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Nanostructured Tendon-derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-derived Stem Cells Eunkyung Ko, Kyle Alberti, Jong Seung Lee, Kisuk Yang, Yoonhee Jin, Jisoo Shin, Hee Seok Yang, Qiaobing Xu, and Seung-Woo Cho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05358 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 21, 2016

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ACS Applied Materials & Interfaces

Nanostructured Tendon-derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-derived Stem Cells

Eunkyung Ko1, Kyle Alberti2, Jong Seung Lee1, Kisuk Yang1, Yoonhee Jin1, Jisoo Shin1, Hee Seok Yang3, Qiaobing Xu2*, Seung-Woo Cho1*

1

Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea

2

Department of Biomedical Engineering, Tufts University, MA 02155, USA

3

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research

Center for Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea

*Corresponding authors: Prof. Seung-Woo Cho Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea; E-mail: [email protected] Prof. Qiaobing Xu Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA; E-mail: [email protected]

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Abstract

Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of crosslinking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-crosslinked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically-committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells. Keywords: nanostructured decellularized tendon scaffolds, human adipose-derived stem cells, osteogenic differentiation, bone formation, mineralization 2 ACS Paragon Plus Environment

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1. Introduction

Bone tissue engineering based on stem cells has recently been viewed as a promising alternative to the conventional use of bone grafts. Current engineered bone constructs require a synergistic combination of biomaterials, stem cells, and bioactive factors to induce functional bone regeneration.1-4 While many natural and synthetic biomaterials have been developed to mimic the structures and properties of native bone tissue and, therefore, facilitate osteogenic differentiation and bone formation of stem cells, there exist various inherent drawbacks and challenges associated with fabrication methodologies and material choice.5 In addition, most scaffolds developed to mimic the natural tissue environment are often oversimplified for effective bone regeneration. Considering that tissues are surrounded by a structurally complex three-dimensional (3D) microenvironment composed of varied collagens and proteoglycans, in which multiple bioactive factors, including growth factors, functional peptides, and integrins, are incorporated, more efficient approaches should be employed to reconstitute such highly complex tissue-specific microenvironments. These aforementioned issues have driven tissue engineers and stem cell researchers to consider increasing the use of biological scaffolds derived from decellularized tissue matrices in both preclinical animal studies6-10 and human clinical applications.11-12 Various types of decellularized matrix have been shown very effective in tissue reconstruction of organs such as the liver,13-16 lungs,17-18 heart,19-21 and blood vessels.22-25 Removal of cells from tissues leaves the complex mixture of structural and functional proteins; in principle, naturally-derived decellularized scaffolds best represent the native tissue-specific cellular microenvironment. Previously, we demonstrated a bioskiving fabrication method that can generate nanofibrous scaffolds 3 ACS Paragon Plus Environment


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based on tendon-derived collagen while retaining the native collagen fiber structure.2629

The scaffolds were produced by decellularizing tendon tissues and sectioning them

into thin sheets using a microtome. This method has several advantages over conventional scaffold nanofabrication technologies such as lithography,30-31 selfassembly techniques,5 and electrospinning;32-33 it is relatively inexpensive, involves simple fabrication steps, and maintains compatibility with biological materials. Tendon was selected as a scaffold material since it contains abundant collagen type I nanofibers with well-aligned structures, thus providing favorable structural microenvironments and mechanical properties for differentiation of stem cells for tissue engineering. Mechanical and biophysical properties of the substrates and scaffolds influence the process of osteogenic differentiation of stem cells via several molecular components of cellular events caused by such mechanical and biophysical cues. Several studies have reported manipulation of cellular behaviors by nanostructured surfaces.34-35 Multiple other studies have proven that topography and stiffness of extracellular matrix (ECM) play significant roles in controlling stem cell differentiation via integrin-mediated mechanotransduction pathways.36-38 Cell adhesion via specific integrin binding onto ECMs with tissue-specific biophysical and mechanical environmental cues (i.e., topography, stiffness) trigger mechanosensitive intracellular signaling cascades through focal adhesion assembly, cytoskeleton reorganization, and activation of downstream signal pathways associated with differentiation.38 A series of these cellular events activate transcriptional activity related to osteogenesis such as Akt/YAP/RUNX2 and RUNX2/Cbfa-1 pathways, which ultimately enhances osteogenesis of stem cells.38-39 Thus, osteogenic differentiation of mesenchymal stem cells (MSCs) has been found to be substantially


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enhanced on the stiff substrates with high elastic modulus and fibrous structures through the aforementioned mechanotransduction signal pathways.38, 40 In this study, we applied decellularized tendon scaffolds with nanofibrous structures to promote osteogenic differentiation and bone formation of human adipose-derived stem cells (hADSCs). The scaffolds were prepared from 2 different parts of bovine tendons [bovine Achilles tendon (BAT) and bovine neck tendon (BNT)] using a bioskiving method, and scaffolds were then subjected to chemical crosslinking to increase their modulus values.37, 41-42 Prepared tendon scaffolds were investigated in terms of mechanical properties, biocompatibility, and osteogenic potential to improve bone formation of hADSCs. We show that decellularized tendon scaffolds could promote focal adhesion, support osteogenic differentiation of hADSCs in vitro, and consequently enhance bone regeneration by hADSC transplantation in a mouse model of critical-sized calvarial bone defect, compared to conventional, commercially-available collagen type I scaffolds. In addition, multilayered stacking of hADSC-seeded tendon scaffolds and human endothelial progenitor cell (hEPC)-seeded tendon scaffolds facilitated a significant enhancement of bone regeneration with vasculature, demonstrating an efficient strategy for constructing vascularized bone formation.

2. Materials and Methods

2.1. Fabrication and Characterization of Decellularized Tendon Scaffolds. Decellularized tendon sections were fabricated as previously reported.27 Briefly, pieces of bovine Achilles tendon (BAT) (sourced from a local farm) were decellularized using a 1% sodium dodecyl sulfate solution with 1 mM Tris-HCl and 5 ACS Paragon Plus Environment


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0.1 mM ethylenediaminetetraacetic acid for 48 hours. The solution was exchanged after 24 hours, and then tendon sections were washed with diH2O for another 24 hours. Tissue pieces were then frozen and sectioned at 50 µm using a cryomicrotome (Leica CM1950, Leica Microsystems, Buffalo Grove, IL). Individual sections were placed onto a polytetrafluoroethylene block and allowed to dry overnight. Multilayer sections were fabricated by stacking multiple sheets on top of each other, alternating the fiber orientation by 90° in adjacent sheets, and allowed to dry overnight. Both types of samples were then rinsed 3 times with diH2O and allowed to dry again. Samples were then crosslinked in either glutaraldehyde (GTA) or 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide (EDC/NHS). GTA samples were crosslinked by immersing samples in the indicated concentration (0.2%, 0.78%, or 1.56%) of GTA for 20 minutes, followed by 3 rinses with diH2O. EDC samples were crosslinked by immersing the samples in EDC and NHS at EDC concentrations of 1, 4 or 16 mM EDC with a 2.5:1 molar ratio of EDC:NHS in phosphate buffered saline (PBS) for 2 hours while being protected from light. These samples were then rinsed 3 times with diH2O and allowed to dry. All samples were rehydrated prior to mechanical testing and tested on an Instron 3366 (Instron, Norwood, MA) at a crosshead speed of 5 mm/min. Samples were tested until rupture, and samples that failed at the grips were excluded from analysis. Ultimate tensile strength was calculated from recorded load and initial crosssectional area, and modulus values were calculated via linear regression of the linear portion of the stress-strain curve. Scanning electron microscope (SEM) images of tendon surfaces were taken by dehydrating samples in graded ethanol followed by hexamethyldisilazane incubation;


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then samples were sputter coated with 3 nm platinum-palladium (PtPd) and imaged with a Zeiss Ultra55 FE-SEM (Oberkochen, Germany).

2.2. Cell Culture. hADSCs purchased from Invitrogen (Invitrogen, Carlsbad, CA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco), with exposure to 5% CO2 at 37°C. Culture medium was changed every 3 days, and cells at passages 5-6 were used for both in vitro and in vivo experiments. hEPCs isolated from human cord blood were cultured in Endothelial Cell Growth Medium (EGM-2, Lonza, Walkersville, MD) in humidified air with 5% CO2 at 37°C as previously described.43 Culture medium was changed every 3 days, and cells at passages of 5-7 were used for the experiments.

2.3. Focal Adhesion Staining. hADSCs were seeded on tendon scaffolds (1.5 × 1.5 cm) with a seeding density of 1.0 × 105 cells/ml. After 3 days of culture, cells were stained with TRITC-conjugated phalloidin, vinculin antibodies, and 4′,6-diamidino-2phenylindole (DAPI, Sigma) for F-actin, vinculin, and nuclei, respectively, using Actin Cytoskeleton and Focal Adhesion Staining Kit (FAK100) (Millipore, Temecula, CA). Stained samples were observed under a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany). Focal adhesion was quantified with the density of focal adhesion complex present in cells by measuring the intensity of vinculin-positive signals in cells.



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2.4. Biocompatibility Assay. The biocompatibility of tendon scaffolds was evaluated by examining immunostimulatory effects of prepared scaffolds. To investigate secretion of inflammatory cytokines from macrophages interacting with scaffolds, tendon scaffolds were cultured with cells of the RAW 264.7 mouse macrophage cell line (TIB-71, ATCC, Manassas, VA, USA). RAW 264.7 cells were seeded on a 24well plate at a density of 1.0 × 106 cells/ml, and sterilized tendon scaffolds were placed on transwell membranes that were then placed into the culture plates seeded with RAW 264.7 cells. On the next day, supernatant from each sample was retrieved to quantify the amount of tumor necrosis factor-α (TNF-α) secreted by cells. Supernatants from cells cultured without scaffolds were used as a negative control, and 10 ng/ml of lipopolysaccharide (LPS, Sigma) was used as a positive control. The amount of secreted TNF-α in each group was measured using enzyme-linked immunosorbent assay (ELISA, Koma Biotech Inc., Seoul, Korea) according to the manufacturer’s protocol.

2.5. Osteogenic Differentiation of hADSCs on Tendon Scaffolds. hADSCs were seeded on different tendon scaffolds (1.0 × 1.0 cm) with a density of 1.0 × 105 cells/ml. Cells were cultured in DMEM for the first 3 days, and then media was exchanged with osteogenic media [DMEM supplemented with 10% (v/v) FBS, 0.1 µM dexamethasone (Sigma), 50 µM ascorbic acid (Sigma), and 10 mM β-glycerol phosphate (Sigma)]. Cells were newly supplemented with osteogenic media every 3 days during osteogenic induction (21 days total). Calcium deposition of osteogenically-differentiated hADSCs for 21 days was examined by Alizarin Red S staining as described in our previous study.1 After fixation with 2.5% (v/v) glutaraldehyde (Sigma) and washing with PBS (pH 4.2), 8 ACS Paragon Plus Environment

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cells were immersed in 2% (w/v) Alizarin Red S solution at 37°C for 20 min. Finally, samples were rinsed with acidic PBS and observed under a light microscope (Olympus CKS41SF, Olympus, Tokyo, Japan). For immunocytochemistry of an osteogenic marker, cells that were cultured for 21 days were first fixed with 4% (w/v) paraformaldehyde (Sigma) for 15 min and washed twice with PBS. Cells were then permeabilized with Triton X-100 (Sigma) for 5 min and incubated in 2% (v/v) goat blocking serum (Sigma) for 30 min to prevent nonspecific primary antibody binding. The samples were then incubated with mouse monoclonal anti-osteopontin (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. On the next day, cells were treated with secondary antibodies conjugated to fluorescent dyes (Alexa Fluor-488 goat anti-mouse IgG, Invitrogen) for 45 min at room temperature and washed with PBS. Last, nuclei were stained using DAPI, and cells were observed under a ﬂuorescence microscope (Olympus IX 71, Olympus).

2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). qRT-PCR was performed after culturing cells on scaffolds for 21 days to determine osteogenic gene expression levels as previously described.2 Briefly, total RNA was extracted from differentiated cells (n = 3 per group) using an RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA). The concentration of extracted RNA was confirmed by measuring the absorbance of each sample at 260 nm using a spectrophotometer (Nanodrop ND-1000, Thermo Scientific, Waltham, MA). A reverse transcription reaction to synthesize complementary DNA (cDNA) from RNA samples and qRTPCR to quantify the gene expression of each osteogenic marker were conducted according to the protocol from our previous study.1 TaqMan Gene Expression Assays



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(Applied Biosystems) were used for quantifying gene expression profiles of human osteopontin (OPN) (Hs00959010_m1), human RUNX2 (Hs00231692_m1), and human collagen type I (COL I) (Hs00164004_m1). Endogenous reference (human glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Hs02758991_g1) was used to normalize the expression of each sample.

2.7. Transplantation of hADSC-seeded Tendon Scaffolds into a Mouse Model of Calvarial Bone Defect. To confirm the ability of tendon-derived scaffolds to support hADSC-mediated osteogenesis, scaffolds cultured with hADSCs were transplanted into a mouse model of critical-sized bone defect, which was prepared in accordance to a previously described protocol.44 All animal experiments were performed according to the Korean Food and Drug Administration (KFDA) guidelines. Protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei Laboratory Animal Research Center (YLARC) (permit number: 2011-0059). Briefly, undifferentiated hADSCs were seeded at a density of 1.0 × 106 cells/scaffold (n = 6 per each group) onto the non-crosslinked BAT scaffolds and collagen scaffolds (CollaCote, Zimmer Dental, Carlsbad, CA), which were both 4 mm in diameter. Cells on scaffolds were maintained in growth media for 3 days to ensure adhesion onto scaffolds, and then cells were cultured in osteogenic media to induce commitment of hADSCs into the osteogenic lineage for 7 days. When scaffolds with osteogenicallycommitted hADSCs were transplanted into defects of the model (defect size: 4 mm), two pieces of tendon scaffolds each containing 1.0 × 106 cells were placed in 1 defect, and fibrin glue (Greenplast kit, Green Cross Corp., Seoul, Korea) supplemented with hydroxyapatite (HA, 1 mg/ml suspended in fibrin glue) was then applied to the scaffolds to prevent dislocation of scaffolds from defects. For multi-layered


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vascularized bone formation, cells were seeded onto the scaffolds at a density of 1.0 × 106 cells/scaffold for hADSCs and 2.0 × 106 cells/scaffold for hEPCs (n = 8 per each group). Culturing and osteogenic commitment of hADSCs on scaffolds were performed following the protocol described above. hEPCs on scaffolds were maintained in EGM-2 media for 5 days prior to transplantation. For transplanting multi-layered scaffolds, hEPC-containing scaffolds and hADSC-containing scaffolds were alternatively placed into defects. Scaffolds were fixed into defect regions using fibrin glue containing HA (1 mg/ml). Calvarial bone samples were collected 4 or 8 weeks after transplantation and bone regeneration in the defect area was analyzed using a micro-computed tomography (Micro-CT) system (SkyScan-1172, SkyScan, Kontich, Belgium).

2.8. Histology and Immunohistochemistry. Calvarial bone samples were stained using the Goldner’s Trichrome method, as previously described.2 Briefly, newly formed collagen in bone tissue indicated by green staining was quantified as a percent based on the ratio of regenerated bone area to total defect area using Image J software (National Institutes of Health, Bethesda, MD). Immunohistochemistry was performed to observe newly formed bone tissue as well as newly formed vasculature. Sections were immunofluorescently stained with primary anti-OPN (1:100; Santa Cruz Biotechnology) and anti-von Willebrand factor (vWF, 1:200; Abcam, Cambridge, UK). Signals for each marker were indicated by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen). Stained samples were examined using a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany).



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2.9. Statistical Analysis. Statistical data in this study are expressed as average values ± standard deviation. Statistical analyses were performed using an unpaired Student’s t test using Prism software (GraphPad Software Inc., San Diego, CA). Values of p < 0.01 and p < 0.05 were considered statistically significant.

3. Results and Discussion

3.1. Characterization of Tendon-derived Scaffolds. The bioskiving method for fabrication of tendon-derived nanofibrous scaffolds to enhance hADSC-mediated osteogenesis is illustrated in Figure 1. Prepared decellularized tendon-derived scaffolds were characterized in terms of structural features, mechanical properties, and biocompatibility. Because stiff substrates that mimic the mechanical properties of developing bone increase the commitment of osteogenic stem cell differentiation compared to soft substrates,37, 45-47 we increased the mechanical properties of scaffolds, which were derived from bovine Achilles (BAT) and bovine neck (BNT) tendons, by facilitating bond formation between collagen molecules48-49 via crosslinking with either GTA or EDC. The ultrastructural characteristics of bovine tendon scaffolds crosslinked with GTA treatment (BAT-GTA, BNT-GTA) and non-crosslinked (NC) tendon scaffolds (BAT-NC, BNT-NC) (without GTA treatment) were examined by scanning electron microscopy (SEM) (Figure 2A). SEM images of both BAT and BNT scaffolds showed aligned bundle structures consisting of striated collagen nanofibers (Figure 2A), which is observed specifically in natural collagen fibers.49-50 Crosslinking with GTA did not create structural changes in either type of tendon scaffold.


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Next, the mechanical properties of crosslinked BAT scaffolds were evaluated by measuring ultimate tensile strength (UTS) and moduli as a function of various concentrations of crosslinking agents (Figure 2B). The UTS and moduli of BAT scaffolds increased in proportion to increases in crosslinking agent concentrations (GTA and EDC) (Figure 2B). Overall, GTA treatment appeared to offer higher mechanical strength than EDC treatment. Then, the moduli of 2 different types of tendon scaffolds (BAT and BNT) were compared after fixation with either EDC or GTA (Figure 2C). BAT scaffolds crosslinked with EDC or GTA exhibited higher moduli compared to BNT scaffolds treated with the same crosslinking agents (Figure 2C). Although mechanical properties serve as a key factor to determine if a scaffold is appropriate for bone regeneration, biocompatibility is another significant factor to consider. Thus, we quantified the level of TNF-α secretion from RAW 264.7 macrophages cocultured with tendon scaffolds to assess the immunostimulatory effects of scaffolds (Figure 2D). Cells cultured with tendon scaffolds showed higher levels of secreted TNF-α, compared to cells without the scaffolds, but TNF-α levels were significantly lower than TNF-α secretion from cells cultured in the presence of lipopolysaccharide (LPS) (Figure 2D). Crosslinking by GTA treatment did not increase the secretion of TNF-α in both BAT and BNT groups, indicating that the fixation process does not cause additional inflammatory responses.

3.2. Enhanced Focal Adhesion and Osteogenic Differentiation of hADSCs on Tendon-derived Scaffolds. Nanostructured tendon-derived scaffolds with wellaligned collagen fibrils may promote cellular alignment and focal adhesion formation, enabling efficient differentiation of stem cells. Several studies have demonstrated that 13 ACS Paragon Plus Environment


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cytoskeleton rearrangement and focal adhesion development induced by internal structures or surface topographies of substrates and scaffolds could enhance stem cell differentiation into specific lineages.51-54 For example, in our previous studies, it was shown that groove-shaped nanopatterned substrates induced alignment, extension, and rearrangement of F-actin, and significantly promoted focal adhesion formation and differentiation of stem cells into neuronal or osteogenic lineages.51, 53 Other studies also reported surface topography-mediated focal adhesion regulation and enhancement of MSC commitment to the osteogenic lineage during differentiation.5556

Thus, we performed dual-immunostaining of F-actin and vinculin to check

cytoskeleton alignment and focal adhesion of hADSCs grown on BAT and BNT scaffolds. Figure 3A shows the representative images of cellular morphology after 3 days of hADSC culture on BAT and BNT scaffolds with or without GTA treatment. In general, cells grown on GTA-treated scaffolds (BAT-GTA and BNT-GTA) showed reduced focal adhesion compared to cells on non-crosslinked scaffolds (BAT-NC and BNT-NC) (Figure 3A), which may indicate that fixation by GTA treatment impedes cellular adhesion on scaffolds. Non-crosslinked BAT scaffolds showed the greatest focal adhesion of hADSCs among all tested groups. The quantification of focal adhesion by measuring the intensity of vinculin-positive signals in hADSCs confirmed that focal adhesion in cells was much greater on non-crosslinked tendon scaffolds than on GTA-treated scaffolds (Figure 3B). Next, we investigated whether tendon scaffolds could enhance osteogenic differentiation of hADSCs during in vitro osteogenesis. After 21 days of culture under osteogenic media conditions, Alizarin Red S staining indicated greater calcium deposition and mineralization in the non-crosslinked tendon scaffolds (BAT-NC and BNT-NC groups) than in the crosslinked scaffolds (BAT-GTA and BNT-GTA


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groups) (Figure 3C). Immunofluorescent staining of an osteogenic marker, osteopontin (OPN), showed the highest expression of OPN in the BAT-NC group (Figure 3D). qRT-PCR analysis for several osteogenic markers 21 days after osteogenic induction confirmed that mRNA levels of osteogenic markers (RUNX2, OPN, and COL I) were generally higher in the cells differentiated on non-crosslinked tendon scaffolds (BAT-NC and BNT-NC groups) than in cells on crosslinked tendon scaffolds (BAT-GTA and BNT-GTA groups) and on commercially available collagen type I scaffolds (Figure 3E-G). Taken together with focal adhesion data, differentiation studies confirmed that non-crosslinked tendon scaffolds (e.g., BAT-NC scaffolds) with nanofibrous structures are generally more favorable for focal adhesion formation and differentiation of hADSCs into the osteogenic lineage, even though their mechanical properties are low compared to those of crosslinked scaffolds; hence, we decided to use non-crosslinked scaffolds for hADSC transplantation to induce bone formation in vivo. Several studies have elucidated potential mechanisms for promotion of lineage-specific differentiation of stem cells by enhanced focal adhesion.39, 52-54, 57 Regarding osteogenesis of stem cells, Salasznyk et al. revealed that activation of focal adhesion kinase (FAK Y397) signal pathway mediated by ECMs, including collagen I, vitronectin, and laminin, stimulates downstream intracellular signaling cascades (e.g., ERK1/2) and subsequent phosphorylation of the RUNX2/Cbfa-1 transcriptional factor coupled to osteogenic gene expression, which ultimately promotes osteogenic differentiation of human MSCs.39, 57 In the current study, we did not clarified important mediators between increased focal adhesion and enhanced osteogenesis of hADSCs on tendon-derived nanofibrous scaffolds, but similar pathways to previous reports such as ERK1/2 may be involved in enhanced osteogenic differentiation on



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the tendon-derived scaffolds. We need to further elucidate how focal adhesion enhanced by our scaffolds affects stem cell differentiation by conducting inhibition experiments with ERK inhibitors. In addition to enhanced focal adhesion, cytoskeleton organization induced by varied biophysical factors of culture substrates or matrices (i.e., topography, modulus, stiffness) could also be one of the main causes for promotion of stem cell differentiation. Seo et al. reported that micropit surfaces increased actin polymerization and traction forces, ultimately promoting osteogenic differentiation of MSCs.58 They revealed that micropit surface-induced acceleration of osteogenic differentiation was caused by the Rho-associated kinase (ROCK) and non-muscle myosin II by conducting inhibition studies for cytoskeleton organization.58 Thus, cytoskeleton reorganization of hADSCs along the aligned nanofibrous structures in the tendon-derived scaffolds may also contribute to enhanced osteogenesis of hADSCs in our study. Definitely, stem cell differentiation is influenced by various biochemical signals as well as biophysical factors. Biochemical signals provided from surrounding environments such as proteins, peptides, or soluble chemical factors also play significant roles in directing stem cell differentiation into specific lineages. In the context of combined signals of biophysical and biochemical cues, our previous study reported that nanopatterned topographical structures immobilized with bone morphogenetic protein 2 peptides substantially enhanced osteogenic differentiation and calcium deposition of hADSCs during osteogenesis process.51 Our current study suggested the advantages of nanoscale structures over micro-scale structures for enhanced cellular alignment and faster regeneration effects. Several studies have highlighted that the substrates with nano- to submicron-scale topographical structures enhance cellular alignment and subsequently improve


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differentiation and regenerative potential of stem cells.52, 54, 59-60 Interestingly, Watari et al. reported that nanoscale groove-patterned structure with 400 nm pitch was more effective for promoting osteogenic differentiation and mineralization of human MSCs than submicron-scale groove structures with either 1.4 µm or 4 µm pitch.59 Considering that the scale of collagen I fiber, one of the most abundance ECMs in bone tissue, ranges from 10 nm to 300 nm,59 the tendon-derived nanostructured fibrous scaffolds developed in our study can mimic more closely bone-specific ECM environments compared to the scaffolds with microstructures, thus inducing better alignment and increased regenerative potential of hADSCs for osteogenesis both in vitro and in vivo conditions.

3.3. Nanostructured Tendon-derived Scaffolds Promote Bone Regeneration of hADSC Transplantation in a Critical-sized Bone Defect. To demonstrate the potential of stem cell-laden tendon scaffolds for bone repair and regeneration, hADSC-seeded BAT-NC scaffolds were transplanted into a mouse model of calvarial bone defect. For osteogenic commitment of hADSCs, stem cells were maintained on scaffolds under osteogenic media for 7 days prior to transplantation. When the scaffolds cultured with osteogenically-committed hADSCs were transplanted into defects, fibrin hydrogel glue supplemented with hydroxyapatite (HA) particulates, a highly osteoinductive material, was applied to transplanted scaffolds to prevent dislocation of scaffolds from defects. Bone formation in defects was evaluated by micro-CT (Figure 4) and histological analyses (Figure 5) 4 and 8 weeks after hADSC transplantation. Application of tendon-derived scaffolds for hADSC transplantation (BATNC-HA) resulted in significantly enhanced bone regeneration in critical-sized bone



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defects, compared to commercially available control scaffold groups, including fibrin matrices with HA (fibrin-HA) and collagen type I scaffolds supplemented with fibrin glue and HA (collagen-HA). As shown in Figure 4, the no treatment and fibrin-HA groups did not show noticeable calcium deposition or mineralization in the defect area 4 and 8 weeks after transplantation (Figure 4A). On the other hand, the BAT-NC-HA and collagen-HA groups exhibited enhanced bone regeneration in defects on week 4 compared to no treatment and fibrin-HA groups, and tissue formation was further increased in BAT-NC-HA and collagen-HA groups 8 weeks after transplantation (Figure 4A). In particular, the defect was almost closed in the BAT-NC-HA group by reaching a bone regeneration rate up to 86%, while the collagen-HA group showed a bone regeneration level of only 52% (Figure 4B). Enhanced bone formation using BAT-NC scaffolds for hADSC-mediated osteogenesis was also confirmed by histological and immunohistochemical analyses (Figure 5). Goldner’s Trichrome staining indicated that the level of bone-specific collagen regeneration in defects was relatively higher on week 8 than on week 4 (Figure 5A, B). The BAT-NC-HA group showed the highest collagen formation, compared to control groups, including the collagen-HA scaffold group (Figure 5B, C). Immunohistochemistry of the osteogenic marker (OPN) indicated higher osteogenic differentiation of hADSCs in the BAT-NC-HA group than in other control groups on both 4 and 8 weeks after transplantation (Figure 5D).

3.4. Multi-layered Vascularized Bone Formation of hADSC Transplantation by Stacked Tendon-derived Scaffolds in a Critical-sized Bone Defect. To investigate the applicability of tendon scaffolds for generation of multiple tissue-like constructs, tendon scaffolds seeded with 2 different types of stem cells were applied into multiple


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layers in bone defects. BAT-NC scaffolds seeded with osteogenically-committed hADSCs and human cord blood-derived EPCs (hEPCs) were stacked layer-by-layer for multi-layered, vascularized bone formation. As a critical step for fine bone regeneration is vascularization that precedes bone formation,61 we expected that stacking of osteogenically-committed hADSC-seeded BAT-NC scaffolds (B) with hEPC-seeded BAT-NC scaffolds (E) would enhance bone formation in the defect area. Eight weeks after transplantation, alternatively-stacked hADSC-seeded BAT-NC scaffolds (B) and hEPC-seeded BAT-NC scaffolds (E) in 3 or 5 layers resulted in greater mineralization and bone formation, as shown in micro-CT images (Figure 6A). Quantification of mineralized bone formation indicated that the no treatment group showed the lowest regeneration level of 19%, while the BAT-NC scaffold alone, 3 B layers (B/B/B), 3 alternative layers of B, E, and B (B/E/B), and 5 alternative layers of B, E, B, E, and B (B/E/B/E/B) groups showed enhanced bone regeneration by 66%, 84%, 87%, and 98%, respectively (Figure 6B). Interestingly, using only tendon scaffolds (3 stacked layers of BAT scaffolds) without stem cell seeding also resulted in a relatively high level of bone regeneration compared to no treatment (Figure 6A, B), indicating a high osteogenic potential of the tendon scaffolds. This may also result from the use of fibrin glue with angiogenic potential and highly osteoinductive inorganic HA particulates, which may contribute to vascularization as well as mineralization in the bone defect. Histological analysis also revealed the effectiveness of using alternative layers of hADSC-seeded scaffolds and hEPC-seeded scaffolds in bone formation (Figure 7). While the no treatment group showed scar formation in the defect area, bone-specific collagen formation was significantly increased in other groups treated with BAT-NC scaffolds (Figure 7A). In particular, the application of alternative scaffold layers of



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hADSCs and hEPCs contributed to more extensive, compact bone formation with collagen regeneration levels of 52% (B/E/B) and 67% (B/E/B/E/B) (Figure 7A, B). Immunofluorescence staining using an osteogenic marker (OPN) and endothelial marker (vWF) revealed bone regeneration with vascularized tissue layers in the B/E/B and B/E/B/E/B groups (Figure 7C, D). These results indicated that vascularization is critical for efficient bone formation, and tendon-derived scaffolds can support regeneration of bone tissue-like structures with vascularized tissue layers.

4. Conclusions

In summary, our study suggests that decellularized tendon-derived scaffolds that possess natural nanofiber structural features can provide favorable, advantageous microenvironments for enhancing osteogenic differentiation of hADSCs both in vitro and in vivo. The bioskiving method allows for easy, reliable scaffold fabrication with well-preserved ECM structures that can guide osteogenic differentiation of hADSCs. Transplantation of hADSCs with nanostructured tendon scaffolds significantly improved bone regeneration in the mouse model of critical-sized calvarial bone defects, compared to a commercial collagen scaffold, and multi-layered stacking of osteogenically-committed hADSC- and hEPC-cultured scaffolds further enhanced regeneration of bone tissue with improved vascularization. Nanostructured tendon scaffolds may be applicable for engineering other types of tissues, such as blood vessel, skin, muscle, or nerve tissue. For example, thin sheet types of tendon scaffolds are high tunable, such that they can be rolled to form a tubular structure or stacked to form multi-layered structures that incorporate various cell types. Therefore,


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decellularized tendon scaffolds can provide a versatile biomaterial platform to reconstruct diverse tissues or organs.

Acknowledgments

This work was supported by a grant (2009-0083522) from the Translational Research Center for Protein Function Control (TRCP) funded by the Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea. This work was also supported by a grant (NRF-2013R1A1A2A10061422) from the National Research Foundation of Korea (NRF). Q.X. acknowledges the NIH grant (1R03EB017402-01) and K.A. acknowledges the IGERT fellowship from NSF.



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Figure 1. Schematic illustration to describe the fabrication of nanofibrous tendonderived scaffolds and hADSC transplantation using fabricated tendon scaffolds in a critical-sized calvarial bone defect.


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Figure 2. Characterization of tendon-derived scaffolds. (A) SEM images of bovine Achilles tendon (BAT) and bovine neck tendon (BNT) with or without fixation using glutaraldehyde (GTA). (B) Changes in mechanical properties (ultimate tensile strength and modulus) depending on varying concentrations of different fixatives (EDC, GTA) (**, p < 0.01 versus 1 mM EDC, ##, p < 0.01 versus 0.20% GTA). (C) Moduli of BAT and BNT sections (thickness; 50 µm) with or without fixation (*, p < 0.05 versus untreated BNT, **, p < 0.01 versus untreated BAT, ##, p < 0.01 versus BNT-EDC). (D) Evaluation of the immunostimulatory effect of tendon scaffolds, using ELISA to measure secreted TNF-α from macrophages (cell line: RAW 264.7) cultured with scaffolds (**, p < 0.01 versus control, ##, p < 0.01 versus LPS).



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Figure 3. Focal adhesion and osteogenic differentiation of hADSCs on tendon scaffolds in vitro. (A) Staining for focal adhesion (vinculin; green) and alignment of cytoskeleton (F-actin; red) of hADSCs on BAT and BNT scaffolds with or without GTA fixation after 3 days of culture in growth media. (B) Quantification of focal adhesion in hADSCs on each scaffold by measuring the relative intensity of vinculinpositive signals in cells (n = 6~8, ##, p < 0.01 versus BAT-GTA group, ++, p < 0.01 versus BNT-GTA group). (C) Alizarin Red S staining of hADSCs differentiated on tendon scaffolds under osteogenic media conditions for 21 days. (D) Immunofluorescence staining for osteopontin (OPN) of hADSCs on tendon scaffolds after osteogenic differentiation for 21 days. qRT-PCR analysis for osteogenic markers


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(E) RUNX2, (F) OPN, and (G) collagen type I (COL I) to analyze the osteogenic differentiation of hADSCs on BAT-GTA, BAT-NC, BNT-GTA, and BNT-NC scaffolds for 21 days (n = 3, *, p < 0.05, **, p < 0.01 versus collagen group).



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Figure 4. Micro-CT analysis of the calvarial bones retrieved 4 and 8 weeks after hADSC transplantation using tendon scaffolds (BAT-NC-HA scaffolds) into a mouse model of critical-sized bone defect. (A) Micro-CT images of retrieved calvarial bones in each group (no treatment, fibrin-HA, collagen-HA, and BAT-NC-HA) to examine mineralized bone formation in defects. (B) Quantification of bone regeneration in each group measured from micro-CT images. Bone regeneration is presented as a percentage ratio of regenerated bone area to total defect area [*, p < 0.05, **, p < 0.01 versus no treatment (4 weeks), #, p < 0.05, ##, p < 0.01 versus no treatment (8 weeks), ++, p < 0.01 versus fibrin-HA (4 weeks), ||, p < 0.01 versus fibrin-HA (8 weeks) , §, p < 0.05 versus collagen-HA (4 weeks), $$, p < 0.01 versus collagen-HA (8 weeks)].


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Figure 5. Histological and immunohistochemical staining of calvarial bones 4 and 8 weeks after transplantation of osteogenically-committed hADSCs. Goldner’s Trichrome staining of retrieved calvarial bone samples (A) 4 weeks and (B) 8 weeks after hADSC transplantation using tendon scaffolds (BAT-NC-HA). Black arrowheads indicate the defected region. (C) The percentage area of collagen regenerated in the defect site, which was quantified from Goldner’s Trichrome27 ACS Paragon Plus Environment


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stained images (n = 5) [**, p < 0.01 versus no treatment (4 weeks), ##, p < 0.01 versus no treatment (8 weeks), ++, p < 0.01 versus fibrin-HA (4 weeks), ||, p < 0.01 versus fibrin-HA (8 weeks), §, p < 0.05 versus collagen-HA (4 weeks), $$, p < 0.01 versus collagen-HA (8 weeks)]. (D) Immunohistochemistry for detection of OPN expression in calvarial bone tissues retrieved 4 and 8 weeks after osteogenicallycommitted hADSC transplantation with BAT-NC-HA scaffolds. White arrowheads indicate the defect site.


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Figure 6. Micro-CT analysis of calvarial bones 8 weeks after transplantation of multiple-layered BAT-NC scaffolds seeded with osteogenically-committed hADSCs (B) or hEPCs (E). (A) Micro-CT images of calvarial bone samples in each group [no treatment, BAT-NC only, BAT-NC (B/B/B), BAT-NC (B/E/B), BAT-NC (B/E/B/E/B)]. (B) Quantification of regenerated bone tissue in the defect 8 weeks after transplantation [**, p < 0.01 versus no treatment, #, p < 0.05 versus BAT-NC only, +, p < 0.05 versus BAT-NC (B/B/B)].



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Figure 7. Histological and immunohistochemical staining of calvarial bones 8 weeks after transplantation of multiple-layered BAT-NC scaffolds seeded with osteogenically-committed hADSCs (B) or hEPCs (E). (A) Goldner’s Trichrome staining of retrieved calvarial bone tissues to detect newly formed collagen in the defects. The black arrowheads indicate the defect region. (B) The percentage area of newly formed collagen tissue in the defect area (n = 6) [**, p < 0.01 versus no treatment, #, p < 0.05, ##, p < 0.01 versus BAT-NC only, ++, p < 0.01 versus BATNC (B/B/B), ||, p < 0.01 versus BAT-NC (B/E/B)]. (C) Immunofluorescence staining to detect the expression of an osteogenic marker (OPN; green) in calvarial bone tissues 8 weeks after transplantation. The white arrowheads indicate the defect area. (D) Immunofluorescence staining of calvarial bone tissues (B/E/B group and B/E/B/E/B group) to detect expression of an endothelial marker (vWF; red) 8 weeks after transplantation. The white arrows indicate the newly formed blood vessels.


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