Synergetic Cues of Bioactive Nanoparticles and Nanofibrous Structure

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Synergetic Cues of Bioactive Nanoparticles and Nanofibrous Structure in Bone Scaffolds to Stimulate Osteogenesis and Angiogenesis Jung-Ju Kim, Ahmed El-Fiqi, and Hae-Won Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12089 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016

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ACS Applied Materials & Interfaces

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Synergetic Cues of Bioactive Nanoparticles and Nanofibrous Structure in Bone 7 8

Scaffolds to Stimulate Osteogenesis and Angiogenesis 9 10 1 12 13

Jung-Ju Kim1,2, Ahmed El-Fiqi1,2, Hae-Won Kim1,2,3,* 14 15 16 17 18 1

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Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Republic of Korea

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Republic of Korea

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Department of Biomaterials Science, College of Dentistry, Dankook University, Republic of Korea

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*Corresponding author: Prof. H.-W. Kim (e-mail: [email protected]; tel: +82 41 550 3081; fax: +82 41 550 3085)

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ACS Paragon Plus Environment


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Abstract 3 5

4 Providing a nanotopological physical cue in concert with a bioactive chemical signal within 3D scaffolds, while it being

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considered a promising approach for bone regeneration, has yet to be explored. Here, we develop 3D porous scaffolds

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that are networked to be a nanofibrous structure and incorporated with bioactive glass nanoparticles (BGn) to tackle this

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issue. The presence of BGn and nanofibrous structure (BGn+nanofibrous) substantially increased the surface area,

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hydro-affinity and protein loading capacity of scaffolds. In particular, the BGn released Si and Ca ions to the levels

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known to be biologically-effective, offering the bone scaffold an ability to deliver therapeutic ions. The mesenchymal

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stem cells (MSCs) from rats exhibited significantly accelerated adhesion events including cell anchorage, cytoskeletal

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extensions, and the expression of adhesion signaling molecules on the BGn/nanofibrous scaffolds. The cells gained a

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more rapid proliferation and migration (penetration) ability over 2 weeks within the BGn+nanofibrous scaffolds than

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within either nanofibrous or BGn scaffolds. The osteogenesis of MSCs, as confirmed by the expressions of bone-

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associated genes and proteins, as well as the cellular mineralization was significantly stimulated by the BGn and

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nanofibrous topology in a synergistic manner. The behaviors of endothelial cells (HUVECs) including cell migration

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and tubule networking were also enhanced when influenced by the BGn and nanofibrous scaffolds (but more by BGn

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than by nanofiber). A subcutaneous tissue implantation of the scaffolds further evidenced the in vivo stimulation of neo-

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blood vessel formation by the BGn+nanofibrous cues, suggesting the possible promising role in bone regeneration.

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Taken together, the therapeutic ions and nanofibrous topology implemented within 3D scaffolds are considered to play

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synergistic actions in osteogenesis and angiogenesis, implying the potential usefulness of the BGn+nanofibrous

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scaffolds for bone tissue engineering.

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41 Keywords: Bone scaffolds; Nanofibrous structure; Bioactive glass nanoparticles; Topological cue; Ionic cue;

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Osteogenesis; Angiogenesis

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1. Introduction 3 4 5 Three dimensional (3D) scaffolds have played significant roles in hard tissue engineering1, 2. Their roles include i)

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7 providing a matrix for cellular anchorage, migration, and growth, ii) guiding neo-vessel formation through nutrients and

9 oxygen supply, and iii) supplying therapeutic molecules for osteogenic differentiation and bone formation3, 4. Therefore,

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1 the physico-chemical and biological properties of scaffolds either intrinsically or extrinsically endowed, e.g., nano-

13 /micro-topology engineered on the surface, adhesion proteins tethered, and bioactive drugs incorporated, should be

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carefully tuned to synergize the functions of scaffolds 5.

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18 Among the properties, nanostructures have special implications in altering cell fate, including angiogenesis and

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osteogenesis of stem cells6-8. Nanofibrous structure is unique to provide an environment mimicking extracellular matrix

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(ECM). For example, the native bone ECM is comprised of collagen fibrous network with embedded hydroxyapatite

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24 inorganic nanocrystals9. For this reason, various types of biomaterials (biopolymers, bioactive inorganics, and their

26 composites) have been developed into nanofibrous scaffolds for bone10, 11. The most common method to generate the

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nanofibrous structure is electrospinning although self-assembly and phase-separation have also been developed12-15.

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30 However, electrospinning is a line-of-sight method, thus creates psudo-3D structure with small pore spacing, which

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32 limits primarily the cellular recognition of 3D networks and rapid cellular penetration. The phase-separation method, on

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34 the other hand, can generate 3D porous scaffolds with networks highly nanofibrous structured. The phase-separated

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36 nanofibrous 3D scaffolds have shown to provide nanofibrous surface topological cue to the cells while allowing cellular

38 penetration through the macropore channels16. The MSCs cultured on the nanofibrous-surfaced poly(lactic acid)

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scaffolds were stimulated to express higher osteogenic differentiation levels than those on the dense-surfaced scaffolds17.

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43 Along with the nanostructural tailoring, the incorporation of bioactive materials or molecules to the scaffolds is another

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effective strategy to stimulate bone regeneration18-21. Bioactive glasses (BG) have been one of the most attractive

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components used in bone regeneration, finding potential clinical applications. The nanoparticulate form of BG, namely

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49 ‘BGn’, is thus an ideal bioactive ‘nanocomponent’ to be added to the bone scaffolds. BGn, mainly composed of silicon

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51 and calcium ions, can be developed into less than a few hundreds of nanometers. Furthermore, the sol-gel process

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53 enables highly mesoporous structure, which increases the surface area and nanospace, ultimately allowing for the

5 loading and delivery of drugs and genes22-24. The release of ions (Si4+ and Ca2+) has also therapeutic functions such as

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the stimulation of cell proliferation, angiogenesis and osteogenesis 25-27 even more, other therapeutic ions (such as Sr, Co,

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59 F, Ag, and Zn) can be easily doped to the glass network, possibly regulating cellular functions more intensely and diversely28-32. 3



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1 Here we aim to synergize the nanotopological cue (nanofibrous structure) and the bioactive nanoparticle signal (BGn)

3 within scaffolds targeting bone. Scaffolds of poly(lactic acid) (PLA) were incorporated with BGn up to 20wt%, and the

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nanofibrous structure was created through the phase-separation method using camphene, which was subsequently salt-

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leaching processed into macroporous foam scaffolds. We hypothesize that the BGn-incorporating nanofibrous scaffolds

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might be effective in the angiogenesis and osteogenesis of cells which ultimately contributes to the successful

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regeneration of bone defects. To prove the hypothesis, we first observe significant alterations in physico-chemical

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properties of the developed scaffolds, then examine their interactions with mesenchymal stem cells and endothelial cells

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implicated in osteogenesis and angiogenesis, respectively, and finally test the tissue invasion and neo-blood vessel

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formation.

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2. Materials and methods 25 26 28

27 2.1. Materials

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31 Poly ethylene glycol (PEG; (C2H4)nH2O, Mn = 10,000), calcium nitrate tetrahydrate (CN; Ca(NO3)2∙4H2O), methanol

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(anhydrous, 99.8%, CH3OH), ammonia (NH4OH) and tetraethyl orthosilicate (TEOS; Si(OCH3)4) were purchased from

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Sigma to fabricate the bioactive glass nanoparticles. Poly-L/D-lactide acid (PLDLA; LESOMER@LR 708, L-

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lactide:D,L-lactide molar ratio = 70:30, Mn = 910,000, Evonic, Germany) was used as a matrix for 3D scaffold. 1,4-

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39 dioxane (C4H8O2, Sigma) and chloroform (CHCl3, Dae-Jung) were used as the co-solvent, and camphene (2,2-dimethyl-

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3-methylene-bicyclo[2.2.1]heptanes; C10H16, Sigma) was used to generate a nanofibrous structure within scaffolds.

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Sodium chloride (NaCl, Dae-Jung) was used as a porogen to generate macropores. Alizarin Red S (ARS; C14H7NaO7S,

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45 Sigma) was used to stain calcium, and cetylpyridinium chloride (CPC; C21H38ClN·H2O, Sigma) was used to eliminate

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47 the ARS stain for the quantification of calcium amount.

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50 2.2. Preparation of bioactive glass nanoparticles

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54 BGn (85%SiO2-15%CaO by mol%) were produced by a sono-assisted sol-gel method, with slight modifications from a

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previous report33. In brief, CN was first dissolved in PEG-methanol alkaline solution, within which TEOS (considering

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58 a molar ratio of Ca/Si=15/85) in methanol solution was added drop-wise while applying a high-power ultrasound with

60 10-s on/10-s off cycle using a sonoreactor (LH700 W ultra-sonic generator, Ulsso Hitech). The white precipitates 4


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1 formed were collected by a centrifugation at 5000 rpm (Mega 17 R centrifuge, Hanil Science), and washed fully with

3 distilled water and ethanol. The powder was heat-treated at 600oC in air with a ramping rate of 1 oC/min.

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6 2.3. Preparation of scaffolds

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10 Four types of 3D scaffolds were developed; dense surface (Den), BGn contained dense surface (Den(B)), nano-fibrous

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12 surface (Fib), and BGn contained nano-fibrous surface (Fib(B)). In particular, the nano-fibrous surface structured

14 scaffolds were produced by using camphene during the phase-separation process, as described in our previous work34.

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16 In brief, 0.3 g of PLDLA was dissolved in 10 ml of co-solvent (1,4-dioxane/chloroform ratio of 4 by weight) and then

18 1.2 g of camphene was added to produce the nano-fibrous structure. For the BGn/PLDLA composite scaffolds, 60 mg of

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BGn were well dispersed within the co-solvent and then followed by the addition of camphene and PLDLA. The

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mixture slurry was poured into a cylindrical plastic vial and then packed with the NaCl particles of 200-500 m in size

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which were collected by sieving. The samples were frozen at -20oC for overnight and then lyophilized for 3 days. The

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26 NaCl particles were completely leached out from the scaffolds in distilled water under shaking at 100 rpm with

28 refreshing the distilled water every day. The ratio of each component used for the preparation of different scaffolds is

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summarized in supporting information (Table S1).

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3 2.4. Characterizations

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37 The morphology of BGn was observed by transmission electron microscopy (TEM; JEM-3010, JEOL). The

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39 mesoporous structure and pore size distribution were analyzed by Brunauer–Emmett–Teller (BET; Quantachrome)

41 methods using N2 adsorption/desorption isotherm. The morphology of scaffolds was determined by scanning electron

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microscope (SEM; S-3000H, Hitachi). Fourier transform infrared spectroscopy (FTIR; 640-IR, Varian) were used to

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determine the chemical bond status. Energy dispersive X-ray spectroscopy (EDX; Bruker) was used to detect the atomic

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composition of the samples. The micro/nanopore structural properties of scaffolds were analyzed using a mercury

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porosimeter (PM33, Quantachrome). The surface area of the scaffolds was assessed by the BET method, as described

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above. The hydro-affinity property of the samples was investigated by the water contact angle measurement using a

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Phoenix300 analyzer. For the measurement of contact angle, samples were prepared in a membrane shape. For this, the

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mixture solutions were cast onto a mold without the use of NaCl particles, and then followed by a freezing at -20oC and

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then a lyophilization for 3 days to obtain thin membranes (thickness of ~1 mm). Images of a water droplet were

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obtained using CCD camera. The compressive mechanical properties of the scaffolds were evaluated using a universal testing machine (Instron 5966, USA) with a 500 N load cell under a cross-head speed of 0.5 mm/min. Each specimen 5



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1 prepared with a cylindrical shape (10 mm diameter x 20 mm height) was tested in wet condition after immersion in

3 distilled water (n = 5).

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6 The capacity to load proteins of the scaffolds was tested using Cytochrome C (Cyt C) as the model protein. Each

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scaffold was immersed in 1 ml distilled water which containing 100 μg of Cyt C. After 1 day, the supernatant was

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collected and quantified by the BCA assay kit (Thermoscientific Co.).

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13 2.5. Ionic release test

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The release amount of silicon and calcium ions from the scaffolds was detected by inductively coupled plasma atomic

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emission spectrometry (ICP-AES; OPTIMA 4300 DV, Perkin-Elmer). For this, 20 mg of scaffold was immersed in 10

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ml of deionized water buffered at pH 7.4 with tris(hydroxymethyl)aminomethane and 1 N HCl (Tris–HCl buffer) and

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kept at 37°C for up to 28 days. At every time point (1, 3, 7, 14, 21, and 28 days), supernatants were collected for the

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analysis.

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28 2.6. Rat mesenchymal stem cells

30 31 32 The MSCs were isolated from 5-week-old male Sprague Dawley rat. Bone marrow was aspirated from the tibiae and

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femurs of rats using Hank’s Buffered Salt Solution (HBSS; Gibco) containing 0.1% collagenase Type I. The

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mononuclear cells were then collected from the enzyme solution by centrifugation at 1500 rpm and then cultured in α-

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Minimal Essential Medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone, Thermo), 100

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U/mL penicillin, and 100 μl/mL streptomycin (all from Sigma) at 37°C in a humidified atmosphere containing 5% CO 2

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for 7 days. The culture medium was changed every 2-3 day. All protocols involving animals were conducted according

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to the standards of IRB in the Dankook University regulations.

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47 2.7. Cell viability

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50 Prior to determining the cell behaviors of the 3D scaffolds, the toxicity of BGn was briefly studied. 5 x 103 MSCs were

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seeded to each well of 96 well plates. After 24 h, unattached cells were washed out and then the BGn were treated to

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each well at varying concentrations of 0, 20, 40, 80, 160, and 320 μg/ml. Cell counting kit (CCK) assay was used to

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measure the cell viability at 24 and 48 h. In brief, 100 μl of working solution (1:10 ratio of CCK-solution:medium) was

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treated to each well and then incubated for 2 h in dark. 80 μl of working solution was transferred to new plates and the optical density was detected at 450 nm using an iMark microplate reader (BioRad). 6


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1 For the tests with scaffolds, 1 x 105 MSCs were seeded onto each scaffold (5 mm diameter x 3 mm height) and cultured

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in growth medium (α-MEM containing 10% FBS and 10% P/S) in an incubator at 37 ºC humidified with 5% CO2. For

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all tests, MSCs in passage three were used and the culture medium was refreshed every 2-3 days until harvest. After 24

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h, scaffolds were transferred to a new well of 96 well plates and cultured over a period of 3, 7, and 14 days. At each

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time point, scaffolds were washed twice with PBS, and the CCK assay was conducted, as described above.

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12 2.8. Cell adhesion

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15 To determine the attachment behaviors of MSCs, the morphology, spreading area, viability, and the expression of

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adhesion molecules, i.e., focal adhesion kinase (FAK), and integrin β1, was investigated at the very early stage of

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culture (4 h). To observe the morphology of cells, the cells attached to scaffolds were fixed in 4% paraformaldehyde

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(PFA) for 10 min at room temperature, and then permeabilized with 0.1% triton X 100, followed by washing 3 times in

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PBS and blocking with 1% BSA for 30 min. Cells were stained with phalloidin for 45 min at room temperature

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followed by washing 3 times with PBS. Nuclei were stained by DAPI (Invitrogen) for 5 min at room temperature and

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then observed with confocal laser scanning microscopy (CLSM; Zeiss 700). The images were used to measure the cell

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spreading area and number by Image J software (NIH). The cell viability was also assessed by the CCK assay, as

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described above.

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34 The expression of adhesion molecules (FAK and its phosphorylated form pFAK, and integrin β1) was examined by the

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Western blot analysis using ibindTM western blot system according to the manufacturer’s instructions. Primary

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antibodies used were mouse anti-β-actin (sc-47778, Santa Cruz), rabbit anti-FAK (sc-557, Santa Cruz), goat anti-pFAK

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(sc-16662, Santa Cruz), and mouse anti-Integrin β1 (sc-13590, Santa Cruz). The blots were then incubated with HRP

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(Horseradish peroxidase)-conjugated secondary IgG (Immunoglobulin G) and band signals were detected using

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enhanced chemiluminescent (ECL, Invitrogen) detection reagent. The ECL treated membrane was visualized by LAS-

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1000 mini image analyzer (GE). Quantitative densitometry was also performed from the band images by Image J

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software (NIH).

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51 2.9. Osteogenesis

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5 The osteogenic induction of MSCs was performed by culturing in osteogenic medium (growth medium plus 10 mM -

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glycerophosphate, 50 µg/ml ascorbic acid and 10 nM dexamethasone, all from Sigma). The osteogenesis of cells was

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characterized by the quantitative real time polymerase chain reaction (qRT-PCR), immunostaining, and ARS. After 7



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1 culturing the MSCs within the scaffolds for 7 and 14 days, the expression of the bone associated genes including

3 alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcine (OCN) was confirmed by

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qRT-PCR. In brief, the first strand cDNA was synthesized from the 500 ng of RNA using a SuperScript first strand

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synthesis system for PCR (Bioneer) according to the manufacturer's instruction. The reaction mixture was made up to

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50 μl. The qRT-PCR was conducted using SYBR GreenER qPCR SuperMix reagents (Invitrogen) using 50 ng of total

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cDNA. The relative transcript quantities were calculated using the ΔΔCt method, with glyceraldehyde 3-phosphate

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dehydrogenase (GAPDH) as the endogenous reference amplified from the samples. Fold change was subsequently

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determined from 2- ΔΔCt .The primer sequences of the genes are summarized in supporting information (Table S2).

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18 To confirm the protein expression, the immunostaining was performed at 7 and 14 days. The samples were incubated in

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20 the primary antibody anti-rabbit (OPN sc-20788 and BSP sc-292394) overnight at 4ºC followed by washing twice in

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2 PBS and then treated with secondary anti-rabbit-FITC antibody. Cells were co-stained with phalloidin and DAPI. The

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24 fluorescence signals were detected by the CLSM.

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27 The cellular mineralization was assessed by the ARS at 21 and 28 days. The fixed cells were immersed in 2% w/v of

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aqueous ARS solution (pH 4.1–4.3) for 30 min at room temperature. After complete washing with distilled water, the

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stained samples were captured using digital camera. Afterward, ARS stain was eluted by 10% w/v CPC in 10 mM

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sodium phosphate (pH 7) for 1 h. The absorbance of eluents was then read at 595 nm using an iMark microplate reader.

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Scaffolds w/o cells served as controls for the optical quantification.

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38 2.10. Angiogenesis of endothelial cells

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42 The angiogenesis of cells was analyzed by using human umbilical vein endothelial cells (HUVECs). Cells were cultured

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in endothelial cell growth kit - VEGF medium (PCS-100-041; ATCC primary cell solutions) at 37 °C in a humidified

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atmosphere containing 5% CO2. The effects of scaffolds on the angiogenic behaviors of HUVECs were investigated by

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an indirect assay using Transwell insert (Corning, 353097).

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51 The migration ability of cells was assessed by means of scratch test. 1 x 105 HUVECs were seeded onto each well of 24

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well plates. After 1 day, attached HUVECs were scratched out using a yellow tip and then followed by placing the

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transwell insert containing each scaffold of different types. After culturing for 24 h, the cells were photographed using

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an inverted light microscope (IX-71, Olympus) and the number of migrated cells toward the scratched area was counted.

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1 A tubular network formation ability of HUVECs was assessed using Matrigel (356234, BD Bioscience) matrix. The

3 Matrigel was coated onto each well of 24 well plates for 1 h at 37°C according to the manufacturer’s specifications. 1 x

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105 cells were seeded onto the Matrigel coated well and then a transwell insert containing scaffold was placed. The cells

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were photographed at 3 and 6 h using an inverted light microscope. Ten random microscopic fields were taken for the

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measurement of the number of branch points (nodes) and mesh-like circles (circle). The analyses were performed using

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Image J software.

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14 2.11. In vivo angiogenesis in rat subcutaneous tissue

16 17 18 The 12-week-old male Sprague-Dawley rats were used in the experiment to evaluate the in vivo tissue compatibility and

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neo-vessel forming ability. The animal experiment was approved by the Institutional Animal Care and Use Committee

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of Dankook University (no. DKU-11-028). Experimental scaffold groups were Den, Den(B), Fib, and Fib(B) and four

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samples per group were implanted. The scaffolds were sterilized using ethylene oxide gas prior to use. A cylinder shape

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of a scaffold (5 mm x 3 mm) was implanted subcutaneously at the mid-spine region of rats. Animals were anesthetized

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by an intramuscular injection of Ketamine HCl (80 mg/kg body weight) and Xylazine (10 mg/kg body weight). The

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skin on the dorsal region was shaved and treated with alcohol and povidone solution. A 2 cm long incision was made

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with a # 10 blade mounted onto a bard-parker scalpel and then four pockets were formed subcutaneously with baby

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metzenbaum scissors. The samples were implanted into the prepared pocket and closed with 4-0 nonabsorbable

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monofilament suture material (Prolene). After 2 and 4 weeks, the animals were sacrificed, and the implanted scaffolds

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and surrounding tissues were extirpated for histological analysis. The harvested tissue samples were immersed in 4 %

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buffered formaldehyde for 24 h at room temperature, treated in a graded series of ethanol, embedded in paraffin,

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sectioned with a microtome, and then stained with hematoxylin and eosin (H&E). Based on the observation under

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optical microscopy, the responses of cells to the scaffolds including cell and tissue invasion (the level of cellular and

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secreted ECM components stained) and the neo-blood vessel formation (noted as the red blood cells collected in a

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circular form tissue) were analyzed and compared. The tissue samples were also immunohistochemically stained with

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CD31 (sc-376764, Santa Cruz) to confirm the newly formed blood vessels.

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2.12. Statistical analysis

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56 The data were averaged and presented as mean ± one standard deviation. A one-way analysis of variance (ANOVA) was

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used to determine the significance of the differences between the groups. The significance was considered at p < 0.05

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1 (*) or p < 0.01(**). In addition, the one-way ANOVA with Bonferroni post hoc test was used for the statistical analysis

3 of multiple comparisons.

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3. Results and Discussion 3 4 5 6

3.1. Scaffolds incorporating bioactive glass nanoparticles are porous and nanofibrous 7 8 9 10 As a bioactive nanocomponent for the scaffolds, BGn, composed of 85% silicon and 15% calcium, were produced by a

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sono-assisted sol-gel process using PEG template for the nanosphere formation under basic conditions. TEM image

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(Figure 1a) of BGn showed a nanosphere form with less than 100 nm in diameter (84.1 nm on average), and a higher

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magnification image revealed a highly mesoporous structure throughout. The N2 adsorption/desorption curve of the

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nanoparticles showed a hysteresis loop, which is considered a type IV isotherm, typically found in mesoporous

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materials23 (Figure 1b). The distribution of meso-pore diameter of BGn showed an average value of 3.8 nm (as shown

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in inset). The -potential value of the BGn was highly negative; -27 mV, due to the presence of a bunch of Si-OH

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groups on the surface35 (Figure 1c).

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41 Figure 1. Characteristics of BGn. (a) TEM images, showing nanospheres with a mesoporous structure. (b) N2 adsorption/desorption curve, and pore size distribution in inset, by BET method. (c) Summary of the BGn properties.

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48 For the preparation of 3D scaffolds incorporating the BGn, we introduced a salt-leaching technique; furthermore, the

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nanofibrous structure was generated by the camphene-assisted phase separation method. First, the BGn were emulsified

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homogeneously in a solvent, which was stable for ~15 min (enough for working time); and the BGn solution was mixed

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with PLA to provide a nanocomposite solution which was stable over days to weeks without a sediment-down of the

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nanoparticles (Figure S1). This fact signifies the possible dispersion of the BGn uniformly within the PLA matrix in the

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nanocomposite scaffolds. The morphology of the scaffolds produced by a salt-leaching process was observed by SEM (Figure 2). All types of scaffolds were macroporous, with pore diameters hundreds of micrometers (mostly in 200-300 11



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1 μm; as pore size distribution shown in Figure S2, measured by mercury immersion method) - a structure considered

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favorable for bone tissue ingrowth16, 36. The porosity of all the scaffolds was almost similar with mean values of 90.0 ~

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91.2%. The surface microstructure of the scaffolds revealed a striking difference between the dense and fibrous groups;

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the ‘Fib’ and ‘Fib(B)’ scaffolds produced with camphene were highly nano-fibrous, which however, was not readily

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observed in ‘Den’ and ‘Den(B)’ scaffolds. The result indicated that camphene played a decisive role in creating the

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nanofibrous network in the scaffolds. A close look at the surface clearly demonstrated the nano-fibrous structure (for Fib

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and Fib(B)) and the presence of BGn on the surface (for Den(B) and Fib(B)). The atomic signals of Si and Ca, which

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assigned to BGn, were well revealed on the EDS mapping and signal peaks (Figure S3a). The FT-IR analysis (Figure

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S3b) showed Si-O-Si band at 465 cm-1 for Den(B) and Fib(B) scaffolds, along with PLDLA-related bands at 1182 cm-1

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(C-O), 1360-1450 cm-1 (C-O-H), and 1752 cm-1 (C=O). These results demonstrate the nanocomposite scaffolds

21

20

incorporated BGn and are highly porous; especially those prepared with camphene are nanofibrous structured, which is

23

2

considered to provide unique physico-chemical properties to the scaffolds. The mechanical properties of the scaffolds

25

24

were then evaluated by applying a compressive load in wet conditions (Figure S4). All groups showed similar

27

26

behaviors, i.e., initial liner elasticity, plateau, and densification, a trend typically observed in sponge type polymeric

29

28

scaffolds37. The mechanical properties obtained based on the stress-strain curves (elastic modulus and stress and strain

31

30

value at densification point) were similar for all groups with only slight differences, i.e., dense scaffolds showed slightly

3

32

higher densification stress than fibrous scaffolds, and BGn-incorporating scaffolds showed slightly higher elastic

35

34

modulus than pure polymer scaffolds.

36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 57

56 Figure 2. Morphologies of the scaffolds observed by SEM (at low and high magnification).

58 59 60

12


Page 13 of 35


2

1

3.2. Nanofibrous bioactive scaffolds present high surface area, water affinity and ionic release 3 5

4 The surface area property of the scaffolds due to nanostructure was examined by the N 2 adsorption/desorption isotherm

7

6

curves (Figure 3a). All the scaffolds showed a clear difference in the adsorption and desorption volume, presenting a

9

8

hysteresis loop-like pattern. Compared to dense surface scaffolds (Den and Den(B)), nanofibrous structured scaffolds

1

10

(Fib and Fib(B)) exhibited larger loop area and higher adsorption/desorption volume level, indicating larger

13

12

nano/micropore structure and higher surface area38-40. The BET measurement of the surface area revealed significantly

15

14

higher values for nanofibrous scaffolds; 12.8 m2/g for Fib(B), 10.2 m2/g for Fib, 3.7 m2/g for Den(B), and 4.2 m2/g for

17

16

Den.

18 20

19 As another experiment to reflect the enhanced surface area related with nanofibrous structure and BGn presence, the

2

21

protein adsorption study was carried out. Cyt C was chosen as a model protein as it is highly positive-charged which is

24

considered to have ionic interactions with the surface of scaffolds 25. The nanofibrous structure or BGn significantly

26

25

23

increased the adsorption of Cyt C to the scaffolds, and the highest level was achieved when both were involved in the

28

scaffolds; 46 g for Fib(B), 19 g for Fib, 18 g for Den(B), and 3 g for Den (Figure 3b). More obvious was noticed

30

29

27

on the protein adsorption than on the gas adsorption as to the enhancement effect of nanofibrous topology and BGn

32

31

presence; this may be due to the ionic nature of the protein (vs. inert N2 gas) and thus the possible specific chemical

34

3

interactions. Interestingly, the role of BGn in increasing the protein adsorption was evident (both dense and fibrous),

36

35

which was not readily noticed in the gas adsorption. The more charged characteristic of BGn (due to a bunch of silanol

38

37

groups) may attribute to this, and this finding implies the possible role of BGn on the surface of scaffolds in the

40

39

biological interactions with proteins.

41 42 43

Next the water affinity of the scaffolds was examined by the contact angle test (Figure 3c). The contact angle was 73.5°

45

4

for Den > 67.1° for Fib > 65.8° for Den(B) > 61.5° for Fib(B), demonstrating the nanofibrous structure and BGn

47

46

component played a key role in enhancing hydrophilicity of scaffolds. Previous studies have reported the water contact

49

48

angle reduced to ~60-65° could provide favorable surface conditions for cell adhesion41, 42. In fact, the water uptake

51

50

capacity of the scaffolds when measured at 24 h of soaking in water also showed significantly increased water soaking

53

52

to the nanofibrous and BGn-incorporated scaffold (data not shown here). The results demonstrate that the nanofibrous

5

54

structured scaffolds have greatly enhanced surface area and hydrophilicity, and the incorporated BGn increased further

57

56

the water affinity, suggesting the Fib(B) scaffolds can favor the water diffusion and possibly hydrolytic degradation and

60

59

58

the related ionic releases.

13



Page 14 of 35

1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 28

27 Figure 3. Nano-structure and hydro-affinity properties of scaffolds: (a) N2 adsorption/desorption isotherm by BET

30

29

measurement. The measured surface areas are noted in the graph. (b) Protein (Cyt C) loading amount onto scaffolds. (c)

32

31

Hydrophilicity measured by a water contact angle. Contact angle recorded at 20 s using samples prepared in a

34

3

membrane type.

35 36 37 38 39 41

40

43

42

The release of ions (Ca and Si) from the scaffolds incorporating BGn was measured in Tris-buffer (pH 7.4, 37oC) for up to 28 days. The cumulative amount of released ions was analyzed by ICP-AES (Figure 4). The Si ion was released

45

4

gradually with time for up to 28 days, and the higher amount was recorded for Fib(B) than for Den(B) through the

47

46

period; the total amount released up to 28 days was approximately 0.88 mM (24.64 ppm) and 1.08 mM (30.24 ppm) for

49

48

Den(B) and Fib(B), respectively. The Ca ion release was also observed to follow a similar pattern to Si ion, but the Ca

51

50 ion released amount was higher than the Si ion through the period; the total amount released up to 28 days was

53

52 approximately 2.15 mM (85 ppm) and 2.50 mM (100 ppm) for Den(B) and Fib(B), respectively. This ionic release

54 behavior has been well recognized in other bioactive glasses including 60% SiO2 / 36% CaO / 4 % P2O5 (mol %), 75%

5 56

SiO2 / 25% CaO (mol %), and 66% SiO2 / 27% CaO / 7% P2O5 (mol %) composition particles26, 43, 44, where cations

57 59

58 (like Ca) released more progressively than Si that is a glass network former. The ion release was then converted to daily

60 release amount (as shown below the graphs). In case of Fib(B), Ca ion was released ~220 M daily up to 7 days, and 14


Page 15 of 35


1 then ~90 M up to 28 days; Si ion was released ~90 M daily up to 7 days, and then ~40 M up to 28 days. The release

2 4

3 rate of those ions has been reported to stimulate cellular responses, including the proliferation of osteoblasts and MSCs,

5 and their subsequent osteogenic differentiation, and the angiogenesis of endothelial cells31, 45, 46. Specifically, silicon

6 7

ions were widely reported to induce both osteogenesis and angiogenesis process47-49. Therefore, the BGn-incorporating

8 10

9 scaffolds are considered to have therapeutic potential attributed to the Si and Ca ionic releases.

1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 Figure 4. Ion release (silicon and calcium ions) from the scaffolds, due to the presence of BGn, as measured by ICPAES. Ions released for each time summarized as a table below.

37

36 38 39 40 41 42

Collectively, the Fib(B) scaffolds, developed with nanofibrous structure and to incorporate BGn, have enhanced surface

43 4

area, protein adsorption, water affinity, and the capacity to release therapeutic ions, and these assets suggest the possible

45 46

stimulation of favorable responses of cells, such as cell adhesion, osteogenesis and angiogenesis.

47 48 49 50 51 53

52

3.3. Nanostructured bioactive scaffolds stimulate MSCs adhesion and growth 54 5 57

56 MSCs derived from rat bone marrow were cultured on the nanofibrous bioactive scaffolds, and the initial adhesion

60

59

58

behaviors were investigated. The morphology of cells attached to the scaffolds at 4 h was observed by confocal microscopy (Figure 5a). Compare to the dense scaffolds, the nanofibrous scaffolds (both w/ and w/o BGn) showed 15



Page 16 of 35

2

1 larger number of cells with better cytoskeletal extensions. Based on the images, the cell number and the spreading area

3 were quantified (Figure 5b). The nanofibrous scaffolds (Fib and Fib(B)) showed significant higher number of cells

4 5

(19.6 and 26.5 cell number per field) than the dense-structured scaffolds (14.1 and 16.4 cells per field). Moreover, the

6 7

cell spreading area was significantly larger on the nanofibrous scaffolds than on the dense ones. Interestingly, the BGn

9

8

incorporation increased the cell spreading area further. As a result, the area is in the order; 987.9 μm2 for Fib(B) > 653.0

1

10

μm2 for Fib > 358.6 μm2 for Den(B) > 156.1 μm2 for Den. The results evidenced the nanofibrous structure could

12 13

enhance initial cell anchorage and the subsequent spreading; as well, the cells adhered were stimulated to spread by the

14 15

BGn incorporated. A closer examination showed more significant influence on the spreading process than on the

16 17

anchorage by the nanostructure and BGn composition.

18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5

60

59

58

57

56 Figure 5. MSC adhesion behaviors to the scaffolds. (a) CLSM images showing adhered cells onto the scaffolds for 4 h (DAPI for nuclei in blue and Phalloidin for F-actin in red). (b) Cell number (blue bars) and spreading area (red dotted line) quantified from the images. (c) Cell adhesion assayed by CCK method. (d) Western blot analysis of p-FAK, FAK, and integrin 1. Band intensities normalized to -actin intensity. Significant difference noted between groups (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 3). 16


Page 17 of 35


1 2 4

3 The viability of cells at this initial time was also examined by CCK assay (Figure 5c). Cells showed significantly

6

5

higher viability on the nanofibrous structure than on the dense structure, a similar result to the cell number. The

8

7

expression of key molecules involved in the adhesion event, including FAK (also phosphorylate form pFAK) and

10

9

integrin β1 was then analyzed, by Western blotting (Figure 5d). FAK is known to play a pivotal role in intracellular

12

1

adhesion signaling of MSCs, and integrin subset β1 (mainly in α5β1) is one of the major receptors for MSCs perception

14

13

of a surrounding matrix50. The band intensities of all the molecules were expressed higher in the cells when cultured on

16

15

the scaffolds with nanofibrous structure and/or BGn. The quantified data normalized to β-actin revealed the expression

18

17

more clearly, in the descending order of Fib(B), Fib, Den(B), and (Den). In particular, the difference between groups

20

19

was clear in pFAK and β-actin expression while only a slight difference was noticed in FAK; the stimulation by the

2

21

Fib(B) was as high as 11 (β-actin) and 4 (pFAK) times of that by the Den. The nanofibrous topology implemented

24

23

herein is considered to enhance the adhesion events of MSCs through a mechanism similar to the surfaces reported

25 previously, although those studies used 2D substrates instead of 3D scaffolds51, 52. Compared to the topological effects,

26 27

the ionic role in cell adhesion is relatively much less known. Some metallic cations (Ca2+, Mn2+, and Mg2+) were

28 29

reported to stimulate the expression of α5β1 integrin in MSCs, implying the role of ions in cellular adhesion53-56.

30 32

31 Furthermore, the bioactive glass added scaffolds were found to have higher cell adhesion; although the mechanism of

3 the enhancement was not elucidated50, those studies imply the possible role of ions released on the cell adhesion.

34 35 37

36 Along with the adhesion, the proliferation and migration of cells - events necessary to achieve high population and

39

38

uniform engraftment of cells through the scaffolds - are thus important for the MSC/scaffold-based bone tissue

41

40

43

42

engineering53, 57, 58. Cells grew actively on the scaffolds either nanofibrous- or dense-surfaced, or incorporating BGn or not, with time up to 14 days. In particular, Fib(B) scaffolds promoted the cell proliferation more significantly (Figure

45

4

6a). The cell penetration was observed by the z-stack fluorescence images at days 7 and 14 (Figure 6b). When

47

46

quantified, the BGn-containing scaffolds showed significantly higher penetration depth of ~450 μm for Fib(b) and ~350

49

48 μm for Den(B) at day 14, as compared to those w/o BGn (~150-200 μm) (Figure 6c). The more viable and highly

51

50 proliferating cells particularly on the Fib(B) can penetrate better through the pore channels; the BGn embedded

53

52 nanofibrous networks of Fib(B) are considered to provide more effective substrate for filopodial development due to

5

54 both BGn and nanofibrous effects

34, 59-61

, enabling cells to cross and migrate quickly. The nanosized fiber and BGn are

57

56 thus considered to provide a sort of nano-recognition sites for filopodial processes. This event observed herein suggests

59

58 the Fib(B) scaffolds can prime the stem cell engraftment, enabling cellular multiplication and distribution

60 homogeneously. 17



Page 18 of 35

1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 28

27 Figure 6. MSC proliferation and migration (penetration) within scaffolds for up to 14 days. (a) Cell proliferation by CCK assay. (b) Cell penetration analyzed by means of confocal z-stacking as illustrated, and (c) the penetration depth quantified. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 3).

31

30

29

32 3 34 35 37

36

3.4. Nanostructured bioactive scaffolds accelerate osteogenesis and maturation of MSCs 38 39 41

40 Next, the osteogenic differentiation of MSCs cultured on the scaffolds was assessed at both gene and protein levels. The

43

42

expression of bone-associated genes, including ALP, OPN, BSP, and OCN, was quantitatively analyzed by RT-PCR at

45

4 days 7 and 14 (Figure 7a). Among other groups, Fib(B) showed the highest level for all genes particularly at day 7; the

47

46 stimulation was as high as 5.3-, 3.2-, 2.4-, and 1.8-fold, for ALP, OPN, BSP, and OCN, respectively, with respect to

49

48 other groups. The secretion of osteogenic proteins (OPN and BSP chosen representatively) was confirmed qualitatively

51

50 by immunocytochemistry at days 7 and 14 (Figure 7b). For OPN, presence of BGn (Den(B) and Fib(B)) enabled the

53

52 secretion of higher protein levels, and the nanofibrous structure (Fib and Fib(B)) stimulated the secretion further. Day

5

54 14 showed more significant stimulation than day 7. The secretion of BSP behaved in a similar trend to that of OPN,

57

56 with regard to the scaffold type and culture time; Den < Fib < Den(B) < Fib(B).

58 59 60

18


Page 19 of 35


1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 48

47 Figure 7. MSC osteogenesis within the scaffolds. (a) Expression of osteogenic-related genes, including OCN, OPN, BSP, and ALP, by quantitative PCR at 7 and 14 days. (b) OPN and BSP protein secretion assayed by immunocytochemistry at 7 and 14 days. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 3).

51

50

49

52 53 54 56

5 Finally, the maturation of osteogenesis was assessed by the cellular mineralization. Cell cultured scaffolds were stained

58

57

with ARS at days 21 and 28 and then optically visualized (Figure 8a). Although the ARS also stained somewhat pure

60

59

scaffolds in pale red (shown for comparison), the mineralized cells were shown to be stained completely different (in 19



Page 20 of 35

2

1 dark red). Apparently, Den(B) and Fib(B) scaffolds revealed dark red stains and the stain density was enhanced with

3 increasing culture time. The stains were then eluted to quantitatively compare the groups; the scaffolds incorporating

4 5

BGn significantly enhanced the cellular mineralization at both periods (Figure 8a).

6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 27

26 Figure 8. MSC mineralization within the scaffolds, measured by the calcium deposition level during culture for 21 and 28 days using alizarin red S (ARS) staining: (a) Photograph of cell-scaffolds after ARS staining, and (b) the quantification after eluting the ARS stain. The results normalized to scaffold only (w/o cell) for each group. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 3).

31

30

29

28

32 3 35

34 Collectively, the osteogenesis and maturation of MSCs were proven to be highly dependent on the type of scaffolds, as

37

36

analyzed by the expression of bone-related genes and proteins and the calcium mineralized levels. Clearly, the

39

38

nanofibrous and BGn-incorporating scaffolds stimulated progressively MSCs to switch into osteoblastic cells and their

41

40

subsequent calcification. This was well demonstrated in time-sequenced processes of cells that experience substantial

43

42

osteogenesis, i.e., mRNA levels at 7 days, protein secretion at 14 days, and calcium deposits at 21-28 days. While the

45

4

nanofibrous structure appeared to play positive roles in enhancing osteogenesis, this was particularly obvious when

47

46

BGn were involved in, which implies their synergistic roles. Furthermore, the effects of BGn were shown to be more

49

48

substantial at later mineralization stage, when the culture periods were 3-4 weeks. The release of ions from BGn, such

51

50

as Ca and Si, continues at this culture period (based on Figure 4), providing cells enough ionic source for the

53

52 accelerated mineralization. The released amounts of Ca and Si ions, as deduced from ICP analysis, indeed position at

54 the therapeutic window that can stimulate the osteogenesis of stem/progenitor cells62, 63. While Ca ions can be a direct

5 57

56 source for the formation of calcium phosphate, Si ions are known to speed up the mineral formation or to stabilize the

58 mineralized products64, 65, which is particularly meaningful in the in vivo bone regenerating conditions to facilitate

59 60

accelerated bone formation with a high quality, and further studies remain to confirm the in vivo efficacy. 20


Page 21 of 35


1 2 3 4 5

3.5. Nanostructured bioactive scaffolds stimulate endothelial cell functions and in vivo angiogenesis 6 7 8 9 The biological effects of nanofibrous BGn-incorporated scaffolds were then investigated in the angiogenic events.

10 1

Endothelial cells (HUVECs) were cultured indirectly with the influence of the scaffolds, and the cellular migration and

12 13

the tubular networking on a Matrigel - well-known angiogenesis assays of endothelial cells - were examined. For 24 h,

15

14

the cells migrated toward a scratched area more actively by the BGn-containing scaffolds (Den(B), and Fib(B)) than

17

16

those w/o BGn (Den, and Fib) (Figure 9a). A quantification of the migrated cells showed clearly the significant

19

18

difference between the groups with and w/o BGn; 225 ± 27.5 for Fib(B) ≈ 191 ± 31.8 for Den(B) > 147.2 ± 21.1 for Fib

20 21

≈ 138.6 ± 13.9 for Den (Figure 9b). The endothelial cell migration, initiating by the polarization of cells, is a

2 23

prerequisite of the blood vessel formation where the migrated endothelial cells form tubular networks and mature into

25

24

vasculatures66. The endothelial tubular networks formed on Matrigel were further assayed. The optical images taken at 3

26 27

and 6 h showed a more profound vessel-like networking in the BGn-containing scaffold groups (Den(B) & Fib(B)) than

28 29

those w/o BGn (Figure 9c). Some key parameters of tubular networking, including circle and node number were

30 31

measured to estimate the angiogenesis ability quantitatively. The BGn-containing scaffold groups presented

3

32

significantly higher circle and node number than those w/o BGn at both periods (Figure 9d). The results demonstrate

35

34

that the indirect cue of BGn (ionic release) from the scaffolds plays a critical role in stimulating in vitro endothelial

37

36

functions in angiogenesis, such as cellular migration and tubular network formation.

38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

21



Page 22 of 35

1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 46

45 Figure 9. Endothelial cell (HUVEC) responses to the scaffolds. (a) Cell migration assay by a scratch test using a transwell membrane at 24 h, and (b) the quantification of cells migrated toward the scratched area (n = 7). (c-e) Tubular networking assay at 3 and 6 h, showing the tubule-like network formation of cells supported on a Matrigel (c), and the quantification of circle number (d), and node number (e); data counted from 10 random fields. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 10).

51

50

49

48

47

52 53 5

54 Next, the scaffolds were implanted in rat subcutaneous tissues and the tissue responses and in vivo blood vessel

57

56

formation were analyzed. H&E stain images acquired at 2 and 4 weeks showed the in vivo cell and tissue reactions

60

59

58

within the different types of scaffolds (Figure 10a). All the scaffolds showed excellent tissue compatibility, showing a very limited number of inflammatory cells but a major population of tissue-forming cells like fibroblasts and 22


Page 23 of 35


2

1 endothelial cells throughout the scaffolds. A clear difference between groups was noted in the tissue/cell invaded areas.

3 The tissue invasion of the fibrous scaffold groups (Fib & Fib(B)) was remarkably rapid, showing almost 60.4 - 70.3%

4 5

of invaded area as early as within 2 weeks, which being in contrast to that of dense counterparts (around 25.0 - 28.8%)

6 7

(Figure 10b). After 4 weeks the tissue invasion became substantially enhanced; the most significant increase was

9

8

noticed for ‘Den(B)’ group (~74%), and almost ~87% area was invaded with tissues for ‘Fib’ and ‘Fib(B)’ groups.

1

10

However, ‘Den’ group still showed very limited tissue invasion (~32%), with little improvement from the level

12 13

observed at 2 weeks. The results support that the nanofibrous structure and BGn component help the cell migration and

14 15

tissue formation. Several reasons are possibly attributed to this. The enhanced hydro-affinity of scaffolds (as shown in

16 17

Figure 3) may increase the conductance of biological proteins and cells (as proven by the enhanced cellular migration

19

18

within scaffolds in Figure 6). Also the provided cues (particularly BGn) may play a chemoattractant role in homing cells

21

20

through the release of Si and Ca ions (as demonstrated by the indirect effect on endothelial migration in Figure 9).

23

2

Although the degradation of scaffolds can be issued to affect the calculation of the cell/tissue invaded area, this might

25

24

be minimal because the scaffold part takes only ~10% (as the porosity levels ~90% for all groups). Conclusively, the

27

26

tissue invasion result is considered to reflect the capacity of scaffolds to facilitate in vivo cell engraftment and tissue

29

28

integration.

30 31 32

Another notable finding in the histological images is the formation of neo-blood vessels (Figure 10c). A closer

3 34

examination of the newly formed tissue areas revealed dark red staining of aggregated red blood cells enclosed by

35 36

circular tubule-like tissues (as indicated with arrows), a typical feature of blood vessels found in H&E stained tissue

38

37

samples67. To further confirm the neo-blood vessel formation in the scaffolds, CD31 immunohistochemical staining was

40

39

also carried out68. As shown in the representative images (Figure 10d) the blood vessel densities and distributions were

42

41

similar to those observed in the H&E staining images. The number of neo-blood vessels quantified from H&E stains

4

43

showed significant differences between groups (Figure 10e). While the nanofibrous scaffolds (Fib & Fib(B)) formed

46

45

more blood vessels at an early phase (2 weeks), the dense scaffold incorporating BGn (Den(B)) also reached a level

48

47

similar to those nanofibrous at 4 weeks, and among all groups the nanofibrous and BGn-containing scaffolds showed

50

49

the most rapid formation and the highest number of neo-blood vessels. The in vivo blood vessel formation in

52

51

subcutaneous tissue supports the in vitro role of BGn in stimulating endothelial functions (migration and tubular

54

53

networking); furthermore, the in vivo finding also underscores the effects of nanofibrous morphology on the

56

5

angiogenesis. As discussed in the in vitro angiogenic effects of BGn, the ions (particularly Si) released should also play

58

57

a similar role in the in vivo blood vessel formation. As to the nanofibrous structure however very limited works have

60

59

been found. One recent study has reported that the nanofibrous structure of titanium oxide surface (on titanium metal) 23



Page 24 of 35

2

1 had positive effects on the in vitro capillary-like tube formation of endothelial cells, including enhanced adhesion,

3 proliferation, tubular formation, and expression of angiogenic factors of HUVECs with respect to dense surface,

5

4

although the mechanism on this was not clear69. Here, we also found the nanofibrous structured surface of scaffolds

6 7

accelerated the blood vessel formation in vivo, and notable was the early angiogenic stimulation at 2 weeks of

8 9

implantation. It is possible that the nanofibrous morphology might enhance the in vivo cellular (endothelial and/or

10 1

progenitor cells) migration and tubule network formation through the pore channels; although the phenomenon could

12 13

not be supported in vitro using endothelial cells here due to the limitation of HUVEC cultures, the stimulating role of

14 15

the nanofibrous structure in cellular migration is envisaged (based on the MSCs studied herein and other cells reported

17

16

elsewhere70), and more studies on this are thus considered to remain in the future.

18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

24


Page 25 of 35


2

1 Figure 10. In vivo tissue responses to the scaffolds, examined by implantation in rat subcutaneous tissue for 2 and 4 weeks: (a) H&E staining images, which used to measure (b) the cell and tissue invasion to the scaffolds by quantifying the stained cell/tissue area %, and (c) enlarged images to show neo-blood vessels. (d) Immunohistochemical staining images of CD31, a marker for neo-blood vessels (red). (e) Number of blood vessels by quantifying the blood cell site/tissue area. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 4).

7

6

5

4

3

8 9 1

10

4. Concluding remarks 12 13 15

14 As witnessed, the BGn/nanofibrous scaffolds demonstrated excellent osteogenesis and angiogenesis of cells. The series

17

16 of biological events explicit in the scaffolds in relation with the physico-chemical properties are schematically

18 illustrated in Figure 11. The two components implemented in 3D macroporous scaffold – nanofibrous topology and

19 20

BGn – represent biophysical and biochemical signals, respectively. On the one hand, the nanofibrous surface provides

21 23

2 ultrahigh surface area, leading to protein adsorption and rapid cell anchorage and spreading, which ultimately help the

25

24 osteogenesis of MSCs. The nanofibrous structure also accelerates the cell and tissue invasion and the neo-blood vessel

27

26 formation. On the other hand, the ion-releasing bioactive nanoparticles not only offer hydrophilic nano-sites initially for

28 more biological interactions but also deliver Si and Ca ions stably. The biological effects are multi-faceted; stimulating

29 30

MSCs adhesion and osteogenesis as well as activating endothelial cell functions such as cell migration, tubular

31 32

networking, and neo-blood vessel formation. Those events were shown to occur mostly in a synergistic manner through

34

3

both cues. The findings observed in the osteogenesis and angiogenesis – two key events in bone regeneration – support

35 36

the developed BGn+nanofibrous scaffolds may be potentially useful in bone tissue engineering.

37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

25



Page 26 of 35

2

1 Figure 11. Schematic illustration of the currently developed 3D scaffolds with BGn biochemical and nanofibrous topological (biophysical) cues that can synergistically action in angiogenesis and osteogenesis of cells, eventually helpful for bone formation.

5

4

3

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

26


Page 27 of 35


2

1

Supporting information 3 5

4 Summary of the ratio of components used to prepare scaffolds; Summary of primer sequences used for quantitative RT-

7

6

PCR; Images of BGn and BGn-PLA solutions; Pore size distribution, SEM-EDX atomic mapping, and FT-IR spectra of

9

8

scaffolds; Mechanical properties of the scaffolds.

10 1 12 13 15

14

Funding sources 16 18

17 Global Research Laboratory (GRL) Program (Grant no. 2015032163), Priority Research Centers Program (Grant no.

20

19

2009-0093829), National Research Foundation (NRF), Republic of Korea.

21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

27



Page 28 of 35

2

1

Figure Captions 3 5

4 Figure 1. Characteristics of BGn. (a) TEM images, showing nanospheres with a mesoporous structure. (b) N2

7

6

adsorption/desorption curve, and pore size distribution in inset, by BET method. (c) Summary of the BGn properties.

8 9 1

10 Figure 2. Morphologies of the scaffolds observed by SEM (at low and high magnification).

12 14

13 Figure 3. Nano-structure and hydro-affinity properties of scaffolds: (a) N2 adsorption/desorption isotherm by BET

16

15

measurement. The measured surface areas are noted in the graph. (b) Protein (Cyt C) loading amount onto scaffolds. (c)

18

17

Hydrophilicity measured by a water contact angle. Contact angle recorded at 20 s using samples prepared in a

20

19

membrane type.

21 23

2 Figure 4. Ion release (silicon and calcium ions) from the scaffolds, due to the presence of BGn, as measured by ICP-

25

24

AES. Ions released for each time summarized as a table below.

26 27 28

Figure 5. MSC adhesion behaviors to the scaffolds. (a) CLSM images showing adhered cells onto the scaffolds for 4 h

30

29

(DAPI for nuclei in blue and Phalloidin for F-actin in red). (b) Cell number (blue bars) and spreading area (red dotted

32

31

line) quantified from the images. (c) Cell adhesion assayed by CCK method. (d) Western blot analysis of p-FAK, FAK,

34

3

and integrin 1. Band intensities normalized to -actin intensity. Significant difference noted between groups (*p < 0.05

35 36

& **p < 0.01, by one-way ANOVA, n = 3).

37 38 40

39 Figure 6. MSC proliferation and migration (penetration) within scaffolds for up to 14 days. (a) Cell proliferation by

42

41 CCK assay. (b) Cell penetration analyzed by means of confocal z-stacking as illustrated, and (c) the penetration depth

4

43 quantified. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 3).

45 47

46 Figure 7. MSC osteogenesis within the scaffolds. (a) Expression of osteogenic-related genes, including OCN, OPN,

49

48

BSP, and ALP, by quantitative PCR at 7 and 14 days. (b) OPN and BSP protein secretion assayed by

51

50

immunocytochemistry at 7 and 14 days. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 3).

52 54

53 Figure 8. MSC mineralization within the scaffolds, measured by the calcium deposition level during culture for 21 and

56

5

28 days using alizarin red S (ARS) staining: (a) Photograph of cell-scaffolds after ARS staining, and (b) the

58

57

quantification after eluting the ARS stain. The results normalized to scaffold only (w/o cell) for each group. (*p < 0.05

60

59

& **p < 0.01, by one-way ANOVA, n = 3). 28


Page 29 of 35


2

1 Figure 9. Endothelial cell (HUVEC) responses to the scaffolds. (a) Cell migration assay by a scratch test using a

3 transwell membrane at 24 h, and (b) the quantification of cells migrated toward the scratched area (n = 7). (c-e) Tubular

4 5

networking assay at 3 and 6 h, showing the tubule-like network formation of cells supported on a Matrigel (c), and the

6 7

quantification of circle number (d), and node number (e); data counted from 10 random fields. (*p < 0.05 & **p < 0.01,

8 9

by one-way ANOVA, n = 10).

10 1 13

12 Figure 10. In vivo tissue responses to the scaffolds, examined by implantation in rat subcutaneous tissue for 2 and 4

15

14 weeks: (a) H&E staining images, which used to measure (b) the cell and tissue invasion to the scaffolds by quantifying

17

16 the stained cell/tissue area %, and (c) enlarged images to show neo-blood vessels. (d) Immunohistochemical staining

19

18 images of CD31, a marker for neo-blood vessels (red). (e) Number of blood vessels by quantifying the blood cell

21

20 site/tissue area. (*p < 0.05 & **p < 0.01, by one-way ANOVA, n = 4).

2 24

23 Figure 11. Schematic illustration of the currently developed 3D scaffolds with BGn biochemical and nanofibrous

26

25

topological (biophysical) cues that can synergistically action in angiogenesis and osteogenesis of cells, eventually

28

27

helpful for bone formation.

29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

29



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Synergetic Cues of Bioactive Nanoparticles and Nanofibrous Structure

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