{'fraction_non_alphanumeric': 0.0351872348275226, 'fraction_numerical': 0.02494927135214905, 'mean_word_length': 4.841864224137931, 'pattern_counts': {'":': 0, '<': 6, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Myogenic Differentiation of Human Myoblasts and Mesenchymal Stromal Stem Cells under GDF11 on Poly-(cid:0)-Caprolactone-Collagen I-Polyethylene-Nanobers

Primary myoblasts (Mb) and adipose derived mesenchymal stromal cells (ADSC) can be co-cultured and myogenically differentiated in the process of skeletal muscle tissue engineering. Electrospun composite nanober scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining biocompatibility and stability. Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11. Therefore the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolacton (PCL)-collagen I-polyethylene oxide (PEO)-nanobers. Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanobers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunouorescence staining showed myosin heavy chain expression in all groups after 28 days of differentiation. Gene expression of myosin heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone. The results of this study show that PCL-collagen I-PEO-nanobers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.


Introduction
Muscle regeneration is orchestrated by muscle satellite cells, adult stem cells, located in a niche between the basal lamina and the sarcolemma. Those satellite cells form a pool of myogenically committed cells, the myoblasts, which fuse and differentiate into multinucleated myotubes and eventually into myo bers 1,2 . In the event of volumetric muscle loss, the natural regeneration capacity of skeletal muscle tissue is exceeded 3 . To overcome the issue of donor site morbidity when using autologous muscle tissue to reconstruct the resulting defect, tissue engineering approaches for creating functional skeletal muscle tissue have been investigated thoroughly [3][4][5][6] . Since skeletal muscle represents a complex tissue with hierarchically organized bers, a matrix, mimicking those properties is needed 7 . A prior study demonstrated that poly-ε-caprolacton (PCL)-collagen I nano bers, electrospun with a benign solvent system, served as a suitable platform for those tissue engineering purposes due to their parallel alignment, stability, and biocompatibility 4,8,9 . However, high density packed bers might result in poor cell in ltration 10 . One method to improve cell permeability into the scaffolds is to integrate polyethylene oxide (PEO) as sacri cial bers to increase porosity of nanoscaffolds 11,12 .
I a more recent study, mesenchymal stromal cells (MSC) from adipose tissue, or so-called adipose derived mesenchymal stromal cells (ADSC) were co-cultured with primary myoblasts (Mb) three-dimensionally (3D) on PCL-collagen I-nanoscaffolds and myogenically differentiated 4 . To eliminate potential inconsistencies and safety concerns, a serum-free differentiation medium was established. The establishment of this biocompatible system has been a crucial step in terms of future translational applications. However, gene expression of late myogenic markers was still higher after long term stimulation with donor horse serum (DHS) compared to serum-free differentiation 4 . Thus, the optimization of a serum-free medium for myogenic differentiation is crucial in terms of clinical translation of models of skeletal muscle tissue engineering.
Growth differentiation factor 11 (GDF11), a member of the transforming growth factor (TGF)-beta superfamily, has shown bene cial effects on skeletal muscle regeneration, restoring satellite cell regenerative function in aged muscle cells and mice 13 and was identi ed as a rejuvenation factor in heterochronic parabiosis experiments 14,15 . It was suggested that GDF11 declines with age, and that restoration of systemic GDF11 to "youthful" levels is bene cial for several age-related conditions 16 . Sinha et al. were able to show that supplementation of GDF11 reversed functional impairments and restored genomic integrity in aged muscle stem cells and mice, suggesting rejuvenating effects of GDF11 on aging skeletal muscle. The authors concluded that GDF11 acts both directly and indirectly to restore satellite cell regenerative function 13 . Similarly, ADSC have shown rejuvenating properties by a GDF-11 dependent mechanism, at least in case of human skin 17 . Contrary to those results, others showed that GDF11 inhibited muscle regeneration and decreased satellite cell expansion in mice 18 and that GDF11 exposure in mice induced whole body wasting and profound loss of function in cardiac and skeletal muscle 19 . All in all, con icting results as to the effect of GDF11 on muscle regeneration exist.
In order to better understand the effect of GDF11 on myogenic differentiation, the aim of this study was to analyze its effect on ADSC co-cultured with Mb on PCL-collagen I-PEO-nano bers under serum-free conditions.

Human myoblast and ADSC characterization
Primary human Mb from P6 showed > 95% positive staining for the muscle-speci c marker desmin. After 7 days of myogenic differentiation, there was evidence of multinucleated myotube formation (Figure 1a and b).
Optimal GDF11 concentration for myogenic differentiation of Mb and ADSC co-cultures Co-cultures of Mb and ADSC were seeded as monolayers and stimulated with serum free differentiation medium supplemented with 3 different concentrations of GDF11: 25 ng/ml, 0.1 µg/ml, and 0.5µg/ml. After 3 days of myogenic differentiation, there was decreasing CK activity with increasing GDF11 concentration ( Figure 3a). Statistical analysis showed signi cant differences between all groups with highly signi cant differences between 25 ng/ml GDF11 and all other GDF11 concentrations, including serum-free media without addition of GDF11 (p < 0.001). There was no difference between serum-free media and 500 ng/ml GDF11 (p = 0.4). After 7 days, pairwise comparisons showed a signi cant decrease of CK activity after serum-free differentiation (p = 0.0037) and with addition of 100 ng/ml GDF11 (p = 0.0071), but not with 25 ng/ml GDF11 (p = 0.11) and 500 ng/ml GDF11 (p=0.25), although there was a tendency towards lower CK activity for both concentrations. Similarly, desmin staining after 7 days of myogenic differentiation showed formation of multinucleated myotubes under serum-free + 25 ng/ml GDF11 and serum-free + 100 ng/ml GDF while serum-free and serum-free + 500 ng/ml GDF11 led to demin positive staining of cells without any clear evidence of myotube formation (Figure 3b). Based on those results, a GDF11 concentration of 25 ng/ml was chosen for further experiments.

Cell viability on PCL-collagen I-PEO-nanoscaffolds
Cell viability of 3D co-cultures of Mb and ADSC on PCL-collagen I-PEO-nanoscaffolds was assessed after 7, 14, and 28 days of myogenic differentiation. While there was no difference between group 1 (serumfree) and group 2 (serum-free + GDF-11) (p = 0.952), cell viability was higher in group 3 (standard) compared to group 1 (p = 0.048) and group 2 (p = 0.034). With increasing differentiation period, this difference became more pronounced: After 14 days, cell viability of group 3 increased highly signi cantly compared to group 1 (p = 0.010) and group 2 (p = 0.008). There was no difference between group 1 and 2 (p = 0.984). After 28 days, the difference in cell viability increased even more between group 3 and group 1 (p = 0.001) and between group 3 and group 2 (p = 0.0003). Over time, cell viability did not increase within groups except within 7 to 14 days for group 1 (p = 0.030). There was a trend towards higher cell viability after 28 days compared to 7 days for group 3 (p = 0.080) ( Figure 4).
There was no difference between group 1 and group 2 during all time periods (p > 0.999 after 7 days, p = 0.986 after 14 days, p = 0.991 after 28 days). Over time, CK activity did not change within groups, except in group 3, where CK activity increased from 7 days to 28 days (p = 0.031) ( Figure 5).
With scanning electron microscopy (SEM) images, con guration of attached cells on PCL-collagen I-PEOnanoscaffolds could be analyzed ( Figure 6). After 28 days of myogenic differentiation, co-cultures of Mb and ADSC showed parallel alignment on the scaffolds under all three differentiation conditions. While in group 2 (serum-free medium + 25 ng/ml GDF11) and group 3 (standard), cells covered almost the entire surface of the scaffold, the cells in group 1 (serum-free) were not as densely packed. Fluorescence microscopy showed a similar trend after 28 days of serum free, serum free + GDF11, and standard myogenic differentiation. All groups led to positive expression of myosine heavy chain (MHC) of differentiated Mb and ADSC co-cultures ( Figure 6).

Discussion
The ndings of this study implicate that Mb and ADSC co-cultures showed adequate viability and adherence on PCL-collagen I-PEO-nano bers. Myogenic differentiation over a period of 28 days led to the expression of myogenic key markers and parallel arrangement along the aligned nano bers. A low concentration of 25 ng/ml GDF11 led to positive MHC expression as shown by immunostaining and enhanced MYH2 gene expression of Mb and ADSC co-cultures on PCL-collagen I-PEO-nanoscaffolds at least under serum-free differentiation conditions. On the other hand, GDF11 was not bene cial in terms of cell viability and myogenic differentiation, assessed by CK activity and even inhibited those parameters when compared to standard serum-containing differentiation.
GDF11 has been reported to rejuvenate aged skeletal muscle tissue 13 . However, these ndings remain controversial as GDF11 has shown to induce skeletal muscle atrophy and inhibition of skeletal muscle growth 18 . Jin et al. were able to show that GDF11 propeptide, an inhibitor of GDF11 leads to skeletal muscle hypertrophy and improvements in muscle strength 20 . Others have proven that GDF11 exposure in mice led to whole body wasting and loss of function in cardiac and skeletal muscle 19 . This might be explained by GDF11 being closely related to GDF8 or myostatin, which is a potent inhibitor of skeletal muscle growth 21 . Both myostatin and GDF11 act via the activin type II receptor, activating the Smad2-/Smad3-complex, and thereby mediating downstream signaling 22,23 . The effect of GDF11 on MSC has also been studied intensely. For instance, GDF11 inhibited MSC to differentiate into the adipogenic lineage via the TGF-beta signaling pathway 24 . GDF11 also inhibited osteoblast and chondrogenic differentiation of bone marrow-derived MSC 25,26 . The in uence of GDF11 on myogenic differentiation capacity of MSC let alone co-cultures of primary Mb and ADSC on PCL-collagen I-PEOnanoscaffolds has not been investigated so far.
The results of the present study re ect a similar trend, but they do not clearly support the previously mentioned studies. CK activity, which has been used as a biochemical marker for direct measurement of myogenic differentiation 19,27 , decreased over time after induction of serum-free myogenic differentiation, which is in accordance to a previous study 4 . As described in the aforementioned study, decrease in CK activity could have been evoked by a decline in growth factors by using serum-free medium 4 . Additional GDF11 did not lead to an increase in CK activity in the present study. When using GDF11 at a high concentration of 500 ng/ml, CK activity even decreased over time, which could be explained by an inhibiting effect of GDF11 on myogenic differentiation. More recent ndings support this theory and demonstrate that GDF11 is not the long-sought rejuvenating factor, opposed to the theory of Sinha et al. 13,28 . Egermann et al. showed that previously used reagents to detect GDF11 were nonspeci c and that GDF11 rather increases with age and inhibits muscle regeneration and myoblast differentiation 18 .
On the other hand, no difference in GDF11 concentration could be detected via enzyme-linked immunosorbent assay between plasma of younger compared to older individuals 29 . In a recent study, low concentrations of 1 ng/ml and 10 ng/ml of GDF11 have not led to a decrease in myogenic differentiation but rather to an increase in nuclear density and myotube length and width of different myogenic cells 30 . Similarly, in the present study, the addition of GDF11 did not lead to a decrease of CK activity and even upregulated MYH2 compared to serum-free differentiation alone. However, both groups were still not as stimulating as the standard serum-containing differentiation medium. As described in a previous study, myogenic differentiation of MSC necessitates growth factors, which might not be contained in Ultroser® G 4 . Other growth factors besides GDF11 might be necessary for an increase of myogenic differentiation to the level of stimulation with serum-containing differentiation medium. At least, GDF11 did not lead to a signi cant decrease of myogenic key markers as opposed to the ndings by Egermann et al. 18 .
Electrospun PCL-collagen I-nanoscaffolds have proven to be a suitable and biocompatible matrix for long-term differentiation of co-cultures of Mb and ADSC 4 . The integration of PEO sacri cial bers is supposed to increase porosity of nanoscaffolds, facilitating cell in ux 11,12 . Unfortunately, we did not compare the newly established PCL-collagen I-PEO-nanoscaffolds to PCL-collagen I-nanoscaffolds. Thus, conclusions about a more e cient myogenic induction of either of those blended ber types cannot be made. Yet, we were able to demonstrate positive MHC expression in this setting of 28 days of myogenic differentiation. Furthermore, the PCL-collagen I-PEO-nano bers of this study promoted cell adherence and viability as shown by SEM and wst-8-assay. A prior analysis of 3D vascularization of PCL-collagen Inano ber scaffolds versus PCL-collagen I-PEO-nano ber scaffolds in the arteriovenous loop model of the rat showed better integration into the host organism for the PEO-blended bers 12 . Taking into account an in vivo translation of this 3D model, which is crucial for the creation of large-scale skeletal muscle constructs, the implementation of PCL-collagen I-PEO-nano bers seems superior to PCL-collagen Inano bers.
There are several limitations of this study. First, standard deviations were sometimes high which might be explained by the use of primary human cells of different subjects. The purchased myoblasts were all from the quadriceps muscle and human subjects were all male to limit heterogeneity. Other factors like age are known to in uence the degree of myotube formation 31,32 . Furthermore, maintaining and myogenic differentiation of primary cells isolated from adult muscle tissue is a larger challenge than culturing immortal cell lines like C2C12 without contact inhibition. However, cell lines are not suitable for establishing a model of skeletal muscle engineering which can be translated into clinical application. Second, Mb monocultures were not analyzed, so it is uncertain what effect GDF11 has on Mb in our experimental setting. On the other hand, Mb alone might not be promising for engineering large-scale tissue, which can eventually be applied in the patient since they have a limited proliferation capacity 7,33,34 . In several studies, Mb and ADSC co-cultures were differentiated into the myogenic lineage 4 Given the results of the present study, we propose that PCL-collagen I-PEO-nano bers are a viable option for myogenic differentiation of Mb and ADSC co-cultures. In this setting, GDF11 alone might be promoting myogenic differentiation of Mb and ADSC co-cultures under serum-free conditions. We were not able to prove a harming effect of the myostatin homologue as proposed by recent studies. A combination of GDF11 with other proteins, particularly those secreted by ADSC might be interesting to analyze in terms of the effect on myogenesis under serum-free conditions. Regardless of the effects of GDF11, this biocompatible 3D model, consisting of primary Mb and ADSC co-cultured on PCL-collagen I-PEO-nano bers can serve as a platform for skeletal muscle tissue engineering, which can be transferred to further in vivo and translational research.

Conclusion
We have established electrospun aligned PCL-collagen I-PEO-nano ber scaffolds as a novel biocompatible matrix for skeletal muscle tissue engineering purposes. Mb and ADSC showed adequate cell adherence, viability, and myogenic differentiation when co-cultured on those matrices. In this setting of serum-free myogenic differentiation, GDF11 had a promoting effect on myogenic differentiation of Mb and ADSC co-cultures.
ADSC were enzymatically isolated from human adipose tissue as described previously 40  collagen I-PEO-nanoscaffolds at 3 x 10 5 cells in a ratio of 1:1. After 7 days of proliferation in expansion medium, differentiation was induced and continued for 28 days. As negative control for immuno uorescence staining, broblasts in P9 were seeded onto the nanoscaffolds as described above and allowed to proliferate in DMEM + 10% fetal calf serum (FCS) + 1% P/S (all from Biochrom GmbH) for 7 days.

Myogenic differentiation conditions
To con rm myogenic differentiation potential of Mb in P6, monolayers were seeded in 48-wells at a cell density of 20.000 and allowed to proliferate in expansion medium for 2 days until cells reached con uence. Afterwards, differentiation was induced by switching to standard differentiation medium, containing 2% donor horse serum (DHS, Biochrom GmbH) ( Table 1). Co-cultures of ADSC and Mb were seeded similarly into 48-wells and were allowed to differentiate with serum free differentiation medium + 25 ng/ml GDF11 (Table 1) for 7 days. Differentiation was continued for 7 days prior to desmin staining as described in detail under "Immuno uorescence".

RNA isolation and quantitative PCR analysis
In 3D co-cultures (n = 3), the gene expression rate of the late myogenic markers MYH2 (myosin heavy chain 2) and ACTA1 (skeletal alpha actin) was analyzed as previously described 4 . As housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as internal control. RNA of the samples was extracted using the RNeasy micro kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocols. RNA was reverse-transcribed into cDNA using a QuantiTect Reverse Transcription Kit and a Sensiscript Reverse Transcription Kit (both from Qiagen GmbH). cDNA was ampli ed in triplicates through quantitative real-time PCR using SsoAdvanced Universal SYBR Green PCR Supermix (Bio-Rad, Hercules, CA, USA) and Light Cycler (Bio-Rad CFX96 Touch TM ). Evaluation of gene expression was performed using the 2 −ΔΔCt method. RNA from human muscle tissue served as control sample. The primer sequences used are given in Table 2. ADSC and Mb were 3D co-cultured on PCL-collagen I-PEO-nanoscaffolds (n = 3) for 7 days before expansion medium was switched to differentiation medium (serum-free, serum-free + GDF11, standard).
After 4 weeks, scaffolds were xed, washed, blocked, and stained with anti-fast myosin skeletal heavy chain (MHC) antibody (ab91506, Abcam) at 5 µg/ml for one hour at room temperature.
Alexa Fluor 594 goat anti-mouse IgG1 cross-adsorbed secondary antibody (A-21125, Thermo sher Scienti c Inc.) was used as secondary antibody at 4 µg/ml for 30 minutes at room temperature for desmin stained cells and Alexa uor 594 goat anti-rabbit IgG (H+L) (Thermo sher Scienti c Inc.) was used as secondary antibody for MHC stained cells at the same conditions. After counterstaining with DAPI 1 µg/ml (diamidine-phenylindole-dihydrochloride, Thermo sher Scienti c Inc.) for 5 minutes, cells were subsequently analyzed and digitally photographed with a uorescence microscope (IX83, cellSens, software, Olympus, Hamburg, Germany).
Human primary broblasts in P9 as monolayers or on nanoscaffolds served as negative control.

Figure 4
Wst-8-assay after 7, 14, and 28 days of myogenic differentiation of 3D co-cultures of Mb and ADSC on PCL-collagen I-PEO-nano bers. Multiple comparisons between different groups within the same time period were performed with one-way ANOVA with Tukey's multiple comparisons test (n = 3). Levels of signi cance were * p < 0.05, ** p < 0.01, *** p < 0.001. Repeated measures ANOVA with Tukey's multiple comparisons test was used to compare same groups over different time periods (n = 3). Level of signi cance was # p < 0.05.   Gene expression of the late myogenic markers myosin heavy chain (MYH2) and actin, alpha skeletal muscle (ACTA1) in co-cultures of Mb and ADSC after 28 days of myogenic differentiation. Expressions are demonstrated in x-fold difference compared with RNA from human muscle tissue using the 2 -ΔΔCt method. GAPDH was used as housekeeping gene in co-cultures and muscle tissue. Comparisons between groups were performed with one-way ANOVA with Tukey's multiple comparisons test or Kruskal-Wallis test with Dunn's correction as appropriate (n = 3). Level of signi cance was * p < 0.05.