Skeletal Muscle Patch Engineering on Synthetic and Acellular Human Skeletal Muscle Originated Scaffolds Birol Ay1,2,*, Erdal Karaoz1,2,**, Cumhur C. Kesemenli3, Halime Kenar1,4,5 1

Kocaeli University, Institute of Health Sciences, Stem Cell Department, Kocaeli, Turkey

2

Kocaeli University, Center for Stem Cell and Gene Therapies Research and Practice, Kocaeli,

Turkey 3

Kocaeli University, Faculty of Medicine, Department of Orthopedics and Traumatology,

Kocaeli, Turkey 4

BIOMATEN Center of Excellence in Biomaterials and Tissue Engineering, METU, Ankara,

Turkey 5

Kocaeli University, Experimental and Clinical Research Center, Kocaeli, Turkey

*Current Address: University of Toronto, Institute of Biomaterials and Biomedical Engineering, Toronto, ON, Canada ** Current Address: Liv Hospital Center for Regenerative Medicine and Stem Cell Research and Manufacturing, Đstanbul, Turkey

Short Running Title: Tissue engineered human skeletal muscle patch Corresponding author: Halime Kenar, Kocaeli University, Experimental and Clinical Research Center, +90 2623038423, [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35948 This article is protected by copyright. All rights reserved.

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Abstract The reconstruction of skeletal muscle tissue is currently performed by transplanting a muscle tissue graft from local or distant sites of the patient`s body, but this practice leads to donor site morbidity in case of large defects. With the aim of providing an alternative treatment approach, skeletal muscle tissue formation potential of human myoblasts and human menstrual blood derived mesenchymal stem cells (hMB-MSCs) on synthetic (poly(L-lactide-co-caprolactone), 70:30) scaffolds with oriented microfibers, human muscle extracellular matrix (ECM), and their hybrids was investigated in this study. The reactive muscle ECM pieces were chemically crosslinked to the synthetic scaffolds to produce the hybrids. Cell proliferation assay WST-1, Scanning Electron Microscopy (SEM), and immunostaining were carried out after culturing the cells on the scaffolds. The ECM and the synthetic scaffolds were effective in promoting spontaneous myotube formation from human myoblasts. Anisotropic muscle patch formation was more successful when human myoblasts were grown on the synthetic scaffolds. Nonetheless, spontaneous differentiation could not be induced in hMB-MSCs on any type of the scaffolds. Human myoblast-synthetic scaffold combination is promising as a skeletal muscle patch, and can be improved further to serve as a fast integrating functional patch by introducing vascular and neuronal networks to the structure.

KEYWORDS: myoblasts; mesenchymal stem cells; oriented-microfiber scaffolds; extracellular matrix scaffolds; skeletal muscle patch

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1. Introduction Restoration of skeletal muscle tissue due to loss by traumatic injury or tumor excision ismanaged by transplantation of an autograft from local or distant sites of the body. This traditional method causes loss of healthy muscle tissue from the donor site, while trying to heal the main defect(1). Therefore, this method does not represent an efficient clinical treatment. Skeletal muscle tissue engineering aims at producing functional skeletal muscle patches in vitro for tissue replacement, in order to provide a potential therapeutic solution to this unmet medical problem(2). Since the last decade, skeletal muscle patch development by tissue engineering methods has been studied progressively. Early studies necessarily focused on identifying the growth factors and hormones effective in differentiation of stem cells into skeletal muscle cells in 2-dimensional (2D) tissue culture systems(3),(4). Since the 2-D tissue culture systems do not provide an effective means of treatment for the patients, 3-dimensional (3D) culture techniques have been developed. In this context, most of the 3D cultures of mesenchymal stem cells and myoblasts have been performed using synthetic polymers(5),(6), gel-based matrices(7), and biologic scaffolds(8),(9) as carriers. Moreover, synthetic and natural polymer blends, such as PCL/collagen, have been used to produce nanofibrous scaffolds to improve scaffold features(10). Since myotube formation is the hallmark of muscle tissue regeneration, Insulin-like growth factor 1 (IGF-1) gene was transferred to the myoblasts to enhance myotube formation(11). Furthermore, the role of endothelial cells in myogenesis was demonstrated by co-culture of myoblasts with endothelial cells(12). Integration ofa physical stimulus, such as electrical or mechanical, to the cell culture environment is necessary to improve cellular functionality. In vitro mechanical stimulation of myoblasts on acellular biological scaffolds improved the contractility of engineered skeletal muscle after in vivo implantation(13),(14). In another study, a porous membrane-based cell culture device was used

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to electrically stimulate a confluent monolayer of mouse myoblast cell line (C2C12) derived myotubes. It was demonstrated that a continuous stimulation considerably increased both the number of stimuli-responding myotubes and the magnitude of the contraction(15). As a means to simultaneously provide topographical and electrical cues, electrically conductive nanofibers with highly oriented structures were produced by electrospinning for stimulation of C2C12 mouse myoblasts and found to be effective in enhancing myotube maturation(16). It was reported in another study that polyethylene glycol-linked multi-walled carbon nanotubes (PEG-CNT), which have nanoscale surface roughness and an orderly arranged structure, increased myogenic differentiation of human bone marrow derived MSCs(17). In addition to human bone marrow and adipose tissue derived MSCs, hMB-MSCs may have a potential use in skeletal muscle tissue engineering. It was demonstrated that when induced with specific chemicals and growth factors, these cells can differentiate into skeletal myocytes(18). It was also shown that hMB-MSCs can efficiently transdifferentiate into myoblasts/myocytes in the coculture with C2C12 mouse myoblasts and can express human dystrophin. In the same study, hMB-MSCs could restore the sarcolemmal expression of dystrophin by fusing to the host myotubes following their injection into the dystrophic muscles of Duchenne muscular dystrophy (DMD) model mdx-scid mice(19). Since vascularization is crucial for graft survival, 3D vascularization of scaffolds is highly required for functional integration(20). Improved vascular organization of skeletal muscle patches was demonstrated with triculture system of endothelial cells (ECs), myoblasts, and foreskin fibroblasts on biodegradable scaffolds(21). Even though these strategies can provide several significantly important features, the above mentioned 3D constructs do not possess the extracellular matrix proteins and mechanical properties of the natural muscle tissue(22). Several 4 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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studies aimed implantation of the decellularized skeletal muscle pieces into the recipients on the purpose of reorganization, but complete integration of these 3D ECM scaffolds could not be achieved(23),(24). There is significant number of attempts to produce functional skeletal muscle substitutes in the literature, yet a commercially available tissue engineered skeletal muscle patch suitable for human use is still not present. Therefore, the main goal of this study was to investigate the possibility of using human muscle extracellular matrix alone or in combination with synthetic scaffolds as a carrier for human myoblasts and human menstrual blood derived mesenchymal stem cells (hMB-MSCs) in the context of developing a skeletal muscle patch. Adhesion, growth, and myotube formation potential of human myoblasts and hMB-MSCs on the aforementioned scaffolds was studied. Mitochondrial activity of the cells on different types of scaffolds wasassessed after 21 days of culture. Immunohistochemical staining and SEM analysis of the cell-laden scaffolds were performed to reveal the best cell-matrix combination for production of an aligned (anisotropic) human skeletal muscle patch.

2. Materials and Methods 2.1. Preparation and characterisation of the scaffolds 2.1.1. ECM Scaffolds. Ethical approval for collection of muscle pieces was obtained from the Ethical Committee for Clinical Researches of Kocaeli University. During orthopedic surgery, non-pathologic and disposed human muscle pieces were obtained. The muscle pieces were collected into phosphate-buffered saline (PBS) with 1% penicillin/streptomycin (Gibco, Invitrogen, CA, USA) in falcon tubes. The tubes were incubated in a shaker at room temperature (RT) overnight. After rinsing in PBS, the muscle pieces were placed in 10 ml of 1% sodium

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dodecyl sulfate (SDS) (Sigma-Aldrich, Switzerland) in PBS and agitated in the shaker for two days at RT. The SDS solution was changed twice a day. After this decellularization step, the muscle pieces were transferred into 1% Triton X-100 (Merck Milipore, MA, USA) and agitated in the shaker overnight. The remaining extracellular matrix (ECM) was washed in a large volume of PBS with antibiotics at least twice a day to wash away the cellular components and detergents. The resulting muscle ECM was sectioned and stained with Hematoxylin and Eosin (H&E) and Masson’s trichrome to verify complete decellularization and the preservation of the collagen structure. The decellularized skeletal muscle pieces were also sectioned in 5 µm thickness with a cryomicrotome (Leica, CM1850, Germany), and the cryosections were dried on glass slides and used as ECM scaffolds. 2.1.2. Synthetic scaffolds. Poly(L-lactide-co-caprolactone) (70:30, Purac, Purasorb PLC 7015, GMP grade, used for medical device applications) copolymer was dissolved in chloroform:N,Ndimethyl formamide (DMF) (95:5 v/v)) to obtain a 15% (w/v) polymer solution. For the process of electrospinning into aligned microfibers, the polymer solution was placed in a 10 ml plastic syringe with a tip diameter of 1 mm. The home-made electrospinning setup used in this study consisted of a high voltage supply (Gamma High Voltage Research, FL, USA), a syringe pump (New Era Pump Systems, NE 1800), a 10 mL syringe capped with a blunt end needle, and a grounded copper frame collector. The electrospinning process was applied under optimized conditions given below to obtain minimum fiber fusion and minimum bead formation. The voltage applied between the needle tip and collector was 15 kV. The needle was placed at a distance of 17.5 cm from the grounded copper frame collector. The flow rate was kept constant at 30 µl/min.

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The morphology, orientation, and diameter of the aligned microfibers (synthetic scaffolds) were determined by using a scanning electron microscope (JEOL JSM-6060, Japan). The fiber diameters were presented as average ± standard deviation. Mechanical properties of the synthetic scaffolds were determined by using a mechanical analysis device (Tinius Olsen H5KT, PA, USA) according to the standart ASTM D882 in a waterbath at 370C with 25.4 mm/min draw ratio.

2.1.3. Hybrid scaffolds. The ECM scaffolds were placed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution and homogenized in the gentleMACS Dissociator (Miltenyi Biotec, Germany) at the program `protein_01` for 3.5 min. Thereafter, homogenized solution was filtered through 70-µm cell strainer (BD Bioscience, USA) to get a size cut off for the tissue pieces. The filtered homogenate was mixed with 1% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Thermo Scientific, IL, USA) and 0.2M N-Hydroxysuccinimide (NHS) (Thermo Scientific, IL, USA) solutions, and agitated gently in the shaker for 15 min at RT to obtain the NHS-EDCmuscle protein linkages. After 15 min incubation, 0.14% 2-mercaptoethanol (at a final concentration of 0.14%) was added to the mixture to block this reaction cascade. By this way, semi-stable amine reactive NHS-esters were obtained on the muscle ECM proteins. Meanwhile, the aligned microfibrous synthetic scaffolds (1x1 cm2) previously produced by electrospinning were treated with 1% 1,6-hexanediamine (Sigma-Aldrich, Missouri, USA) in 2-propanol (SigmaAldrich, Missouri, USA) for 3 min at 37oC to form amine groups on the fibers. Afterwards, the suspension of reactive muscle ECM pieces and synthetic scaffolds were combined and agitated gently in the shaker for 16 hours at RT to produce the hybrid scaffolds. At the end of this incubation period, uncrosslinked muscle proteins were washed away, and the unreacted amine

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reactive groups on the hybrid scaffolds were blocked by incubation in 1M glycine (SigmaAldrich, Missouri, USA) for 1 hour at RT. The hybrid scaffolds were observed by SEM and also stained with Commassie Brillant Blue (Merck, MA, USA) as a supporting method to prove the crosslinking of muscle proteins.

2.2. Isolation of hMB-MSCs and human myoblasts 2.2.1. hMB-MSCs. Ethical approval for menstrual blood collection was obtained from the Ethical Committee for Clinical Researches of Kocaeli University. Menstrual blood samples were collected from volunteers at the second day of the menstruation and mixed with high-glucose DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) (Gibco, Invitrogen, CA, USA). The mixture was filtered through a cell strainer (pore size: 70 µm, BD Bioscience, USA). The filtrate containing the hMB-MSCs was transferred into T25 tissue culture polystyrene flasks (BD Bioscience, CA, USA) and maintained at 37°C in a humidified atmosphere containing 5% CO2 for 48 h to allow cell attachment. Nonadherent cells were removed by renewal of the culture medium. When the culture reached 70% confluence, the cells were harvested with 0.25% trypsin and 1 mM Ethylene diamine tetra acetic acid (EDTA) (Gibco, Invitrogen, CA, USA) and passaged. 2.2.2. Human myoblasts. Ethical approval for collection of muscle pieces was obtained from the Ethical Committee for Clinical Researches of Kocaeli University. Non-pathologic and disposed muscle pieces were taken from human quadriceps muscle during hip-joint implant surgery. The muscle pieces were (ca. 3x3x3 mm3 in size) obtained from adults independent of sex and age. All muscle pieces were transported in low-glucose DMEM with 1% penicillin/streptomycin and stored at RT until processing within 3 h after surgery. The muscle pieces were first rinsed in PBS

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and cleaned by removing adherent fat tissues by a scalpel. The pieces were then minced in smaller parts of 1x1x1 mm3 and digested in a 10 mL of 0.1% collagenase Type I (w/v) (Gibco, Invitrogen, CA, USA) and 0.1% dispase (w/v) (Gibco, Invitrogen, CA, USA) enzyme cocktail in Hank's Buffered Salt Solution (HBSS) for 2 h at 37°C. After digestion, the solution was filtered through a strainer with a pore size of 100 µm and centrifuged at 1500 rpm for 5 min. The remainder of the enzymes was removed through repetitive washes with low-glucose DMEM medium. Thereafter, the pellet was resuspended in the growth medium composed of Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12) (Gibco, Invitrogen, CA, USA) medium with 20% FBS, 2.5 ng/mL human basic Fibroblast Growth Factor (bFGF), 10 ng/mL human Epithelial Growth Factor (hEGF), and 0.1% (v/v) Primocin (Invivogen, CA, USA). Resuspended cells were precultured in 1% (w/v) gelatin (BD, Clontech, MA, USA) coated T25 flasks. After 1 h of preculture, the medium of preculture flask that contained the human myoblasts was transferred into uncoated T25 tissue culture flasks and incubated at 37°C in a humidified atmosphere containing 5% CO2.

2.3. Characterization of hMB-MSCs and human myoblasts Immunophenotypic characterization of the cells was carried out with flow cytometry to analyze MSC-specific markers. Flow cytometric analyses were performed with undifferentiated hMBMSCs and human myoblasts at the passage numbers of 3 to 5. The cells were harvested and resuspended in their own culture medium at a concentration of 1×106cells/mL. The cytometric data were analyzed with CellQuest software (BD Biosciences, CA, USA), and the forward and side scatter profiles were gated out of debris and dead cells. Immunophenotyping of hMB-MSCs and human myoblasts was performed with antibodies against the following antigens: CD29

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(Integrin β1 chain; PE), CD34 (Hematopoietic Progenitor Cell Antigen; PE), CD44 (hyaluronate/lymphocyte homing-associated cell adhesion molecule-HCAM; PE), CD45 (Protein tyrosine phosphatase receptor type C, PTPRC; FITC), CD90 (Thy-1/Thy-1.1; FITC), CD146 (melanoma cell adhesion molecule, MCAM; PE), HLA ABC (MHC class I antigen receptor; PE), and HLA-DR (MHC class II; FITC). All of the antibodies were purchased from BD Biosciences. More than 10% staining was considered as positive. To demonstrate the adipogenic and osteogenic differentiation capability of hMB-MSCs, the cells were chemically induced and stained with Oil Red O and Alizarin Red (Sigma-Aldrich, Missouri, USA). Osteogenic differentiation was induced with 10mM Glycerol 2-phosphate, 50 µg/ml ascorbate-2-phosphate, 10 nM dexamethasone, 10% FBS, and 0.1% Primosin in Minimum Essential Medium (MEM) for 4 weeks. The Alizarin Red staining, adipogenic differentiation, and the Oil Red O staining were performed as previously described elsewhere (25). The human myoblasts were analyzed for the expression of muscle specific proteins, such as desmin and myoD. Immunocytochemical staining was carried out using the streptavidin– peroxidase method (UltraVision Plus Large Volume Detection System Anti-Polyvalent, HRP immunostaining Kit; Thermo Scientific, UK) to reveal the desmin positive myoblasts. Briefly, cultured cells were fixed in ice-cold methanol with 0.3% hydrogen peroxide for 15 min and allowed to dry. After washing with phosphate buffer saline (PBS), the cells were incubated with Ultra V Block for 5 min at room temperature, then, with the primary antibody for desmin (Santa Cruz Biotechnology sc-14026) overnight at 4oC. The following day, cells were incubated with the biotinylated secondary antibody for 15 min at room temperature. Then, streptavidin peroxidase treatment was carried out for 15 min at room temperature. Signal was detected with the AEC kit (Zymed Laboratories/Invitrogen, Carlsbad, CA). After being counter-stained with

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hematoxylin (Santa Cruz Biotechnology), cells were examined under a light microscope. Myotubes formed by the myoblasts in the culture dish were immunostained for α-actinin and also stained with phalloidin to visualize their actin filaments. Immunofluorescent staining of myoblasts for MyoD and myotubes for α-actinin was performed as described previously (25). For staining with Phalloidin, the cultured cells were fixed with cold methanol for 10 min and permeabilized with 0.1% Triton-X 100 in PBS for 5 min. The samples were then incubated for 1 h in Alexa Fluor® 488 Phalloidin (Thermo Scientific, IL, USA) (1:100) at 370C. Nuclei were counterstained using 40,6-diamidino-2-phenylindole (DAPI).

2.4. Scaffold Disinfection Synthetic scaffolds were placed into the petri dishes and ultraviolet (UV-C) light was applied to both sides of synthetic scaffolds for 15 min in a biological safety cabinet (HERA Safe, Germany).

The

ECM

scaffolds

were

incubated

within

PBS

containing

penicillin/streptomycin for disinfection during the decellularization process.

1%

Since both

synthetic scaffolds and muscle pieces used to produce hybrid scaffolds were disinfected during processing, the hybrid scaffolds were not disinfected furthermore.

2.5. Cell culture on the scaffolds Prepolymer of the Poly(dimethyl siloxane) (PDMS) (Dow Corning Sylgard 184, MI, USA) was mixed with its catalyst and poured into the wells of 12-well plates. The plates were exposed to vacuum until all the air bubbles were removed and were then incubated at 37oC overnight for polymerization of the PDMS. The synthetic and hybrid scaffolds were strained on the PDMS

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surface through sterile needles to keep their fibers aligned. Meanwhile, ECM scaffolds on the glass slides were directly placed into 12-well plates. The cells were resuspended in DMEM/F12 medium with 10% FBS and 0.1% (v/v) primocin and seeded on the scaffolds at a density of 2x104 cells/cm2, and the cell-laden scaffolds were incubated for 24 hours in a humidified incubator to allow cell attachment. Afterwards, the media were changed with low-glucose DMEM with 10% FBS and 0.1% primocin for hMB-MSCs and high-glucose DMEM with 10% FBS and 0.1% primocin for human myoblasts to promote ECM-induced differentiation of these two different cell types. The media were changed twice a week during incubation.

2.6. Mitochondrial activity and myotube formation on the scaffolds 2.6.1. WST-1 assay. The mitochondrial activity of hMB-MSCs and human myoblasts on the scaffolds were assessed with cell proliferation reagent WST-1 (Roche, Germany) on day 21 of incubation. The scaffolds (n=3, for each type) were rinsed two times with low-glucose DMEM with 0.1% primocin and incubated for 2 h in a 10% WST-1 solution (v/v) at 37°C in a humidified atmosphere with 5% CO2. Absorbance of the incubation medium was measured at 480 nm by a UV–visible spectrophotometer (VersaMax, Molecular Devices, CA, USA). The WST-1 assay was also applied to the synthetic scaffolds without cells, which served as controls. 2.6.2. Immunofluorescence staining. hMB-MSC and human myoblasts cultured on ECM scaffolds for 3 weeks were immunostained for desmin to demonstrate the myotube formation. Samples were rinsed briefly in PBS and fixed in 4% paraformaldehyde (PFA) (Merck Milipore, Germany) for 15 min at RT. After washing three times with PBS, the samples were blocked with goat serum (Santa Cruz Biotechnology, Texas, USA,3:200 in PBS) for 30 min at RT. The cells were labeled with a primary antibody for Desmin (Santa Cruz Biotechnology, Texas, USA)

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(1:100) for 1 h at 37°C followed by incubation with goat anti rabbit IgG-FITC (Santa Cruz Biotechnology, Texas, USA) (1:100) for 45 min at RT. Digital images were acquired under a fluorescence microscope (Leica DMI 4000B, Germany). Immunofluorescence staining was also performed to visualize the myotube formation of human myoblasts on synthetic and hybrid scaffolds. After a culture of 14 days, cells were rinsed briefly in PBS and fixed in 4% PFA for 15 min at RT. Thereafter, samples were rinsed three times in PBS to remove the residual PFA and incubated with goat serum (Santa Cruz Biotechnology, Texas, USA,3:200 in PBS) for 30 min at RT to suppress non-specific binding. After blocking, cells were immunostained with anti-α actinin (Sigma-Aldrich, Missouri, USA) (1:800) for 1 h at 37°C. Immunofluorescence labeling was completed using goat anti mouse IgG (Thermo Scientific, IL, USA) (1:100) as a secondary antibody for 45 min at RT. 2.6.3. Scanning electron microscopy (SEM) analysis The morphology of hMB-MSCs and human myoblasts on the scaffolds was observed by SEM at day 21. The samples were fixed with 2.5% glutaraldehyde (Merck Milipore, Germany) for 20 min and dehydrated in ethanol series (i.e., 35%, 50%, 70%, 85%, 95% and 100%) for 15 min each. After becoming completely dry, the samples were coated with gold using a table-top sputter coater (Bal-tec SCD005) under 21 mA current for 90 sec. and examined in a JEOL JSM6060 SEM.

2.7. Statistical analysis Statistical analysis was performed to evaluate the statistical significance of mitochondrial activity on different types of scaffolds on day 21. Minitab 13 (Minitab Company, USA) was used

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to perform one-way analysis of variance (ANOVA), followed by Tukey’s test to determine the significant differences among the groups at significance level of p < 0.05.

3. Results 3.1. Phenotypic characteristics of hMB-MSCs and human myoblasts Both hMB-MSCs and human myoblasts had a fibroblastic morphology and formed colonies in their primary culture. In the upcoming passages, human myoblasts demonstrated higher proliferation rate compared to hMB-MSCs in the culture. Flow cytometric analyses revealed that both hMB-MSCs and myoblasts remarkably expressed MSC characteristic surface antigens, such as CD29 (Integrin beta-1), CD44 (Hyaluronate/lymphocyte homing-associated cell adhesion molecule-HCAM), CD90 (Thy-1/Thy-1.1), and HLA-ABC (major histocompatibility complex, MHC class I, cell surface receptor for cytotoxic T-cells). Nonetheless, they did not display significant expression of CD34 (Hematopoietic Progenitor Cell Antigen) or CD45 (leucocyte common antigen/cell marker of hematopoietic origin). 42% of the myoblasts expressed HLA-DR (major histocompatibility complex, MHC class II, cell surface receptor) (Fig. 1A, F). The absence of HLA-DR in hMB-MSCs in this study implies that these cells will be less prone to immune rejection in case of allogenic transplantation, because it is known that HLA-DR antigens are responsible for triggering humoral immune response(26). CD 146 (melanoma cell adhesion molecule; MCAM/a marker for endothelial cell lineage) was expressed by almost 80% of the hMB-MSCs indicating their pericytic origin(27) while the myoblasts were negative for this surface marker. Furthermore, since The International Society for Cellular Therapy (ISCT) suggests that one of the main characteristics of MSCs is the ability to differentiate into different

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cell types(28), differentiation into osteogenic and adipogenic cells confirmed the mesenchymal stem cell characteristics of hMB-MSCs (Fig. 1B-E). The flow cytometric analysis indicated that the myoblast population isolated by enzymatic digestion contains cells with multipotent differentiation capabilities as shown before by other researchers(29)(Fig. 1F). Moreover, immunostaining of desmin and myoD revealed that isolated human myoblast population also contains cells that express muscle specific proteins (Fig. 1G, H). Furthermore, myotubes formed by the human myoblasts in the culture dish were visualized with anti-α actinin and phalloidin staining (Fig. 1I, J). Thus, immunostaining and flow cytometry results indicated that human myoblast population, used in this study, contains muscle protein expressing myoblasts as well as multi-potential MSCs. 3.2. Physical characteristics of the scaffolds 3.2.1. ECM Scaffolds. Natural skeletal muscle ECM contains collagen Type I and III, and conservation of collagen structure is the key event for stem cell differentiation on ECM scaffolds(30). ECM scaffolds were produced in this study to reveal the effects of muscle proteins and conserved collagen matrix on stem cell differentiation. Masson’s trichrome staining of the decellularized samples demonstrated that collagen was conserved at the dark blue stained areas (Fig. 2F). Moreover, preventing immune rejection is an important consideration to obtain a desired tissue integration upon transplanting the scaffold(31). Removal of donor cells from the tissue is a way of considerable reduction in immune rejection. Here, donor cells were removed from human muscle pieces, which became yellowish after the detergent based decellularization process (Fig. 2B). Hematoxylin and eosin staining after the decellularization process confirmed the absence of cell nuclei meaning that muscle pieces were successfully cleaned off the donor`s cells (Fig. 2D).

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It was hypothesized in this study that the ECM scaffolds would be the best choice for induction of stem cell differentiation due to their inborn biological cues. Human myoblasts cultured on the ECM scaffolds for 3 weeks were able to fuse with each other and form the myotubes, while only several desmin positive single cells were accounted among the hMB-MSCs cultured under the same conditions (Fig. 3). 3.2.2. Synthetic scaffolds. PLC (poly(L-lactide-co-caprolactone) copolymer based scaffolds are frequently used in tissue engineering applications(32),(33). In one of the studies, the PLC, modified with collagen and fibronectin, was succeffully used as a scaffold in esophageal tissue engineering(34). Here, we demonstrated that electrospun, oriented PLC scaffolds alone are also appropriate to use in skeletal muscle tissue engineering, since myotube formation from primary human myoblasts is achieved on the PLC scaffolds even in the absence of electrical or mechanical stimuli. The electrospun PLC scaffolds produced in this study had an elastic modulus of 4.6 ± 0.7 MPa, stress at break value of 15.7 ± 0.6 MPa, and a strain at break of 246 ± 1 %. The diameter of aligned microfibers was measured as 4.53 ± 1.6 µm from the scanning electron micrographs. 3.2.3. Hybrid scaffolds. These new generation scaffolds were obtained by combining synthetic scaffolds (Fig. 4A) with acellular muscle components. These scaffolds were designed to promote cell alignment due to their oriented structure and stem cell differentiation via their natural muscle components. Even though cross-linking of muscle pieces was determined to be successful by SEM analysis (Fig. 4B) and Coomassie Brilliant Blue staining (Supplementary figure 1), the distribution of acellular muscle pieces on the hybrid scaffolds was not uniform.

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3.3. Cell Culture Studies 3.3.1. Mitochondrial activity of hMB-MSCs and human myoblasts on the scaffolds. Mitochondrial activity of hMB-MSCs and human myoblasts on all types of scaffolds was evaluated by WST-1 assay on day 21. Metabolic activity of hMB-MSCs was statistically higher on ECM scaffolds compared to their activity on the other scaffold types. It was clearly revealed that hMB-MSCs were unable to adhere to the synthetic scaffolds. hMB-MSC adhesion was improved on the hybrid scaffolds due to the presence of ECM pieces but was still lower than on ECM scaffolds (Fig. 5A). For myoblasts, the absorbance value obtained from WST-1 assay was not significantly different among different types of scaffolds (Fig. 5B). They were able to adhere to all types of scaffolds. This difference in adhesion to synthetic scaffolds between the two cell types may imply a difference in their cell surface molecules responsible from cell adhesion. 3.3.2. Myotube formation potential of hMB-MSCs and human myoblasts on the scaffolds. Although there are some differentiation inducing strategies for

hMB-MSCs(35) and human

myoblasts(10) to the muscle cells, we aimed to investigate spontaneous differentiation of these cells on the scaffolds, as it is not possible to extend the growth factor supply in in vivo unless there are some sustained release particles incorporated into the scaffolds. During the preliminary culture of hMB-MSCs with low glucose DMEM and 10% FBS on tissue culture polystyrene, multinucleated cell formation was observed within 6 weeks. Although these multinucleated cells were not desmin positive, we hypothesized that hMB-MSCs may undergo spontaneous differentiation in the presence of muscle ECM, which may serve as an inducing microenvironment. In contrary to hMB-MSCs, human myoblasts spontaneously formed desmin positive multinucleated myotubes in less than two weeks when cultured in high-glucose DMEM

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and 10% FBS on the tissue culture polystyrene; we could induce expression of muscle specific proteins necessary for cell fusion(36). Human myoblasts were incubated for 14 days on the synthetic and hybrid scaffolds with their own growth media. At the end of the incubation period, the cells were immunostained with antiα actinin antibody (Fig. 6). The best myotube formation was shown by human myoblasts on the synthetic scaffolds (Fig. 6C). These results showed that myoblasts have a high myotube formation potential which is evident even on synthetic scaffolds in a medium that does not contain any additional growth factors except the ones coming from FBS. hMB-MSC attachment to the synthetic scaffolds failed (Fig. 7A-C), whereas human myoblasts were robust on the synthetic scaffolds (Fig. 8A-C). Although the crosslinking process of acellular muscle pieces to the synthetic scaffolds was successful, unexpected results were faced upon their incubation with cells. Even though the hybrid scaffolds possessed the advantages of both synthetic and ECM scaffolds, neither high differentiation ratio nor myotube alignment could be obtained on them. The surface chemistry and the scaffold microstructure acquired upon use of the cross-linking procedure did not promote myotube formation. Myotube alignment could not be achieved on the hybrid scaffolds seeded with either hMB-MSCs (Fig. 7D-F) or human myoblasts (Fig. 8D-F) possibly due to the loss of fiber alignment. Moreover, any obvious myotube structure could not be observed on ECM scaffolds seeded with hMB-MSCs (Fig. 7G-I), while a few elongated myotube-like structures were demonstrated on the ECM scaffolds with human myoblasts (Fig. 8G-I).

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4. Discussion Remarkable studies on decellularizing cadaveric heart37 and liver38 has been recently published, however, recellularization of these 3D biologic scaffolds is still a significant issue. In the case of skeletal muscle replacement, massive amount of human muscle tissue would be required to form a 3D muscle patch for treatment of large muscle defects and sophisticated perfusion bioreactors would be required to provide a homogeneous cell distribution and growth within the dense ECM scaffold. Even though myotube formation by human myoblasts was successful on muscle ECM scaffolds in our study, these constructs may not be the best choice to use in skeletal muscle tissue engineering due to the inability of the cells to penetrate the dense structure and form aligned muscle bundles. Hybrid scaffolds could be reasonable alternatives for ECM scaffolds to overcome all these problems. In case of large muscle defects, a small skeletal muscle biopsy from the patient will be enough to serve as the ECM component of the hybrid scaffold of proper size, eliminating a donor site morbidity. Still as another advantage, the cells can better penetrate into the 3D hybrid scaffold through the space between the oriented microfibers, as compared to the dense ECM of acellular muscle scaffolds. In addition to all these, the hybrid scaffolds were produced in this study to take advantage of biological cues of natural skeletal muscle ECM scaffolds that can initiate spontaneous stem cell differentiation and advantage of oriented fibrous structure of synthetic scaffolds to encourage cell alignment. Nonetheless, SEM images revealed that our crosslinking method somehow over-crosslinked the parallel fibers and impaired their oriented structure. To overcome this issue, the incubation time of 1,6-hexanediamine, the chemical agent used to crosslink acellular muscle pieces to the synthetic scaffolds, can be reduced, since it is reported that long time incubation of 1,6-hexanediamine may lead to its reaction with the ester

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groups of adjacent polymer molecules in a PLC membrane(34). Moreover, some other strategies, such as muscle piece adsorption by drying on the synthetic scaffolds or cross-linking them with a more natural molecule like genipin(39) to the synthetic scaffolds, may help improve or preserve the aligned fiber structure of these scaffolds. Acellular muscle pieces did not show a uniform distribution on the hybrid scaffolds as a result of our crosslinking method. It seems that the size of the acellular muscle pieces (less than 70 µm) was too large to achieve a homogenous distribution, as agitation causes large pieces to accumulate at the center. Increasing the duration of homogenization process to have smaller acellular muscle pieces may improve the outcome. The hybrid scaffolds improved the attachment of the hMB-MSCs compared to the synthetic scaffolds, but could not support the myotube formation by either type of cell. Although they had the biological cues, either their modified surface chemistry due to excessive cross-linking or the excess -NH2 groups introduced to the scaffolds by the 1,6-hexanediamine may have caused such a problem. It is reported by Chieh et al. (2013) that adipose tissue derived mesenchymal stem cells adhere and spread very well on the -NH2 terminated self-assembled monolayers, but this leads to decrease in their migration and proliferation rate(40). Immunocytochemical staining results of our study revealed that myoblasts and multinucleated myotubes are very low in number on the hybrid scaffolds. This may be attributed to the lowered proliferation and migration rate in the presence of -NH2 groups on the scaffold surface since migration of myoblasts is crucial for myoblast fusion in the process of myotube formation(41). As cell carriers in tissue engineering applications, synthetic scaffolds have some indispensable advantages like reproducibility in desired form and quantity, long shelf life, suitability for clinical use, and in addition to all these, their chemistry supports effective imaging(42). The synthetic scaffolds used in this study supported adhesion of human myoblasts, although not

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adhesion of hMB-MSCs, and having an oriented fiber structure, contributed to the formation of aligned myotubes. As is evident from the flow cytometry analysis, human myoblasts are HLA-DR positive, which means that these cells can be used only in autologous transplantation. Nonetheless, they can be effectively isolated from the needle biopsy samples and used for personalized cell therapy in the prospective clinical tissue engineering applications of this study. hMB-MSCs, on the other hand, are HLA-DR negative, thus hMB-MSCs are suitable for use in a larger population compared to human myoblasts. However, their inability to attach to the synthetic scaffolds and their inability to undergo spontaneous myogenic differentiation may limit their use in muscle patch formation. A self-differentiating microenvironment, which does not require the use of any additives like hormones, growth factors or horse serum, is the simplest and preferred one to use in tissue engineering applications and can be considered more economical. Thereby, we did not induce the cells with chemicals or growth factors in order to be able to observe spontaneous differentiation led by the biological cues on the hybrid and ECM scaffolds. Moreover, we did not include mechanical or electrical stimulation in this study since our aim was to reveal, specifically, the effects of biological cues on myogenic differentiation. Further studies could investigate improvement of myogenic differentiation with mechanical or electrical stimuli on our scaffolds. The proposed muscle cell-matrix combination (human myoblasts-synthetic scaffolds) in this study can be improved further by addition of vascular and peripheral nervous system components to the structure to produce a functional skeletal muscle patch. To produce vascular network on the skeletal muscle patches, human muscle progenitor cells can be co-cultured with human umbilical vein endothelial cells (HUVECs). By this way, HUVECs will organize to 21 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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produce vascular structures on the skeletal muscle patches(43). Innervated muscle patch can also be obtained by coculturing motoneurons derived from fetal spinal stem cells and myoblasts from skeletal muscle(44). It is also possible to differentiate myoblasts into neuron-like cells by inducing with Neurococktail, therefore myoblasts can also be used to grow peripheral nervous system in a skeletal muscle patch(45).

5. Conclusion To the best of our knowledge, this is the first study demonstrating spontaneous differentiation of primary human myoblasts on synthetic scaffolds produced from GMP grade Poly(L-lactide-cocaprolactone), which is suitable for medical device applications, in an attempt to produce a skeletal muscle patch. We also investigated the potential of hMB-MSCs as a cell source to use in skeletal muscle tissue engineering for the first time. We conclude that human myoblasts on muscle ECM and synthetic scaffolds were effective in producing a muscle tissue patch. Producing an anisotropic muscle tissue substitute was more successful when myoblasts werecultured on the synthetic scaffolds with parallel microfiber arrangement. The hybrid scaffolds, which had both synthetic and acellular muscle components, did not promote myotube formation of myoblasts, possibly due to the 1,6-hexanediamine impaired surface chemistry, but could promote hMB-MSC attachment. The acellular muscle tissue itself was not effective in inducing hMB-MSCs to form multinucleated myotubes. Our prospective studies will focus on organizing vascular and peripheral nerve networks in the human myoblast seeded synthetic scaffolds to produce a functional skeletal muscle patch.

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Acknowledgements Research fund from The Scientific and Technological Research Council of Turkey (111S248) is acknowledged. The authors have no conflict of interest.

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Figure Legends FIGURE 1. Phenotypic characteristics of hMB-MSCs and human myoblasts: Flow cytometry histograms of hMB-MSCs (A) and human skeletal muscle derived cells (F); Oil Red O (B,C) and Alizarin

Red

(D,E)

stained

control

and

differentiated

hMB-MSCs,

respectively; 29

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Immunophenotypic characteristics of human myoblasts stained for Desmin (G) and myoD (H); α -actinin positive myotubes (I) formed by the human myoblasts and their sarcomeric F-actin (J) revealed by staining with phalloidin. Blue: DAPI, (A,F) Green line: histogram of isotype control immunoglobulin. FIGURE 2. Macroscopic and histochemical evaluation of native and acellular muscle tissues. Macroscopic view of native (A) and acellular (B) muscle tissue obtained via detergent treatment of the native tissue; Native (C) and acellular (D) muscle tissue stained with H&E; Native (E) and acellular (F) muscle tissue stained with Masson's trichrome. Dark blue zones demonstrate collagen (E,F). FIGURE 3. Myotube formation capacity of hMB-MSCs and human myoblasts on the ECM scaffolds: Brightfield (A, B) and fluorescence microscopy (C, D) images of hMB-MSCs (A, C) and human myoblasts (B, D) on the ECM scaffolds after a 21-day culture. Blue: DAPI, Green: Desmin. FIGURE 4. Scanning electron micrographs of synthetic (A) and hybrid (B) scaffolds. Crosslinked acellular muscle pieces on the PLC microfibers are evident in the scanning electron micrographs of hybrid scaffolds. FIGURE 5. Mitochondrial activity of hMB-MSCs (A) and human myoblasts (B) on the scaffolds on day 21, determined by Cell Proliferation Reagent WST-1. While the mitochondrial activity of hMB-MSCs was significantly higher on ECM scaffolds than synthetic and hybrid scaffolds, mitochondrial activity of human myoblasts did not show any scaffold dependent difference . * denotes statistically significant differences (ANOVA, p < 0.05, n = 3). FIGURE 6. Fluorescence micrographs of human myoblasts on synthetic (A-C) and hybrid scaffolds (D-F) on day 14. Blue: DAPI, Red: α-actinin.

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FIGURE 7. SEM images of hMB-MSCs on the synthetic (A-C), hybrid (D-F), and ECM (G-I) scaffolds after 21 days of culture. a, d, g –X200; b, e, f -X1000; c, f, i – X1500. FIGURE 8. Scanning electron micrographs of human myoblasts on the synthetic (A-C), hybrid (D-F), and ECM (G-I) scaffolds on day 21. a, d, g –X200; b, e, f -X1000; c, f, i – X1500. White arrows point tentative myotube structures. SUPPLEMENTARY FIGURE 1: Brightfield images of Coomassie Brilliant Blue stained synthetic (A) and hybrid (B) scaffolds. While the dark blue dots, representing human acellular skeletal muscle pieces, are evident on the hybrid scaffold, there is no such staining on the synthetic scaffold.

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FIGURE 1. Phenotypic characteristics of hMB-MSCs and human myoblasts: Flow cytometry histograms of hMB-MSCs (A) and human skeletal muscle derived cells (F); Oil Red O (B,C) and Alizarin Red (D,E) stained control and differentiated hMB-MSCs, respectively; Immunophenotypic characteristics of human myoblasts stained for Desmin (G) and myoD (H); α -actinin positive myotubes (I) formed by the human myoblasts and their sarcomeric F-actin (J) revealed by staining with phalloidin. Blue: DAPI, (A,F) Green line: histogram of isotype control immunoglobulin. Fig.1 62x39mm (300 x 300 DPI)

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FIGURE 2. Macroscopic and histochemical evaluation of native and acellular muscle tissues. Macroscopic view of native (A) and acellular (B) muscle tissue obtained via detergent treatment of the native tissue; Native (C) and acellular (D) muscle tissue stained with H&E; Native (E) and acellular (F) muscle tissue stained with Masson's trichrome. Dark blue zones demonstrate collagen (E,F). Fig.2 99x99mm (300 x 300 DPI)

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FIGURE 3. Myotube formation capacity of hMB-MSCs and human myoblasts on the ECM scaffolds: Brightfield (A, B) and fluorescence microscopy (C, D) images of hMB-MSCs (A, C) and human myoblasts (B, D) on the ECM scaffolds after a 21-day culture. Blue: DAPI, Green: Desmin. Fig.3 66x44mm (300 x 300 DPI)

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FIGURE 4. Scanning electron micrographs of synthetic (A) and hybrid (B) scaffolds. Crosslinked acellular muscle pieces on the PLC microfibers are evident in the scanning electron micrographs of hybrid scaffolds. Fig.4 39x16mm (300 x 300 DPI)

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FIGURE 5. Mitochondrial activity of hMB-MSCs (A) and human myoblasts (B) on the scaffolds on day 21, determined by Cell Proliferation Reagent WST-1. While the mitochondrial activity of hMB-MSCs was significantly higher on ECM scaffolds than synthetic and hybrid scaffolds, mitochondrial activity of human myoblasts did not show any scaffold dependent difference . * denotes statistically significant differences (ANOVA, p < 0.05, n = 3). Fig.5 40x16mm (600 x 600 DPI)

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FIGURE 6. Fluorescence micrographs of human myoblasts on synthetic (A-C) and hybrid scaffolds (D-F) on day 14. Blue: DAPI, Red: α-actinin. Fig.6 49x24mm (300 x 300 DPI)

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FIGURE 7. SEM images of hMB-MSCs on the synthetic (A-C), hybrid (D-F), and ECM (G-I) scaffolds after 21 days of culture. a, d, g –X200; b, e, f -X1000; c, f, i – X1500. Fig.7 70x49mm (300 x 300 DPI)

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FIGURE 8. Scanning electron micrographs of human myoblasts on the synthetic (A-C), hybrid (D-F), and ECM (G-I) scaffolds on day 21. a, d, g –X200; b, e, f -X1000; c, f, i – X1500. White arrows point tentative myotube structures. Fig.8 70x49mm (300 x 300 DPI)

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SUPPLEMENTARY FIGURE 1: Brightfield images of Coomassie Brilliant Blue stained synthetic (A) and hybrid (B) scaffolds. While the dark blue dots, representing human acellular skeletal muscle pieces, are evident on the hybrid scaffold, there is no such staining on the synthetic scaffold. Supp. Fig.1 39x16mm (300 x 300 DPI)

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Skeletal muscle patch engineering on synthetic and acellular human skeletal muscle originated scaffolds.

The reconstruction of skeletal muscle tissue is currently performed by transplanting a muscle tissue graft from local or distant sites of the patient'...
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