Macromolecule Biomaterials Promote the Osteogenic Differentiation Capacity of Equine Adipose -Derived Mesenchymal Stem Cells

Background: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. In the present study, the effect of biomaterial bone substitutes on equine adipose derived MSCs morphology, viability, adherence, migration and osteogenic differentiation were investigated. Methods: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials in osteogenic differentiate medium either under static or mechanical uid shear stress (FSS) culture conditions. The data were generated by histological means, life cell imaging, cell viability, adherence and migration assays. Osteogenic differentiation was detected by semi-quantication of alkaline phosphatase (ALP) activity, matrix mineralization using Alizarin Red S (ARS) staining and quantication of the osteogenic markers; runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression using RT-qPCR. All data were statistically analyzed using ANOVA. Results: The data revealed that combined mechanical stress with MC but not B30 enhanced MSCs viability and promoted their migration. Combined osteogenic medium with MC, B30 and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30 and B30Str resulted in diffused matrix mineralization by means of ARS. FSS increased the viability in the presence of the osteogenic medium with MC but not B30 or B30Str. FSS enhanced osteogenic differentiation in the presence of B30Str. Upregulation of Runx2 and ALP expression was detected with osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. Conclusions: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects. investigate the effect of biomaterial substitutes on equine adipose derived mesenchymal cells (MSCs) morphology, viability, adherence, migration ability and osteogenic differentiation potential. MSCs were co-cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials. These combinations were allowed to differentiate into the osteogenic fate up to day 21 either under static (ST) or mechanical uid shear stress (FSS) culture conditions. Data were generated by histological investigations as well as by evaluation of cell viability, cell adherence and the migratory potential. Osteogenic capacity was assessed by semi-quantication of the alizarin red S staining (ARS), alkaline phosphatase (ALP) activity and quantication of the osteogenic markers runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression. Our data revealed the inuence of various scaffolds biomaterials on stem cells criteria including morphology, attachment and cell migration. We show enhanced cell viability in the presence of MC and mechanical FSS. An increased ALP activity in the osteogenic differentiation together with B30 under static culture could be shown. We report that mechanical FSS together with MC improves matrix mineralization. We show that combined mechanical FSS and B30/B30Str increases osteogenic differentiation indicated by upregulated ALP and Runx2 expression. All together underpinned the effect of various biomaterials on osteogenic commitment of MSCs which suggest their usefulness for regenerative applications in veterinary medicine. with attached different time point was performed. The analysis revealed that signicant increase in the CSA of B30/MSCs complex observed after 36 h (p< 0.01) compared to T0. Then, a gradual increase of the size of the B30/MSCs complex found up to 72 hours (p< 0.001) compared to 36 h. In contrast, the cell migration out of the MC showed different patterns considering the same time scale. MSCs/MC complex demonstrated a in the already after h (p< 0.01) compared to T0. Interestingly, The CSA of a signicant 36 h (p< 0.01) compared remains to h (g. 1p). No in of pellet for B30 and in initial 12 h of the complex Further data analysis revealed a signicant linear correlation between the of MSCs out of pellet and the These data point out the pattern of cell migration out of the pellet in a


Introduction
Cell-based stem cell therapy together with biomaterials is a rapidly growing approach for tissue regeneration. The aim is to repair and restore normal tissue function by means of stem cells together with biological macromolecules as scaffold materials. Such approach may provide a new therapeutic option for the treatment of chronic degenerative disorders that are currently incurable with ordinary routine applications.
Osteoporosis is a disease causing bone fragility, risk of fracture and insu cient bone healing due to deterioration of the bone microarchitecture [1,2]. Scarcity of effective treatment has encouraged seeking for alternative therapeutic approaches [3][4][5]. Currently, autologous and allogenic bone grafts are the most appropriate approach to restore bone defects [6]. However, the limited bone availability and the necessity of an invasive and expensive surgical intervention have fostered the promotion of the development of substitute materials.
Using biomaterials substitutes for bone tissue engineering allows developing biological scaffolds capable of restoring, maintaining and improving tissue function [7]. Developing a biological substitute to re ll bone defects requires a combination of a proper scaffold biomaterial that maintain a suitable niche for mesenchymal stem cell differentiation [8,9]. Certain criteria have to be considered to select the biomaterial; (1) it should mimic the target tissue components in terms of biocompatibility and biodegradability, (2) it has biomechanical and osteoinductive properties and (3) the biomaterial must be easy to establish, e cient and economical to facilitate clinical application [6,8]. A novel biomimetic silica-collagen hybrid nanocomposites has been developed that meet the required criteria [10]. Inspired by the siliceous basal spicules of marine glass sponges, it represents a combination of the organic bovine tropocollagen type I and inorganic silicate [11]. Both ingredients of this nanocomposite are physically found in bone tissue and have been proven to exert a positive in uence on bone healing. Collagen is the most abundant framework protein in mammalian tissues which provides a promising component of a biomaterial due to low antigenicity, low in ammatory and low cytotoxicity. Moreover, it has the ability to promote cell adhesion, proliferation and differentiation and it is responsible for biocompatibility, controlled biodegradation and the maintenance of signal transduction [11][12][13]. Silicon is one of the most abundant trace element in living organism. A group bunch of evidence demonstrated the important role of silicon for bone metabolism and formation that have been proven under in vitro and in vivo investigation [14,15]. It has been found that silicon not only increased the collagen synthesis within the extracellular matrix of human osteoblasts but also it promoted their proliferation and differentiation [16][17][18]. In addition, it plays an important role as an initiator for bone mineralization and formation [19,20].
Furthermore, Silici cation reinforces the collagen brils leading to improved mechanical stability of the biomaterial [21]. The combined silica and collagen provides inorganic/organic composite with promising characteristics regarding chemical, structural and technological parameters for the biomaterial suitable for bone engineering [13,21,22]. Currently, a group of evidence have shown the effectiveness of collagen based microcarrier scaffold for MSCs proliferation and chondrogenic differentiation [23]. Similar study has combined MC with chitosan hydrogel revealed an enhanced metabolic activity and surface adhesion of chondrocytes suggesting the usefulness of the composite for tissue regeneration [24]. The criteria of cell viability, adhesion and detachement would determine the quantity of cells available for regeneration and production of the therapeutic effect. Additionally, the porosity of MC and surface adhesion support cell attachement, nutrient transfer and a reservoir system to deliver active biological molecules [25].
Mesenchymal stem cells (MSCs) are multipotent undifferentiated adult stem cells, extractable from various tissues including bone marrow and adipose tissue. These cells have the ability for self-renewal and differentiation potential into bone, fat and cartilage [26]. The osteogenic differentiation of MSCs provides a valuable tool for bone tissue engineering. Furthermore, the accessibility, straightforward isolation and immunomodulatory properties of MSCs demonstrate their usefulness in cell-based therapy for bone regeneration [27]. However, the effect of biomaterials on the cell viability, adherence, migration and osteogenic differentiation of MSCs are not fully elucidated yet. Thus, the present study aims to investigate the effect of biomaterial substitutes on equine adipose derived mesenchymal stem cells (MSCs) morphology, viability, adherence, migration ability and osteogenic differentiation potential. MSCs were co-cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials. These combinations were allowed to differentiate into the osteogenic fate up to day 21 either under static (ST) or mechanical uid shear stress (FSS) culture conditions. Data were generated by histological investigations as well as by evaluation of cell viability, cell adherence and the migratory potential. Osteogenic capacity was assessed by semi-quanti cation of the alizarin red S staining (ARS), alkaline phosphatase (ALP) activity and quanti cation of the osteogenic markers runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression. Our data revealed the in uence of various scaffolds biomaterials on stem cells criteria including morphology, attachment and cell migration. We show enhanced cell viability in the presence of MC and mechanical FSS. An increased ALP activity in the osteogenic differentiation together with B30 under static culture could be shown. We report that mechanical FSS together with MC improves matrix mineralization. We show that combined mechanical FSS and B30/B30Str increases osteogenic differentiation indicated by upregulated ALP and Runx2 expression. All together underpinned the effect of various biomaterials on osteogenic commitment of MSCs which suggest their usefulness for regenerative applications in veterinary medicine. The silica-collagen nanocomposites were kindly provided from the Max Bergmann Center of Biomaterials and Institute of Materials Science from Dresden University of Technology and preparation was carried out as described by [11,22]. Our investigations were performed with xerogel granules. Therefore, the silicacollagen monoliths were ground into a powder and then classi ed into different particle fractions. In these investigations fractions with a particle size < 0.125 mm were examined.

CultiSpher-S Microcarrier (MC)
Preparation of the CultiSpher macroporous gelatin microcarriers was performed according to the company instruction (Percell, Biolytica AB, Sweden). Brie y, the dry MC were rehydrated in phosphate buffered saline (PBS, 1g/50 mL) for one h then were autoclaved at 121 °C for 15 min. The PBS was removed and fresh PBS was added then MC were washed three times in culture medium. Generally, 1x 10 5 cells were mixed with 0.1 % MC in DMEM for all experimental setups.

Isolation of adipose tissue derived MSCs
Experiments were accomplished from equine adipose mesenchymal stem cells MSCs (N=8; aged 7.66 ± 1.34 years). Equine MSCs of both sex were examined as previously reported by (Elashry et al., 2018). Brie y, the adipose tissue was obtained from the subcutaneous fat from horses being slaughtered at the local abattoir and the Institute of Veterinary Pathology, Justus-Liebig University in Giessen. MSCs were transferred in cold PBS supplemented with 1 % penicillin-streptomycin (P/S, Gibco ® life technologies, Germany). Adipose tissue samples were cut into 1mm 2 pieces using sterile scalpel then were washed twice in PBS for 5 min. The samples were digested under shaking in 0.1 % collagenase I (Biochrom AG, Germany) in 1 % bovine serum albumin dissolved in PBS for 60 min at 37 °C. The homogenates were ltered in 70 µm cell strainer and were centrifuged at 240 g for 5 min. The supernatant was discarded and the pellets were suspended in fresh Dulbecco's Modi ed Eagle's Medium (1g/L, DMEM, Gibco ® life technologies, Germany). The cells were counted using a hemocytometer, were expanded in DMEM containing 10 % foetal bovine serum (FBS, Biocell, Germany) and 1 % P/S growth medium within the T75 culture asks. The cells were incubated at 37 °C, 5 % CO 2 and 90 % humidity up to con uency. In the current study, MSCs were characterized either by ow cytometry using the surface marker CD90 also using PCR for the expression of stem cell markers CD90, CD 105 and Oct 4 as previously reported by our group [28,29]. Upon con uency, cells were detached using TryplE (Gibco ® life technologies), were counted using a hemocytometer and were stored in 1 mL aliquots at -160 °C. Medium change was carried out twice a week and cells of passage 2-4 were used for the following experiments. After expansion, 4x10 6 cells and 20 mg of MC and B30 biomaterials were cultivated in 10 mL growth medium using two different settings: a static culture in falcon tubes (Sarstedt, Germany) and a rotating bioreactor (Rotary cell culture systems, Synthecon Inc., Houston, TX, USA). To enable proper gas exchange, the falcon lid was perforated before incubation. In the rotating culture, the vessel was allowed to perform 11 cycles/min. Both settings were incubated at 37 °C with 5 % CO 2 for four days without carrying out a medium change.
Afterwards, the medium with biomaterial-cell-complexes (from both experimental settings) was carefully transferred to a new 15 mL falcon tube. Two washing steps followed by 5 min interval until all complexes were precipitated. The latter was suspended in 10 mL fresh medium for 5 min to select all non-attached cells using various sedimentation speed. The harvested complexes were used for the consecutive experiments.

Histological staining
The biomaterial-cell-complexes were xed with 4 % paraformaldehyde (PFA, Roth, Germany) for 20 min at 4°C followed by two washing steps in distilled water. The cell biomaterials complexes were dehydrated in ethanol (Roth, Germany), cleared in xylene (Roth, Germany) and were processed for para n embedding.
Sections of 6 µm were produced using a microtome(Leica, Germany), were transferred to glass slides (Roth, Germany) and were processed for hematoxylin and eosin histological staining to get an overview of cell-biomaterial interaction. The sections were mounted using Eukitt mounting media (HICO-MIC, Hirtz & Co, Germany) and were examined using light microscopy microscope equipped with a digital camera and the LAS V4.4 software (Leica, Germany).

Phalloidin staining
Complexes were xed in 4 % PFA, were washed twice for 5 min interval and were then incubated with phalloidin, an actin laments cytoskeleton staining (2, 5 %, Sigma-Aldrich, Germany) for 30 min. The nuclei were counterstained with 0. 05 % Hoechst dye in TBS for ve min (Invitrogen, USA). After the complexes were stained, they were transferred in PBS buffer on a microscopic slide and were examined using uorescence microscope (Axio Observer.Z1, Carl Zeiss, Germany).

Scanning electron microscopy
To examine the morphology of the biomaterial surface and the appearance of attached cells, a scanning electron microscopy (SEM) was performed. Cell-biomaterial complexes were xed in 2 % glutaraldehyde in cacodylate buffer (Merck, Germany). The specimens were dehydrated in ethanol gradient for ten min and afterwards, were coated by hexamethyldisilazane (Merck) overnight. After drying, they were sputtercoated with Au/Pd with a Balzer sputter coater (SCD 020, Balzers Union, Germany) and examined by Digital Scanning Microscope (DSM 940, Zeiss at 15kV and 16 mm working distance). The cell biomaterial complexes were photographed using a Digital Image Scanning System 5 and Digital Image Processing System 2.9 (Point Electronic, Germany).

Transmission electron microscopy (TEM)
To assess the ultramicrocellular morphology of the cell-biomaterial in combined culture, the complexes were xed in cacodylate buffer solution (pH 7.2) containing 2 % PFA, 0.02 % picric acid (Fluka, Germany) and glutaraldehyde (Sigma Aldrich, Germany) for 24 hours at 4°C. After that, the complexes were post xed in 1% osmium tetroxide (Sigma Aldrich, Germany) in 0.1 M cacodylate buffer for two hours at room temperature. The complexes were counterstained with 0.5 % uranyl acetate (Delta microscopy) for 30 min and 0.2 % lead citrate for 80 sec. Then, complexes were dehydrated and embedded in Peon (Sigma-Aldrich). Ultrathin 70nm sections were generated using an ultramicrotome (Reichert -Jung Ultracut, Leica Microsystems) and were examined using a TEM (EM 109, Zeiss, Germany).

Life cell imaging
To investigate the cell viability and the capacity to spread out from the biomaterial after having attached on their surface, life cell imaging was carried out. Cell-biomaterial complexes were placed into a 35x10 mm culture dish (VWR, Germany) with 3 mL basic medium and then incubated for 60 min under standard culture conditions. Life cell imaging was implemented using Axio Observer Z1, Temp Module S, CO 2 Module S, Zeiss, Germany). Incubation parameters were established at 37 °C and 5 % CO 2 . The images were taken every 15 min up to 72 h. The analysis was performed using the Axiovision Imaging Software (Zeiss, Germany). Furthermore, the complexes were seeded in 24-well plastic dishes and were incubated for 24 h under standard conditions with basic medium. After this, cells were xed in 4 % PFA for 20 min and were stained in phalloidin.

MTT cell viability assay
To evaluate the viability of cells already attached on the biomaterial, a colorimetric MTT assay was performed. Viable cells are able to reduce the yellow substrate MTT (3-4, 5-Dimethylthiazol-2yl -2, 5diphenyltetrazoliumbromid, Sigma-Aldrich, Germany) to the insoluble blue formazan, the later can be solubilized and measured by a spectrophotometer reader at a speci c wavelength. Therefore, 2x10 6 cells with MC, B30 and B30Str were incubated in the two mentioned settings for 4 days. After washing twice in basal medium (BM), the cells and biomaterials were incubated with MTT for 12 h. The cells were lysed with 200 µL of Dimethylsulfoxid (DMSO, AppliChem, Germany) together with a tissue Lyser in oscillation frequency of 50 HZ (Quiagen, Germany) for 5 min. Hereafter, the color intensity was measured by a microplate reader at 570 nm (Tecan, Germany) and was analysed with Magellan TM -Data Analysis Software (Crailsheim, Germany).

Osteogenic differentiation induction
To examine the osteogenic differentiation capacity of combined cultivation of MSCs together with biomaterial (B30, B30Str and MC), the cells were cultivated in growth medium for 48 h to facilitate cell attachment on biomaterials. The cells were allowed to differentiate in osteogenic medium containing DMEM, 5 % FBS, 0.05 mM ascorbic acid-2-phosphate (Sigma-Aldrich, Germany), 10 mM βglycerolphosphate (Sigma-Aldrich, Germany) and 0.1 µM dexamethasone (Sigma-Aldrich, Germany) up to 21 days. Cells were provided with fresh medium twice a week. Cells were incubated in parallel either in BM (5 % FBS in DMEM and 1 % P/S) or osteogenic differentiation medium without biomaterials were served as control (NC). Osteogenic differentiation was assessed by evaluating the morphological alteration, histological examination using alizarin red S (ARS) staining, alkaline phosphatase (ALP) activity and osteogenic relative markers at day 7, 14 and 21 post induction.

Mechanical uid shear stress
MSCs were seeded in combination with MC, B30 and B30Str in a ratio 1x10 5 per well in a 24-well culture plates (VWR, Germany. After 48 h, the growth medium was replaced by the osteogenic differentiation medium (OD). The plates were divided into two experimental groups; static (ST) and mechanical uid shear stress (FSS) culture conditions. In the latter, the cells were incubated under a regular vertical rocking pattern. To optimize the mechanical stress, a rocking angle of 10° with a frequency of 40 turns/min and a uid depth of 3.13 mm were generated. The formula for the FSS calculation was previously described by [30]. The assumed uid viscosity of 10 -3 Pa s, the FSS was 77.21 mPa (in non SI-units 0.77 dyn/cm²) at the center of the dish bottom. The experimental setup guaranteed that the cells were covered with medium during plates cycling. In parallel, cells were grown in parallel in BM served as negative control.

Alizarin Red S staining (ARS)
ARS speci c dye with a nity to "Ca 2+ " used as an indicator for Ca 2+ deposition in the mineralized matrix. Fixed cells in 4 % PFA were washed in distilled water three times at 5 min interval. The cells were incubated with 1 % ARS staining solution (pH 4.2) (Roth, Germany) at room temperature for 30 min. Excess of staining was washed three times in distilled water for 5 min to remove the unbound dye. The morphological alterations following each time point were examined and were photographed using light microscope equipped with a digital camera and the LAS V4. 4 Software (Leica, Germany).

Semi-quanti cation of ARS staining
ARS stained cells were washed twice in distilled water then were incubated with a volume of 2 mL of 10 % Cetyl Pyridinium Chloride (CPC, Roth Germany) with a moderate shaking for 60 min. An equal volume from each experimental group was transferred to a 96-well microplate. The absorbance was measured in triplicates at 562 nm using microplate reader (Tecan, Germany).

Measurement of alkaline phosphatase (ALP) activity
Cells were incubated with 500 µL of 1 % Triton TM X-100 in 10 mM Tris (pH 7.4) at 4 º C for 60 min. The cells/biomaterials complexes were transferred into a 1.5 mL Eppendorf tube. Cell lysates were centrifuged at 28400 g for two min at room temperature and kept on ice. P-Nitrophenylphosphate (2 mg/mL) was dissolved into buffer solution containing 1M Tris and 5 mM MgCl 2 (pH 9.0). A volume of 50 µL of cell lysate were incubated with 150 µL of p-Nitrophenylphosphate solution for up to 30 min. The mixtures were loaded in triplicates into a 96-well microplates. The standard of p-nitrophenol solution was used to prepare the standard curve. ALP potency to hydrolyze p-Nitrophenylphosphate into p-Nitrophenol (PNP) was measured as previously reported [31]. The absorbance was measured at 405 nm by using a microplate reader. The rate of ALP activity was measured using the equation, Y = mx+b. Where, x= sample absorbance value and Y= pNP concentration of samples.
2.14. Real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from cells cocultivated with MC, B30 and B30Str at day 7, 14 and 21 post osteogenic induction using an innuPREP RNA mini kit (Analytik, Jena AG, Germany) and was quanti ed using a UV-Spectrophotometer (Bio-Photometer, Eppendorf AG). Cells in parallel were examined either incubated in BM or osteogenic medium without biomaterials were served as controls (NC). Brie y, RNA samples were incubated with 3.9 µL per sample DNase mix containing 1.

Effect of biomaterials on MSCs viability and migration potential
To get further insight into cell-scaffold interaction, cell motility was assessed following 4 days of SC and RC conditions using life cell imaging. In this study the migration capacity can also be used as a quantitative tool for cell viability. Motility of cells that had attached to the different scaffold materials was studied at 15 min intervals over 72 h using the life cell imaging set up. Following the establishment of biomaterial/cell-cell attachment, cells started to grow out of the biomaterials and to migrate in a radial fashion into the vicinity of the scaffolds via developing long cytoplasmic processes. In addition, those cells cultivated in conjunction with MC demonstrated enhanced viability and proliferation rate under RC in comparison to SC condition ( g. 1k, l). Interestingly, within the cytoplasm of stem cells migrating away from B30 granules small particles can be detected. The quantity of internalized particles per cell is variable. Given the fact that B30/B30Str granules show a strong auto uorescence using the appropriate wave length suitable for the detection of the phalloidin staining, the assumption suggests, that the cells were indeed able to incorporate parts of the B30 granules via autophagy in order to gradually degrading them. This observation was con rmed by TEM of the cells and pellet complexes ( g. 1m, n). In some cases, after having migrated away from the scaffold material, MSCs were observed to re-invade the scaffold particles.

Combined MSCs with Osteogenic differentiation under static culture
To understand the effect of various biomaterial compounds on MSCs fate, the cells were allowed to differentiate into the osteogenic lineage using a standard osteogenic induction cocktail (OD) under static culture condition. Evaluation of the alkaline phosphatase activity (ALP) as an indicator for osteogenic differentiation was performed up to 21 days post induction.

Evaluation of the osteogenic differentiation marker expression
In order to evaluate the effect of combined biomaterials together with osteogenic medium on MSCs differentiation, osteogenic relative marker expression was analyzed at days 14  Furthermore, comparing the osteogenic induction using various biomaterials, Runx2 expression displayed two-three folds increase in the B30 and B03Str-based osteogenic induction in comparison to either osteogenic differentiation alone or with MC biomaterials ( g. 4f). The effect of FSS on ALP expression was also monitored at day 14 and 21. The data showed that ALP expression was only upregulated under combined osteogenic differentiation together with B30Str at both time points either compared to BM (p<0.01) or compared to other experimental protocols (p<0.001) ( g. 4g, h).

Discussion
Mesenchymal stem cells and biological bone substitute material offer a promising approach to restore bone defects. Such combination requires a proper selection of scaffold biomaterial that maintain a suitable niche for mesenchymal stem cell proliferation and differentiation. The ideal scaffold should provide a suitable vehicle for cell viability, adhesion, migration, proliferation and differentiation.
Optimization of such criteria would impact on the selection of the superior combination for regenerative medicine. In the current study the effect of biomaterial bone substitutes on equine adipose derived mesenchymal stem cells morphology, viability, adherence, migration and osteogenic differentiation were investigated.
The data revealed that scaffolds including MC, B30, and B30Str demonstrate suitable delivery vehicles for MSCs in terms of cell adhesion, viable networking and migration within the pores of the biomaterials as shown by histological staining. In addition, MC biomaterial promoted cell viability compared to B30 and B30Str under both static and mechanical FSS. These data suggest that the construction of various biomaterials is a determining factor for MSCs growth and therapeutic potency. It has been found that combined cultivation of chondrocytes with microcarriers in a 3D fashion facilitates phenotype maintenance by delivering mechanical stimulation [33]. Furthermore, MC have the ability to support cell viability and cell adhesion [34]. Although biomaterials play a role for cell survival and proliferation [35,36], it is questionable whether they also support MSCs differentiation. There are some negative examples.
A study has shown that despite the silicon rubber microcarrier promoted cell growth and attachment, the functional differentiation of gastric stem cells was abolished [37].
In our study we could show that MSCs detach from the biomaterial and migrate in a radial-pattern. In some instances cells internalize biomaterial particles into their cytoplasm during the detachment process as could be shown by TEM and phalloidin staining. These data are important criteria indicating and con rming the biocompatibility and biodegradability of the scaffold material under investigation. It has been documented that combined cell culture with collagenous microcarriers are favorable for three important aspects including cell attachment, cell proliferation and cell detachment [38,39]. In order to evaluate the cell migration out of the biomaterial, we quanti ed the CSA of complexes with MSCs and biomaterials in the life cell imaging set up. The analysis revealed a faster cell detachment from MC compared to the B30 biomaterial in an observation period of up to 72 h. These results could be referred to the morphological differences and basic construction of MC compared to B30. In the rst biomaterial, the spherical form, and the micropores provide stable cell adhesion conditions so that cells spread on the whole surface of MC. Thus, there is a large number of cells that may detach after the adhesion of the MC/ cell conglomerate on the culture plate. In contrast, the sharp granular surface of the B30 biomaterial leads to an unstable attachment and thus an impaired cell detachment. These data are congruent with the enhanced cell viability on MC. In agreement with our hypothesis, it has been documented that MC should provide a suitable cell attachment to tolerate mechanical stress but also not to impair cell detachment at the end of the regenerative process as previously reviewed [40]. Thus, our data point out the suitable support of cell migration out of the biomaterial, which should be the basis for the differentiation fate and an e cient tissue regeneration.
Our data revealed no signi cant alteration in the CSA of the combined cell pellet together with B30 and MC in the rst 12 h of the cultivation period. This aspect might refer to the establishment of the proper cell niche with a stable and secure cell adhesion on the surface of the appropriate biomaterials before the induction of further cell networking and cell proliferation.
The data of the presented study revealed that the ALP activity at day 7 and day 21 is increased by the osteogenic induction under static culture conditions in all experimental setups with biomaterials. The osteogenic commitment was indicated by ARS semi-quanti cation particularly in the presence of combined MC and osteogenic medium. These data are in the same line with previous report that showed a rapid osteogenic differentiation, matrix mineralization and upregulation of Runx2 and ALP expression in periodontal stromal stem cells after cultivated with alginate-based MC [41]. The osteoinductive effect of MC could deliver stable cell adhesion via the gelatin coat and induction of matrix mineralization. In a similar study comparing the effect of enzymatic harvested and MC bound cells regarding the osteogenic differentiation of human fetal MSCs, there was an upregulation of the osteogenic markers as well as matrix mineralization of MC bound cells [42]. Additionally, our data point out that there might be a link between the osteogenic commitment of cells and the structure of MC. It has been reported that gelatin coated surfaces or MC fully generated from collagen enhance the osteogenic differentiation and matrix mineralization of human bone marrow MSCs as indicated by enhanced ALP activity, ARS staining and upregulated expression of ALP and bone sialoprotein [42]. On the other hand, the analysis of the osteogenic speci c markers have shown that under static culture conditions in the presence of an osteogenic induction medium, B30Str induced an early upregulation of Runx2 and ALP expression at day 14. However, MSCs cultivated in combination with B30 biomaterial showed a later upregulation of Runx2 at day 21. These data highlight the potential of biomaterials on the osteogenic induction of MSCs in a time wise comparison. In agreement with our data, an earlier study has shown that calcium phosphate based hydroxyapatite and a composite of degradable gel (BONITmatrix) have promoted the osteogenic differentiation of human bone marrow derived MSCs even without the addition of any osteogenic induction medium as could be shown by an enhanced ALP activity and an upregulation of osteogenic markers [43]. The authors of this study concluded that the surface interaction of MSCs with a certain and suitable biomaterial already act as an osteogenic stimulant even in the absence of an osteogenic induction medium. Furthermore, by integrating strontium within the B30 biomaterial, the osteogenic commitment was even promoted as shown by the upregulation of Runx2 and ALP expression. In the same line, several reports revealed that strontium enhances the osteogenic differentiation and matrix Ca 2+ deposition which can be a useful tool for bone repair [44]. In another study from the same group reported about an enhancement of the osteogenic potential of human MSCs by a collagen-strontium based hydroxyapatite scaffold. In the latter material the included strontium activates bone formation via the β-catenin signaling pathway [45].
The data revealed that the combination of B30Str and the osteogenic medium enhanced the ALP activity indicating the osteogenic commitment. This could even be enhanced under mechanical FSS. These results indicate a double osteoinductive effect in which the applied FSS might mimic the invivo bone formation under mechanical loading and the additional presence of strontium promotes Ca 2+ deposition in the vicinity of cells. These ndings con rm reports suggest that strontium promotes osteoblasts viability and reduces the osteoclasts activity as previously reviewed [46]. Moreover, strontium has shown to promote the matrix mineralization by increasing the Ca 2+ level in the extracellular microenvironment which modulates the activity of a Ca 2+ sensory receptor [47].
Surprisingly, the expression of Runx2 and ALP was only weak under conditions of the osteogenic induction using MC under static culture conditions. However, the combined osteogenic medium with MC under FSS increased Ca 2+ deposition as shown by CPC analysis not only compared to standard protocol without biomaterials but also in comparison to a B30 and B30Str based protocol. The data clearly indicate that FSS enhances the osteogenic capacity of MC compared to static culture condition. This is also underlined by the increased Runx2 expression under the stimulation of FSS in most experimental setups independent of the biomaterial used. In this context previous studies by our and other groups have shown that the ectopic stimulation using Ca 2+ [48] and/or mechanical FSS [49] promoted osteogenic differentiation of MSCs.

Conclusion
The present study investigates the impact of various biomaterials on the osteogenic differentiation potential of equine adipose tissue MSCs under static and mechanically stimulated culture conditions. We provide evidence that especially the collagenous MC promoted cell viability, migration as well as matrix mineralization suggesting the usefulness of MC as a scaffold for cell-based therapy. All biomaterial constructs promoted ALP activity in the presence of osteogenic medium already under static nonstimulated culture conditions. We found that FSS increases cell viability of those cells cultivated on MC in the osteogenic medium which might be an important factor to enhance tissue regeneration. Our data revealed that mechanical FSS enhanced osteogenic differentiation of MSCs as shown by increased ALP activity and upregulated Runx2 and ALP osteogenic markers expression which could be a target to promote bone regeneration. The upregulation of the osteogenic marker Runx2 was found to be B30 and B30Str biomaterials dependent.

Declarations
Ethics approval and consent to participate All the standard procedures were approved by Justus-Liebig-University of Giessen and the local authorities (RP Giessen) regarding animal care and use.

Consent for publication
Not applicable Availability of data and material The data collected and the analysis performed to generate the manuscript results are available from the corresponding author on reasonable request.

Competing interests
All the authors have declared no con ict of interest regarding the publication of this article.

Funding
This work receives no funding to report or Not applicable.
Authors' contributions MIE, MCK and FG have collected the raw data set, analyzed the results and wrote the original draft of the manuscript. SA has interpreted the results, revised and nalized the submitted version of the manuscript.    GAPDH was used as an endogenous reference. The data presented as mean ± SEM. *= p<0.05, **= p<0.01, ***= p<0. 001).

Supplementary Files
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