In Vitro Comparison of PU, PU-CA, and PU-CA-GO scaffolds to evaluate osteogenic differentiation of mesenchymal stem cells

doi:10.21203/rs.3.rs-4509036/v1

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Research Article

In Vitro Comparison of PU, PU-CA, and PU-CA-GO scaffolds to evaluate osteogenic differentiation of mesenchymal stem cells

https://doi.org/10.21203/rs.3.rs-4509036/v1

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Background

Nanoscale scaffolds play a significant role in bone tissue engineering due to their appropriate biological activity which could be increased by the presence of nanoparticles such as graphene oxide (GO). This study aimed to develop a scaffold based on polyurethane (PU), cellulose acetate (CA), and GO nanoparticles for bone tissue engineering by electrospinning technique.

Methods

In this study, the scaffolds including PU, PU-CA, and PU-CA-GO, were prepared to assess the differentiation of human adipose-derived mesenchymal stem cells (AD-MSCs) into bone. The scaffolds were characterized via scanning electron microscopy (SEM), FTIR, water contact angle, and tensile test. After cell culture, SEM, DAPI staining, and MTT assay were performed. Then, alizarin red staining (ARS), alkaline phosphatase (ALP) activity, and calcium maps were employed to evaluate the differentiation potential of MSCs.

Results

The results showed that GO nanoparticles were successfully distributed with PU-CA polymer without any agglomerations. The PU-CA-based scaffolds created hydrophilic surfaces that supported cell attachment and proliferation and provided a better milieu for cells. Also, GO increased the strain and Young modulus of the scaffolds and also, better osteogenic differentiation. ALP activity, calcium deposition, and ARS indicated that PU-CA-GO scaffold enables a suitable matrix for the osteogenic differentiation of AD-MSCs.

Conclusion

In general, the porous electrospun PU-CA-GO scaffold with desirable mechanical properties, could increase osteogenic differentiation of AD-MSCs. Hence, this scaffold could be considered as a favorable biodegradable candidate for bone tissue engineering. However, animal evaluations should be considered in future investigations.

Cellulose acetate

Polyurethane

Graphene oxide

Osteogenic differentiation

Mesenchymal stem cell

Recently, people older than 50 years old in developed countries have suffered from musculoskeletal diseases, including a wide majority of chronic pain. Based on research, bone tissue injuries can cause trauma, infection, tumors, and local disorders (1). Treatment of bone lesions through surgery, autotransplantation, and allotransplantation has a lot of problems, such as limited supply, donor site morbidity, unsuitability for large bone grafts, and unpleasant feelings at the injury site (2). World Health Organization (WHO) officials have introduced 2000 to 2010 as the golden time for developing bone defect regeneration in worldwide tissue engineering studies. Accordingly, tissue engineering was considered to be interdisciplinary knowledge in sciences such as chemistry, physics, engineering, biology, and medicine to solve musculoskeletal problems (3). Nowadays, adipose-derived mesenchymal stem cells (AD-MSCs) have been utilized in various tissue engineering applications. These cells have a high potential for repairing and regenerating damaged tissues. Adipose tissue is a suitable source of MSCs because of the relative abundance of AD-MSCs, multipotential ability, and easy harvest. Since MSCs are highly active primary cells, they can be differentiated into various cells, such as adipocytes, osteoblasts, and chondrocytes (4). The self-renewal potential of MSCs increases the number of isolated AD-MSCs for researchers in an administered procedure at in vitro conditions. Since the amount of tissue essential during the primary extraction is restricted, cell proliferation develops after culturing on scaffolds (5).

Nanofibers are desirable candidates for providing a simple system in vitro experiments. Using nanofibers in scaffolds is a natural imitation of tissue in which fibers are embedded in a matrix. Electrospinning, with the ability to create micro-nano polymeric fibers similar to the extracellular matrix (ECM) structure, has attracted much attention in tissue engineering. In this line, electrospinning is an easy, scalable, versatile, and cost-effective method to develop fibers with various diameters, from nanometers to a few micrometers, in different forms (6). Recently, electrospinning nanofibers have been used as a suitable clamp in bone regeneration and repair studies (7). These scaffolds used to support bone cell growth could improve the mechanical properties, act as structural support for cell proliferation, show morphological behavior similar to ECM, and help the cultured cells maintain their phenotype (8).

Cellulose acetate (CA) is a synthetic polymer that comes from esterifying acetic acid with cellulose. The acetate ester of cellulose imparts several benefits such as biodegradability, high affinity with other substances containing hydroxyl groups, biocompatibility, and adequate flexural and tensile strength, which develops its practicability for the production of nanofiber mats. CA is a widely applied biopolymer for bone ingrowth due to its characteristics, such as hydrophilicity, biodegradability, and mineralization. Taken together, CA demonstrates great potential in tissue engineering due to its biocompatibility and its ability to advance osteogenic cell differentiation and osteoblast proliferation (9). However, due to its high modulus, low breaking stress, poor resistance, and strain, CA has typically been used as a portion of complex composites; thereby, it cannot be used alone as a biomedical material for load-bearing applications (6).

Polyurethane is one of the most widely used polymers in bone tissue engineering, which has been used in combination with CA to augment its biocompatibility and mechanical properties (10). In this line, thermoplastic polyurethane (TPU) is a category of PU with linear molecular chains defined by a wide range of mechanical, physicochemical, structural, and morphological attributes that are used more extensively than biodegradable polymers in the biomedical field. Depending on the raw materials used to produce the PU, it is a biocompatible, biodegradable, and non-toxic polymer with a high tensile strength that causes more resistance. Polyurethanes originate from three portions: 1) the polyol, 2) the chain extender, and 3) the diisocyanate. Polyol is a soft part with hydroxyl (-OH) end groups. The chain extender contains a small molecule with amine terminal or hydroxyl groups. Diisocyanate is a low molecular weight segment that reacts with either a chain extender or polyol (11). PU is utilized with a broad range of mechanical attributes, from tough to flexible, in different chemical compositions. A broad category of products, such as coatings, foams, films, fibers, etc., can be gained from PU in bone regeneration (12). In this line, many researchers represented that PU is one of the most important materials in bone tissue engineering applications, while its hydrophobic nature limits its ability to stimulate cell proliferation and adhesion activities (10), (13). Hence, combining CA and PU can reduce the restriction of both polymers.

Researchers to improve the mechanical strength combined with graphene derivatives within the scaffolds. Besides increasing the physical attributes, the graphene derivatives also improve cell differentiation and proliferation due to their great biocompatibility under specified and restricted concentrations. It is also believed that graphene could provide the differentiation of cells by changing the surface of the materials. In this way, graphene enhances osteoblast growth, proliferation, differentiation, and adhesion (14). Graphene oxide (GO) is a type of graphite sheet with the thickness of an atom, which contains groups of reactive oxygen at the edges (Carbonyl and Carboxyl) and the surface (hydroxyl and epoxide). According to its unique chemical, physical, and mechanical attributes and good biocompatibility, GO creates modern bone regeneration trends (14). Due to stimulating the differentiation process of cells bone-like stem cells, this nanoparticle can be considered a suitable candidate for bone tissue engineering (15). This research aimed to evaluate the effects of these three scaffolds on the regeneration, proliferation, and osteogenic differentiation of ADMSCs. Taken together, the present study was used to realize the definite effect of GO in electrospun scaffolds for the regeneration and repair of bone tissues. Figure 1 represents the graphical abstract of the study.

Synthesis and Characterization of GO

Modified Hummer’s technique was utilized to create GO nanoparticles (16). First, 23 mL sulfuric acid 98% and 0.5 g graphite powder were mixed, then 3 g KMnO4 and 0.5 g NaNO3 were combined gently with the mixture. After stirring the solution for 4 hours at 36°C, 3 mL H2O2 30% v/v and 40 mL deionized water (DW) were added to the solution. In order to dispel impurities, the solution was washed and purified frequently. The laminate graphene nanosheets were acquired by sonication (100 kHz, 120 W, 30 minutes) and centrifugation (4000 rpm, 20 minutes). Ultimately, transmission electron microscopy (TEM) (JEOL, JEM-2000EXII) was applied for the characterization of GO nanoparticles.

Preparation of the polymer solution

PU solution containing 6 wt% (Sigma Aldrich, USA) was dissolved in the 1:3 ratio of tetrahydrofuran (THF) (Merck, Germany) and N, N dimethylformamide (DMF) (Merck, Germany) for 14 hours at room temperature (RT) on a stirrer to procure a uniform solution. Also, for fabrication of the PU-CA scaffold, we used a CA solution containing 18 wt% (Sigma Aldrich, USA) and PU 6 wt% (mentioned above). In this line, CA was dissolved in H2O and acetic acid solutions in a ratio of 3 to 1 for 14 hours at RT on a stirrer; after that, CA and PU solutions were mixed in 1:3 ratios. For the preparation of PU-CA-GO nanocomposite, 0.001 gr of the graphene oxide was sonicated in the THF solution for 1 hour and then added to the mixed solution of PU-CA. Finally, these three solutions moved to distinct syringes and ran with an electrospinning device.

Fabrication of the electrospun scaffolds

Each kind of these three solutions was transferred to separated syringe tubes and was run by the electrospinning device (Nanoazma, Iran) at a 19 cm distance with two nozzles under 15 kV voltage at a drum speed of 300 rpm and a flow rate of 0.2 mL/h between the collector and needles. The solution of each scaffold was electrospuned for 7 hours in the manner mentioned. Then, fibers were accumulated on the drum and utilized for characterization and cell seeding. To increase the surface hydrophilicity of the scaffolds, they were placed in a plasma cleaner for 3 minutes. Finally, for cell seeding, the fabricated scaffolds were rinsed with PBS and treated with 70% ethyl alcohol under UV radiation for 20 minutes. The sterilized scaffolds were placed in DMEM culture media with 10% FBS and incubated at 37°C to identify microbial contamination.

Characterization of electrospun nanofibers

SEM (Philips XL30; Philips, Netherlands) has been applied to determine the morphology, structure, and size distribution of electrospun scaffolds that coat with a 10 nm layer of gold under a 25 kV accelerated voltage for each specimen. Image j software (National Institute of Health, USA) was utilized to assess the fiber diameter from images obtained by SEM. Also, Fourier transform infrared spectroscopy (FTIR) was employed to recognize molecular components, functional groups, bonding configurations, and structures in the wavenumber range of 4000 − 400 cm^− 1. The contact angle (Krüss, Hamburg, Germany) was investigated to measure the hydrophilicity of the scaffold surface (scaffold groups with and without plasma treatment). The scaffolds were cut 1×1 cm² to characterize water contact angle values and placed on a mainstay where 2 µL of the DW was dropped. The pictures were taken at 0, 5, and 10 seconds. Mechanical properties of scaffolds (1 × 4 cm) were determined by tensile measurement on an Instron Universal Testing Machine (Model STM-20, SANTAM, Iran) with a force of 50 N. These scaffolds were cut (0.5 × 3 cm), fixed on the paper framework, and subjected to tensile force for fracture. Finally, strain at break, the elastic modulus, final tensile strength, and stress-strain curves were determined.

MTT assay

The viability of AD-MSCs confirmed by flow cytometry (17, 18) was investigated via MTT (3–(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide; Sigma, Germany) assay on each scaffold. The cytocompatibility of PU, PU-CA, and PU-CA-GO scaffolds was evaluated on days 1, 3, 7, and 14 by MSCs seeding. The same manner was used as a control group, tissue culture polystyrene (TCPS), for the specimen without a scaffold. In this way, the cell media was removed and washed with PBS buffer. Then, the culture medium containing 10% MTT was used on the cultured cells and incubated for 3.5 hours at 37°C. During this time, mitochondrial dehydrogenases of living cells changed MTT into formazan crystals. Dimethyl sulfoxide (DMSO) was used to dissolve formazan crystals. Then, scaffolds of each well, along with DMSO, were transferred to the microtube and vortexed to dissolve crystals. Ultimately, solutions were accumulated, and their absorbance was investigated through a microplate reader (BioTek, USA) at 570 nm.

Cells culture and adhesion analysis

The attachment of AD-MSCs on scaffolds was assessed via SEM images and the 4,6-Diamidino 2-phenylindolestaining (DAPI) (Sigma-Aldrich, UK) test. Therefore, scaffolds were cultured with 1 × 10⁴ cells/well of density for three groups, and DAPI staining was performed on days 1, 14, and 21. In this way, the media was removed, specimens were washed with PBS and incubated with 4% glutaraldehyde for 40 minutes, and washed with PBS three times. Then, a DAPI stain was employed to stain the cell’s nuclei in the dark for 12 hours. The samples were kept in dark and cold conditions before photographs were taken with a fluorescent microscope (Nikon Instruments, USA). Furthermore, SEM images taken on day one approved the cell’s attachment. In this line, the cultured scaffolds were washed with PBS, cell fixation was done by glutaraldehyde (4%) for 2 hours, and a serial dilution of ethanol (50–100%) was utilized for dehydration treatment. Finally, the scaffolds were coated with a golden layer on the surface and examined by Philips SEM.

Alizarin Red staining (ARS)

ARS is used to approve the mineralized matrix performed on days 14 and 21 of cell seeding. In this regard, the cell-seeded scaffolds were rinsed with PBS, fixed with 4% glutaraldehyde for 40 minutes, and sectioned. After cross-section, 100 µl of alizarin red was added to each specimen for 20 minutes at RT and washed with PBS. Then, the samples were photographed by an Olympus BX46 microscope (Japan).

Alkaline phosphatase activity

ALP enzyme plays a role in the mineralization process and is suggested as an osteogenic marker. In order to measure ALP activities, the ALP assay kit (Man, Iran) was used to confirm the ALP synthesis on cell culture. First, 600 µL of RIPA buffer was used for lysing cells on days 7, 14, and 21. Then, scaffolds were transferred to the microtube, sonicated for 2 minutes, and centrifuged at 12000 rpm at 4°C for 5 minutes. The supernatants that contained total proteins were acquired, and 50 µL of supernatant was mixed with 50 µL of ALP substrate solution (p-nitrophenyl phosphate) and incubated at 37°C for 90 minutes. A microplate reader (BioTek, USA) was used to represent the adsorption of ALP activity in the cell lysates (yellow) at 405 nm. Normalization of data was simultaneously performed against total proteins.

Calcium map and SEM for bone differentiation of AD-MSCs

Calcium deposition was evaluated for differentiation of seeded MSCs on the scaffold by SEM and energy-dispersive X-ray spectroscopy (EDS) to accomplish EDS mapping on day 21. EDS can create illustrations recognized as digital mapping, which are compositional maps of the specimens (19). For SEM images, scaffolds on days 7 and 14 were washed with PBS, fixed by 4% glutaraldehyde for 2 hours, dehydrated by a serial dilution of ethanol, and examined by Philips SEM.

Statistical analysis

This study represented the data as means ± standard deviation (SD). Two-way ANOVA and T-test were employed to compare the means between multiple and two groups, respectively (Graph Pad Prism 4.0, San Diego, California USA, www.graphpad.com).

Physical and Morphological characteristics of scaffolds

The morphology of GO synthesized by the modified Hummer method was investigated by TEM image. The results show that the synthesized GO nanoparticles are layered and wrinkled (Fig. 2). SEM images show the fibers are regular, uniform, and willow-free. Based on findings, the average diameter of the PU (Fig. 2a, d), PU-CA (Fig. 2b, e), and PU-CA-GO (Fig. 2c, f) nanofibers of scaffolds were 1370 (nm), 1407(nm), and 1578 (nm), respectively. Mechanical attributes, such as strain at break, Young modulus, and ultimate tensile strength, were surveyed by acquiring the stress-strain curve of scaffolds. Table 1 shows the results of the tensile strength test on electrospun scaffolds. The PU scaffold had the higher strain among other groups (230%) (Fig. 3a). In this way, the existence of GO in the PU-CA scaffold significantly increased the strain and ultimate tensile strength at the fracture of the scaffold, while there was no remarkable difference in Young modulus (Fig. 3b). To characterize the chemical bonds of polymers and nanoparticles, FTIR spectra of the PU (Blue), PU-CA (Red), and PU-CA-GO (Yellow) samples were evaluated (Fig. 4). The spectroscopy of PU demonstrated the characteristic peaks at 600–620 correspond to the benzene ring in polyurethane, and the characteristic peaks at 1057, 1597, and 1459 wavenumbers represent ester, carbonyl, and amine (20, 21). The adsorption peaks of CA were at 1743 cm^− 1 and 1235 cm^− 1, corresponding to hydroxyl and ether (21). Also, the bands at 1072 and 1622 cm^− 1 are characteristic of the epoxide and C – C groups of graphene oxide nanoparticles (16).

Table 1

Mechanical attributes of PU, PU-CA, and PU-CA-GO scaffolds.
Scaffolds\ property	strain (%)	Ultimate tensile strength (MPa)	Young modulus
PU	230	3/68	1/024
PU-CA	8/54	4/87	1/29
PU-CA-GO	9/37	5/98	1/38

Water contact angel

The wettability attributes of scaffolds were evaluated via contact angles. The hydrophilicity rate of PU, PU-CA, and PU-CA-GO scaffolds before plasma treatment was 94, 62, and 60 degrees, respectively. The contact angle of the mentioned scaffolds after plasma treatment was 92, 33, and 50 degrees, respectively (Fig. 5).

MTT assay

A viability test on days 1, 3, 7, and 14 was done to evaluate the growth and proliferation of cells on TCPS, PU, PU-CA, and PU-CA-GO scaffolds. The absorbance values were evaluated over 14 days. Cell proliferation and survival in all groups have an increasing trend over time. The PU-CA-GO scaffold represented the highest cell activity and viability during 14 days of the assay (Fig. 6). The number of active cells on day one in the TCPS group was enhanced compared to other groups, particularly in the PU scaffold (** P ≤ 0.05). On day 14, the PU-based scaffolds had significant differences from the control group (TCPs) (****P ≤ 0.001, ***P ≤ 0.01, ** P ≤ 0.05). Evaluation of isolated MSCs from abdominal adipose tissue for survival and biocompatibility of electrospun scaffolds was performed using MTT assay. The results of the MTT assay on AD- MSCs displayed a suitable growth with a sensible rate on scaffolds until day 14.

Cell adhesion characterization

On days 1, 14, and 21, the cells were prepared for DAPI staining. The attachment of AD-MSCs with an intact nucleus to all scaffolds was illustrated in blue color with a dark field through fluorescence microscopy (Fig. 7). Although MSCs were attached and spread on scaffolds well, these cells had better attachment and expansion on the nanofibrous surface of PU-CA-GO. On days 14 and 21, MSCs cultured on the GO scaffold showed proliferation, adhesion, and orientation compared to other scaffolds. The results of the SEM microscope for cell adhesion and proliferation of cells on electrospun scaffolds were consistent with DAPI staining. As shown in Fig. 8a, b, and c, morphology, overlapping, and the interaction between cells and nanofibers are well demonstrated after one day. However, compared to other groups, the PU-CA-GO scaffold provided more cell identification and attachment sites to promote biocompatibility (Fig. 8c). SEM images and DAPI staining were applied for cell adherence. Based on the tissue design method, many studies showed the production of cartilage tissue using AD-MSCs. Due to MSCs’ ability to create cell colonies similar to cartilage and bone tissue, they received much attention (22). DAPI staining observations were consistent with MTT and SEM results, confirming the cells’ connection in all three groups of scaffolds.

Bone differentiation of AD-MSC

To demonstrate the calcium deposition as an osteogenic marker, an ARS stain was used on days 14 and 21 (Fig. 9). As clearly represented in the images, calcium deposition was shown on PU-CA and PU-CA-GO scaffolds at two-time point measurements. Over time, more mineral deposits were shown in the GO-containing scaffold. ALP activity was evaluated on TCPS and three scaffolds as one of the primary markers of bone differentiation on days 7, 14, and 21. The consequence of ALP showed a higher activity pattern on day 7, and the highest ALP activity was demonstrated on the GO-containing scaffold compared with other groups on all three days. Significant differences existed between the PU-CA-GO scaffold and the control group (Fig. 10, *** P ≤ 0.01). SEM was used to show the mineralization and formation of calcium deposits on days 14 and 21. A higher rate of mineral crystals on days 14 and 21 was observed on GO-containing scaffolds compared with other groups (Fig. 11).

Energy dispersive X-ray spectroscopy (EDS) analysis

EDS results indicated that the PU-CA-GO has greater calcium deposition than other nanofibrous scaffolds (Fig. 12a, b, and c). The findings show that the presence of C and Ca ions in the PU-CA-GO scaffold was more significant compared to PU-CA and PU nanofibers. The results of the calcium map showed the amount of calcium in the PU-CA-GO scaffold was higher than in other groups. The formation of crystals and mineral particles was indicated by SEM imaging to evaluate the mineral particles.

This study targeted to construct and characterize scaffolds with a material mixture containing PU, CA, and GO nanoparticles via electrospinning to evaluate whether PU-CA with or without compilation of GO nanoparticles ameliorated the interaction of PU-based scaffolds to AD-MSCs attachment, differentiation, and proliferation. The electrospinning technique provided similar conditions to the normal ECM for MSCs to evaluate cell differentiation. Recently, nanoparticles have been used in tissue engineering studies to achieve a better mechanical and biological performance of scaffolds. These nanoparticles are used in various medical fields as engineered scaffolds in different types and sizes. Using nanoparticles in scaffolds opens a new way to repair defective tissue and the goals of tissue engineering (23). GO nanoparticles enhance the bony differentiation of progenitor cells and help bone regeneration. These nanoparticles were successfully created through a modified Hummer technique with wrinkled and lamellar structural attributes. According to other studies, the wrinkled surface developed the nanoparticles’ consistency, appropriate interactions with other polymers, and increased scattering in the polymers (17, 24). It was defined that the GO nanoparticles represented high interfacial reactions with PU-CA polymer, which is an excellent symptom that likely created GO were interspersed on polymers (1). Prior to the viability test, the properties of PU, PU-CA, and PU-CA-GO scaffolds were investigated. According to SEM images for evaluation of scaffolds, the morphology of electrospun fibers was nano and homogeneous in all groups (25). FTIR results showed principal chemical bands corresponding to the employed material for each scaffold. The index peaks of CA and PU polymers were detected in scaffolds. Moreover, index peaks of GO nanoparticles in the PU-CA-GO scaffold were also observed.

Mechanical features of biomimetic polymers, such as stiffness, fibers, pores, and porosity, alter cell manners like proliferation, differentiation, attachment, migration, and spreading, which, following it, impresses tissue ingrowth (26). The biochemical, mechanical, and viability properties of scaffolds evaluated by different tests showed good signs that probably synthesized PU, PU-CA polymers, and proper dispersion of GO nanoparticles on the PU-CA scaffold. The metamorphosis manner of the scaffolds was assessed through compression tests. PU, PU-CA, and PU-CA-GO scaffolds represented compressive modulus of 1024, 1290, and 1380 kPa, which propounds that all scaffolds were stiff. Similar to prior results, increasing the modulus of scaffolds was imputed to the augmenting function of GO nanoparticles. Outstandingly, scaffold stiffness plays a critical role in cell behaviors, containing migration, proliferation, differentiation, adhesion, and spreading (17). Also, the results of the water contact angle represented that the scaffolds before plasma treatment were in the range of hydrophobic substrates, and after plasma treatment, they were included in the range of hydrophilic substrates. Also, the addition of CA and GO nanoparticles shows that hydrophilic groups, including oxygen and hydrogen bonds, increase surface polarity to enhance their hydrophilicity. After plasma treatment, the contact angle of the scaffolds increased the hydrophilicity surface of the scaffolds. These hydrophilic nanofibers promote cell adhesion and proliferation compared to hydrophobic nanofibers. Hydrophilicity improves the uniform distribution of cells at the surface of scaffolds. In this way, the contact angle of water droplets on the scaffold can be used as an important measure for investigating the hydrophilicity or hydrophobicity of the scaffold. The lower contact angle showed the more hydrophilic surface of the fabricated scaffold, and the larger degree indicated the surface’s resistance to water. Based on the results, PU was more hydrophobic than PU-CA and PU- CA- GO scaffolds. Therefore, owing to the possibility of hydrogen bonding and the presence of hydrophilic groups such as oxygen, scaffolds containing CA and GO nanoparticles increase the surface polarity to enhance hydrophilicity (14). In this line, Aidun et al. showed that the addition of GO nanoparticles to polycaprolactone/chitosan/collagen scaffolds increases the hydrophilicity of the scaffold surface (27). The observations of MTT confirmed that all scaffolds, particularly PU-CA-GO nanocomposite, had suitable biocompatibility and were not toxic for AD-MSCs, as indicated by other reports (6, 28–30). Thus, GO could increase the biocompatibility and bioactivity properties of the MSCs.

As well, viability results could be related to the higher hydrophilicity and biocompatibility of the scaffold (31). The SEM findings confirmed that the number of AD-MSCs that adhered to the PU-CA-GO scaffold was more than the PU-CA and PU groups due to the higher surface stiffness value of nanofibers to suitable proliferation in stem cells (32). In this regard, Fu et al. showed that the proliferation and attachment of E1-MC3T3 cells on the Gel-PLGA-GO nanofiber scaffold were much higher than those on the pure PLGA nanofiber scaffold. In particular, GO nanofiber on Gel-PLGA scaffold significantly increased ALP activity and the calcium deposition of E1-MC3T3 cells (28). Osteogenic differentiation of AD-MSCs on PU, PU-CA, and PU-CA-GO scaffolds was investigated by illustrating the calcium deposition via ARS staining, assessing the amount of ALP activity, and EDS analysis. ALP is an enzyme that indicates cell mineralization for assessing bone differentiation of primary cells. Considerable differences were found in calcium deposition and ALP activity corresponding to seeding on PU-CA-GO scaffolds compared to other scaffolds in all-time point quantifications. Hence, GO-containing scaffold was introduced as an influential group for inducing bone differentiation. These data were confirmed further by alizarin red staining, the degree of mineralization analysis, and the calcium map test. On days 14 and 21, the results of the ALP were consistent with alizarin red staining. Elkhenany et al. also focused on bone differentiation using tricalcium phosphate (TCP) and GO nanoparticles. They reported that these nanoparticles could improve osteogenic differentiation of MSCs isolated from bone marrow after 21 days in vitro. Since the ARS showed significant calcium in differentiated cells, they demonstrated that GO as a non-toxic and mimetic nanomaterial could cause osteogenic and mineralize bone marrow (33). In this regard, Fu et al. conformed that GO is the nature donor to increase mineralization (28). Furthermore, it was proved that MSCs provided an osteogenic phenotype when seeded on a strong substitute similar to pre-mineralized bone (34).

This study was one of the first in vitro studies on the bone differentiation of AD-MSCs on the electrospun nanofibers through GO nanoparticles without any osteogenic growth factors. As CA ameliorated the hydrophilicity of the PU-based scaffold, GO nanoparticles also boosted the hardness and wettability of the scaffold. GO nanoparticles and polymeric materials can lead to bone differentiation of AD-MSCs without environmental differentiation growth factors. Therefore, this study can provide novel intuitions for the fabrication of PU-CA scaffold and GO nanoparticles with appropriate compatibility to imitate the ECM of bone tissue. This finding suggested that the electrospun nanofibers with GO nanoparticles could provide optimal economic conditions for bone tissue engineering by reducing costs; thereby, this scaffold could be offered as a favorable candidate for bone reconstruction and repair studies. Although the presence of GO nanoparticles showed an exciting result for bone differentiation in vitro, evaluation of further parameters along with in vivo studies is required.

AD-MSCs

adipose-derived mesenchymal stem cells

ALP

alkaline phosphatase

ARS

Alizarin Red stain

cellulose acetate

DAPI

4,6-Diamidino 2-phenylindolestaining

DMF

N, N dimethylformamide

DMSO

Dimethyl sulfoxide

deionized water

EDS

energy-dispersive X-ray spectroscopy

FTIR

Fourier transform infrared spectroscopy

Graphene oxide

MTT

3–(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide

room temperature

SEM

scanning electron microscopy

TCP

tricalcium phosphate

TCPS

tissue culture polystyrene

TEM

transmission electron microscopy

THF

tetrahydrofuran

TPU

thermoplastic polyurethane

Acknowledgment:

This study was done by a grant of shahid beheshti university of medical sciences with the ethical code of IR.SBMU.REC.1401.026 and ID number of 43002437.

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Editorial decision: Major revisions
08 Aug, 2024
Reviewers agreed at journal
24 Jul, 2024
Reviewers invited by journal
22 Jul, 2024
Editor assigned by journal
31 May, 2024
First submitted to journal
31 May, 2024

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In Vitro Comparison of PU, PU-CA, and PU-CA-GO scaffolds to evaluate osteogenic differentiation of mesenchymal stem cells

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Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Synthesis and Characterization of GO

Preparation of the polymer solution

Fabrication of the electrospun scaffolds

Characterization of electrospun nanofibers

MTT assay

Cells culture and adhesion analysis

Alizarin Red staining (ARS)

Alkaline phosphatase activity

Calcium map and SEM for bone differentiation of AD-MSCs

Statistical analysis

Results and discussion

Physical and Morphological characteristics of scaffolds

Water contact angel

MTT assay

Cell adhesion characterization

Bone differentiation of AD-MSC

Energy dispersive X-ray spectroscopy (EDS) analysis

Discussion

Conclusion

Abbreviations

Declarations

Acknowledgment:

References

Status:

Version 1