vivo bone regeneration, various IPN gel formulations (i.e., (1) control, (2) only hydrogel, (3) hydrogel with bolus BMP-2, (4) hydrogel with BMP-2-loaded Coa, and (5) hydrogel with BMP-2loaded GMPs) were bilaterally implanted into 5 mm-sized rat calvarial defects. After 4 weeks, micro-CT and histological analysis were performed to evaluate new bone formation. Significantly higher scores for bony bridging and union were observed in BMP-2-loaded Coa and BMP-2-loaded GMP groups as compared to other formulations. In addition, rats treated with BMP-2-loaded GMPs showed a significantly higher ratio of bone volume/total volume and lower trabecular separation scores than others. Finally, rats treated with either Coa or GMP groups exhibited a significant increase in bone formation area, as assessed via histomorphometric analysis Taken together, it could be concluded that Coa and GMPs were 2


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effective carriers to maintain the bioactivity of cargo BMP-2 during its sustained release. Consequently, our IPN composite hydrogel system that combines such BMP-2 carriers could effectively promote skull bone regeneration.

3



INTRODUCTION Large craniofacial defects due to trauma, congenital disease, or cancer resection pose serious difficulties for reconstructive surgeons. Such critical sized defects prevent spontaneous bone regeneration and often require a complex reconstruction approach.1 Conventionally, a variety of sources including skulls, ribs, tibia, and iliac crest are used in the replacement of defective areas.2,3 In addition, one of the disadvantages of existing implantable metallic devices including stainless steel and titanium-based materials is a need for secondary surgeries to remove metallic implants due to infection, extrusion, bone loss around the implant, and subsequent device failures.4,5 To date, bone tissue engineering applications have been developed to substitute these traditional medical approaches. For the guided bone regeneration (GBR) of skull bone, osteoconductive and osteoinductive bone substitute materials that fill the bone defect have been developed.6,7 A variety of biopolymerbased scaffold materials, especially hydrogels, have been used as effective carriers for stem cell population, vascular endothelial growth factor (VEGF) or bone morphogenetic protein-2 (BMP-2) to promote host cell osteogenesis and bone regeneration in bone defect sites.8,9 Since BMPs have a short half-life from about several minutes to hours in the body, a large amount of BMP is usually required to obtain a desired bone formation.10-12 However, direct administration of high dose BMP-2 can have various clinical side effects including osteoclast activation, inflammation, and bone cyst formation.13 Herberg et al. also demonstrated that overdose of BMP-2 could decrease in-vivo skull bone regeneration (i.e. over 10 µg of BMP-2 in 8 mm defect size in a male Sprague-Dawley rat model).14 Therefore, the IPN composite hydrogel described herein could be utilized as an effective BMP-2 carrier for sustained and controlled release, as well as a bulk bone substitute material in the craniofacial/skull region. So in this study, we develop interpenetrating (IPN) composite 4


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hydrogels containing BMP-2 as implantable growth factor (GF) delivery systems for skull bone regeneration. IPN composite hydrogels are composed of natural gelatin and synthetic poly(ethylene glycol)-diacrylate (PEGDA). Gelatin is biodegradable, biocompatible, nonimmunogenic, and widely used for pharmaceutical and medical purposes.15,16 However, gelatin-based gels often exhibit low stability, mechanical strength, and elasticity, all of which lead to limit their use in the biomedical field.16 For instance, physically entangled gelatin rapidly loses more than 70% in mass at physiological temperature within 48 hours in-vitro.17 To overcome this problem, gelatin is often chemically modified with other moieties to enhance crosslinking properties. In addition, composite hydrogels with biocompatibility and good mechanical strength are utilized.18-20 PEGDA is a particularly well-known synthetic polymer with excellent hydrophilicity and controllability for IPN hydrogel fabrication.21,22 By modulating the concentration and chain length of PEGDA, the mechanical strength and stiffness of the resulting hydrogel could be increased for bone implant applications. Therefore, our gelatin/PEGDA IPN hydrogel could be utilized as an implantable BMP-2 delivery system. BMP-2-mediated bone regeneration approaches have been intensively studied for decades due to the osteoinductive capacity of BMP-2 (i.e. accelerated osteogenic differentiation of progenitor mesenchymal stem cells and enhanced bone regeneration).23,24

In order to achieve sustained release of cargo BMP-2 from IPN composite hydrogels, we incorporated two biodegradable carriers in a gel: coacervate (Coa) and gelatin microparticles (GMPs). Both carrier systems protect the GF cargo from both in-vitro and invivo environments and maintain their bioactivity for tissue regeneration.25,26 Coa is selfassembled liquid droplets held together by Coulombic forces between macromolecules of opposite charge, such as polycationic polymers and heparin.27 A synthetic polycation, 5



poly(ethylene argininylaspartate diglyceride) (PEAD), interacts with anionic heparin and cargo BMP-2 to form a biocompatible complex of BMP-2-loaded Coa.28 PEAD-based coacervation has been used as an excellent GF delivery vehicle for multiple types of tissue regeneration. For instance, Coa -mediated delivery of stromal cell-derived factor-1α (SDF-1α) effectively facilitates stem cell recruitment and enhances vascular regeneration.29 Administration of fibroblast growth factor-2 (FGF-2) Coa ameliorate the ischemic injury caused by myocardial infarction. Additionally, dual delivery of FGF-2 and interleukin-10 (IL10) via Coa improves ischemic heart repair in an rat infarct model.30 Co-administrated VEGF and transforming growth factor (TGF)-β3 enhances neovascularization and subsequent skin regeneration in mouse model.26 Finally, BMP-2, which is protected and released from Coa, effectively enhances osteogenic differentiation of muscle-derived stem cells both in-vitro and in-vivo.28 Like Coa, GMPs are biocompatible and enzymatically degradable GF carriers, which incorporate cargo GFs via physical adsorption.31,32 Crosslinking processes via glutaradehyde increase thermal and mechanical stability of GMPs.33 Kim et al. showed that implantation of bilayered hydrogels containing insulin-like growth factor-1 (IGF-1) and TGFβ3-loaded GMPs improved osteochodral tissue regeneration in a rabbit model.34 Lu et al. investigated that oligo(poly(ethylene glycol) fumarate) (OPF) bilayered hydrogels containing BMP-2 and IGF-1-loaded GMPs could be used as a spatially-guided dual GF delivery system for osteochondral tissue regeneration.25 Moreover, this protective capability of GF carriers has been also reported in other studies. For instance, Chu et al. demonstrated that Coa could protect cargo GF from enzymatic degradation. After 2 hrs of trypsin treatment, which is a broad spectrum protease, 86.1% of FGF-2 was present in the Coa, whereas bolus free FGF-2 was completely degraded within 0.5 hr.35 TGF-β3 encapsulated in Coa also more effectively inhibited in-vitro CCL-64 proliferation even with a relatively lower concentration, as 6


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compared with administration of bolus TGF-β3.26 A similar protective effect of Coa was also found in epidermal growth factor-incorporated Coa for improved in-vitro migration of keratinocytes36, and a series of enhanced in-vivo tissue regeneration via Coa -mediated GF delivery, as compared to non- Coa protected cargo GFs.28, 35-41 In the case of GMPs, bioactive VEGF was released from GMPs over 3 weeks, demonstrated by a transwell migration assay using human umbilical cord blood endothelial progenitor cells.42 Several in-vivo studies also demonstrated that GMPs in hydrogels effectively delivered TGF-β1 and IGF-1 or BMP-2 + IGF-1to promote osteochondral tissue regeneration of rabbit cartilage tissues.25, 34

A global hypothesis of this study is that a release profile of BMP-2 from two distinguish GF carries of colloidal electrolyte complex (i.e., coacervate) and particulate (i.e., GMP) platform, and subsequently available optimal dose of BMP-2 in a physiological defect environment could effectively regulate guided skull bone regeneration, especially when using IPN composite hydrogel. Herein, we design a macropore structure of thiolated gelatin (GelSH)/PEGDA IPN hydrogels which have consolidated mechanical strength driven by the disulfide bond between gelatin fibers and carbon-sulfied between polymeric chains of gelatin and PEGDA for sustained BMP-2 delivery to promote skull bone regeneration in a rat calvarial defect model. To this end, we first characterized the effect of Gel-SH and PEGDA concentrations on the morphology and degradation characteristics of IPN hydrogels. Subsequently, we assessed the BMP-2 release profile from either Coa or GMPs, and evaluated the bioactivity of BMP-2 releasate via osteogenic differentiation of human mesenchymal stem cells (hMSCs). Finally, BMP-2-loaded IPN hydrogels were implanted in rat calvarial defects to determine their in-vivo regenerative capacity. In this study, Coa and GMP, which are used in the field of tissue engineering, were used as carriers of BMP-2, 7



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suggesting which of the two mediators delivered by different mechanisms is the most effective to critical sized calvarial bone regeneration.

2. EXPERIMENTALSECTION 2.1. Materials Gelatin,

ethylenediaminetetraacetic

acid

(EDTA),

γ-thiobutyrolactone,

β-

mercaptoethanol, imidazole, poly(ethylene glycol) diacylate (PEGDA) (Mw 2,000), ammonium persulfate (APS), N, N, N', N'-tetramethylethylenediamine (TEMED), pnitrophenyl phosphate (pNPP), glutaraldehyde, Tween 80, glycine, cysteine, and Ellman’s reagent were obtained from Sigma-Aldrich. BMP-2 and human BMP-2 enzyme linked immunosorbent assay (ELISA) development kits were received from PeproTech. Dulbecco’s modified Eagle’s medium (DMEM), Penicillin-Streptomycin antibiotics, and trypsin/EDTA were purchased from Wisent Inc. Fetal bovine serum (FBS) was obtained from Corning. Quant-iT PicoGreen dsDNA reagent kit was purchased from ThermoScientific.

2.2. Synthesis of thiolated gelatin For gelatin thiolation, 1% of gelatin solution was prepared by dissolving gelatin in degassed deionized water

43

. After that, 3 mL of 0.62% (w/v) of ethylenediaminetetraacetic

acid (EDTA) solution and 3 mL of 22.67 % (w/v) of imidazol solution were added into gelatin solution and stirred at 40 ℃for 5 min. After 5 min, 840 µL of γ-thiobutyrolactone was added to gelatin solution and stirred at 40 ℃ for 24 hr. After reaction, this solution was dialyzed using dialysis bag (molecular weight cut-off 6,000 ~ 7,000) (Spectra/Por) against 0.2 % (v/v) β-mercaptoethanol in degassed DW and stirred at 40 ℃ for 24 hr. This product, GelSH, was lyophilized and stored at 4 ℃ before use. All of the steps were done under nitrogen

8


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atmosphere in order to prevent oxidation of free thiol moieties. The thiol group content of Gel-SH was 1.858 mM, which was measured by Ellman’s reagent and calculated by using a cysteine standard curve (Figure S1 and S2).44,45

2.3. Fabrication of coacervates and gelatin microparticles PEAD was synthesized as previously described.46,47 PEAD and heparin solution were prepared in DW at 10 mg/mL and sterilized by passing through 0.22 µm syringe filter. Initially, heparin was complexed with 0.2 µg/µL of BMP-2 and mixed well. Then, PEAD solution was added into the heparin/BMP-2 solution, upon which coacervation was observed immediately. PEAD, heparin, and BMP-2 was mixed at a 500:100:1 PEAD:heparin:BMP-2 mass ratio. The coacervation was confirmed via the transition of transparent solution to a turbid solution. The solution of BMP-2 loaded Coa was centrifuged and the supernatant containing any unincorporated BMP-2 was used to determine initial loading efficiency of BMP-2 into Coa with ELISA. GMPs were fabricate via a water in oil emulsion technique.48-50 Gelatin solution was prepared by dissolving 5 g of gelatin in 45 mL of DW. This gelatin solution was added dropwise to 200 mL of chilled olive oil and overhead stirred at 500 rpm for 1.5 hr. Formed GMPs were filtered and washed by chilled acetone. For the crosslinking of GMPs, they were reacted with 500 mL of solution containing 1g of glutaraldehyde and 0.5 mL of Tween 80 overnight and the reaction was terminated by adding 937.5 mg of glycine while stirred at 500 rpm for 1 hr surrounded by ice. The crosslinked GMPs were washed using acetone and collected by filtration and lyophilization. The average diameter of dried GMPs was determined by image analysis (n=5). Resulted GMPs (Average diameter: 61.3 ± 4.2 µm) were sterilized under UV-light over night. Then, 5 µL of BMP-2 solution (0.37 µg/µL) was 9



loaded in mg of sterile GMPs at 4℃ and stored for 16 hr. Initial loading efficiency of BMP-2 into GMPs was determined by ELISA.

2.4. Fabrication of interpenetrating composite hydrogels Gel-SH was dissolved in degassed DW at 2.5% (w/v) of low concentration Gel-SH (LGS) and 5% (w/v) of high concentration Gel-SH (HGS), respectively. PEGDA was prepared to 12.5% (w/v) of low concentration PEGDA (LPEGDA) and 25 % (w/v) of high concentration PEGDA (HPEGDA). And 10% (w/v) of ammonium persulfate (APS) and 5% (w/v) of N, N, N', N'-tetramethylethylenediamine (TEMED) that used to initiator were prepared. Prepared gelatin solution was mixed with PEGDA solution at a 5:4 volume ratio and these mixtures were prepared to three different concentrations of gelatin and PEGDA (i.e., (1) HGS-HPEGDA, (2) HGS-LPEGDA, (3) LGS-LPEGDA). After then, APS and TEMED solution added into gelatin/PEGDA mixture at a 1:1:18 volume ratios. Additionally, BMP-2 loaded Coa or BMP-2 loaded GMPs were added to gelatin / PEGDA mixture before addition of initiators. This solution was casted in the polytetrafluoroethylene mold (8 mm in diameter and 2 mm in height for in-vitro degradation property tests, 30 mm × 30 mm × 5 mm for a mechanical test, and 4.5 mm in diameter and 2 mm in height for a release study and in-vivo implantation) and incubated at 37℃for 30 min.

2.5. Hydrogel characterization For the degradation and swelling studies, three different concentrations of gelatin and PEGDA hydrogel were fabricated as previously described. The composite hydrogel placed in 4 mL of PBS, and incubated at 37 ℃ for 14 days. At 6 hr, 1, 3, 7 and 14 days, the swelling ratio, sol fraction and mass remaining were calculated using the following equations:% 10


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swelling ratio = (Ws−Wd)/Wd × 100 (%), % sol fraction = (Wi −Wd)/Wi × 100 (%), and % mass remaining =Wd/Wi × 100 (%) (Wi: weight of initially dried hydrogel, Ws: weight of swollen hydrogel at each time point, and Wd: weight of dried hydrogel after swelling at each time point).51 For SEM imaging, hydrogels were prepared and lyophilized. Pre-platinum coated hydrogel observed using FE-scanning electron microscope (SEM) (S-4300SE, Hitachi, Japan) at 15 kV accelerating voltage. The elastic modulus values of hydrogels were measured using an indentation instrument (Softmeasure, HG1003-SL, Horiuchi Electronics Co., Ltd.). We conducted indentation tests for three samples of each hydrogel type. An indenter of 10 mm diameter was loaded to five points of 30 mm × 30 mm × 5mm hydrogel samples with indentation speed of 0.2 mm/s and the maximum indentation force of 0.45 N. Young’s modulus of hydrogel samples was obtained by the analysis software (Horiuchi Electronics Co., Ltd.).

2.6. Quantification of in vitro BMP-2 release kinetics Hydrogels + bolus, hydrogel + Coa, and hydrogel + GMPs groups were prepared using HGS-HPEGDA to determine BMP-2 release kinetics from IPN composite gels. Each group contained total 980 ng of BMP-2 per gel. Samples were placed in 500 µL of PBS, and incubated 37 ℃ for 28 days. At day 1, 3, 5, 7, 14, 21, and 28, supernatant was collected and replenished with fresh PBS solution. After that, collected supernatant were stored at 80 ℃.The amount of BMP-2 released into the supernatant was quantified by an human BMP2 enzyme linked immunosorbent assay (ELISA) development kit, according the manufacturer’s procedure. And the optical density was measured by UV-visible spectrophotometer (Thermo scientific).

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2.7. In-vitro osteogenic differentiation study. Early osteogenic differentiation of human mesenchymal stem cells (hMSCs) (LONZA, Walkersvile, USA, Passage 4) was determined by alkaline phosphatase (ALP) activity. Osteogenic differential (OS) media was prepared with Dulbecco’s modified Eagle’s medium (DMEM) (89 % (v/v)), Penicillin-Streptomycin antibiotics (1 %), fetal bovine serum (FBS) (10 % (v/v)), 50 µg/mL ascorbic acid, 0.01 M glycerol-2-phosphate, and 10-7 M dexamethasone. For the coacervation, PEAD and heparin solution were prepared at 10 mg/mLwhile BMP-2 was prepared at 0.2 µg/µL. In order to achieve 500:100:1 mass ratio of PEAD: heparin: BMP-2, 2 µL of heparin solution and 1 µL of BMP-2 solution (containing 200 ng of BMP-2) were first mixed, then 10 µL of PEAD solution was added into heparin:BMP-2 mixture. This total 13 µL of coacervate solution was applied into the each well of hMSCs. 10,000 hMSCs were seeded onto 24well culture plates under OS media. After 24 hr, old media removed and washed using PBS, and bolus BMP-2 or BMP-2 loaded Coa or BMP-2 loaded GMPs added with OS media to achieve 200 ng of BMP-2 per well. BMP-2 administrated hMSCs were incubated at 37 ℃, 5 % CO2, and 95 % humidity. After 5 days, ALP activity was studied using the p-nitrophenyl phosphate (pNPP) assay according to the manufacturer’s protocol. The hMSCs were detached using trypsin/EDTA and lysed using a homogenizer. Lysed cells were centrifuged at 13,000 xg for 30 min, and 10 µL of supernatants were mixed with 200 µL of pNPP solution and incubated at 37 ℃ for 30 min. After stopping the reaction using 50 µL of 3 N NaOH, the optical density was measured at 405 nm by UV-visible spectrophotometer. DNA amount was detected using Quant-iT PicoGreen dsDNA reagent kit according to the manufacturer’s protocol with same set of samples. DNA amount was used to normalize ALP activity.

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2.8. Rat bilateral calvarial defect model for bone regeneration For in-vivo tests, HGS-HPEGDA hydrogel containing 980 ng of BMP-2/gel was used in the calvarial defect. IPN hydrogel were manufactured for calvarial implantation in the five experimental groups (n=4 for each groups). (1) Control, (2) Hydrogel, (3) Hydrogel + bolus, (4) hydrogel + Coa and (5) hydrogel + GMPs. 8-week-old twenty Male Sprague Dawley rats weighing approximately 280 g were used for present experiment. Male Sprague Dawley rats were purchased from Nara Bio Animal Center (NARA biotech, Seoul, Korea). The rats were housed in a conventional housing facility. The temperature of the breeding facility was maintained at 21 degrees with a 12-hour day/night cycle. Water and food were supplied daily enough and bedding was replaced every two days. All rats were acclimatized for 1 week before the experiments. After the operation, all rats were disinfected with betadine twice a day. After the experiment, animals were euthanized by cervical dislocation under anesthesia with isoflurane. In-vivo experiments were performed in compliance with the guideline outlined by the Kangwon National University Animal Care Committee (KW-170801-1). All operations were performed with general anesthesia via intraperitoneal injection of xylazine (5 mg/kg), zolazepam (20 mg/kg), and tiletamine (20 mg/kg), and for an analgesic, tramadol (12.5 mg/kg) was injected subcutaneously. To prevent corneal dessication, ophtalmicointmet was used prior to skin incision. After disinfection of calvarial skin with alcohol based hexidine, a midline skin incision from the nasal bone to middle sagittal bregma was made. The periosteum covering the calvarium was incised carefully, elevated and retracted laterally along with skin to visualize the calvarium. 5-mm trephine bur was used to make two bilateral full-thickness 5-mm diameter circular defects on both side of parietal bone under the saline irrigation. Care was taken not to damage dura matter. The full thickness of the bone was removed by using elevator blade, the defect area was filled with each hydrogel composite and 13



defect was left empty in a control group. After confirming that the IPN hydrogel was inserted into the defect, the periosteum was sutured as continuous suture with absorbable surgifit 3-0 and skin was sutured with simple interrupted suture using nylon 3-0. All rats were sacrificed after 4 weeks, then the skull caps were extracted and fixed in 10% neutral buffered formalin at 4℃.

2.9. Micro-computed tomography After 4 weeks of implantation, all rats were scanned using a micro-computed tomography (micro-CT) scanner (VivaCT 80, Scanto medical AG, Bruttisellen, Switzerland) at a voltage of 70 kV and current of 114 µA at Chuncheon Center of the Korea Basic Science Institute with inhalation anesthesia. Data were obtained at an isotropic resolution of 48.5 µm and a threshold range of 220-1000 was used. Coronal view, oblique view and sagittal view were obtained through the three-dimensional (3D) reconstruction. Bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separatioin (Tb.Sp) were investigated in a region of interest (ROI) (5 mm diameter; 53 slide at 48.5 µm = 2.5 mm) by using HP DECwindows Motif for OpenVMS v1.7. Established scoring guide was used to determine the extent of bony union and bridging within defect.52,53 Three blinded reviewers evaluated coronal view of the 3D reconstructed micro-CT images separately. The scores for each sample were averaged to give an overall score via scoring guide.52 Additionally, area of unhealed defect was measured by using ImageJ software (1.51v, National Institutes of Health, Bethesda, MD, www.nih.gov) at coronal view.

2.10. Histological and histomorphometrical analysis

14


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After micro-CT scanning, rats were sacrificed, and skull caps were harvested to use for histological analysis. Skull caps were fixed in 10% neutral buffered formalin for 24 hours and decalcified in decalcifying solution (Hydrochloric acid 40 ml, 96% Formic acid 50 ml and distilled water 500 ml) for 3 days. After dehydration in 70% ethanol, samples were embedded in paraffin and sectioned transversely at the middle part of the defects at a thickness of 5 µm. The sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome for the detection of cells and bone structures. Digital images of stained sections were obtained by scanners. The area of the new bone was calculated by measuring the area of the newly generated bone from the defect margin. Total defect area was measured as 5 mm x the height of defect (about 1 mm) of each rat by using I-solution software. The percentage of newly bone formation within the calvarial defect area was calculated using Isolution software as (new bone area/total defect area) x 100.54

2.11. Statistical analysis Quantitative data were reported as mean ± standard deviation. Statistical analysis was performed using Graphpad Prism V 7.0 (Graphpad Software Inc., San Diego, CA, USA). Data in degradation properties (Figure 1C), in-vitro osteogenic differentiation (Figure 2), and BMP-2 release profile (Figure 3) were analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test. For analysis of in-vivo results (Figure 4-6), kruskalWallis one-way ANOVA test was performed to assess the statistical significance. Furthermore, The Mann-Whitney test was performed to evaluate the differences between two groups. Statistical significance was accepted for a value of p < 0.05.

3. RESULTS AND DISCUSSION 3.1. Hydrogel characterization 15



In this study, the hydrogel was prepared through a simple crosslinking method by thermal initiation. Disulfide bond formation between Gel-SH and crosslinking between diacrylates in PEGDA influences the overall gelation process on resulting IPN hydrogels as well as subsequent mechanical properties. Surface morphology and cross sections of the hydrogel were observed by SEM (Figure 1A and B). Pore size of hydrogels was reduced when the concentration of PEGDA decreased from 25% to 12.5%. However, hydrogels containing LPEGDA exhibited similar macropores despite the differences in gelatin concentration. In addition, the in-vitro degradation properties of IPN hydrogles in PBS were examined for 14 days (Figure 1C, D, and E).The swelling ratio of hydrogels containing HPEGDA remained stable over 14 days compared to other groups (Figure 1C). Sol fraction of hydrogels containing LPEGDA started to increase after 7 days and showed a significant difference with those containing HPEGDA at 14 days (Figure 1D). Percent of initial mass for all three groups showed a steady trend for the first 3 days and started to decrease after 7 days. Specifically, the mass remaining for HGS-HPEGDA hydrogels after 7 days showed significant stability compared to other groups (Figure 1E). Furthermore, elastic modulus of hydrogels was evaluated by indentation instrument (Figure 1F). HGS-HPEGDA hydrogel exhibited the highest Young's modulus of approximately 53 kPa than HGS-LPEGDA and LGS-LPEGDA. In addition, although the difference between HGS-LPEGDA and LGSLPEGDA was not statistically significant, HGS-LPEGDA showed higher elastic modulus than LGS-LPEGDA by increasing disulfide bond, and the Young’s modulus were 12 kPa and 3 kPa, respectively. These results demonstrate that microstructures and degradation properties of IPN hydrogels could be effectively manipulated by the concentration of incorporated PEGDA; other studies also indicated that the porosity of hydrogels depends on the molecular weight and concentration of PEGDA.55 Reducing the concentration of PEGDA can reduce 16


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the cross-linking density of the hydrogel, which increases the swelling capability.

Figure 1. Characterization of Gel-SH/PEGDA IPN hydrogels. Structural morphologies of (A) surface and (B) cross-section of hydrogels were obtained using SEM. Degradation properties of hydrogel including (C) Swelling ratio, (D) Sol fraction, and (E) Mass remaining were evaluated for 14 days. (F) Young’s modulus indicates mechanical strength of hydrogels with different formulation. (*) indicates a significant difference as compared with all other groups. 17



(p

Enhanced Skull Bone Regeneration by Sustained Release of BMP-2

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