Article ECM Decorated Electrospun Nanofiber for Improving Tissue Regeneration

Yong Fu 1,*, Lili Liu 2, Ruoyu Cheng 2 and Wenguo Cui 2,3,* ID 1 Department of ENT and Head & Neck , The Children’s Hospital Zhejiang University School of Medicine, 3333 Bingsheng Road, Hangzhou 310051, China 2 Orthopedic Institute, Soochow University, 708 Remin Road, Suzhou 215006, China; [email protected] (L.L.); [email protected] (R.C.) 3 Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, China * Correspondence: [email protected] (Y.F.); [email protected] (W.C.); Tel.: +86-571-8667-0860 (Y.F.)

Received: 25 January 2018; Accepted: 26 February 2018; Published: 6 March 2018

Abstract: Optimization of nanofiber surface properties can lead to enhanced tissue regeneration outcomes in the context of bone tissue engineering. Herein, we developed a facile strategy to decorate elctrospun nanofibers using (ECM) in order to improve their performance for bone tissue engineering. Electrospun PLLA nanofibers (PLLA NF) were seeded with MC3T3-E1 cells and allowed to grow for two weeks in order to harvest a layer of ECM on nanofiber surface. After decellularization, we found that ECM was successfully preserved on nanofiber surface while maintaining the nanostructure of electrospun fibers. ECM decorated on PLLA NF is biologically active, as evidenced by its ability to enhance mouse bone marrow stromal cells (mBMSCs) adhesion, support cell proliferation and promote early stage osteogenic differentiation of mBMSCs. Compared to PLLA NF without ECM, mBMSCs grown on ECM/PLLA NF exhibited a healthier morphology, faster proliferation profile, and more robust osteogenic differentiation. Therefore, our study suggests that ECM decoration on electrospun nanofibers could serve as an efficient approach to improving their performance for bone tissue engineering.

Keywords: elctrospun nanofibers; extracellular matrix; biologically active; osteogenic differentiation; scaffolds

1. Introduction Large-sized bone defects cannot heal by themselves, due to the limited regenerative capability of bone tissue; therefore, a variety of bone regeneration strategies have been developed in the past 30 years to address this critical clinical problem [1–3]. Among these approaches, bone tissue engineering, which uses a combination of scaffolds, cells, and growth factors to regenerate new bone, still holds the promise of fully solving this important matter [4,5]. As one of three key components for bone tissue engineering, scaffolds play a pivotal role in successfully regenerating bone. Researchers have developed various approaches to fabricating scaffolds for bone regeneration, such as salt-leaching, freeze-drying, gas foaming, phase separation, and sintering, etc. [6–11]. For instance, Ma et al. developed a series of scaffolds with controllable pore size, porosity and other parameters using a phase separation method [6]. Bone tissue is generally considered to be a nanomaterial with nanoscale fibers and organized into a hybrid structure [12,13]. However, most of these methods are capable of making scaffolds with micrometer features and fail to mimic the natural structure of bone. is a facile and versatile method for fabricating fibrous scaffolds from a rich variety of materials [14–16]. Electrospun scaffolds possess very high surface-to-volume ratio, pore sizes ranging from several to tens of micrometers, and adjustable porosities of up to more

Polymers 2018, 10, 272; doi:10.3390/polym10030272 www.mdpi.com/journal/polymers Polymers 2018, 10, 272 2 of 12 than 90% [17]. Therefore, electrospun fibrous scaffolds have been successfully applied in neural, skin, cardiovascular, heart and bone tissue engineering [18–21]. In in the context of bone tissue engineering, electrospinning can be easily leveraged to make nanofibers similar to those of collagen fiber in , thus precisely emulating the natural structure of bone nanostructure [22–24]. Poly (L-lactic acid) (PLLA) is an FDA-approved that possesses excellent biocompatibility and biodegradability; therefore, it has been extensively used for bone repair [25,26]. For instance, PLLA has been successfully electrospun into nanofibers to mimic the nanostructure of collagen fibers in natural bone and was able to promote bone formation [27]. One of the advantages of using nanofibrous materials is that they can effectively interact with stem cells and stimulate autocrine/paracrine growth factor signaling pathways [28]. A study led by Laurencin showed that delivery of rat MSC along with biomimetic electrospun matrices enhanced ligament repair by showing increased mechanical strength and improved tissue organization [29]. However, structural similarity does not provide all properties needed for bone regeneration. For instance, the surface properties of the electrospun nanofibers also play a crucial role in their performance once implanted in a bony environment [30]. Unfortunately, the surface properties of most electrospun nanofibers cannot meet the requirement for optimal bone regenerative performance. These materials are usually made of natural or synthetic polymers, which are not capable of directing cellular differentiation down to the bone lineage [19,31]. Therefore, a surface modification strategy combined with electrospun nanofiber scaffolds is highly desirable in bone tissue engineering. Herein, we report an extracellular matrix (ECM) based surface modification approach for electrospun nanofibers to substantially improve their performance during bone repair and regeneration. ECM is composed of structural and functional molecules secreted by the resident cells of each tissue; therefore, ECMs are tissue specific, suggesting ECM secreted by cells from different tissues might have unique functions [32–34]. Due to these desirable properties of ECM, various medical devices, such as wound dressings and hernia patches, have been developed and have demonstrated satisfactory clinical outcomes [35]. Thus, we hypothesized that by modifying the material surface with a specific type of ECM, the cell behavior on the surface may be tailored by controlling the presentation of ECM deposition. In this study, in order to enhance the osteogenic differentiation of /osteoprogenitor cells on electrospun nanofiber, we used MC3T3-E1 cells as a tool to deposit osteogenic ECM on electrospun PLLA surface by culturing the cells on PLLA nanofiber followed by a decellularization process to remove the cellular components. ECM decoration on PLLA nanofiber significantly enhanced cell attachment, as well as cell growth and osteogenic differentiation of mouse bone marrow stromal cells (mBMSCs). This work should set up an example of using ECM as a surface modification approach to obtain desirable biological properties in electrospun materials.

2. Experimental

2.1. Electrospinning

To prepare a PLLA nanofibrous scaffold, PLLA (Mn = 60.145 kDa, MWD = 1.64) pellets were dissolved in dichloromethane (DCM)/N,N-dimethylformamide (DMF) (50/50 vol %) solvent system at 12 wt %. The PLLA solution was fed into a plastic syringe connected to a stainless steel capillary tube at a feeding rate of 2.0 mL/h, controlled by a micro infusion pump (WZ-50C2, Zhejiang University Medical Instrument Co., Hangzhou, China). A positive DC high-voltage power supply (DW-P303-1AC, Tianjin Dongwen High-Voltage Power Supply Factory, Tianjin, China) was connected to the capillary tube to generate a high electric field of 2.0 Kv/cm between the capillary tube and the grounded collector, extending for a distance of 10 cm. A flat plate wrapped in aluminum foil was used as the collector to spin scaffolds with a random fibrous structure. The electrospun scaffolds were dried under vacuum at room temperature for 72 h. The morphology of the electrospun scaffolds was observed using field emission scanning electron microscopy (Zeiss DSM 982, FESEM, Oberkochen, Germany) at 3 keV. Polymers 2018, 10, 272 3 of 12

2.2. Cell Culture MC3T3-E1 cells were cultured in alpha minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) (Corning, New York, NY, USA) and 1% pen-strep (Corning, New York, ◦ NY, USA). Cells were grown in a humidified atmosphere of 5% CO2 at 37 C. The culture medium was changed every other day. An osteogenic medium, consisting of α-MEM plus 10 mM β-glycerol phosphate and 50 mg/mL-ascorbic acid (Sigma, St. Louis, MO, USA), was used for osteogenic differentiation of the cells. Mouse bone marrow stromal cells (mBMSCs) were derived from 6–8-week-old mice. The study was conducted in accordance with relevant national legislation on the use of animals for research and the protocol was approved by the Ethics Committee of Zhejiang University Laboratory Animal Center. Briefly, mice were euthanized using CO2 asphyxiation. Femurs and tibias were dissected from surrounding tissues. Bone marrow was harvested by flushing bones with α-MEM medium containing 10% FBS using a 25-G needle. The cells were filtered through a 70 µm cell strainer and plated at a density of 1 × 108 cells in 100 mm culture dish. Half of the culture medium was replaced by fresh medium to remove the non-adherent cells at day 4. The cells were allowed to grow for 7–8 days in α-MEM containing 10% FBS and then passaged. mBMSCs were used at passage 2 for all the experiments performed in this study.

2.3. ECM Decoration on Electrospun Nanofibers PLLA nanofibrous (PLLA NF) was cut into 8.0 mm circular samples using a biopsy punch with a diameter of 8.0 mm. All the samples were then sterilized using ethylene oxide gas before cell culture. Sterile PLLA NF samples were glued to the bottom of a 24-well plate using pharma-grade Vaseline to prevent floating and soaked in cell culture medium for 12 h. After that, MC3T3-E1 cells were seeded on PLLA NF at 5 × 104 cells/cm2. MC3T3-E1 cells were allowed to grow on PLLA NF for 2 weeks with osteogenic medium described previously. The culture was terminated after 2 weeks by washing the samples with PBS once and all the samples were fixed in 4% paraformaldehyde and buffered with PBS for 30 min. After that, all samples were collected in conical tubes and frozen in a −20 ◦C freezer overnight and then thawed in a 37 ◦C water bath. To remove all the cellular components of MC3T3-E1 cells, the cells were treated with 1% SDS (Life Technology, Waltham, MA, USA) in deionized water for 30 min (Figure S1). The decellularized samples were washed with PBS extensively to remove any detergent residue and cellular components. The obtained ECM decorated PLLA NF (ECM/PLLG NF) were kept in PBS at 4 ◦C for further use.

2.4. Cell Attachment and Morphology mBMSCs were seeded on both PLLA NF and ECM/PLLA NF in a 24-well plate at a seeding density of 2 × 104 cells/cm2 in 1.0 mL medium (n = 10). The cells were allowed to adhere to the test materials for 2 h. After that, all the cell culture medium was slowly aspirated, and all the samples were washed gently with PBS twice. Half of the samples were then fixed in 2.5% glutaraldehyde for 1 h. Then they were incubated in 0.1 mol/L sodium cacodylate buffer for 1 h. The specimens with fixed cells were dehydrated in graded ethanol series and critical point dried. All specimens were then sputter coated with gold–palladium. The cell morphology on different substrates was observed using FESEM (JEOL 6335F, Akishima, Japan) at an acceleration voltage of 2 keV. The other half samples were then washed by PBS three times to remove the unattached and loosely bound cells from material surfaces. CellTiter-Blue® assay was used to measure the density of cells left on the samples. 0.5 mL fresh medium containing 10% CellTiter-Blue® dye (Promega, Madison, WI, USA) was added to each well and incubated for 2 h. Aliquots of 200 µL from each well were transferred into a 96-well plate and read at (λex = 570 nm/λem = 590 nm) using a fluorescence microplate reader (Bio-tek MQX, Winooski, VT, USA). The cell number on each sample was calculated based on a calibration curve prepared with predetermined cell numbers. Polymers 2018, 10, 272 4 of 12

In another set of experiments, mBMSCs were seeded on both PLLA NF and ECM/PLLA NF in a 24-well plate at a seeding density of 2 × 104 cells/cm2 in 1.0 mL medium (n = 5). The cells were allowed to grow on the specimens for 7 days. After that, cells were fixed by 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min. To visualize both actin and the nucleus, tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and 0.5 µg/mL 40,6-diamidino-2-phenylindole dihydro-chloride (DAPI) were added to the cells and incubated for 30 min. After extensive washing with PBS, the cells were imaged using a fluorescent microscope (Zeiss Axiovert 200M, Oberkochen, Germany) with filters appropriate for TRITC and DAPI.

2.5. Cell Metabolism In another set of experiments, mBMSCs were seeded onto the scaffolds (PLLA NF and ECM/PLLA NF) at a density of 2 × 104 cells/cm2 in 1.0 mL of medium (n = 8). At each predetermined time points (day 3, 7 and 14), each scaffold was carefully transferred into another 24-well. 0.5 mL 10% CellTilter Blue in α-MEM was added into each well and incubated at 37 ◦C for 2 h. Aliquots of 200 µL from each well were transferred into a 96-well plate and read at (λex = 570 nm/λem = 590 nm) using a fluorescence microplate reader (Bio-tek MQX, Winooski, VT, USA). The cell number on each sample was calculated based on a calibration curve prepared with predetermined cell numbers. Finally, the cell morphology on different substrates was examined using field emission scanning electron microscopy (FESEM, LEO/Zeiss DSM 982, Oberkochen, Germany). To assess the cell distribution and extracellular matrix deposition, mBMSCs were seeded and cultured on PLLA NF and ECM/PLLA NF for 14 days. After that, all the specimens were prepared as described earlier in this section and then subjected to FESEM observations.

2.6. ALP Activity The osteogenic differentiation of mBMSCs was determined by measuring their alkaline phosphatase (ALP) activity. Briefly, after 3, 7 and 14 days of culture, all media were removed, and each specimen was washed with PBS twice. 200 µL lysis buffer containing 0.5% Triton X-100 was added into each well and incubated for 20 min. The cell lysis was then centrifuged at 3000× g at 4 ◦C for 5 min. Aliquots of supernatants were subjected to a total protein assay using a BCA assay kit (Thermo Scientific, Waltham, MA, USA). The ALP activity was measured by colorimetric assay with reagent mixture composed of 5 mM p-nitro-phenol phosphate disodium (p-NPP), 1 mM MgCl2, and 0.15 M 2-amino-2-methyl-1-propanol (AMP) (Sigma, St. Louis, MO, USA) together with an equal volume amount of nitrophenyl phosphate (10 mM). The optical density of the solution was measured at 405 nm using a microplate reader (Biotek, MQX200, Winooski, VT, USA). The ALP activity was expressed as total activity per microgram protein per sample.

2.7. Characterization of Mineralized Tissue Formation Formation of mineralized tissue was assessed by Alizarin Red-S (ARS) staining at 14 days of osteogenic culture. The cultured specimens were washed with PBS and fixed in 10% (v/v) formaldehyde at RT for 30 min. The cells were then washed twice with excess dH2O prior to addition of 1 mL of 40 mM ARS (pH 4.1) per well for 30 min. After aspiration of the unincorporated ARS, the wells were washed four times with 4 mL dH2O while shaking for 10 min. The specimens were then imaged using a Cannon scanner (Tokyo, Japan). To quantify the amount of calcium deposited on each type of specimen, the mineral component was dissolved in 1.0 M HCl and the calcium concentration was measured as follows: 5 µL of sample solution was mixed with 195 µL of a 0.4 mM Arsenazo III prepared in 0.02 M Tris buffer (pH = 7.40). The amount of calcium quantitatively detected by measuring the absorbance of the 615 nm band on a microplate reader (Biotek, MQX200, Winooski, VT, USA) and compared it to a set of standards with known calcium concentrations. Polymers 2018, 10, 272 5 of 12

Polymers 2018, 10, x FOR PEER REVIEW 5 of 12 2.8. Mineralization 2.8.To Mineralization test the apatite formation capability on both PLLA NF and ECM/PLLA NF, the nanofibrous ◦ mats wereTo incubatedtest the apatite in modified formation simulated capability body on fluidboth (mSBF)PLLA NF at 37and CECM/PLLA 7 days with NF, continuous the nanofibrous rotation. Themats mSBFs were were incubated prepared in bymodified adding simulated the following body reagents fluid (mSBF) into deionized at 37 °C water7 days in with the ordercontinuous shown: 141rotation. mM NaCl, The4.0 mSBFs mM were KCl, prepared 0.5 mM MgSOby adding4, 1.0 the mM following MgCl2, reagents 4.2 mM into NaHCO deionized3, 20.0 water mM HEPES,in the 5.0 mMorder CaCl shown:2, and 141 2.0mM mM NaCl, KH 4.02PO mM4. TheKCl, pH 0.5 ofmM mSBF MgSO was4, 1.0 then mM adjusted MgCl2, 4.2 to mM 6.80 NaHCO using HCl/NaOH.3, 20.0 mM In particular,HEPES, 5.0 each mM nanofibrous CaCl2, and mat 2.0 wasmM incubatedKH2PO4. The in 50 pH mL of mSBF mSBF in was a conical then tubeadjusted to form to 6.80 the mineralusing coatings.HCl/NaOH. The mSBFs In particular, were refreshed each nanofibrous daily throughout mat was theincubated entire coatingin 50 mL process mSBF in ordera conical to maintaintube to consistentform the mineral concentrations coatings. The for mSBFs mineral were coating refresh growth.ed daily After throughout 7 days the of incubation,entire coating the process scaffolds in wereorder rinsed to withmaintain deionized consistent water ion and concentrations lyophilized. After for that,mineral the specimenscoating growth. were gently After washed7 days withof de-ionizedincubation, water the andscaffolds air dried. were Therinsed formation with deionized of the apatitewater and coating lyophilized. on the After surface that, of the titanium specimens disks waswere then gently observed washed using with FESEM de-ionized (JEOL water 6335F and Akishima, air dried. Japan) The formation at 5 kV. of the apatite coating on the surface of titanium disks was then observed using FESEM (JEOL 6335F Akishima, Japan) at 5 kV. 2.9. Statistical Analysis 2.9. Statistical Analysis All quantitative data are given as mean ± standard deviation. The numerical results obtained in All quantitative data are given as mean ± standard deviation. The numerical results obtained in this study were subjected to a two-tailed Student’s t-test. The significance of the results was evaluated this study were subjected to a two-tailed Student’s t-test. The significance of the results was evaluated at a significance level of p < 0.05 based on the p value of comparison. at a significance level of p < 0.05 based on the p value of comparison. 3. Results and Discussion 3. Results and Discussion 3.1. Generation and Characterization of ECM Decorated PLLA NF 3.1. Generation and Characterization of ECM Decorated PLLA NF Similar to decellularized tissues, cell-derived ECM provides a natural scaffold for cell attachment, Similar to decellularized tissues, cell-derived ECM provides a natural scaffold for cell proliferation and differentiation as it is a bioactive materials containing a series of fibrillary protein, attachment, proliferation and differentiation as it is a bioactive materials containing a series of matrix molecules, and embedded growth factors [36]. Therefore, the utility of cell-derived ECM for fibrillary protein, matrix molecules, and embedded growth factors [36]. Therefore, the utility of cell- enhancing the performance of electrospun PLLA nanofibers is confirmed to be a feasible approach. derived ECM for enhancing the performance of electrospun PLLA nanofibers is confirmed to be a Specifically, we used MC3T3-E1 cell line to generate ECM on PLLA NF surface, as its ECM contains feasible approach. Specifically, we used MC3T3-E1 cell line to generate ECM on PLLA NF surface, as osteogenicits ECM andcontains osteoinductive osteogenic and factors osteoinductive accumulated factors during accumulated its differentiation. during its differentiation.

FigureFigure 1. 1.PLLA PLLA electrospun electrospun nanofibers nanofibers beforebefore andand afterafter ECMECM decoration: (A (A) )Electrospun Electrospun PLLA PLLA Nanofibers,Nanofibers, low mag;low mag; (B) Electrospun (B) Electrospun PLLA Nanofibers,PLLA Nano mediumfibers, medium mag; (C )mag; Electrospun (C) Electrospun PLLA Nanofibers, PLLA highNanofibers, mag; (D) Electrospunhigh mag; ( PLLAD) Electrospun Nanofibers PLLA decorated Nanofibers with ECM,decorated low mag;with (ECM,E) Electrospun low mag; PLLA (E) NanofibersElectrospun decorated PLLA Nanofibers with ECM, decorated medium with mag ECM,;(F) Electrospunmedium mag PLLA; (F) Electrospun Nanofibers PLLA decorated Nanofibers with ECM,decorated high mag; with ( GECM,) Histogram high mag; of (G PLLA) Histogram NF diameter; of PLLA (H NF) Histogram diameter; ( ofH) ECM/PLLAHistogram of NFECM/PLLA diameter; NF diameter; (I) Average diameter comparison between PLLA and ECM/PLLA, * indicates significant (I) Average diameter comparison between PLLA and ECM/PLLA, * indicates significant difference difference (p < 0.05). (p < 0.05).

Polymers 2018, 10, 272 6 of 12

PolymersIn order 2018 to, 10 obtain, x FOR PEER ECM REVIEW decoration on PLLA NF surface, we employed a cell-based strategy6 of 12 to generate ECM coating on the nanofibrous PLLA surface. As shown in Figure1A, nanofibers with uniformIn size order were to formedobtain ECM after decoration electrospinnnig. on PLLA Although NF surface, fiber we size employed variation a cell-based can still be strategy observed to generate ECM coating on the nanofibrous PLLA surface. As shown in Figure 1A, nanofibers with when zoomed in (Figure1B,C,G,H), the average diameter of the nanofibers was around 350 nm based uniform size were formed after electrospinnnig. Although fiber size variation can still be observed on image-based statistics (Figure1I). After decorating with ECM, it was observed that a layer of ECM when zoomed in (Figure 1B,C,G,H), the average diameter of the nanofibers was around 350 nm based uniformly covered on the nanofiber while still preserving the nanoscale morphology of the electrospun on image-based statistics (Figure 1I). After decorating with ECM, it was observed that a layer of ECM fibers. Compared to nanofibers without ECM decoration, it was noticed that the average size of the uniformly covered on the nanofiber while still preserving the nanoscale morphology of the nanofiberselectrospun was fibers. slightly Compared increased, to nanofibers which is probably without ECM due todecoration, the swelling it was of noticed PLLA duringthat the longaverage cell culturesize times.of the nanofibers was slightly increased, which is probably due to the swelling of PLLA during long cell culture times. 3.2. Cell Attachment and Spread 3.2.Although Cell Attachment polyester-based and Spread nanofibers fabricated by electrospinning have been widely used in tissue engineeringAlthough andpolyester-based regenerative nanofibers medicine fabricated [15], the cell by attachmentelectrospinning behaviors have ofbeen these widely materials used arein stilltissue considered engineering to be and suboptimal regenerative due medicine to their high[15], the hydrophobicity. cell attachment Webehaviors hypothesized of these thatmaterials ECM decorationare still considered on electrospun to be nanofiberssuboptimal due could to thei significantlyr high hydrophobicity. improve cell We attachment hypothesized by that providing ECM abundantdecoration adhesive on electrospun components nanofibers such as fibronectin, could sign vitronectin,ificantly improve etc. on cell the attachment materials’ surfaceby providing [37,38 ]. Thisabundant hypothesis adhesive was verified components by showing such as afibronectin, high cell attachment vitronectin, ratioetc. on on the ECM/PLLA materials’ surface NF compared [37,38]. to PLLAThis hypothesis NF (Figure was2G). verified The percentage by showing of a cells high attached cell attachment to ECM/PLLA ratio on ECM/PLLA NF was 30% NF morecompared than for PLLAto PLLA NF. NF The (Figure morphology 2G). The ofpercentage mBMSCs of after cells beingattached seeded to ECM/PLLA on nanofibers NF was is shown30% more in than Figure for2 . CellsPLLA on PLLA NF. The NF exhibitedmorphology a round of mBMSCs morphology after being with limitedseeded on filopodia nanofibers extension is shown (Figure in Figure2A). The2. Cells side viewon of PLLA individual NF exhibited cell showed a round that morphology the cell only with physically limited attachedfilopodia toextension the nanofiber, (Figure and2A). no The further side interactionsview of individual between thecell nanofibersshowed that and the thecell cellonly were physically observed attached (Figure to the2B,C). nanofiber, In contrast, and no rBMSCs further growninteractions on ECM/PLLA between NF the demonstrated nanofibers and a flatthe polygonalcell were observed shape on (Figure the material 2B,C). In surface, contrast, and rBMSCs most of themgrown exhibited on ECM/PLLA multipolar NF extensions. demonstrated As shown a flat polygona in Figurel2 E,shape cells on on the ECM/PLLA material surface, NF tend and to most stretch of them exhibited multipolar extensions. As shown in Figure 2E, cells on ECM/PLLA NF tend to stretch their filopodia along the nanofiber direction, indicating that the ECM coating on these fibers had their filopodia along the nanofiber direction, indicating that the ECM coating on these fibers had remarkably enhanced their interactions with cell anchoring points. This feature of the ECM decorated remarkably enhanced their interactions with cell anchoring points. This feature of the ECM decorated nanofibers was clearly visible in high-magnification SEM images, which clearly showed that the cells nanofibers was clearly visible in high-magnification SEM images, which clearly showed that the cells spread following the orientation of the nanofibers. Therefore, ECM deposited by MC3T3-E1 cells can spread following the orientation of the nanofibers. Therefore, ECM deposited by MC3T3-E1 cells can effectivelyeffectively improve improve cell cell attachment attachment on on electrospun electrospun nanofibers. nanofibers.

Figure 2. mBMSC adhered on PLLA electrospun nanofibers before and after ECM decoration: (A) Figure 2. mBMSC adhered on PLLA electrospun nanofibers before and after ECM decoration: Overview of mBMSCs on PLLA NF; (B) Single cell attached on PLLA NF; (C) Highlight of mBMSCs (A) Overview of mBMSCs on PLLA NF; (B) Single cell attached on PLLA NF; (C) Highlight of mBMSCs attached to PLLA nanofibers; (D) Overview of mBMSCs on ECM/PLLA NF; (E) Single cell attached attachedon ECM/PLLA to PLLA nanofibers; NF; (F) Highlight (D) Overview of mBMSCs of mBMSCs attached on ECM/PLLA to ECM/PLLA NF; (nanofibers.E) Single cell (G attached) mBMSC on ECM/PLLAattachment NF; on (F two) Highlight scaffolds of after mBMSCs 2 h; * indicates attached tosignificant ECM/PLLA difference nanofibers. at p < 0.05. (G) mBMSC attachment on two scaffolds after 2 h; * indicates significant difference at p < 0.05. 3.3. Cell Proliferation and Differentiation To determine the effect of ECM decoration on mBMSCs viability, cell numbers on different treated PLLA nanofibers were recorded at day 3, 7 and 14. As shown in Figure 3A, cell numbers on both tested groups increased significantly as culture time extended. At day 3, the cell numbers on

Polymers 2018, 10, x FOR PEER REVIEW 7 of 12

ECM/PLLA NF were slightly higher than those on PLLA NF, which might be due to the faster cell settlement on ECM-decorated nanofibers. The difference in cell numbers between ECM/PLLA NF and PLLA NF had become even more significant by day 7. At day 14, the cell numbers for ECM treated group were 70% higher than those for PLLA NF, indicating that the presence of ECM not only improved cell attachment at the initial stage, but also provided proliferative signal, stimulating the cells to grow faster. Polymers 2018, 10, 272 7 of 12 ALP activity was evaluated as an early marker of osteogenic differentiation of mBMSCs cells. Although ALP activity in both groups was very low at day 3, it was noticed that ALP on ECM/PLLA NF was still slightly higher than PLLA NF group (Figure 3B). ALP activity on both tested groups 3.3. Cell Proliferationincreased and gradually Differentiation between 3 and 7 days, and there is a noticeable difference between ECM/PLLA NF and PLLA NF, suggesting that ECM presence on nanofiber surface could increase ALP activity, To determinewhich the may effect be further of ECM assigned decoration to mineral deposition on mBMSCs during viability,MC3T3-E1 differentiation. cell numbers A dramatic on different treated increase in ALP activity was observed during the second week of culture due to the switch of culture PLLA nanofibersmedium were from recorded basal medium at day to osteogenic 3, 7 and medium. 14. As Although shown the in ALP Figure activity3A, on cell PLLA numbers NF surfaces on both tested groups increasedincreased significantly by over 3 folds, as culture the ALP activity time on extended. the ECM-treated At daysurfaces 3, were the still cell much numbers higher than on ECM/PLLA the untreated group, which indicates that the ECM decoration approach used here is more favorable NF were slightlyfor higher osteogenic than differentiation. those on Similar PLLA results NF, have which also been might observed be dueby other to investigators the faster in cell the settlement on ECM-decorated nanofibers.context of bone Thetissue differenceengineering. For in instance, cell numbers Sutha et al. between showed in ECM/PLLA their study that embryonic NF and PLLA NF had become even morestem significant cell-derived ECM by possessed day 7. Atosteoinductive day 14, thefactors, cell which numbers promoted for ectopic ECM bone treated formation group in were 70% vivo [39]. Since MC3T3-E1 cell is an osteoblastic cell line which can differentiate into osteoblast, higher than thoseosteogenic for PLLA factors NF, can indicating be secreted during that thetheir presencedifferentiation of [40]. ECM Thus, not the only enhanced improved osteogenic cell attachment at the initial stage,differentiation but also of provided mBMSCs on proliferativeECM/PLLA NF might signal, be attributed stimulating to the osteogenic the cells signals to contained grow faster. in MC3T3-E1 derived ECM.

Figure 3. mBMSCs proliferation and osteogenic differentiation on PLLA NF and ECM/PLLA NF for Figure 3. mBMSCs3, 7 and proliferation 14 days: (A) The and cell number osteogenic was significantly differentiation higher onECM/PLLA on PLLA NF than NF on and PLLA ECM/PLLA NF (p NF for 3, 7 and 14 days:< (0.05)A) at The all time cell points. number (B) ALP was activity significantly normalized to total higher protein onECM/PLLA content of mBMSCs cultured NF than on on PLLA NF PLLA NF and ECM/PLLA NF for 3, 7 and 14 days. ALP activities were significantly higher on (p < 0.05) at all timeECM/PLLA points; NF (thanB) on ALP PLLA activity NF (p < 0.05) normalized at all time points. to total * indicates protein significant content differences. of mBMSCs cultured on PLLA NF and ECM/PLLA NF for 3, 7 and 14 days. ALP activities were significantly higher on 3.4. Cell Morphology and Mineral Deposition ECM/PLLA NF than on PLLA NF (p < 0.05) at all time points. * indicates significant differences. Figure 4 shows the mBMSC cells growing on the two types of surface after 14 days of culture; however, cell coverage on ECM/PLLA NF appears to be more uniform compared to PLLA NF. Similar ALP activityresults was were evaluated also observed as in anFigure early 5 using marker SEM. Both of surfaces osteogenic were covered differentiation with a layer of cells. of In mBMSCs cells. particular, cells on PLLA NF exhibited a flatter morphology and tended to fully spread out. However, Although ALP activitythere are still in small both gaps groups between was cells, very and nanofib lowers at were day still 3, exposed it was without noticed cell thatlayers ALPcovering on ECM/PLLA NF was still slightlythem. Meanwhile, higherthan mBMSCs PLLA on ECM/PLLA NF group NF grew (Figure tightly3 toB). each ALP otheractivity and tended on to form both cell tested groups increased graduallycolonies between (Figure 5D). 3 andMoreover, 7 days, these andcells demon therestrated is a noticeablea spindle-shaped difference morphology, between which is ECM/PLLA more similar to the morphology of . At a high magnification, although cells on PLLA NF NF and PLLA NF,showed suggesting a healthy morphology, that ECM the presencesurface was only on covered nanofiber a single surface layer of cells. could In comparison, increase ALP activity, which may be furtherECM/PLLA assigned NF surface to was mineral heavily depositioncovered by multiple during layers MC3T3-E1 of mBMSCs, differentiation. which displayed A dramatic numerous interactions between different cells (Figure 5B,E). Furthermore, we also found that new increase in ALP activity was observed during the second week of culture due to the switch of culture medium from basal medium to osteogenic medium. Although the ALP activity on PLLA NF surfaces increased by over 3 folds, the ALP activity on the ECM-treated surfaces were still much higher than the untreated group, which indicates that the ECM decoration approach used here is more favorable for osteogenic differentiation. Similar results have also been observed by other investigators in the context of bone tissue engineering. For instance, Sutha et al. showed in their study that embryonic stem cell-derived ECM possessed osteoinductive factors, which promoted ectopic bone formation in vivo [39]. Since MC3T3-E1 cell is an osteoblastic cell line which can differentiate into osteoblast, osteogenic factors can be secreted during their differentiation [40]. Thus, the enhanced osteogenic differentiation of mBMSCs on ECM/PLLA NF might be attributed to the osteogenic signals contained in MC3T3-E1 derived ECM.

3.4. Cell Morphology and Mineral Deposition Figure4 shows the mBMSC cells growing on the two types of surface after 14 days of culture; however, cell coverage on ECM/PLLA NF appears to be more uniform compared to PLLA NF. Similar results were also observed in Figure5 using SEM. Both surfaces were covered with a layer of cells. In particular, cells on PLLA NF exhibited a flatter morphology and tended to fully spread out. However, there are still small gaps between cells, and nanofibers were still exposed without cell layers covering them. Meanwhile, mBMSCs on ECM/PLLA NF grew tightly to each other and tended to form cell colonies (Figure5D). Moreover, these cells demonstrated a spindle-shaped Polymers 2018, 10, 272 8 of 12 morphology, which is more similar to the morphology of osteoblasts. At a high magnification, although cells on PLLA NF showed a healthy morphology, the surface was only covered a single layer of cells. In comparison, ECM/PLLA NF surface was heavily covered by multiple layers of mBMSCs, which displayedPolymers 2018 numerous, 10, x FOR PEER interactions REVIEW between different cells (Figure5B,E). Furthermore,8 of 12 we also found that new ECM deposition on ECM/PLLA NF was more abundant than PLLA NF. Mineralized nodules canPolymersECM be deposition 2018 clearly, 10, x FOR seen on PEER ECM/PLLA on REVIEW ECM/PLLA NF was more NF, abundant whereas than they PLLA are NF. sparse Mineralized on PLLA nodules NF can8 (Figureof 12be 5C,F), clearly seen on ECM/PLLA NF, whereas they are sparse on PLLA NF (Figure 5C,F), which implies which implies that decoration of ECM secreted by MC3T3-E1 cells enhanced newly deposited ECM by ECMthat decorationdeposition ofon ECM ECM/PLLA secreted NF by was MC3T3-E1 more abundant cells enhanced than PLLA newly NF. deposited Mineralized ECM nodules by BMSC can cells be BMSC cellsclearlyand and osteoblastic seen osteoblastic on ECM/PLLA cells. cells. NF, whereas they are sparse on PLLA NF (Figure 5C,F), which implies that decoration of ECM secreted by MC3T3-E1 cells enhanced newly deposited ECM by BMSC cells and osteoblastic cells.

Figure 4. Immunostaining of actin, and nuclei showing mBMSC cells cultured on PLLA NF and Figure 4. Immunostaining of actin, and nuclei showing mBMSC cells cultured on PLLA NF and ECM/PLLA NF, respectively: (A–C) actin staining, DAPI staining, and merged image of mBMSCs on ECM/PLLAFigurePLLA NF, NF4. respectively: Immunostainingat day 7; (D–F) (actinA of– C actin,staining,) actin and DAPI staining, nuclei staining, showing DAPI and mBMSC staining,merged cellsimage and cultured of mergedmBMSCs on PLLA on image ECM/PLLA NF of and mBMSCs on PLLA NFECM/PLLA atNF day at day 7; 7; ( DNF, Red:–F respectively:) actin; actin Blue: staining, (nuclei.A–C) actin DAPI staining, staining, DAPI and staining, merged and merged image image of mBMSCs of mBMSCs on on ECM/PLLA NF at dayPLLA 7; Red: NF at actin; day 7; Blue: (D–F) nuclei.actin staining, DAPI staining, and merged image of mBMSCs on ECM/PLLA NF at day 7; Red: actin; Blue: nuclei.

Figure 5. SEM micrographs showing mBMSCs morphologies cultured on PLLA and ECM/PLLA NF for 14 days. (A) PLLA (at low mag); (B) PLLA (at high mag); (C) PLLA (matrix deposition); and (D) FigureECM/PLLA 5. SEM (at micrographs low mag); (showingE) ECM/PLLA mBMSCs (at morphologieshigh mag); (F cultured) ECM/PLLA on PLLA (matrix and deposition). ECM/PLLA ItNF is Figure 5. forSEMshown 14 days. micrographsthat (mBMSCsA) PLLA grown(at showing low on mag); ECM/PLLA mBMSCs(B) PLLA NF (at deve morphologies highloped mag); multilayer (C) PLLA cultured structure (matrix on deposition);and PLLA ECM deposition andand ( ECM/PLLAD) NF for 14ECM/PLLAon days. this surface (A (at) PLLA islow more mag); (atrobust. ( lowE) ECM/PLLARed mag); arrow: ( Bmineralized(at) PLLAhigh mag); (atnodule ( highF) ECM/PLLAformation mag); after ((matrixC) culture. PLLA deposition). (matrix It deposition);is and (D) ECM/PLLAshown that mBMSCs (at low grown mag); on ( ECM/PLLAE) ECM/PLLA NF deve (atloped high multilayer mag); ( structureF) ECM/PLLA and ECM (matrixdeposition deposition). It is shownonWe this that also surface mBMSCsstudied is more the grownrobust.mineral Red ondeposition arrow: ECM/PLLA mineralized in terms NF ofnodule calcium developed formation on both after multilayer surfac culture.es after structure two weeks. and It ECM was found that both groups were mineralized, showing a layer of calcium mineral on the surface deposition on this surface is more robust. Red arrow: mineralized nodule formation after culture. (FigureWe 6).also Nevertheless, studied the mineral it was founddeposition that calciumin terms depositionof calcium on bothECM/PLLA surfaces NF after was two about weeks. 12.59 It wasmg/sample, found that which both is groups much werehigher mineralized, than that ofshowing PLLA NFa layer (8.72 of mg/sample); calcium mineral thus, on decoration the surface of We also(FigureMC3T3-E1 studied 6). Nevertheless, derived the mineral ECM itsubstantia depositionwas foundlly increasedthat in calcium terms calcium ofdeposition calcium deposition on on ECM/PLLA bothduring surfaces mBMSC NF was afterdifferentiation. about two 12.59 weeks. It was mg/sample, which is much higher than that of PLLA NF (8.72 mg/sample); thus, decoration of found that both groups were mineralized, showing a layer of calcium mineral on the surface (Figure6). MC3T3-E1 derived ECM substantially increased calcium deposition during mBMSC differentiation. Nevertheless, it was found that calcium deposition on ECM/PLLA NF was about 12.59 mg/sample, which is much higher than that of PLLA NF (8.72 mg/sample); thus, decoration of MC3T3-E1 derived ECM substantially increased calcium deposition during mBMSC differentiation. Polymers 2018, 10, 272 9 of 12

Polymers 2018, 10, x FOR PEER REVIEW 9 of 12

Polymers 2018, 10, x FOR PEER REVIEW 9 of 12

Figure 6. Calcium deposition on PLLA NF and ECM/PLLA NF after mBMSCs culture for 14 days: Figure 6. Calciuminserted deposition images were Alizarin on PLLA Red S NFstaining and of calcium. ECM/PLLA Calcium deposition NF after were mBMSCs significantly higher culture for 14 days: inserted imageson were ECM/PLLA Alizarin NF than Red on PLLA S staining NF (p < 0.05) of calcium. after 14 days. Calcium * indicates depositionsignificant differences. were significantly higher

on ECM/PLLA3.5. Biomineralization NF than on PLLA NF (p < 0.05) after 14 days. * indicates significant differences. Figure 6. CalciumBiomineralization deposition capability on PLLA is closely NF and related ECM/PLLA to the bone-forming NF after mBMSCspotential of culture biomaterials for 14 in days: 3.5. Biomineralizationinsertedvivo; images therefore, were it Alizarin is commonly Red used S staining to characterize of calcium. the bioactivity Calcium depositionof biomaterials were [41]. significantly To investigate higher on ECM/PLLAthe effect NFof ECM than decoration on PLLA on NF PLLA (p < nanofiber0.05) after biomineralization, 14 days. * indicates both significantPLLA NF and differences. ECM/PLLA BiomineralizationNF were soaked capability in mSBF is to closely evaluate related their mine toral-forming the bone-forming capability. As potential shown in Figure of biomaterials 7A, in vivo; scattered mineral clusters were formed on PLLA NF after soaking in mSBF for 24 h. When zoomed therefore,3.5. Biomineralization it is commonlyin, it can be noticed used that to these characterize minerals mainly the grew bioactivity along the fiber of directions, biomaterials instead of [ 41uniformly]. To investigate the effect of ECMBiomineralization decorationcoating the surface oncapability PLLA(Figure 7A,B). nanofiberis closely On the relatedother biomineralization, hand, to mineralthe bone-forming growth bothon ECM/PLLA PLLApotential NF of showed and biomaterials ECM/PLLAa in NF were soaked inmuch mSBF faster to evaluatetrend. It was their found mineral-forming that mineral coating capability.had homogeneously As shown formed inalong Figure the 7A, scattered vivo; therefore,nanofibers, it is commonly which increased used the to diameter characterize of the nanofibersthe bioactivity from several-hundred of biomaterials nanometers [41]. To toinvestigate mineralthe clusterseffect ofabout ECM were 1 µmdecoration formed (Figure 7E,F). on on PLLA ThePLLA localization nanofiber NF after of mine biomineralization, soakingral coating in on mSBF nanofibers both for indicates PLLA 24 h. WhenNFthat andmineralzoomed ECM/PLLA in, it can be noticedNF were that soakednucleation these in minerals mainly mSBF occurred to mainly evaluate on the grew nanofibers,their along mine whral-forming theich may fiber be directions,due capability. to the presence insteadAs shownof MC3T3-E1- of in uniformly Figure 7A, coating scattered mineralderived ECM.clusters When were we extended formed the on biomineralization PLLA NF after time soakingto 48 h, it was in mSBFfound that for a 24thick h. mineral When zoomed the surface (Figurecoating7A,B). coveredOn the whole the othernanofiber hand, surface, mineral causing PLLA growth NF to lose on its ECM/PLLA nanoscale features NF (Figure showed a much fasterin, trend. it can Itbe7C,D). wasnoticed However, found that these thatthis situation mineralminerals did mainly coatingnot occur grew wi hadth alongECM/PLLA homogeneously the fiberNF. Instead, directions, formedthe mineral instead along coating of uniformlythe nanofibers, coating thecontinued surface to(Figure grow along 7A,B). the Onnanofiber the other direction, hand, and mineralpreserved growthits nanoscale on ECM/PLLAmorphology (Figure NF showed a which increased the diameter of the nanofibers from several-hundred nanometers to about 1 µm much faster7G,H). trend. The Itresults was from found biomineralization that minera indicatel coating that ECMhad decorationhomogeneously can facilitate formed mineral along the (Figurenanofibers,7E,F). Thedeposition which localization increased on PLLA nanofiber ofthe mineral diameter surface, coatingwh ofich the is beneficial nanofibers on nanofibers for bone from regeneration. several-hundred indicates that mineralnanometers nucleation to mainlyabout occurred 1 µm (Figure on the 7E,F). nanofibers, The localization which may of mine beral due coating to the on presence nanofibers of indicates MC3T3-E1-derived that mineral ECM. Whennucleation we extended mainly the occurred biomineralization on the nanofibers, time wh toich 48 h,may it be was due found to the that presence a thick of MC3T3-E1- mineral coating coveredderived the wholeECM. When nanofiber we extended surface, the causingbiomineralization PLLA NF time to to lose 48 h, its it nanoscalewas found that features a thick (Figure mineral7 C,D). However,coating this covered situation the whole did not nanofiber occur with surface, ECM/PLLA causing PLLA NF. NF Instead, to lose theits nanoscale mineral features coating (Figure continued to grow7C,D). along theHowever, nanofiber this direction,situation did and not preserved occur wi itsth nanoscaleECM/PLLA morphology NF. Instead,(Figure the mineral7G,H). coating The results continued to grow along the nanofiber direction, and preserved its nanoscale morphology (Figure from biomineralization indicate that ECM decoration can facilitate mineral deposition on PLLA 7G,H). The results from biomineralization indicate that ECM decoration can facilitate mineral nanofiberdeposition surface, on PLLA which nanofiber is beneficial surface, for wh boneich is regeneration. beneficial for bone regeneration.

Figure 7. Apatite coating formation on two types of PLLA nanofibers with different treatment: (A) Apatite growth survey on PLLA NF at 24 h; (B) Apatite growth zoom-in on PLLA NF at 24 h; (C) Apatite growth survey on PLLA NF at 48 h; (D) Apatite growth zoom-in on PLLA NF at 48 h; (E) Apatite growth survey on ECM/PLLA NF at 24 h; (F) Apatite growth zoom-in on ECM/PLLA NF at 24 h; (G) Apatite growth survey on ECM/PLLA NF at 48 h; (H) Apatite growth zoom-in on ECM/PLLA NF at 48 h, after soaking in mSBF for different time periods.

Figure 7. Apatite coating formation on two types of PLLA nanofibers with different treatment: (A) Figure 7. ApatiteApatite growth coatingsurvey on formation PLLA NF at on 24 two h; (B types) Apatite of PLLAgrowth nanofiberszoom-in on PLLA with NF different at 24 h;treatment: (C) (A) ApatiteApatite growthgrowth survey survey on on PLLA PLLA NF NFat 48 at h; 24(D) h; Apatite (B) Apatite growth growthzoom-in zoom-inon PLLA NF on at PLLA 48 h; NF(E) at 24 h; (C)Apatite Apatite growth growth survey survey on ECM/PLLA on PLLA NFNF at 24 48 h; h; (F (D) Apatite) Apatite growth growth zoom-in zoom-in on ECM/PLLA on PLLA NF NF at at 48 h; (E) Apatite24 h; ( growthG) Apatite survey growth on survey ECM/PLLA on ECM/PLLA NF at 24 NF h; (atF )48 Apatite h; (H) growthApatite zoom-ingrowth zoom-in on ECM/PLLA on NF atECM/PLLA 24 h; (G) ApatiteNF at 48 growthh, after soaking survey in on mSBF ECM/PLLA for different NF time at periods. 48 h; ( H) Apatite growth zoom-in on

ECM/PLLA NF at 48 h, after soaking in mSBF for different time periods. Polymers 2018, 10, 272 10 of 12

4. Conclusions In conclusion, electrospun PLLA nanofibers were successfully decorated by ECM derived from osteoblastic cells by decellularization of MC3T3-E1 cells grown for two weeks. The results demonstrated that application of ECM on PLLA nanofibers enhanced mouse bone marrow stromal cell (mBMSC) adhesion, supported cell proliferation and promoted osteogenic differentiation of mBMSCs. Therefore, this study provided an alternative platform for directly translating the regenerative potential of ECM into clinical therapeutics and motivating the development of cell-derived ECM materials for broad regenerative medicine applications.

Supplementary Materials: The following are available online at www.mdpi.com/2073-4360/10/3/272/s1. Acknowledgments: We gratefully acknowledge financial support from the National Natural Science Foundation of China (81470686), Zhejiang Provincial Natural Science Foundation of China (LY14H130001), Zhejiang province medical and health science research fund plan, (2014KYB077). Author Contributions: Yong Fu and Wenguo Cui conceived and designed the experiments; Yong Fu performed the experiments; Lili Liu and Ruoyu Cheng analyzed the data; Yong Fu and Ruoyu Cheng contributed reagents/materials/analysis tools; Yong Fu and Wenguo Cui wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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