Enhancing Proliferation and Osteogenesis of Single-cell hBMSCs Encapsulated in Alginate Microgels by Single-Layer Graphene Oxide Nanosheets: In-vitro Droplet Microfluidics Study

doi:10.21203/rs.3.rs-4929938/v1

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Enhancing Proliferation and Osteogenesis of Single-cell hBMSCs Encapsulated in Alginate Microgels by Single-Layer Graphene Oxide Nanosheets: In-vitro Droplet Microfluidics Study

https://doi.org/10.21203/rs.3.rs-4929938/v1

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published 25 Oct, 2024

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Microfluidics cell encapsulation into the alginate droplets offers a way to mimic a three-dimensional (3D) microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Microcapsules offer a new method for creating injectable tissue transplants that are minimally invasive. Several studies have demonstrated the advantages of graphene oxide (GO) in the field of bone tissue engineering. GO has been recently reported as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young's modulus of the alginate and alginate-slGO was obtained as 985.9 kPa and 1414 kPa, respectively. Live/dead assay reveals that slGO maintains hBMSCs viability. Also, fluorescence microscopy images illustrate that slGO can enhance the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the microgel matrix, so that microgels containing hBMSCs gradually become opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration.

Mesenchymal stem cell

Microfluidics single cell encapsulation

Mono-layer graphene oxide nanosheet

Osteogenesis

stiffness

Microfluidics single-cell encapsulation is used as a platform for investigating the effect of single-layer graphene oxide nanosheets on single hBMSC´s fate
slGO nanosheets increase the roughness and decrease the stiffness of alginate hydrogel
Micro-sized slGO nanosheets induces the proliferation and odteogenic differentiation of microencapsulated hBMSCs

Graphene oxide (GO) is a novel nanomaterial that has recently gained significant attention in the field of biomedicine, particularly for the advancement of biosensors, drug delivery systems, tissue engineering, innovative approaches for bioimaging and cancer therapy [1]. It is commonly used in scaffold engineering due to its extraordinary characteristics, including biocompatibility, strong hydrophilicity, and numerous oxygen functional groups (e.g., hydroxyl, carboxyl, and epoxy groups) [2, 3]. In the past decades, there has been a significant increase in the utilization of GO as a promising substance for tissue engineering and regenerative medicine, particularly in stem cell differentiation. Additionally, the electrical conductivity characteristics of GO have a positive impact on enhancing the differentiation of stem cells into different lineages and excitable tissues, like neural, bone, and cardiac muscle [4]. Several studies have shown the benefits of GO in the field of bone tissue engineering. La et al. demonstrated that GO has the ability to decrease the necessary dosage and enhance the delivery of Bone Morphogenetic Protein-2 (BMP-2) in order to promote the osteogenic differentiation of mesenchymal stem cells (MSCs) [5]. Another study has shown that when the GO is present, the 3D bioactive glasses scaffold exhibits improved cytocompatibility and enhanced osteogenesis differentiation ability with rat BMSCs. In addition, GO stimulates the growth of blood vessels and significantly improves the healing of bone at the site of injury in a rat model with cranial defects [6]. One mechanism that has been introduced to explain the effect of GO on bone repair is the enhancement of stiffness in hydrogels that contain stem cells, leading to the promotion of osteogenesis [7]. Another mechanism is enhancing the electrical conductivity of the substrates by stimulating the entry of calcium ions, which facilitates the process of bone differentiation and biomineralization [8]. The findings indicate that the presence of GO greatly enhances the hydrophilicity of culture substrates and improves the adhesion and diffusion capabilities of BMSCs [9]. According to Tabatabaee et al., the presence of GO in gelatin/PHEMA scaffolds enhances the hydrophilicity, electrical conductivity, and compressive modulus of the scaffolds. [10]. A study conducted by ES. Kang et al. revealed that the size of GO particles has a notable impact on the process of hMSc differentiating into osteoblasts. Therefore, micro-sized GO (1–10 µm) promotes osteogenesis more efficiently than nano-sized GO (100–300 nm). [11].

Cell therapy is an innovative approach in tissue engineering and regenerative medicine that involves the transplantation of stem cells into a specific area to promote the development of new tissue. Mesenchymal stem cells (MSCs) possess several advantages compared to other types of stem cells, rendering them highly suitable for cell therapy. These characteristics encompass the capacity for self-renewal, differentiation into various lineages, low immunogenicity, and minimal risk of teratoma development, while avoiding any ethical implications [12, 13]. Human bone marrow-derived mesenchymal stem cells, also known as hBMSCs, have the capacity to differentiate into a wide variety of cell types, including osteoblasts, chondrocytes, adipocytes, neurons, astrocytes, fibroblasts, myoblasts, cardiomyocytes, hepatocytes, endothelial cells, and stromal cells [14]. Nonetheless, the process of implanting hBMSCs into the body is met with notable obstacles, such as immune rejection [15], the development of teratomas [16, 17], and undirected cell differentiation [18]. Therefore, there are presently three main obstacles that the tissue regeneration field is trying to overcome: 1) keeping and growing stem cells in-vitro, 2) controlling stem cell differentiation into particular cell types with in-vivo functionality, and 3) generating multicellular structures that mimic the structure and organization of living tissue [19, 20]. In order to achieve this objective, tissue engineering has utilized novel biomaterials/scaffolds that promote bone formation and stimulate bone cell growth, along with stem cells and growth factors, to enhance the process of bone repair and regeneration [21]. Recent studies demonstrate that the microfluidic single-cell encapsulation technique can enhance stem cell therapy for bone regeneration [22, 23].

Reconstructing the cell's microenvironment using 3D cultures is crucial in order to maintain the survival and differentiation of hBMSCs when the cells are transplanted into target tissues. Cells can maintain their phenotype, adhesion, metabolism, and response to soluble factors, among other effects, within this 3D environment [24]. Several approaches have been developed for growing cells in 3D cultures at a small scale, such as hanging drops, microwells, cellular microarrays, and microfluidic devices [25]. Hydrogels are highly versatile and therefore serve as exceptional scaffolds for tissue engineering [22]. The microfluidics approach for hydrogel microencapsulation provides a highly accurate and precise environment for microscale 3D cell culture, surpassing other methods, as it effectively mimics the ECM. Recent research indicates that stem cells microencapsulation exhibits enhanced viability, proliferation, and differentiation capabilities. These findings can be attributed to the biocompatibility, monodispersity, and efficient exchange of growth factors, respiratory gases, and nutrients facilitated by the microgels. [26–28]. Furthermore, microgels are highly appropriate for bone repair and tissue engineering because of their biodegradability [29], injectability [23], immunoisolation [30], and programmability [31].

Currently, the tissue regeneration field is facing three primary challenges that it is striving to overcome: 1) in-vitro cultivating and expanding of stem cells, 2) regulating the differentiation of stem cells into particular types of cells with in-vivo functionality, and 3) producing multicellular arrangements that mimic the living tissue’s structure and function. [19, 20]. In order to accomplish tissue regeneration using stem cells, the 3D scaffolds that contain the cells must possess both biophysical and biochemical cues that can stimulate the formation of bone tissue. The objective of this study is to encapsulate hBMSCs within homogenous alginate microgels and to investigate the effect of single-layer graphene oxide (slGO) on the viability and differentiation of these stem cells into bone tissue. In order to achieve this objective, hBMSCs and slGO are encapsulated within alginate microgels using a flow focusing single-cell encapsulation design. The study focuses on investigating the behavior of the stem cells and the mechanical characteristics of the microgels.

Chemicals and reagents

All chemicals and reagents utilized in this study were of analytical grade and could be obtained commercially. Acetic acid (≥ 99.5%), calcium chloride, perfluorinated carbon oil, 1H,1H,2H,2H-perfluoro-1-octanol (PFO), and medium viscosity ultrapure medium molecular weight sodium alginate were purchased from Merck. Ethylenediaminetetraacetic acid (EDTA) was purchased from Fluka. Fetal bovine serum (FBS), cell culture α-MEM (α-Modified Eagle’s Medium), and phosphate buffered saline tablet (PBS) were sourced from Life Technologies (Gibco, USA). Penicillin and streptomycin (P/S) (10000 U/ml/10 mg/ml), Trypsin-EDTA (0.25%), and Poly-l-lysine (PLL) were obtained from Sigma-Aldrich. Thiazolyl Blue Tetrazolium Blue (MTT) (0.5 mg/ml) was sourced from Carl Roth (Germany).

hBMSC culture

hBMSCs were purchased from the Royan Institute (Tehran, Iran). hBMSCs were cultured in culture medium (CM) consisting of α-MEM (α-Minimum Essential Medium) supplemented with 1 vol% penicillin/streptomycin, 15 vol% FBS, and 1 vol% l-glutamine (all reagents purchased from Thermo Fisher Scientific), and the medium was changed every two days. Cells were incubated at 37 ˚C with 5% CO₂ and passaged at 80% confluence, and used between passages 3–5 [32].

In order to study the impact of slGO nanosheets on cellular differentiation, hBMSCs were encapsulated within alginate and alginate-slGO microgels and incubated with CM and osteogenic differentiation medium (OM). OM was consisting of α-MEM supplemented with 10 nM dexamethasone, 50 µM ascorbate-2-phosphate, and 10 mM β-glycerophosphate. Encapsulated hBMSCs were induced in OM for 21 days, and 50% of the media was changed every 48 hours. Cultures in CM and OM in the absence of slGO were kept as negative and positive controls, respectively.

slGO suspension preparation

slGO nanosheet powder was purchased from Nanoshel Company, USA. The slGO powder was dispersed in distilled water at a concentration of 100 µg/mL and ultrasonicated for 30 min at room temperature. The functional groups of slGO were determined by Fourier transform infrared (FTIR) spectroscopy (Bruker Tensor 27 Spectrometer, Bruker, Japan) from 4000 − 400 cm^− 1 at room temperature using dried slGO samples. The surface topography and morphology of slGO were examined by atomic force microscope (AFM, CoreAFM, NanoSurf, Switzerland) and scanning electron microscope (FESEM, TESCAN VEGA 3 LMH SEM) after gold coating. The Zetasizer Nano ZS95 (Malvern Instruments Ltd., UK) was used to assess the size distribution and zeta potential of slGO. The measurements were conducted at a temperature of 25°C using a red laser with a wavelength of 663 nm.

MTT assay

A thiazolyl blue colorimetric assay (MTT) was performed to determine the potential toxicity of slGO nanosheet suspension toward the hBMSC cell line quantitatively. Toxicity of the slGO nanosheets at various concentrations of 0, 0.002, 0.02, 0.2, 2, and 20 µg/mL (final concentrations) was investigated separately for different time durations (24 and 48 h) [details in SI].

Microfluidic device fabrication

The microfluidic design was created using AutoCAD software and then produced through the process of soft lithography, as mentioned earlier [33]. In summary, the silicon wafer was coated with a layer of negative photoresist SU-8 2050, which was deposited using a spin-coating technique to achieve a thickness of 50 µm. The coated wafers were then subjected to a hard-baking at a temperature of 150 ºC for a duration of 3 minutes. Subsequently, the wafers were exposed to UV light by aligning them with a transparent photo-mask for a period of 70 seconds. A silicon wafer was fabricated by immersing it in the developer solution for a duration of 2:30 minutes. Subsequently, the silicon wafer underwent a cleaning process utilizing DI water, followed by a delicate drying procedure using nitrogen gas.

Poly-dimethylsiloxane (PDMS) (Sylgard, USA) was thoroughly combined with a crosslinker in a 10:1 proportion. The mixture was then poured onto the template wafer, subjected to a vacuum for a duration of 30 minutes, and left to solidify over night at a temperature of 65ºC. The PDMS mold was removed from the master, and the channel inlets and outlets were created using a 1 mm diameter biopsy punch (Kai Medical, Japan). The PDMS replica was bound to a glass slide after treating it with a plasma cleaner (Harrick Plasma, USA) to make the surface hydrophilic. The device was initially coated with aquapel for a duration of 10 minutes to render the surfaces of the channels hydrophobic. Afterwards, aquapel was removed by flushing it with nitrogen gas. PTFE tubes, with an outer diameter of 1.2 mm, were utilized to establish connections between the channel inlets and outlets and the syringe pump (Elveflow, France). Prior to use, all devices were sterilized by washing them with 70% alcohol and subjecting them to two hours of UV irradiation.

Microfluidics single-cell encapsulation

The microgel precursor solution was prepared by dissolving 2 wt% alginate and 50 mM Ca-EDTA (in a 1:1 ratio, pH 7.4) solution in α-MEM. A small amount of PLL (0.001%wt) is added to the dispersion phase in order to enhance cell adherence in microgels. The continuous phase consisted of perfluorinated carbon oil with a surfactant concentration of 0.1 wt% and an acetic acid concentration of 0.05 vol%. An additional oil phase was created by adding 20 volume percent of PFO to the perfluorinated carbon oil for on-chip de-emulsification. The continuous oil and precursor solutions were loaded into PTFE tubes and connected to inlets and applied as a continuous oil phase and an aqueous phase, respectively. The flow rate of the continuous oil phase and the aqueous phase were maintained constant at 1000 µL/h, and 200 µL/h, respectively. After the on-a-chip gelation and de-emulsification processes, the microgels were extracted from the outlet. The encapsulated stem cells were harvested, and the pH was neutralized using a solution of α-MEM with PBS buffer at a pH of 7.4. The microgels were ultimately obtained by centrifugation (300 g for 5 min). Subsequently, they were rinsed with PBS three times before being dispersed in the culture medium. The encapsulation process is visualized using an inverted microscope (Olympus IX81, Japan) equipped with a high-speed camera (Olympus DP72, Japan). Each droplet's aspired particle size and cell density were achieved by independently adjusting the flow rates. hBMSCs were utilized at a concentration of 6 × 10⁶ cells per mL for the purpose of single-cell encapsulation. The number of cells within droplets was estimated using the Poisson distribution [34].

Cell imaging and live/dead assay

The visualization of single-cell encapsulated hBMSCs in alginate droplets was performed using Dapi and PI (Thermo Fisher Scientific) solutions on days 1, 7, 14, and 21. In this experiment, a volume of 10 µL of droplets containing cells was utilized. Following centrifugation, Dapi and PI were added to the plate well and allowed to incubate for 20–30 minutes at a temperature of 37˚C in order to effectively stain the enclosed cells. Each sample required a minimum of 30 cells containing droplets. During this experiment, both viable and non-viable cells were isolated based on the presence of blue and red nuclei. A live/dead experiment was performed using trypan blue cell counting by Neubar. The percentage of cell viability was determined for each sample by calculating the ratio of the number of viable cells to the total number of cells. In this test, live and dead cells were captured with blue and red nuclei.

RNA extraction and Quantitative RT-PCR analysis

Live cells were extracted from hydrogels using a degradation solution for further analysis [details in SI]. Total RNA was extracted from hBMSCs after 14 and 21 days of culture using REX solution (Pishgam, Iran) according to the manufacturer´s instructions. Following that, the concentration and the quality of total extracted RNA were measured by using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and 1% agarose electrophoresis gel, and then 10 µg of the extracted RNA was then reversely transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Quantitative PCR was conducted using 2x SyGreen High-ROX (Ampliqon, Denmark) on a Real-Time PCR system (Bio-Rad CFX Manager system, USA) following the manufacturer’s instructions. The PCR reaction was carried out under the following conditions: an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95℃ for 10 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 30 seconds. The expression levels of osteogenic-related marker genes (Runx2, ALP, and OCN) were assessed. The primer sequences can be found in SI-Table 1. The expression of the targeted gene was standardized using GAPDH as an internal reference.

AFM topography and cell stiffness

AFM imaging was used to investigate the single-layer structure of GO nanosheets. For this purpose, slGO suspension in deionized water at concentration of 1 µg/ml was uniformly loaded on the cover slip, and after drying at 50℃, it was used for the AFM test. To obtain the topography of nanoparticles, phase contrast mode and Tap190Al-G cantilever were used [35]. Young’s modulus of living cells and microgels was calculated using AFM spectroscopy mode as described previously [36, 37]. The force-distance curves (F-D curves) were measured in liquid mode [details in SI]. F-D curves were analyzed using AtomicJ software.

Statistical analysis

All results were obtained through more than three iterations, and the values were described as the mean standard deviation. The statistical analysis was conducted using GraphPad Prism software using a one-way and/or two-way ANOVA. The statistically significant differences were defined as the p-value being less than 0.05. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

slGO characteristics

The FTIR spectra of slGO as shown in Fig. 1A shows the peak at 1045 cm^− 1 is attributed as (C-O) stretching. The peak at 1218 cm^− 1 is confirmed as (C-O-C) bending, and (C-OH) bending is observed at 1565 cm^− 1. The carbonyl groups are shown at 1708 cm^− 1 as (C = O) stretching and a broad peak at 3416 cm^− 1 is attributed as (O-H) stretching vibration of the (C-OH) groups and water content in the substrate. The obtained results are consistent with previous studies [38, 39]. The FTIR spectra of alginate-slGO illustrate the increase in water content and higher hydrophilicity due to the presence of GO [40]. SEM image (Fig. 1B) confirms the monolayer structure of slGO. The lateral dimensions of GO sheets varied from several hundred nanometers to several micrometers. The topographical and amplitude images of slGO nanosheets on a glass slide were recorded using an AFM, which is shown in Fig. 1C and D. As shown in the topography, the planar structure of GO is quite clear, and the GO nanosheets are completely single-layer in some parts and show a height below 2 nm (blue arrow). In some parts, several layers are stacked on top of each other, showing a height of 10 nm (red arrow). The average roughness in this topography was calculated to be 5.4 nm (Fig. 1E). Dynamic light scattering (DLS) results showed that the average diameter of slGO after 30 minutes of sonication was 916.9 nm with a standard deviation of 128.1 nm (Fig. 1F). Previous studies have shown that the size of GO particles has a significant effect on the differentiation of mesenchymal stem cells to osteoblasts. Thus, GO nanoparticles with a size of 1–10 micrometers have a much greater effect, especially on days 21–28 of the osteogenesis [11]. Therefore, the one micrometer size of slGO nanosheets can be a suitable option for the differentiation of stem cells in alginate microgels. Zeta potential for graphene nanosheets was also equal to -18.7 mV.

The potential cytotoxicity of various concentrations of slGO was assessed using the MTT test, which quantifies the metabolic activity of cells. The MTT assay is the predominant method employed to assess cell viability, proliferation, and cytotoxicity in research studies. As depicted in Fig. 1G, a low dosage of slGO exhibited comparable patterns of biocompatibility. However, it was observed that when the concentration of slGO increased, the proliferation of hBMSCs decreased. A notable reduction in cell viability was caused by slGO at doses of 2 and 20 µg/mL after 48 hours of exposure, which is similar with the findings reported by Halim et al. in prior research [41]. The statistically significant differences of slGO appeared at concentrations of 2 µg/ml and 20 µg/ml after 24 h exposure with respect to the control group (p < 0.0001).

Microfluidics cell encapsulation

According to ionic crosslinking in a flow-focusing microfluidic device, alginate microgels have been generated (Fig. 2A). Droplet formation, gelation, and de-emulsification are all integrated by this device [33]. The detail of applying the present microfluidic chip to encapsulate hBMSCs at the single-cell level is demonstrated below. This microfluidic chip utilizes a flow-focusing design to produce monodisperse and homogenous droplets (Fig. 2B). It consists of two oil inlets, one water inlet, and one outlet. This design incorporates a channel with a diameter of 60 µm for the disperse phase, which connects to the junction of two vertical oil channels with a diameter of 50 µm. Following the junction, an outlet channel with a diameter of 40 µm was constructed, which was subsequently expanded to 80 µm. In all channels, the chip height is 50 µm. De-emulsification is accomplished by inserting the second oil channel in the center of the outlet channel. Droplet generation within the chip occurs at the point where the disperse and oil phases intersect [SV-1]. The droplet size can be controlled by manipulating the flow rate ratio of the oil phase to the aqueous phase (Q_oil/Q_aqu). The chip described above produces microgels with an average diameter of 57 µm at a fixed Q_oil/Q_aqu ratio of 2 (Fig. 2C). The controlled internal gelation of alginate was initiated by use of the neutral-pH Ca-EDTA complex, which chelates calcium ions so that they are soluble but inaccessible to the alginate polymer chains. Besides, the fluorinated carbon oil was mixed with acetic acid. Following droplet formation, acetic acid diffuses into the droplets, lowering pH and finally causing Ca²⁺ ions to be released, which causes homogenous alginate microgel formation (Fig. 2D). It's possible to remove the oil phase right away after droplet formation because of the internal gelation of the droplets. As a result, the microgels are collected at the outlet after the de-emulsification solution enters the outlet channel directly. After the encapsulation process, it is observed that approximately 22% of the microgels contain a single-cell, while 3% of the microgels have several cells, and the other 75% of the microgels are empty. This result is consistent with previous studies and the mathematical limitations of the Poisson distribution [26, 34]. hBMSCs were encapsulated in alginate and alginate-slGO microgels and cultured in α-MEM and OM for 21 days.

Extensive research has been conducted on the process of encapsulating cells using alginate. The primary difficulty typically lies in preserving the long-term survival of the encapsulated cells both in-vitro and in-vivo. In the present investigation, after exposing hBMSCs to acidic conditions induced by the employed approach, the viability of all four formulations was initially assessed for a period of three weeks. The viability of the encapsulated cells was evaluated using the Trypan blue exclusion method at specific time intervals (1, 7, 14, and 21 days). The mean viability for Ctrl- (alginate) and Ctrl+ (alginate + OM) samples, in the entire duration of the experiment, was calculated to be 76 ± 6% and 74.5 ± 4%, respectively. For microgels containing slGO and slGO + OM, the mean viability was assessed to be 64 ± 3% and 66 ± 5%, respectively (Fig. 2E). Therefore, the cells maintained their viability during the experiment.

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Figure 2

The characteristics of microgels

By adjusting the flow rate ratio of the oil phase to the aqueous phase (Q_oil/Q_aqu), the size of the microgels can be modified. The average diameter of collected microgels was calculated to be 60.85 µm with a given Q_oil/Q_aqu of 2 (Fig. 3A). The brightfield microscope image clearly shows that the microgels have a spherical and uniform structure. SEM was used to examine the surface microstructure of the microgels in order to gain a better understanding of their microenvironment. Figure 3 (B and C) displays the contrasting microscopic composition of alginate and alginate-slGO microgels. The alginate microgel exhibits a homogeneous and smooth structure, but the alginate-slGO microgel displays a coarser texture with more ridges. The SEM images of our microgels exhibit an intense similarity to the SEM photos of alginate and alginate-reduced GO hydrogels, as documented by X. Yang et al [42]. It seems that the 3D structure of alginate-slGO presents a distorted and interwoven structure of GO and the microgels in comparison with the structure of alginate microgels [43]. This structure is advantageous as it enhances the mechanical stability of alginate-slGO microgels and facilitates a substantial porosity, consequently increasing the adsorption efficiency of microgels [40].

The data acquired by AFM topography exhibits similarities with SEM images. The AFM topography analysis reveals a smooth and homogeneous microstructure in the alginate microgels. The results indicate that the alginate-slGO surface structure exhibits increased porosity, as depicted in Fig. 3(E and F). Furthermore, the surface roughness of alginate-slGO exhibits a notable increase compared to the surface roughness of alginate alone, which suggests the enhanced hardness of alginate in the presence of GO. The surface roughness of the alginate hydrogel was determined to be 2.219 ± 113 nm, while the surface roughness of the alginate-slGO was found to be 10.963 ± 571 nm. Recent investigations have additionally highlighted the augmentation of roughness induced by GO in hydrogels [40]. Prior research has consistently demonstrated that a greater roughness of 3D substrates results in enhanced adherence and growth of MSCs, as well as the stimulation of bone differentiation [44, 45]. The Young's modulus of the surface of the hydrogels was determined using the nanoindentation method and force-distance curve analysis. The values are 985.9 kPa for alginate hydrogel and 1414 kPa for alginate-slGO hydrogel (Fig. 3D). The alginate-slGO hydrogel exhibits a notable decrease in stiffness compared to the alginate hydrogel. This decrease in stiffness can be attributed to the enhanced hydration of alginate hydrogels in the presence of graphene oxide [46].

Viability and proliferation of microencapsulated stem cells

Alginate has been demonstrated to be a suitable and biocompatible substance for encapsulating cells and has recently been introduced to enhance the survival of cells immediately after being encapsulated [47]. However, the long-term viability of cells inside alginate microgels is very important. Therefore, the viability of hBMSCs was investigated to assess the biocompatibility of alginate-slGO as a cell encapsulant compared to alginate. The objective of this experiment was to investigate the viability of hBMSCs in four different states: encapsulated in alginate microgels (Alg), alginate microgels in the presence of OM (Alg + OM), alginate-slGO microgels (Alg-slGO), and alginate-slGO microgels in the presence of OM (Alg-slGO + OM) over a period of 21 days. Following a 24-h period of encapsulating the cells, the viability rate exceeds 72% in all samples. This outcome is likewise in agreement with the findings of cell counting (Fig. 2E). The maximum survival rate on the first day of treatment is associated with alginate microgels in α-MEM culture media, which is an appropriate medium for cell proliferation. The cell viability for all samples remains above 65% throughout the 21-day period. Hence, the hydrogel substrate possesses the capacity to sustain the viability of stem cells over an extended duration. The viability of hBMSCs within alginate microgels and α-MEM culture media gradually increases until day 21, potentially attributed to cellular adaptation to the alginate substrate and cell proliferation (Fig. 4A). MSCs divide and devote to actively proliferating pre-osteoblasts, which do not release ECM, during the early stages of differentiation. They develop further into non-proliferating osteoblasts that are involved in the initial mineralization, maturation, and secretion of the matrix [48].

Figure 4(B-E) displays the fluorescence images of the encapsulated hBMSCs on days 21, where the nucleus of living cells is stained with DAPI. As depicted in the image, after 21 days of cell culture within alginate microgels, the cells have grown and filled the whole volume of the microgels, resulting in the development of cell plaques within the microgels. Obtained results illustrated that in the presence of slGO, encapsulated hBMSCs show better proliferation during 21 experiment days (Fig B, and E). It seems that a low dosage of slGO can induce proliferation of stem cells [49].

Differentiation of microencapsulated hBMSCs

The mineralization of the microgel matrix provided additional evidence of the osteogenesis of encapsulated MSCs. The initially transparent alginate microgels underwent a change into gray precipitates after three weeks of osteogenic culture. Further light microscopic investigation revealed that after three weeks, most of the microgels, notably those containing cells, displayed a steadily rising opacity and eventually become entirely opaque as a result of the mineral deposition. More precisely, on day 21, most of the alginate-slGO microgels with cells showed more opacity than empty microgels (Fig. 5A), indicating that the inclusion of slGO inside microgels may promote mineralization. The negative charges of slGO are considered to have the ability to attract calcium ions during the process of osteogenesis. [50].

Genes associated with bone formation and the process of mineralization, such as OCN, Runx2, and ALP, were selected as indicators for the differentiation of osteogenic cells. Osteocalcin (OCN), a protein produced by osteoblasts, attaches to calcium ions on the negatively charged surface of slGO nanosheets. OCN is recognized as an indicator of bone growth, mineralization, and calcium ion balance. Runx2 is a crucial transcription factor that plays a vital role in the differentiation of osteoblasts and the regulation of the production of immature bone. Runx2 induces the expression of many genes involved in bone matrix formation during the early stages of osteoblast development. ALP gene expression changes in hBMSCs serve as a crucial early indicator of osteogenic differentiation. [51].

Figure 5B-D demonstrates notable variations in the expression of the three chosen marker genes among the hBMSCs encapsulated in alginate (Ctrl) with alginate-slGO, alginate + OM, and alginate-slGO + OM microgels at both 14 and 21 days. At day 21, the expression of OCN in the alginate-slGO microgels was 4.27-fold higher than in the alginate microgels. Similarly, the expression of Runx2 in the slGO-treated microgels was 5.87-fold higher than in the alginate microgels. In addition, the level of OCN expression in the alginate-slGO + OM microgels was 1.44 times more than in the alginate + OM microgels. Similarly, the level of Runx2 expression in the slGO-treated microgels was 1.24 times higher compared to the alginate + OM microgels. The alginate-slGO microgels exhibited a notably higher ALP expression compared to both the alginate + OM and Ctrl microgels on days 14 and 21. According to the data obtained, microgels treated with slGO and grown in osteogenic medium (OM) exhibited considerably increased expression of OCN and Runx2 compared to alginate microgels cultured in OM. Based on the RT-qPCR, the expression levels of all the osteogenic markers that were tested were considerably greater in hBMSCs that were encapsulated within alginate-slGO + OM microgels, compared to those encapsulated in alginate + OM and the control group. This overall positive osteogenic impact of slGO nanosheets on hBMSCs was consistent with findings in previous reports [52–54] and indicated that the unique structure and composition of slGO nanosheets can be recognized by the cells, and the cell membrane-slGO interaction can lead to intracellular signaling processes, resulting in enhanced osteogenic differentiation [50, 55].

This paper presents the development of a microfluidics-based method for the continuous encapsulation of single hBMSCs within alginate microgels. This facilitated the efficient production of monodisperse cell-containing microgels while preserving the viability and functionality of the encapsulated cells. Our objective was to investigate the effect of slGO on the viability, proliferation, and differentiation of microencapsulated hBMSCs into bone tissue. Since the micro-sized slGO nanosheets have a notable impact on the process of hMSc differentiating into osteoblasts, hBMSCs are encapsulated within alginate microgels in the presence of micro-sized slGO. The microstructure of slGO nanosheets was characterized using FTIR, SEM, and AFM. Alginate-slGO microgel shows higher porosity and roughness and lower stiffness in comparison to alginate microgel. Bright-field and fluorescence images show that the viability, proliferation, and mineralization of microencapsulated hBMSCs in the presence of slGO nanosheets are higher than cells microencapsulated in alginate microgels. Our RT-qPCR results indicated that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. Also, slGO nanosheets can enhance the osteogenesis of microencapsulated hBMSCs cultured in OM in comparison to microencapsulated cells without slGO. It is generally recommended to utilize encapsulated and differentiated MSCs for implantation in the treatment of bone injuries. This is because they have a reduced risk of immune response and teratoma formation, and a higher probability of complete healing. Thus, during the implantation technique, the MSCs undergo a transformation into osteocytes in order to treat injury to the bone tissue. Therefore, our alginate-slGO microgels have the potential to be utilized in the development of tissue engineering and bone regeneration.

hBMSCs: human bone marrow-derived mesenchymal stem cells

MSC: mesenchymal stem cell

GO: graphene oxide

slGO: single-layer graphene oxide

ECM: extracellular matrix

PLL: poly-l-lysine

α-MEM: alpha-minimum essential medium

OM: osteogenic differentiation medium

Ca-EDTA: calcium-ethylenediaminetetraacetic acid

DI water: deionized water

PDMS: poly-dimethylsiloxane

PTFE: polytetrafluoroethylene

PFO: 1H,1H,2H,2H-perfluoro-1-octanol

PI: propidium iodide

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Q_oil/Q_aqu: the flow rate ratio of the oil phase to the aqueous phase

AFM: atomic force microscopy

F-D curves: force-distance curves

FTIR: fourier-transform infrared spectroscopy

DLS: dynamic light scattering

SEM: scanning electron microscopy

RT-qPCR: real time-quantitative polymerase chain reaction

OCN: osteocalcin

ALP: alkaline phosphatase

Runx2: Runt-related transcription factor 2

Acknowledgment

The authors would like to thank the “Clinical Research Development Center of Baqiyatallah Hospital” for their kind cooperation. We would like to thank the guidance and support from the Research and Technology Comprehensive Laboratory (RTCL) of Baqiyatallah University of Medical Sciences.

Author contributions

The subject design was completed by Hossein Soleymani and Ramezan Ali Taheri. Ramezan Ali Taheri supervised the study and analyzed data. Hossein Naderi-Manesh and Mehrdad Moosazadeh Moghaddam provided the materials and instruments. Hossein Soleymani performed the experiments and analysis. Hossein Soleymani wrote and Ramezan Ali Taheri revised the manuscript. All the authors contributed to discussing the results and approved the final manuscript.

Data availability

Derived data supporting the findings of this study are available from the corresponding author on request.

Competing Interests

The authors declare no competing interests.

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No competing interests reported.

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Enhancing Proliferation and Osteogenesis of Single-cell hBMSCs Encapsulated in Alginate Microgels by Single-Layer Graphene Oxide Nanosheets: In-vitro Droplet Microfluidics Study

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Journal Publication

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Abstract

Figures

Highlights

Introduction

Materials and Methods

Chemicals and reagents

hBMSC culture

slGO suspension preparation

MTT assay

Microfluidic device fabrication

Microfluidics single-cell encapsulation

Cell imaging and live/dead assay

RNA extraction and Quantitative RT-PCR analysis

AFM topography and cell stiffness

Statistical analysis

Results and discussion

slGO characteristics

Microfluidics cell encapsulation

``

The characteristics of microgels

Viability and proliferation of microencapsulated stem cells

Differentiation of microencapsulated hBMSCs

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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