Silk Fibroin Electrospun

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Biological and Medical Applications of Materials and Interfaces

Estradiol Loaded Poly(#-caprolactone)/Silk Fibroin Electrospun Microfibers Decrease Osteoclast Activity and Retain Osteoblast Function Chris Steffi, Dong Wang, Chee Hoe Kong, Zuyong Wang, Poon Nian Lim, Zhilong Shi, Eng San Thian, and Wilson Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01855 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ACS Applied Materials & Interfaces

Estradiol Fibroin Osteoclast

Loaded Electrospun Activity

Poly(ε-caprolactone)/Silk Microfibers and

Retain

Decrease Osteoblast

Function Chris Steffi,1‡ Dong Wang,1,2‡* Chee Hoe Kong,1 Zuyong Wang,3,4 Poon Nian Lim,3 Zhilong Shi,1 Eng San Thian,3 Wilson Wang 1* 1 Department of Orthopaedic Surgery, National University of Singapore, Singapore 119074, Singapore 2 State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University 570228, P.R. China 3 Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore 4 College of Materials Science and Engineering, Hunan University, Changsha 410082, P.R. China

KEYWORDS: Osteoporosis, estradiol, osteoclast, osteoblast, electrospining, silk fibroin, polycaprolactone.

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Estrogen, a steroid hormone, plays an important role in modulating osteoclast proliferation and development. Estrogen deficiency boosts osteoclast activity, leading to osteoporosis in elderly women. In this study, 17-ß estradiol (E2) loaded poly(ε-caprolactone) (PCL)/silk fibroin (SF) electrospun microfibers was developed as a proposed localized E2 delivery system to treat osteoporotic fractures. PCL is a synthetic polymer known for its biocompatibility and excellent mechanical properties. The bioactivity of PCL was enhanced by mixing it with natural silk fibroin (SF) polymer that has low immunogenicity and inherent bioactivity. Different ratios of PCL/SF blends were electrospun and characterized by scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), and water contact angle measurement. PCL and SF at ratio of 50:50 (PCL50/SF50) augmented cell proliferation of murine preosteoblast MC3T3-E1 cells and murine preosteoclast RAW 264.7 cells. Hence, PCL50/SF50 was selected and mixed with three concentrations of E2 to produce electrospun fiber mesh (0.1%E2@PCL/SF, 1%E2@PCL/SF and 5%E2@PCL/SF). Sustained release of E2 was obtained for about 3 weeks at higher E2 concentration 5%E2@PCL/SF. E2 loaded PCL50/SF50 elecrospun microfiber (1%E2@PCL/SF and 5%E2@PCL/SF) reduced tartrate-resistant acid phosphate activity, total DNA and multinucleated cell formation of osteoclasts. On the other hand, the alkaline phosphatase activity and collagen I expression of osteoblasts were retained on all E2 loaded electrospun microfibers. The E2@PCL/SF system shows potential to be used for localized E2 delivery for the treatment of osteoporotic fractures.

1. Introduction Systemic skeletal diseases such as osteoporosis have altered bone metabolism. Microenvironment cues of an osteoporotic bone stimulate bone resorbing osteoclasts over osteoblasts. Imbalanced bone physiology decreases bone mineral density (BMD) and predisposes bone to fragility fractures, which occurs primarily in elderly population 1. 2 ACS Paragon Plus Environment

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Fragility fractures are surgically treated by orthopedic implants. However, low BMD of osteoporotic bone negatively affects the holding power of implant screw and reduces the pullout strength of implants 2. Moreover, impaired micro-architecture and physiology of aged osteoporotic bone decelerate fracture healing

3-4

. These factors contribute to post-surgical

complications and poor life quality for the patients. Considering the challenges in osteoporotic fracture fixations, new surgical strategies are being explored 5. Adjunct to surgery, bone anabolic drugs, such as strontium ranelate, were administered orally to the patients to reduce healing time of osteoporotic fracture 6. Localized release of growth factors like bone morphogenetic protein-2 (BMP-2)

7

and fibroblast growth factor

8

at the fracture

site improved fracture healing in osteoporotic animal models. Clinical trials with local administration of BMP-2

9

and recombinant human BMP-7 (rhBMP-7)

10

exhibited

accelerated fracture healing and reduced post-operative complications. However, growth factors are expensive and not easy to handle due to their instability in chemical environments. Additionally, a growing side effect profile of BMP-2 including post-surgical inflammation, uncontrolled deposition of ectopic bone, accelerated bone resorption, and inappropriate modulation of adipogenesis, has emerged 11. Therefore, investigations of cheaper non-protein alternatives are preferred. Estrogen, a female endogenous hormone, orchestrates bone remodeling

12

. Estrogen

inhibits osteoclast differentiation while boosting osteoblast activity. For instance, 17-ß estradiol (E2), most potent form of estrogen, has been shown to induce apoptosis in murine osteoclasts

13-14

. Apart from the effects on bone resorbing osteoclasts, E2 has also been

shown to enhance rat osteoblast proliferation

15

. A balanced bone remodeling is achieved

with estrogen as it modulates bone metabolism. Post-menopausal decline in estrogen decreases mineralization and escalates the rate of bone resorption causing osteoporosis Apart from increasing the risk of bone fractures

16

.

17

, estrogen deficiency also delays fracture



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healing 18-19. Association of estrogen deficiency and bone health lead to the use of estrogen to treat osteoporosis in women. Systemic administration of estrogen was shown to reverse the post-menopausal decline in BMD and reduced fragility fractures

20-21

. However, life

threatening diseases such as breast cancer, thrombosis and stroke were associated with systemic E2 administration

22

and these risks depend on the dose and length of time for E2

used. Hence, when long-term E2 therapy is indicated, a controlled and sustained localized delivery of E2 to maintain a lowest effective dose is essential to evade systemic side effects. Effective delivery via an implantable device to enhance the concentrations of E2 locally in the bone microenvironment or the surface of implants would be a desirable goal for the treatment of osteoporotic bone fractures, while avoiding the associated problems of systemic drug administration. Few studies have been carried out to develop localized estrogen delivery systems for osteoporotic bone fracture. Yan et al functionalized titanium with E2 loaded mesoporous silica nanoparticles. The E2 modified titanium was shown to improve osteoblast activity 23. However, this system is limited to E2 delivery on the surface of implant. Another group developed E2 loaded poly(lactide-co-glycolide) (PLGA) nanoparticle encapsulated in chitosan-hydroxyapatite scaffolds. Sustained delivery of E2 from this system has been found to stimulate osteogenic differentiation of adipose tissue-derived mesenchymal stem cells both in-vitro 24 and in-vivo 25 systems. Electrospun fibers are one of the most promising platforms to be designed as implantable drug delivery system due to their unique features including ultrathin diameter, high permeability, interconnecting pore structure, and high drug encapsulation efficiency. The biomimetic cellular environment provided by electrospun micro-/nano-fibers resembles the extracellular matrix (ECM) of native tissues, thus can be used as a temporary threedimensional support for cell adhesion, cell proliferation, cell migration, vascularization and formation of new tissues. At the same time, therapeutic molecules could be encapsulated in


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electrospun fibers allowing controlled and sustained local delivery of these molecules to target sites

26

. Localised delivery of therapeutic molecules enables the drug to act on the

specific confined areas, while eliminating the adverse side effects associated with systemic delivery

27

. Anticancer drugs, antibiotics, proteins, various growth factors, and living cells

have been incorporated into electrospun fibers for investigation in tissue engineering

28

.

However, to the best of our knowledge, there is still no relevant study of using electrospun fibers as an implantable drug delivery system for localised E2 delivery to treat osteoporotic bone defects. Appropriate polymer materials need to be identified for making electrospun fibers into implantable drug delivery system. Poly(ε-caprolactone) (PCL) is known for its biocompatibility, biodegradability and favourable mechanical properties, yet it is inert, hydrophobic and lack biological recognition sites. These limit them to provide a desired microenvironment for cell adhesion, proliferation and biomaterial/cell interaction. To increase the hydrophilicity and biocompatibility, natural polymers with inherent bioactivity were considered. Natural polymers such as collagen, chitosan, and gelatin, were electrospun with PCL to produce composites that encouraged cell adherence and spreading 29. Silk fibroin (SF) is a promising biomaterial amongst natural polymers for drug delivery and tissue engineering because of its good biocompatibility, inherent bioactivity, low inflammatory response, slow and controllable biodegradability 30. SF scaffolds have been shown to support mesenchymal stem cells (MSCs) attachment, osteogenesis, and ECM deposition

31

. Silk

fibroin-modified poly(D,L-lactic acid) surface improved the cellular-biomaterial interaction with osteoblasts

32

. However, regenerated silk fibroin has low mechanical property. Thus,

blending of regenerated SF with synthetic polymers is expected to develop composite scaffold with improved processability, while retaining the preferred mechanical and biocompatible characteristics.



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In this study, E2 was incorporated directly into hybrid PCL/SF microfibers via electrospinning process for assessment as E2 delivery system to decrease osteoclast activity. Ethanol vapor treatment method was used to convert the water soluble, random coil configuration of SF to water insoluble, β-sheet conformation. Due to non-contact feature of this post treatment method, 100% encapsulation efficiency could be achieved. The release of E2 from electrospun microfibers could be modified based on the original loading concentration of E2 and other parameters. A sustained release of E2 over an appropriate time frame was obtained through incorporation of 5% E2 into PCL/SF hybrid microfibers. The effects on bone cell function of E2 loaded was studied in-vitro using osteoclasts derived from osteoclast precursor RAW 264.7 cells and murine preosteoblasts MC3T3-E1. 2. Experimental section 2.1 Materials Silkworm cocoons were kindly provided by The Silk Institute, College of Materials and Textiles, Zhejiang Sci-Tech University, China). All additional chemicals were acquired from Sigma-Aldrich (Darmstadt, Germany) unless otherwise specified.

2.2 Synthesis of E2 loaded PCL/SF electrospun microfibers The regenerated SF was prepared as per the protocol described previously

33

. Briefly,

cocoons from silkworm were boiled for 30 min in 0.02 M Na2CO3 aqueous solution, rinsed thrice with distilled water, dried in air overnight, before completely dissolved for 4 h in 9.3 M LiBr solution at 60 °C. Subsequently, the mixture was dialyzed against distilled water for 3 days. Water was replenished every 6 h. The SF solution was collected, centrifuged to remove impurities, and lyophilized to acquire dry SF sponge. PCL/SF emulsion (50:50, w/w) was made by dissolving 0.4 g PCL (MW 80,000) and 0.4 g dry SF together in 9.2 g hexafluoroisopropanol (HFIP) to obtain a mixture with total polymer


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mass of 8 wt%. The polymer mixture was constantly stirred in HFIP at room temperature (RT) for 24 h. To add E2 in PCL50/SF50 solution, E2 was first dissolved in HFIP before mixing with PCL50/SF50 emulsions to obtain 0.1wt%, 1wt% and 5wt% mass ratio of E2 to total polymer (named as 0.1%E2@PCL/SF, 1%E2@PCL/SF and 5%E2@PCL/SF respectively). PCL50/SF50 samples without E2 were prepared as control. The other emulsions with different ratios of PCL to SF (100:0, 75:25, 25:75 and 0:100, w/w) were also prepared according to the same procedure. The blends were named as PCL100/SF0, PCL75/SF25, PCL50/SF50, PCL25/SF75, PCL0/SF100. However, only PCL50/SF50 was mixed with E2. The electrospinning process was carried in a glove box. The blend solutions were pumped through a blunted stainless steel needle (24G) with a flow rate of 5 ml/h and 12 kV was applied to the needle. Electrospun microfibers were collected using a grounded aluminium foil, which was placed 16 cm away from the syringe nozzle. For ease of handling in cell culture studies, the microfibers were collected on round glass coverslips of 13 mm diameter attached to the surface of grounded aluminium foil. The collected microfibers were vacuumdried for 3 days at RT to remove residual HFIP. To obtain water insoluble β conformation of SF from water soluble α secondary structure, an ethanol vapor process was developed for the post-treatment of electrospun microfibers. SF/PCL microfibers were treated with saturated absolute ethanol vapor at 60 °C for 4 h in a sealed container, dried naturally at room temperature.

2.3 Characterization of electrospun microfibers The morphology of PCL/SF electropsun microfibers was observed using a field emission scanning electron microscopy (FESEM, JSM-6701F, JEOL, Tokyo, Japan) with accelerating voltage of 10 kV. The samples were imaged after gold sputter coating treatment. The average



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diameter of microfibers was measured by randomly selecting and measuring the diameter of 50 fibers for each group of sample, respectively, in ImageJ software. Fourier transform infrared spectroscopy (FTIR, Vertex 70 FTIR spectrometer, Bruker, Billerica, MA) was used to analyse the chemical structure of electrospun microfibers. The surface wettability was assessed with a contact angle analyser (Phoenix 300 Touch, Surface Electro Optics, Korea).

2.4 E2 release study E2@PCL/SF microfibers were weighed (0.5 mg) to calculate the loaded amount of E2 (E2 concentrations were either 5%, 1% or 0.1% w/w of the total weight of scaffold). Microfibers were submerged in 1 ml phosphate buffered saline (PBS, pH=7.4), sealed and incubated with gentle shaking at 37 °C. At pre-determined time frames, 1 ml PBS was collected and replaced by an equivalent amount of fresh PBS. The released E2 in PBS was evaluated by E2 ELISA kit (DRG International, Inc. Springfield, NJ) with appropriate dilution.

2.5 Cell culture Murine preosteoclast cell line RAW 264.7 (ATCC, Manassa, VA) was maintained in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Waltham, MA). To induce osteoclast differentiation, 20,000 cells were seeded per microfibrous scaffold on round glass coverslip that was placed in ultra-low adhesion 24-well plates. Cells were cultured in differentiating media composed of phenol red-free Minimum Essential Medium Alpha (MEM-α, Thermo Fisher Scientific) adding 10% heat inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin and 20 ng/ml mouse receptor activator of NF-κB ligand (RANKL, R&D Systems, Minneapolis, MN). Murine presteoblast cell line MC3T3-E1 was


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cultured in MEM-α (Thermo Fisher Scientific) that includes 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cell culture media (phenol red free MEM-α) was supplemented with 10 mM sodium β-glycerophosphate and 50 µg/mL ascorbic acid for osteoblast differentiation studies. Electrospun microfibrous mesh were UV sterilised (30 min) before placing in ultra-low adhesion 24-well plates 40,000 MC3T3-E1 cells were seeded per well. All the cultures were maintained in humid environment at 37 oC with 5% CO2. Media were replaced once every 2-3 days.

2.6 MTT assay Cell toxicity of PCL/SF (100:0, 75:25, 50:50, 25:75 and 0:100) hybrid microfibers was studied using MTT assay. RAW 264.7 and MC3T3-E1 were cultured separately on all the PCL/SF microfibers with different mass ratios for three days. The cell cytotoxicity was studied using MTT reagent as described previously

34

. In brief, cell culture media

supplemented with 10% MTT stock solution (5 mg/ml in phosphate buffer saline, PBS) was given to the cells. Subsequently, the media was removed after 4 h of incubation at 37 oC. DMSO was then added to dissolve the formazan complex. Optical density of 570 nm was measured using a Microplate Reader (Synergy H1, BioTek Instruments Inc, Winooski, VT).

2.7 Cell proliferation assay Cell proliferation study of respective RAW 264.7 and MC3T3-E1 on various microfibrous scaffolds were cultured and assessed at different time points using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) as per manufacturer’s instructions. In brief, CCK-8 mixed in cell culture media (1:10 v/v) was added to the cells. After 4 h incubation at 37 oC, the absorbance of 450 nm was detected with a Microplate Reader (Synergy H1).



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2.8 Tartrate-resistant acid phosphate (TRAP) activity assay In the presence of RANKL for 5 days, RAW 264.7 cells were cultured and TRAP activity was measured with cell culture media using TRAP activity assay kit (Takara Bio Inc, Shiga, Japan) as instructed by the manufacturer. In brief, 50 µl of tartrate supplemented acid phosphatase buffer was mixed with 50 µl of cell culture supernatant and incubated at 37 oC for 1 h. 50 µl of 0.5N NaOH was added to terminate the reaction. Absorbance 450 nm of pnitrophenol (pNP) produced was measured using Microplate Reader (Synergy H1). Amount of pNP produced was calculated from pNP standard curve. The TRAP activity was normalised to total protein and expressed as µM of pNP produced per minute per mg of protein.

2.9 DNA quantification After 5 days of culture, osteoclasts were washed twice with PBS. 0.2% triton X-100 was added to the cells and exposed to three freeze-thaw cycles. The cell lysate was span down at 12 000 g for 20 min at 4 oC. Total DNA in the cell lysate was estimated by nuclear dye Picogreen reagent (Thermo Fisher Scientific, Waltham, MA) as per the manufacturer’s manual. Briefly, cell lysate was mixed with pico green reagent. After 5 min incubation at RT, the samples were excited at 480 nm and the fluorescence was measured at 520 nm using Microplate Reader (Synergy H1).

2.10 Immunofluorescence confocal microscopy of osteoclasts Osteoclasts were cultured on electrospun microfibers for 5 days. Cells were fixed for 15 min using 4% paraformaldehyde and permeabilised by 0.1% triton X-100. 3 % bovine serum albumin (BSA) in PBS was utilised to impede the non-specific binding. Osteoclasts were


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stained for TRAP with anti-mouse TRAP primary antibody (Abcam, Cambridge, UK) for 90 min, and secondary antibody Alexa Fluor 546 tagged anti-rabbit IgG (H+L) (Invitrogen, Waltham, MA) for 30 min. Actin was stained with Alexa Fluor 488 phalloidin (Invitrogen) for another 30 min. Nuclei were counter stained using DAPI. Cell morphology and TRAP staining were observed using Olympus FV1000 confocal laser scanning microscope (CLSM, Olympus, Tokyo, Japan). TRAP positive cells having more than 3 nuclei were considered as osteoclast.

2.11 Osteoblast differentiation MC3T3-E1 cells were cultured on microfibrous scaffolds. Early osteoblast differentiation marker alkaline phosphatase (ALP) activity was estimated in cell culture supernatant after 7 days of culture by QuantiChrom Alkaline Phosphatase Assay Kit (BioAssay Systems, Hayward, CA). ALP activity was calculated as µM of pNP produced per minute per mg of protein. Collagen I was estimated in cell culture media after 21 days of cell culture, using Pro-Collagen I alpha 1 SimpleStep ELISA kit (Abcam, Cambridge, UK). The activity and concentration were normalised to total protein. 2.12 Immunofluorescence microscopy of osteoblasts MC3T3-E1 cells were grown on scaffolds for 21 days. The cells were fixed with 4 % paraformaldehyde and immersed in 0.1% triton X-100 for 5 min to permeabilise cell membrane. The cells were then blocked overnight in 3 % BSA, before incubating with antimouse collagen type I antibody (Merck, Darmstadt, Germany) for 90 min. Cells were then stained with secondary antibody Alexa Fluor 546 tagged anti-rabbit IgG (H+L) (Invitrogen) and Alexa Fluor 488 phalloidin (Invitrogen) for 30 min. DAPI was used to stain nuclei. Olympus FV1000 CLSM (Olympus, Tokyo, Japan) was used to image the cells.



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2.13 Statistics All the experiments were replicated three times. The results are reported as mean±standard deviation (SD). One-way ANOVA was applied for the analysis. Values of p 0.05) and no cytotoxicity was observed for both MC3T3-E1 and RAW 264.7 cells (Figure 5C and Figure 5D).

3.4 Osteoclast proliferation and differentiation To study the potency of immobilized E2 in PCL/SF microfibers on the osteoclast function, RAW 264.7 cells were cultured on the electrospun microfibers in presence of RANKL. DNA quantity of cells is used to assess cell number and cell proliferation

46-47

. After 5 days of

culture, osteoclast proliferation was studied by quantifying total DNA in cell lysate (Figure 6A). Total DNA of osteoclasts cultured on 1%E2@PCL/SF and 5%E2@PCL/SF were significantly reduced as compared to that on 0%E2@PCL/SF and 0.1%E2@PCL/SF (p < 0.01). On the other hand, no decrease in osteoclast DNA was observed in 0.1%E2@PCL/SF. Several studies have reported the apoptotic effect of E2 on osteoclasts

13-14, 48

. E2 negatively

influences the life span of osteoclasts via estrogen receptor alpha and by modulating Fas/FasL signalling

49

. Even though previous reports assessed the effect of E2 in media,


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effect of PCL/SF based E2 delivery system has not been studied. This study is the first to report osteoclast-inhibitory effects of E2 loaded PCL/SF delivery system. TRAP, hallmark of osteoclastogenesis

50

, was estimated to study the potency of

E2@PCL/SF microfiber composites (Figure 6B). A dose dependent decrease of TRAP activity was observed in E2@PCL/SF microfiber composites. TRAP activity of osteoclasts cultured on 0.1%E2@PCL/SF, 1%E2@PCL/SF and 5%E2@PCL/SF was significantly reduced as compared to TRAP activity of osteoclasts cultured on unloaded control (0%E2@PCL/SF, p < 0.01). TRAP activity of osteoclasts on 1%E2@PCL/SF and 5%E2@PCL/SF was significantly less than 0.1%E2@PCL/SF (p < 0.01). No difference was observed between the TRAP activity of osteoclasts cultured on 1%E2@PCL/SF and 5%E2@PCL/SF (p > 0.05). E2 in solution was shown to impede RANKL induced maturation of osteoclast 51. Likewise, the current study also demonstrated E2 loaded PCL/SF microfibers reduce osteoclast differentiation, suggesting that the biological activity of loaded E2 is preserved in the electrospun microfibrous scaffold.

Figure 6. (A) Total DNA of the osteoclasts after 5 days of cell culture on various microfibrous scaffolds loaded with different concentration of E2. (B) TRAP activity of



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osteoclast after 5 days of cell culture. Statistical significant is denoted by (**) p

Silk Fibroin Electrospun

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