Sandwich-Like Nanofibrous Scaffolds for Bone Tissue Regeneration

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Biological and Medical Applications of Materials and Interfaces

Sandwich-Like Nanofibrous Scaffolds for Bone Tissue Regeneration Sarah Yahia, Islam A Khalil, and Ibrahim M. El-Sherbiny ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b06359 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 22, 2019

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ACS Applied Materials & Interfaces

Sandwich-Like Nanofibrous Scaffolds for Bone Tissue Regeneration Sarah Yahia1, Islam A. Khalil 1,2, Ibrahim M. El-Sherbiny*1 1Nanomedicine

Lab, Center of Materials Sciences (CMS), Zewail City of Science and Technology, 6th of October, Giza 12578, Egypt, 2Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy and Drug Manufacturing, Misr University of Science and Technology (MUST), 6th of October, Giza 12566, Egypt

*Correspondence to: Ibrahim M. El-Sherbiny, PhD Zewail City for Science and Technology. Ahmed Zewail Road, October Gardens, 6th of October City, Giza, Egypt. Nanobuilding, Room F001. (Email: [email protected]) Tel: +20238540407, Fax: +20238517181

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Abstract Advanced bone healing approaches included a wide range of biomaterials that mainly mimic the composition, structure and properties of bone extracellular matrix with osteogenic activity. The present study aimed to develop a sandwich-like structure of electrospun nanofibers (NFs) based on polycaprolactone (PCL) and chitosan/ polyethylene oxide (CS/PEO) composite to stimulate bone fracture healing. The morphology of the fabricated scaffolds was examined using SEM. Apatite deposition was evaluated using simulated body fluid (SBF). Physicochemical and mechanical properties of samples were analyzed by FTIR, DSC, TGA and universal testing machine. SEM micrographs exhibited a porous 3D structure with NFs diameters of 307.2-900 nm and 20.5-380.4 nm for PCL NFs layer and the sandwich-like NFs scaffolds, respectively. Deposition of apatite crystal on scaffolds started at week 2 followed by heavy deposition at week 8. This was confirmed by measuring the consumption of calcium and phosphorous ions from SBF. Thermal stability of scaffolds was confirmed using DSC and TGA. Moreover, the PCL NFs layer in the middle of the developed sandwich-structure reinforced the scaffolds with bear load until 12.224 ± 1.12MPa and young’s modulus of 3.97 MPa. The scaffolds porous structure enhanced both cells propagation and proliferation. Besides, presence of CS in the outer NFs layers of the scaffolds increased the hydrophilicity as evidenced by reduction of contact angle from 116.6° to 57.6° which is essential for cell attachment. Cell viability study on mesenchymal stem cells proved the cytocompatibility of the fabricated scaffolds. Finally, in-vivo mandibular bone defect rabbit model was used to confirm the regeneration of a new healthy bone within 28 days. In conclusion, the developed scaffolds could be a promising solution to stimulate bone regeneration. Key words: Electrospun, Nanofibers, Sandwich-structure, Sildenafil, Bone regeneration


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1. Introduction Bone is a natural composite in the body, and it composes of both organic and inorganic phases. Its organic extracellular matrix (ECM) consists of glycosaminoglycans, type І collagen, proteoglycans and glycoprotein, whereas the hydroxyapatite (HA) forms its hard and brittle inorganic matrix

1,2.

Nowadays, osteoporosis is mainly affecting geriatric patients bone density,

where the total hip replacement is the solution for severe osteoporosis and hip fractures treatment. New treatment approaches involve the use of bio-inert materials in fractured bone, which could cause loss of bone mineral density and implant failure. To the same extent, bone restoration in the maxilla or mandible in geriatrics with cysts involvement require bone graft during maxillofacial surgeries. Bone grafted tissues can be either autograft or allograft, with both types facing difficulties like the graft origin and body rejection. This has encouraged orthopedic surgeons and scientists to search for more appropriate alternatives that mimic bone tissue structure and function 3.

Furthermore, blood supply in skeletal system is essential for bone homeostasis 4, where it

provides angiogenesis through preserving oxygen level, supplying biological mediators, nutrients and cells as well as excreting side products 5. On the other hand, different geriatrics concomitant diseases like osteoporosis, rheumatoid arthritis, congenital malformation, acute injuries and deficient osteogenesis could affect the quality of orthopedic surgery. Therefore, the use of biomaterials could be an alternative solution in such cases 6. Generally, orthopedic surgery has a mutual and complex clinical issue for bone repair and regeneration 7. Currently, tissue engineering (TE) is dealing with bone repair, cartilage and intervertebral disc treatment with the aid of cells and biomaterials 8. TE can be defined as restoration or rejuvenation tissue deterioration via the substitution with engineered tissues which assist functions restoration during repair along with conjunction with the body tissue 9. From the biological perspective, bone growth requires both cell-cell and cells-ECM interconnection as well as availability of appropriate growth factors. Besides, fabricated scaffolds should mimic the bone structure and enhance the cell growth in a 3D way 10. Consequently, combining suitable fabrication technique, appropriate biomaterials, suitable cells, and growth factors is crucial for the bone regeneration process 11.



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Biomaterials used for bone regeneration should be non-toxic, biocompatible, biodegradable, highly porous, bioactive, osteocoductive and of tunable shape and size. Besides, they should confer osteointegration and osteoinduction as well as enhancing vascularization

12.

The bioactivity of

bone-related materials usually refers to their ability to interact and bind with the body via supporting the cells to generate apatite 12. Furthermore, adequate scaffold pores size is one of the essential parameters that allow cell seeding as well as cell diffusion through the matrix for bone tissue reconstruction. In addition, the selection of a proper technique for the bone scaffold fabrication represents another critical factor towards enhanced cell attachment, proliferation and differentiation 13. Chitosan (CS), a linear polysaccharide derived from partial deacetylation of chitin, is one of the recently used biopolymers to develop TE scaffolds, where CS polysaccharide backbone has identical structure to that of glycosaminoglycans, which are one of the main ECM components of cartilage and bone. CS-based scaffolds possess another characteristic for bone tissue regeneration which implicates the genesis of scaffolds with high porosity and interconnected pores, osteoconductivity and capability to improve both in vitro and in vivo bone regeneration

14.

Fabrication of CS nanofibers (NFs) via electrospining is restricted by the high repulsive forces resulting from its ionic groups 15,16. Polycaprolactone (PCL) is another example of the currently used polymers for regeneration of both soft and hard tissue. PCL has demonstrated many advantages such as low cost, good mechanical flexibility, stability under ambient conditions, easy processability, low antigenicity, and the low degrees of chronic persistence. However, its high hydrophobicity and low water absorptivity, and low cell attachment and proliferation are limiting the PCL wide usage. Therefore, combining PCL and CS in one composite is recommended

15,17.

For instance, addition of

hydrophilic CS to hydrophobic PCL-based scaffolds has enriched the bioactivity these scaffolds via improving the cell attachment and growth on their surface 18. PCL/CS blend NFs enhanced cell activity of mouse preosteoblast cells and hSaOs-2 osteosarcoma cells as reported by Jing et.al. 19.


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Several techniques have been used, to date, for fabrication of TE scaffolds. These include, for instance, solvent casting-particulate leaching, gas foaming, freeze-drying, emulsion freeze drying, phase separation, electrospinning and 3D bioprinting

12.

Electrospinning (ES) is a multilateral

method for fabrication of NFs matrices, which have been widely used in various fields like wound dressing, TE, drug delivery medias well as biosensing applications 20. ES of different synthetic and natural polymers such as PCL, poly(lactide-co-glycolide), alginate, collagen, CS, and cellulose acetate has been reported. NFs-based scaffolds have several inherited advantages including, but not limited to, good permeability and high porosity with dimensions ranging from nano to microns 21,22.

It is worth to mention that beside scaffold structure and bioactivity, different phases of bone tissue healing should be considered, like inflammation, proliferation (MSCs and chondrocyte differentiation) and remolding

23–25.

Several drugs have been used to treat bone fracture like

sildenafil which has been incorporated in a PCL scaffold to regulate the pro-angiogenic factors known as vascular endothelial growth factor (VEGF), which stimulated the angiogenesis. Also, sildenafil increased the callus formation, probably via the cysteine-rich protein (CYR61) pathway 26,27.

The main objective of the present study was to design, develop and evaluate a series of layer-bylayer (sandwich-like) nanofibrous scaffolds based on PCL, CS and poly(ethylene oxide) (PEO) with tunable angiogenic and osteogenic properties (Figure 1a). PEO was used with CS to particularly improve its electrospinnability 28. The developed LbL-NFs scaffolds were evaluated for their biodegradability, biocompatibility, bioactivity, mechanical properties and drug release. Also, in-vivo bone mandibular defect model was used to investigate the healing efficiency for bone tissue regeneration. 2. Methodology 2.1. Materials Chitosan (CS) of low MW, polycaprolactone (PCL) of MW of 80 kDa, acetic acid, N,Ndimethylformamide (DMF), dichloromethane (DCM), glutaraldehyde (GA), sodium chloride, sodium bicarbonate, potassium chloride, disodium hydrogen phosphate dihydrate, magnesium 5 ACS Paragon Plus Environment


chloride hexahyderate, calcium chloride dihyderate, sodium sulphate, tris(hydroxymethyl) aminomethane were purchased from Sigma-Aldrich (China and Germany). Poly(ethylene oxide) (PEO) (MW 900 kDa) was purchased from Acros , USA. Sildenafil citrate was obtained as gifts from Medical Union Pharmaceuticals, Egypt. GIBCO® RPMI medium with 10% FBS, Iscove's Modified Dulbecco’s Medium (IMDM) and sterile deionized water for cell culture were purchased from Thermo Fisher, Germany. Isolated mesenchymal stem cells (MSCs) were isolated from healthy rat bones for the cell viability experiment. 2.2. Preparation of Electrospun PCL NFs Layer PCL polymer solution (10% w/v) was prepared in a mixture of DMF and DCM (40:60% v/v) at room temperature. A plastic syringe (13.1 mm internal diameter) was filled with the polymer solution followed by its electrospining using NANON-01A electrospining system (MECC Co., LTD). Several electrospining parameters were optimized for non-beaded NFs, where the voltage was maintained at 25-27 KeV with a distance of 15cm between spinneret tip and the grounded receiver and 3.5-4 mL/h feed rate to get a Tylor cone ended with NFs beyond the jet. The same procedure was repeated to obtain sildenafil-loaded PCL NFs, where sildenafil citrate (1% w/v) was dissolved in acidified 10% w/v PCL solution at room temperature (Table 1). 2.3. Preparation of Sandwich-Like Structure of CS/PEO NFs on PCL NFs PEO aqueous solution (7.5% w/v) was obtained and CS solution (3% w/v) was prepared in 1% aqueous acetic acid at room temperature. Then, a homogeneous CS:PEO (1:1 v/v) electrospining solution was obtained via mixing the two polymer solutions with stirring. Electrospinning parameters were adjusted at 0.5-0.8mL/h flow rate, 14-16KeV applied voltage, and 15cm distance between spinneret tip and the ground stationary collector29. For LbL-NFs, CS/PEO nanofibers layer covered PCL NFs layer from both sides to prepare the sandwich structure (Table 1). The process was carried out under humidity of 30-35 %. Then the fabricated specimens were dried in desiccator for 48h to remove the excess solvent. Then, samples were crosslinked using glutaraldehyde (GA) vapor by fixing the dried LbL-NFs on a porous shelf in well-sealed container filled with GA at room temperature. The crosslinked LbL-NFs were transferred to desiccator to remove the residual GA30.


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Table 1. Composition of different LbL-NFs formulation Formulation

CS/PEO (mg)*

PCL (mg)

Crosslinking

Drug (mg)

F1 3.1225 F2 1 F3 3.1225 1 F4 3.1225 1 ✔ F5 3.1225 1 0.1 F6 3.1225 1 0.1 ✔ * CS/PEO weight represent amount of polymers in both CS/PEO layers which covering PCL layer from both sides. 2.4. Chemical and Thermal Characterization of the Developed NFs Characteristic peaks for each polymer were examined using Fourier Transform Infrared (FTIR) spectroscopy (Thermoscientific, USA) at range of 600−4000 cm−1 and 25oC. Besides, both differential scanning calorimetery (DSC; Q20, TA Instruments, USA) and thermogravimetric analysis (TGA; Q500, Thermoscientific, USA) were used to study the thermal behavior of all samples 30. 2.5. Physical and Mechanical Characterization of the NFs The structural characteristics and mechanical properties of the developed NFs were investigated by different techniques. Surface morphology of the developed NFs was examined by a scanning electron microscope, SEM (Nova Nano SEM, FEI, USA). Image-J software analysis was used to measure the NFs diameter. Tensile strength was tested at 5kN with 10 mm/min rate using Universal testing machine (AGX-PLUS, Shimadzu, Japan). Each sample was cut into rectangle with thickness × width × length (0.5 × 5 × 20 mm) 31. Porosity of the fabricated NFs (1cm × 1cm of known weight) was investigated using pycnometer (Ultrapyc 1200e, Quantachrome instruments, USA). Pycnometer contains helium gas that was used to detect the volume of the sample. Then, porosity per volume and porosity percentage were obtained using the following equations: 𝑉𝐹

Porosity per volume =1 ― 𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒

(1)

Porosity% = porosity per volume × 100

(2)

where, VF is the NFs volume that investigated by the pycnometer



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The hydrophobicity of NFs was evaluated by contact angle measurements. The contact angle was measured after 10 seconds by dropping a water drop on the NFs surface, and the angle between the liquid surface and NFs was then calculated using Image-J program. 2.6. In-vitro Mineralization Study Using Simulated Body Fluid (SBF) The developed sandwich-like NFs scaffolds with area of 1 cm2 were immersed in a concentrated simulated body fluid (c-SBF) solution, and incubated in a closed screw-cap tube for a period of 8 weeks at 37°C, followed by examining the NFs at different intervals (1, 2, 4 and 8 weeks). The SBF solution was replaced with the same volume of fresh SBF after each week to mimic the dynamic behavior of bone extracellular matrix. The SBF solution was prepared according to Kukubo and Takadama procedure 28. After each period, the NFs samples were washed three times with deionized water, followed by drying and examining the mineralization by SEM (Nova Nano SEM, FEI, USA). The initial calcium ion concentration in c-SBF along with its concentration after each interval were estimated by atomic absorption spectrophotometry (AA-7000/GFA-7000/ASC7000, Shimadzu, Japan). Then, the calcium deposition concentration was determined by subtracting the two concentrations at each interval. The initial phosphate ion concentration in cSBF along with its concentration after each interval were estimated using UV-visible spectrophotometry (Evolution UV 600, Thermo Scientific, USA) at wavelength of 700 nm, where a colored complex (molybdenum blue) was evaluated through reduction of molybdophosphoric acid. Briefly, 25 g of ammonium molybdate was dissolved in 175 ml distilled water. Then, 280 ml of concentrated sulfuric acid was diluted with 400 ml distilled water, followed by mixing the two solutions at room temperature, and completed to 1 liter of molybedate solution. Afterwards, 2.5 g of stannous chloride was dissolved in 100 ml of glycerin. The colored complex (molybdenum blue) was then formed via mixing 25 ml of SBF sample with 1 ml of molybedate solution and 2 drops of stannous chloride solution. The phosphate deposition concentration was determined by subtracting the phosphate ion concentration in c-SBF after each interval from its initial concentration. Biodegradability and water adsorption for the NFs scaffold samples were evaluated in SBF and distilled water (n=3) according to the following equations: Remaining Weight % = 100 ―

― W2 × 100] [WoWo


(3)

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Water absorption % =

W1 ― W2 W2

(4)

× 100

where Wo is the initial weight of sample before immersion in distilled water/SBF, W1 is the sample weight after removing from distilled water/SBF and blotting its surface using a filter paper, and W2 is the sample weight after removing from distilled water/SBF and complete drying. 2.7. In-vitro Drug Release Study In-vitro drug release study was carried out in a hydroalcoholic PBS solution (pH= 7.4) with 2% w/v tween 80 to maintain sink condition. Typically, 50 mg of the developed NFs sample (n=3) was immersed in 2 ml of release medium in a dialysis membrane (Spectra Por7, 10 kDa) as donor compartment. Then, the donor compartment was placed into 30 ml of release medium maintained at 37 °C with mild agitation (50 rpm). Then, 1 ml of aliquot was withdrawn at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 24 and 48 h, then daily till 28 days) and replaced by fresh medium. The concentration of the withdrawn aliquots was measured at wavelength of 294 nm using UVvisible spectrophotometry (Evolution UV 600, ThermoScientific, USA). Then, the cumulative drug release percentage was determined according to equation 5, and plotted against time. n-1

Cn= Cn-means + A/V ∑s = 1 Cs means

(5)

Where, Cn is the accumulated samples concentration, Cn-means is the estimated concentration, A is the aliquot volume, V is the released medium volume, n -1 is the overall volume of all previous aliquots, and Cs is the added concentration of all previous samples. The kinetics modeling of release profile was also estimated through the previously approved calculations 30. 2.8. Cell Viability Study Cell experiment was carried out to detect the biocompatibility of the fabricated sandwich-like NFs scaffolds. Mesenchymal stem cells (MSCs) was isolated from cord blood mononuclear cells (MNCs) of healthy albino rats and used to detect the proliferative activity of the fabricated NFs scaffolds. Briefly, MSCs were incubated in the growth medium inside CO2 incubator at 37 ⁰C for 24 h, followed by their collection, and re-suspension in the growth medium with a cell count adjusted to be 5 x 103. Then, 100 µl of the MSCs suspension were plated in each well of a 96-well plate and incubated at 37 ⁰C for 24 h. Then, the well plate was investigated under an inverted 9 ACS Paragon Plus Environment


microscope to confirm the successful adherence of the cells at the bottom of the wells. Afterwards, an amount of 10 mg of the NFs scaffolds were added individually to each well. Control was tested with wells containing the cells without scaffolds. The well plate was incubated at 37 ⁰C for 72 h to detect the cytotoxicity of the fabricated scaffolds, and the experiments were performed in triplicates. Afterwards, the well plate was removed, followed by cells trypsinization for counting. Typically, 20 µl of cell suspension was withdrawn from each well individually, mixed with an equal volume of trypan blue dye then the cells were counted using a hemocytometer 32. 2.9. In-vivo Studies 2.9.1. Rabbit Mandibular Defect Model Surgical procedure was carried out according to the guidelines after approval of the Institutional Ethics Committee of Misr University for Science and Technology (Figure 5a). Mandibular defect experiments were performed on white New Zealand male rabbits (5-6 months). All animals were treated with xylazine HCL (30 mg/kg) and Ketamine HCL (50 mg/kg) through IP injection route. The hair covering the ramus of left mandible was pared and sterilized with povidone-iodine solution. Then, spherical bone deficiency (6 mm) was created by a slow-speed drilling surgical osteotomy bur with continuous dropping of 0.9% physiological saline at contact area to avoid temperature raising. Afterwards, animals were classified into four groups (F3, F4, F5 and F6). The scaffolds (5.5 mm diameter × 5 mm thickness) were applied into the defects, and the soft tissue above the defect was sutured. During postoperative suturing, the animals were maintained under diclofenac sodium (10 mg/kg) and ceftriaxone (10 mg/kg) for 3 days. 2.9.2. Cone Beam Computed Tomography (CBCT) Cone beam computed tomography (CBCT, KaVo Dental GmbH, Biberach, Germany) images were obtained after treatment to evaluate the bone healing. Bone cavity volume was measured using OnDemand-3D software in standardized technique. The defect volumes were compared to confirm the regeneration of new bone tissue, where smaller volume means cavity filled with new bone tissue.


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2.9.3. Histological examination The bone samples were removed and fixed in 10% neutral buffered formalin for 48 h, and then decalcified in 10% EDTA. Tissue processing, including dehydration, clearing, impregnation and embedding was done through graded ethanol, xylol and paraffin. Histology sections with a thickness of 6 μm were prepared from each defect containing an intact border of the bone, and then the samples were stained with hematoxylin and eosin 33. Histological grading scale (Table 2) was used to evaluate detects properties (union , spongiosa, cortex and bone marrow) 34. Table 2. Histologic grading system for bone healing evaluation Scale

Category Union

Spongiosa

Cortex

Bone marrow

0

No sign of union

No sign of cellular activity

Absence of cortex

Not available

1

Fiberous union

Early bone formation

Early detection

2

Osteochondral union

3

Bone union

4

Complete reorganization

Active new bone formation Reorganized spongiosa formation Complete organized formation

Intiation of formation Reorganization in majority Complete organization

Detection of fiberous material Defect occupying more than half Fully occupied the red B.M Adult type fatty marrow

2.10. Statistical Analysis All data was expressed as mean ± standard deviation. Significance were tested using one-way analysis of variance (ANOVA) to all data using GraphPad Prism Software Version 6 (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

3. Result and Discussions 3.1. Structural and Thermal Characterizations Chemical structure of polycaprolactone (Figure 1b), chitosan (Figure 1c), and poly(ethylene oxide) (Figure 1d) were investigated using FTIR. FTIR spectra of the developed sandwich-like NFs are illustrated in (Figure 1e). The IR spectrum of CS/PEO (F1) showed a peak at 3379 cm-1 which refers to the stretching vibration of NH2 groups, stretching vibrations of OH groups, and the intermolecular H-bonds, while the peak appeared at 1571 cm-1 corresponds to bending vibration of the NH2 groups

1,2,30.

There is also a band noted at 2883 cm-1 due to the stretching vibration

mode of C-H 35. The bands appeared at 1096 cm-1 and 1271 cm-1 are corresponding to the [υC-OC] stretching and C-O-C bending vibrations, respectively, whereas, the absorption peak observed 11 ACS Paragon Plus Environment


around 1348 cm-1 was related to the (⸹CH2) bending vibration

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36,37.

The peak at 1648 cm-1 is

attributed to the stretching vibration of [amid I υC=O] 1,2. On the other hands, the spectrum of PCL NFs layer (F2) demonstrates two peaks for asymmetric and symmetric stretching υCH2 at at 2939 cm-1 and 2862 cm-1, respectively. The intense peak that correspond to the stretching vibration of ester [amid I υC=O] appeared in the spectrum at 1718 cm-1. The bands noted at 1243 cm-1 and 1103 cm-1 are corresponding to the [υC-O-C] asymmetric and symmetric stretching vibration, respectively. Moreover, the band attributed to (C-O and C-C) was also observed at 1103 cm-1 38– 40.

Spectra of the developed sandwich-like NFs scaffolds (F3 and F4) confirmed that all the

characteristic peaks of the polymeric constituents were observed, but with slight band-shifts and a reduction in peak intensities. Thermal analysis of CS/PEO NFs (F1), PCL NFs (F2), and the sandwich-like NFs (F3 and F4) was investigated using DSC (Figure 1f). As apparent from the figure, the presence of PEO in CS/PEO (F1) was led to appearance of a peak at 57 °C which refers to the PEO melting 30. On the other hand, the PCL NFs (F2) revealed an endothermic transition reaching its maximum at 5960°C which is related to the PCL melting temperature. The PCL thermogram also depicts a sharp endothermic peak at 405°C in addition to an exothermic transition at the range 468 – 479 °C 41. It is also obvious from the thermograms that F3 and F4 NFs maintained the characteristic bands of both PEO/CS and PCL layers. TGA thermograms of the various polymeric constituents of the NFs scaffolds are illustrated in (Figure 1g). As can be noted from the figure, CS/PEO NFs (F1) showed one-step in weight loss of about 80% in the range of 189-389 °C. The reduction of weight above this range can be attributed to the burning out of the sample. On the other hand, the PCL NFs (F2) showed a higher thermal stability as compared to the CS/PEO NFs until they decomposed completely at 570 °C with weight loss beyond 97%. In the case of the sandwich-like NFs (F3 and F4), presence of the PCL has enhanced the overall thermal stability of the CS/PEO NFs layers. Besides, and as obvious from the figure, the TGA patterns of all the investigated NFs are almost similar that confirms the nonchemical interaction between the polymeric constituents in the developed NFs scaffolds 40,42.


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Figure 1. (a) A schematic illustration of the study design, chemical structure of (b) polycaprolactone, (c) chitosan, and (d) poly(ethylene oxide). (e) FTIR of the developed NFs, and (f & g) The thermal characteristics (DSC & TGA, respectively) of the developed NFs,



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3.2. Morphological and Mechanical Characterizations of the Developed NFs Scaffolds Morphology of the developed NFs scaffolds (F2, F3 and F4) was investigated using SEM (Figure 2a-c). As apparent from the figure, the PCL NFs (F2) micrograph (Fig 2a) demonstrated a relatively smooth surface of randomly oriented NFs with diameters ranging from about 514 to 4745 nm (average diameter 1640 ± 940 nm). Furthermore, the F2 micrograph showed a porous surface with interconnected voids through the fibers. The narrow distribution of NFs diameter in F3 outer layer (Fig 2b), ranged from 68 to 786 nm (average diameter 147 ± 93 nm), is due to the presence of CS/PEO which is smaller and denser than PCL NFs middle layer. On the other hand, crosslinking of the outer CS/PEO NFs layers using GA (F4) resulted in formation of CS/PEO hydrogel structure that appeared as a continuous layer in SEM micrograph (Fig 2c). Contact angle is a measure of the degree of wettability, which plays a pivotal role in controlling cell adhesion and proliferation. Figure 2d summarizes the measured contact angles for the various prepared NFs scaffolds (F2, F3 and F4). It is clear from the graph that PCL NFs (F2) possess the highest value of contact angle (116.6°) which confirms the PCL hydrophobicity. On the other hand, coating of PCL with CS/PEO NFs layers has increased the overall hydrophilicity by decreasing the contact angle to 57.6° for F3. Crosslinking of the CS/PEO NFs layer further decreased the angle to 25.16° in F4 due to its conversion into hydrogel

39,43.

These obtained results

are in agreement with the previously reported studies. For instance, Cao et al 2017 have developed a 2-N, 6-O-sulfated CS electrospun fibrous PCL scaffold for bone morphogenetic protein-2 (BMP2) delivery to improve osteoinduction. They reported a significant decrease in the contact angle of hydrophobic PCL fibrous scaffold from 120.6° to 53.3° upon the modification with CS derivative along with a noted increase in mesenchymal stem cells attachment 44. In another study, Hong et al 2011 conducted a comparative study on PCL NFs and PCL/CS biocomposite designed by two methods; two step layer-by-layer and co-electrospining techniques. The study revealed that the contact angle of the co-electrospun composite decreased significantly from 131° to 79°, while the two-step composite decreased from 135° to 117°, which was related to the way of CS distribution over the PCL layer 15. Porous structure of scaffolds improves both cell spreading and attachment as well as facilitating the flow of oxygen and essential nutrients to the cells which consequently enhances the 14 ACS Paragon Plus Environment

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proliferation 39,45. In porosity study (Figure 2e), PCL NFs (F2) showed the highest percentage of porosity of 79.6%. This value decreased after coating of PCL with the CS/PEO layer reaching 69.5% due to increasing the fibers density along with decreasing the pores diameters in the outer layer. Crosslinking of the CS/PEO NFs outer layers by GA further decreased the value to 58.5% as a result of converting these outer layers into hydrogel. The mechanical properties of the developed scaffolds were investigated by stress-strain and young’s modulus measurements, tensile strength as well as elongation at break (Figure 2f-i). Both As apparent from Fig 2f, PCL NFs (F2) have depicted a good mechanical support under elongation. Besides, the PCL NFs sample behaves as ductile material as its curve started with elastic region converted into a plastic one then deformed beyond the ultimate stress, where the PCL inner layer had an ultimate stress of 5.23 ± 1.17 MPa with a maximum strain (extensibility) of 139.5 ± 22.21 MPa. On the other hand, F3 and F4 samples can withstand force more than F2 with higher cohesion force than adhesion as a result of presence of the CS/PEO layers. Moreover, crosslinking affects the elasticity of specimens via reducing their elastic modulus, so both elongation and extensibility have increased

45,46.



Figure 2. Morphological, wettability, porosity and mechanical characterizations of the developed NFs scaffolds: (a-c) SEM micrographs (pale brown fake coloring represents the crosslinked CS/PEO layer), (d) Water contact angle measurements, (e) Porosity%, and (f-i) Mechanical measurements. 3.3. Characterizations during In-vitro Mineralization Study in SBF As apparent from Figure 3, after immersion in SBF, the apatite crystals have commenced to deposit on the surface of all the fabricated NFs scaffolds (F2, F3 and F4) starting from the first week with a significant appearance (violet color) after the second week (Ca/P ratio 1.69). After 8 weeks, dense layers of bone-like apatite have been formed which proves the high bioactivity of the developed scaffolds towards bone regeneration. Crosslinking of CS/PEO layers (F4) seems to relatively decrease the rate of apatite crystal deposition as compared to F3.


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Figure 3. Morphological characterization during the in-vitro mineralization study in SBF over 8 weeks: (a-d) F2 NFs, (e-h) F3 NFs, and (i-l) F4 Sandwich-like NFs. (SEM fake coloring where pale brown color represents the CS/PEO layers and the violet color represents the formed apatite layers). The reduction of calcium and phosphate ions concentration in SBF solutions was used as an evidence on the bioactivity of the developed NFs scaffolds to induce the formation of bone-like apatite on their surface. In Figure 4a,b, the results indicated that both PCL NFs (F2) and the prepared multilayer architecture scaffolds (F3 and F4) have achieved a good nucleation. They can attract calcium ions to form a positively charged complex followed by bonding with biphosphate and carbonate groups to form an appetite layer

47.

Coating the PCL NFs with CS/PEO layers

showed no significant increase in concentration of the deposited calcium and phosphate ions. These results were visually confirmed using SEM as in Figure 3. Figure 4c shows the biodegradability study in SBF of the prepared NFs scaffolds (F2, F3 and F4). As apparent from the figure, the remaining weight (%) for PCL NFs (F2) attained 46.8 ± 4.5% 17 ACS Paragon Plus Environment


after 1 week followed by an increase in weight to almost the original weight of sample. This could be attributed to the increase in apatite deposition with time. After 8 weeks, polymers degradation rate exceeded the apatite deposition rate, so the overall remaining weight decreased again to 57.6 ± 5.7 %. The formulation F3 exhibited a profile similar to F2 but with around 60% of weight remained after the first week followed by an increase in weight due to apatite deposition. The decrease in weight after week 8 could be due to the complete degradation of the outer layers (CS/PEO). On the other hand, crosslinking of CS/PEO layers (F4) leads to a resistance in weight change (around 80% and 50% weight were remained after 4 and 8 weeks, respectively). This could be attributed to the lower degradation rate of the crosslinked NFs layers. On the other hand, the biodegradability study in distilled water showed no significant change in all formulations (F2, F3 and F4) for two weeks (Figure 4d). F2 (PCL) degradation didn’t show the same degradation rate when data of distilled water was compared with SBF after one week. SBF components directly increase degradation rate (46.8 ± 4.5%), while the distilled water slightly affect NFs (84.39 ± 0.85%). This could be attributed to crystallinity of PCL in different buffers 48,49. After two weeks, no weight loss/gain was observed in distilled water samples, while SBF samples showed weight gain due to apatite deposition. To the same extent, F3 showed similar profile like F2. Moreover, F4 showed similar profiles in both media which could be attributed to the effect of the crosslinking NFs layers. Figure 4e demonstrates the water adsorption measurements of PCL NFs (F2) and the different prepared multilayer NFs (F3 and F4). As apparent from the figure, F2 attained water adsorption of 224 ± 43 % over 8 weeks due to the NFs porosity 50. On the other hand, the higher hydrophilicity of CS/PEO NFs layers (F3) increased the water uptake at week 2 followed by a noticeable decrease till week 8 due to the apatite crystals deposition. In the case of F4, crosslinking of CS/PEO layers adsorbed highest percentage of water at the first week, which was stable till week 8. On the other hand, the effect of distilled water on water absorption percentage showed similar profiles to SBF data (Figure 4f) except F3. SBF demonstrated a limited water absorption on F3 after one week in contrary to that noted in distilled water, which could be attributed to the apatite crystal precipitation. 3.4. In-vitro Release Study The release profile of sildenafil from the developed NFs scaffolds was investigated up to 28 days using diffusion cell (Figure 4g). The dissolution of the plain/unloaded sildenafil was fast where a 18 ACS Paragon Plus Environment

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complete drug dissolution was reached within 6 h. The release profiles from both F2 (PCL NFs) showed burst release at first day (~30%), followed by sustain release till 12 days (~60%). A significant increase in release rate was observed after 12 days till 17 days (~95%), which could be attributed to erosion of outer layer of PCL. The release profiles from both F5 (without crosslinking) and F6 (with crosslinking) were almost similar. For instance, both F5 and F6 released about 25% and 60% of drug after 1 and 7 days, respectively, then sustained till 23 days with 75% overall release. The similarity of release profiles could be due to the sandwich-like structure of the developed NFs scaffolds. In both F5 and F6, sildenafil was loaded into the PCL middle-layer, and the difference between both scaffolds is in crosslinking of CS/PEO NFs outer-layers. This didn’t affect the drug release due to hydrophilicity and fast degradation rate in both cases. The difference between CS coating formulations (F5 and F6) and PCL NFs (F2) confirm the effect of coating on sustaining the drug release. The release profiles of sildenafil from F5 and F6 were fitted to several kinetic models including zero order, first order, Higuchi, Korsmeyer–Peppas, Hixson-Crowell, Makoid-Banakar and Hopfenberg. It was found that the suitable model to describe the release profiles from F2 was Makoid-Banakar (r2 0.96) and for F5 and F6 was Korsmeyer–Peppas model (r2 0.95) where n (diffusional exponent value) was 0.318 and 0.316, respectively. The value of diffusional exponent represents fickian diffusion model 51. The design, composition, and surface characteristics of a scaffold are the main players in the generation of a specific tissue. Figure 4h demonstrates the relative MSCs count after 72 h for cytotoxicity and proliferation study. As apparent from the figure, F6 attained the highest cell viability followed by F3, F5 and F4. These results implies an evidence on the role of the scaffolds in enhancing the cells proliferation. In addition, the porosity percentage and pore type (open or closed) affected the distribution and penetration of cells through the scaffold. Besides, the interconnected pores allow the cells interaction and colonization

52,53.

Crosslinking of CS/PEO

layers significantly increased cell viability from 48% in F3 to 67% in F4 and from 52% in F5 to 80.7% in F6. Furthermore, incorporation of drug showed no significant difference when comparing F3 with F5, while F4 with F6 showed significant difference (p < 0.001). In an earlier study, Poornima et al 2017 reported a 90% cell viability for a coaxial electrospun NFs of PCL shell and CS core developed for wound healing applications where the NFs possessed interconnected pores with proper morphology for diffusion and propagation of cells 54. Also, mouse embryo fibroblasts 19 ACS Paragon Plus Environment


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(NIH3T3), murine aneuploid fibro sarcoma (L929), and human osteosarcoma cells (MG63) cells were found cytocompatible with CS/PCL scaffold and supported cells attachment and proliferation as reported in Shalumona et al 2011 55. In the case of F6 NFs, presence of sildenafil significantly increased cell viability to around 85%. Sildenafil is PDE5 inhibitor which increases cGMP

56

which in turn regulate the cellular proliferation and enhance proliferation of endothelial cells 57,58.

Figure 4. (a,b) In-vitro mineralization study in SBF (calcium and phosphate ions concentration), (c) Degradation study in SBF, (d) Degradation study in distilled water, (e) Water absorption in SBF, (f) Water absorption in distilled water, (g) In-vitro drug release study, and (h) In-vitro cytotoxicity assessment. 3.5. In-vivo Study In general, a delayed bone regeneration could be returned to many reasons, including but not limited to, unsuitable mechanical stability, morbid hormone, pathological diseases, age, nutrition, genetic differences and some side effects of some therapeutics. The most appropriate animals to test musculoskeletal problems are rabbits, where mineral density and fracture toughness of mid20 ACS Paragon Plus Environment

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diaphyseal bone of rabbits are analogous to human. Therefore, it is a common choice for orthopedic tissue engineering studies. The sildenafil-loaded and unloaded scaffolds were evaluated using mandibular bone defect rabbit model using procedure illustrated in Figure 5a.

Figure 5. Surgical procedure for bone defect creation, scaffold application and evaluation: (a) Scheme of in-vivo procedure, (b) 3D images of cone beam computed tomography, and (c) defect volume measurements. After 28 days from scaffold application, 3D skull images using cone beam computed tomography (CBCT) were obtained (Figure 5b). Generally, decreasing the linear measurements for the bony defect indicates cavity filling with formation of new bone (Figure 5c). The defect volume of F3 (unloaded and non-crosslinked) was the highest (141.93 ± 20.4 mm3). Crosslinking of scaffold 21 ACS Paragon Plus Environment


(F4) significantly improved the bone healing with only 30.87 ± 9.8 mm3. Also, it was observed that there was no remarkable difference in improving bone generation between F4 and F5. On the other hand, F6 demonstrated a significantly decreased defect size of 15.27 ± 7.3 mm3, which could be attributed to osteoinductivity mechanism of scaffold based on the used polymers and drug. Consequently, F6 could be the optimum scaffold for the regenerations of several types of bone defects. The histological evaluation of bone defect was conducted to confirm the formation of healthy bone in the defected area (Figure 6). As apparent from the figure, there were no inflammatory signs such as lymphocytes, neutrophils, orosteoclasts in the defect site. Usually, two parameters are used to evaluate bone healing which are endochondral and ossification 59. Microscopic section from the healing site of experimental group was stained using hematoxylin and eosin stain (×200) (Figure 6a-p). It was observed that F3 exhibits mild primary woven bone with fibrous union (arrows) near the defect (Figure 6a), while F4 - F6 displayed abundant cartilaginous callus with osteochondral union (Figure 6b-d). In addition, retained granulation tissue in F3 and high percent of primary lamellar bone in F4-F6 were observed, which indicates an increased active bone formation (Figure 6e-h). Furthermore, scoring data confirmed the tendency of F3 to initiate cortex that reorganized in the majority of the rest formulas (Figure 6i-l). Also, there are woven ossification which proven via osteoblastic cells proliferation after F3 implanted. On the other hand, bone cortex and periostium were grown on F4 - F6 (Figure 6i-l). Finally, F3 - F5 showed bone marrow as fibrinous material with black spots (Figure 6m-o), while bone marrow matured to red bone marrow in case of F6 (Figure 6p). A semi-quantitative scoring system was used to numerically compare all scaffolds (Figure 6q-t). The scoring system confirmed the formation of mature bone marrow in F6. These findings were aligned with CBCT images and confirm the healthy bone formation with suitable bone healing rate.


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Figure 6. Histopathological examination of bone defect after 28 days of application: (a-d) for F3, (e-h) for F4, (i-l) for F5 and (m-p) for F6 and semiquantitative scoring of tissue (q-t).

Conclusion The study involved the development of a series of sandwich-like structure NFs scaffolds that provide a combination of the advantages of both synthetic and natural polymers, which improved the bioactivity and offered stable mechanical characteristics. Electrospinning technique provided a good solution to generate a fibrous structure that mimics the organic component of bone ECM, 23 ACS Paragon Plus Environment


which improved the cell to cell and cell to matrices intercommunication. Also, crosslinking of the external NFs layers has enhanced the mechanical properties and hydrophilicity of the developed scaffolds, which are important in osteochondral mechanism. Furthermore, the prepared scaffolds offered a promising opportunity to load drug like sildenafil that enhanced the angiogenesis; essential for oxygen and nutrients support and waste removal, and callus formation. The porous 3D structure with a wide range of pore size helps the attachment, proliferation and differentiation of osteoblasts. The elasticity of the scaffold was suitable for in-vivo environment, which prevents stress shielding phenomena. Also, the bioactivity of scaffolds allowed the generation of apatite crystals which support successful bone healing. In summary, the developed scaffolds with multilayered structure is a promising solution for musculoskeletal problems. Funding No funding was received. Declaration of Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study References (1) Bajpai, A. K.; Singh, R. Study of Biomineralization of Poly(Vinyl Alcohol)-Based Scaffolds Using an Alternate Soaking Approach. Polym. Int. 2007, 56 (4), 557–568. (2) Thein-Han, W. W.; Saikhun, J.; Pholpramoo, C.; Misra, R. D. K.; Kitiyanant, Y. Chitosan–Gelatin Scaffolds for Tissue Engineering: Physico-Chemical Properties and Biological Response of Buffalo Embryonic Stem Cells and Transfectant of GFP–Buffalo Embryonic Stem Cells. Acta Biomater. 2009, 5 (9), 3453–3466. (3) Jones, J. R.; Hench, L. L. Regeneration of Trabecular Bone Using Porous Ceramics. Curr. Opin. Solid State Mater. Sci. 2003, 7 (4–5), 301–307. (4) Ramasamy, S. K. Structure and Functions of Blood Vessels and Vascular Niches in Bone. 24 ACS Paragon Plus Environment

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Figures and Tables Captions Table 1. Composition of different LbL-NFs formulation

Table 2. Histologic grading system for bone healing evaluation Figure 1. (a) A schematic illustration of the study design, (b) FTIR of the developed NFs, and (c & d) The thermal characteristics (DSC & TGA, respectively) of the developed NFs. Figure 2. Morphological, wettability, porosity and mechanical characterizations of the developed NFs scaffolds: (a-c) SEM micrographs (pale brown fake coloring represents the crosslinked CS/PEO layer), (d) Water contact angle measurements, (e) Porosity%, and (f-i) Mechanical measurements. Figure 3. Morphological characterization during the in-vitro mineralization study in SBF over 8 weeks: (a-d) F2 NFs, (e-h) F3 NFs, and (i-l) F4 Sandwich-like NFs. (SEM fake coloring where pale brown color represents the CS/PEO layers and the violet color represents the formed apatite layers). Figure 4. (a,b) In-vitro mineralization study in SBF (calcium and phosphate ions concentration), (c) Degradation study in SBF, (d) Degradation study in distilled water, (e) Water absorption in SBF, (f) Water absorption in distilled water, (g) In-vitro drug release study, and (h) In-vitro cytotoxicity assessment. Figure 5. Surgical procedure for bone defect creation, scaffold application and evaluation: (a) Scheme of in-vivo procedure, (b) 3D images of cone beam computed tomography, and (c) defect volume measurements. Figure 6. Histopathological examination of bone defect after 28 days of application: (a-d) for F3, (e-h) for F4, (i-l) for F5 and (m-p) for F6 and semiquantitative scoring of tissue (q-t).



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Sandwich-Like Nanofibrous Scaffolds for Bone Tissue Regeneration

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