Masoud Ghorbani^1*, Mohammad Reza Nourani¹, Hanieh Alizadeh², Vahabodin Goodarzi¹

¹Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
²Department of Cellular and Molecular, Central Tehran Branch, Islamic Azad University, Tehran, Iran

*Corresponding Author: Masoud Ghorbani, PhD, Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. E-mail: dr.ghorbani62@yahoo.com

Received: June 15, 2021; Published: July 21, 2021

Abstract

Background: Spermatogenesis is the process that spermatogonial stem cells (SSCs) differentiate to spermatozoa in the testis seminiferous tubules. Effective in vitro differentiation of SSCs to sperm can be a promising sign for reconstruction of spermatogenesis disorders. This research was designed to evaluate the effect of a 3D nanofibrous scaffold on culture and differentiation of mouse SSCs.

Materials and Methods: In this research, using electrospinning technique, a nanofibrous polycaprolactone (PCL) scaffold incorporated with multiwalled carbon nanotubes (MWCNTs) was fabricated. The nanofibrous PCL/MWCNTs were assessed using Scanning electron microscopy (SEM) and Fourier‐transform infrared spectroscopy (FTIR).

Results: Then, the SSCs were seeded on the PCL/MWCNTs scaffolds and they had high survival rate and differentiated to subsequent cell lines. Also, molecular result demonstrated that the SSCs on the 3D scaffold overexpressed the C‐kit and SYCP3 proteins.

Conclusion: Finally, this research showed the synergistic effects of 3D scaffolds on proliferation and differentiation of SSCs.

Keywords: PCL; MWCNTs; Scaffold; Spermatogonial Stem Cells (SSCs); Spermatogenesis

Introduction

  About 15% of couples are infertile worldwide [1], that 7-12% of all men complain of infertility in reproductive age [2,3]. Azoospermia is an important male infertility cause that approximately 1% of all men or 10-15% of infertile men suffer from. Sometimes normal volume of a ejaculated semen contains no sperm which is called non-obstructive azoospermia [4,5]. Despite being infertile, these patients have potential to initiate a pregnancy, their testis biopsy revealed that 30-60% of these men have focal areas of spermatogenesis [6]. Severe male infertility occurs in 2/3 of infertile men that have untreatable testicular disorders and induce spermatogenic failure [7]. Spermatogenesis is cornerstone of male fertility through differentiation of spermatogonial stem cells (SSCs). These cells are tissue-specific stem cells with self-renewal and differentiation potentials [8], which could be supported by a long-term culture system [9].

  One of the innovative approaches in medicine to overcome male infertility is in vitro spermatogenesis specially in 3D scaffolds. Engineered 3D scaffolds can mimic the native extracellular matrix (ECM) [10,11] and provide desired biological niche for stem cells to have self-renewal or differentiation [12]. Being biocompatible, a suitable scaffold meets specific criteria that allow the cells to migrate, attach, proliferate, and differentiate to the desired fate [13,14]. Many polymers can been used to synthesis tissue engineering scaffolds in order to provide the necessary physical and chemical signals for cells to reside and spread in the porous structure [15]. Among polymers, properties of synthetic polymers could be easily tailored to achieve unique architecture and mechanical characteristic for different tissue engineering applications [16]. The most synthetic polymers in tissue engineering are Polylactic acid (PLA), poly glycolic acid (PGA), polyurethane (PU), polylactic acid, polycaprolactone (PCL), and poly (l­lactide­co­ ε­caprolactone) [17-22]. PCL is a linear synthetic biodegradable aliphatic polyester and it is one of the most popular polymers among the researchers for tissue engineering applications. PCL is inexpensive, has a controllable degradation kinetics and mechanical properties and could be easily shaped and manufactured [23,24]. This biocompatible polymer is FDA-approved [25] and exhibits appropriate mechanical, structural [26] and thermal stability. Electrospun PCL scaffold has shown promising results in different tissue engineering applications [27]. Having a porous interconnected structure, nanofibrous scaffolds can mimic the filamentous structure of ECM, facilitating optimal cell growth [27].

  In tissue engineering nanocomposites are designed to improve matrix and scaffold’s properties. Carbon based nanoparticles such as graphene and carbon nanotube (CNT) are highly versatile in biomedicine and tissue engineering [28]. CNTs are tubular nanoparticles composed of carbon atoms which have specific characteristics such high mechanical strength and high electrical conductivity [29]. It has been proved that they could support adhesion and proliferation of different cell types such as osteoblasts [30] and neuronal cells [31] and induce stem cell differentiation to different cell lineage [32]. It has been reported that, spermatogonial cells also remained viable and adherent up to 21days when seeded on a CNT-based scaffold [33].

  According to the high efficiency of 3D nanofibrous scaffolds in male reproductive systems, a PCL scaffold incorporated with Multiwall CNT(MWCNT) scaffold were synthesized using electrospinning technique. Beside morphological and compositional characteristics of the electrospun scaffolds, viability, and spermatogenesis potential SSCs seeded on the designed scaffold were evaluated.

Materials and Methods

  MWCNTs (Nanocyl Korea Ltd) were prepared using Xiao., et al. protocol to create active sites on its surface for more reactions [34]. Briefly, MWNTs were dissolved into 70 ml hydrochloric acid (HCl) (36.5 wt%) solution while slowly being stirred until 2 h. Afterward, it diluted by water, refined by filter, and were dehydrated in vacuum at 40°C for 12h. Thereafter, MWNTs were mixed with 50 ml nitric acid (HNO3) (65 wt%) and warmed up to 140°C inside nitrogen atmosphere until 4 h and finally cooled at 25°C.

  In the next step, PCL (Mw 5 100 000, Chemiekas, Vienna, Austria) was mixed (15%w/v) with N, N‐dimethyl formamide (DMF) and this mixture was stirred for 6 hours at room temperature. Then, for preparation of PCL/MWCNT solution, 3%w/v of MWCNT was mixed with the pure PCL solution and the mixture was ultra‐sonicated for 1h. Finally, for electrospinning, PCL/MWCNT solution was put in a 10 mL syringe and an 18‐gauge metal needle was used. Electrospinning process was done on an aluminum rotating plate as collector with the rate of 200 rpm for 8 hours and 20 kV voltage, the flow rate of 3 mL/h, and distance of 20 cm.

  The electrospun scaffolds morphological analysis was done using scanning electron microscopy (SEM, Seron Technology, South Korea). The scaffolds were covered with gold for 3min by a sputter coater (Quorum Technologies, England) and SEM result were recorded at 20 kV voltage. The fibers mean thickness was analyzed by a image analyzer (Image‐J National Institutes of Health, USA) using measuring diameter of 20 randomly selected fibers and thickness of fibers were then represented.

  The scaffolds structure was evaluated using Fourier transform infrared (FTIR) spectroscopy (EQUINOX 55, Germany). The scaffolds were grinded with KBr and samples were studied in the 400 to 4000 cm−1 wavelength range.

  All animal studies were conducted with approval of the Ethical Committee of Baqiyatallah University of Medical Sciences, Tehran, Iran. Firstly, neonatal mice (3-5 day-old) testes were collected.

  The testes were decapsulated and cut into small fragments and then, the testis tissues were washed with Dulbecco's Modified Eagle medium (DMEM; Gibco, UK), containing 100 IU/mL penicillin, 100 μg/mL streptomycin, and 40 μg/mL gentamycin (all from Gibco, UK). The testis pieces were put in DMEM, containing 0.5 mg/mL collagenase/dispase, 0.5 mg/mL trypsin, and 0.08 mg/mL DNase and the suspension was shaken for 60 minutes at 37°C. Then, the mixture was washed three times in DMEM and most of the interstitial cells were removed. Afterwards, DMEM supplemented with fresh enzymes was added to the seminiferous cord fragments for second digestion step (45 min at 32°C). The cells were washed using staining buffer and then were fixed and permeabilized in 4% paraformaldehyde and in 0.5% Triton X-100 (Darmstadt, Germany) respectively. For blocking of the nonspecific antibody binding, 10% heat‐inactivated goat serum with staining solution buffer was used. For each sample ~1.5 × 10⁵ cells were utilized. The cells were incubated with primary PLZF (ab189849, Abcam) antibody. The coated cells with species‐specific secondary antibodies were put in a staining buffer and they incubated for 30 minutes at 4°C. The flow cytometric analysis was done by a fluorescence‐activated cell sorting (FACS, Sysmex Partec CyFlow Space). Finally, ~1.5 × 10⁴ cells/cm² of the isolated SSCs were seeded on 2D culture vessels (without scaffold) and 3D (with scaffolds) groups. The samples and 2D culture group were incubated in DMEM/F12 containing 10% FBS for 7 and 14 days at 34°C.

  The cells were fixed on fibers using 2.5% glutaraldehyde/PBS solution at 25°C for 30 minutes and the seeded cells were analyzed after 7 and 14 days. The samples using a gradient of ethanol (30%, 50%, 70%, and 100% v/v) were dehydrated. Finally, the cells were covered with gold and then the morphological analysis of spermatogonial stem cells was performed using SEM.

  To proliferation assessment of the SSCs on PCL/MWCNTs scaffolds, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Carl Roth, Germany) assay was performed in 3 times (1, 3, and 7 day). Briefly, after washing the seeded samples by PBS, they incubated with serum‐free DMEM and composed with 5 mg/mL MTT powder at 37°C in a dark place. The medium was removed 4 hours later and for dissolving the formazan crystals, DMSO was added. The Spectrophotometric measurements at 570 nm was performed using a microplate reader (Biochrom, Berlin, Germany).

  Total RNA was obtained from seeded SSCs on 2D and 3D groups by QIAzol (Qiagen, Germany). To remove genomic contamination, RNA was treated with Deoxyribonuclease (DNase I) enzyme (Fermentas, Vilnius, Lithuania). RNA concentrations were measured by Ultraviolet (UV) spectrophotometry (Eppendorf, Germany). The cDNAs were made from 500 ng DNase-treated RNA samples with a RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Germany) by oligo (dT) primers. PLZF, C‐kit, Id4, and SYCP3 expression was analysed and GAPDH was used as a housekeeping gene. For Polymerase Chain Reaction (PCR), the primers gene sequences were got from the National Center for Biotechnology Information (NCBI) database and their exons and introns sequence was determined and primes were designed using the Primer3 online software. The primers are blasted to approve their accuracy and reproduce only the genes’ mRNA sequences and synthesized by Cinnagen company (Table 1). qRT-PCR were done by Master Mix and SYBR Green I (S7563, Thermo Fisher) in a StepOne™ thermal cycler (Applied Biosystems, USA).

Table 1: The list and details of RT-PCR primers that were used to evaluate proliferation and differentiation of spermatogonial stem cells (SSCs).

  The program initiated with a melting cycle at 95°C for 5 minutes to activate the polymerase, followed using 40 melting cycles (30 seconds at 95°C), annealing (30 seconds at 58°C) and expanse (30 seconds at 72°C). The PCR reactions quality was confirmed using melting curve assessments and efficiency of each gene was determined by a standard curve. The reference gene and target gene for each sample were amplified in the same run. All runs were performed in triplicate. The target genes were standardized with the reference gene and expression of the gen was evaluated with the ΔΔCT method.

  The result were described as the mean ± standard error. Analysis of variance (ANOVA) was utilized to compare result by using the Statistical Package for Social Sciences (SPSS) software, Version 18.0 (SPSS Inc., USA). The P value < 0.05 was considered statistically significant (Figure 1).

Figure 1: A scheme of 3D scaffold synthesis process for spermatogonial stem cells (SCCs) isolation, culture and differentiation.

Results

  Base on SEM images of the PCL/MWCNTs (Figure 2), the randomly oriented nanofibers formed a porous micro and nano structure. The fibers diameter average in this scaffold reported 792 ± 37 nm.

Figure 2a and 2b: The SEM of PCL/MWCNTs scaffold (scale bars: 5 and 30 μm). The magnification shows the intra fiber porosity of PCL/MWCNTs scaffold.

  FTIR is a method to diagnose the chemical groups of the composite fibrous scaffolds. Figure 3A shows PCL/MWCNTs scaffold spectra. The PCL FTIR peaks is associated with the existence of C═O, C─O, ─CH3 asymmetric, and ─CH₃ symmetric bonds. The peak of absorbance C═O stretching bonds was detected at 1758 cm⁻¹ and stretching absorbance peaks in 1086, 2943, and 2944 cm⁻¹ were observed associated with the C─O, ─CH₃ asymmetric, and ─CH3 symmetric bonds in PCL nanofibers, respectively. The peaks in 1455 and 1366 cm⁻¹ are related to the presence of ─CH3 asymmetric and ─CH₃ symmetric bending PCL peaks were recognized. Furthermore, some MWCNTs characteristic peaks were observable in the spectra and were attributed to the existence of COOH and ─OH bonds. Also, another absorbance peak in 3450 to 3550 cm⁻¹ was detected which belong to the stretch bending of O─H of the MWCNTs. Moreover, the peak in 2994 cm⁻¹ can be related to C─H stretching vibration in the aromatic structure of MWCNTs (Figure 3).

Figure 3: FTIR spectra of PCL and PCL/MWNCTs composite scaffolds.

  To confirmed the SSCs isolation flow cytometry technique was done, 99.39 ± 2.7% of isolated cells expressed PLZF marker (Figure 4a). After 2 weeks culture, the viability of SSCs on feeder cells (Sertoli cells) in 2D and 3D culture showed in figure 5, the attached and distributed in 3D scaffold was similar to 2D scaffold. The 2D culture phase contrast images showed that SSCs had intend to be aggregated and create colony‐ shaped clusters (Figure 5a and 5b). Generally, PCL/MWCNTs could provide a suitable 3D environment to SSCs differentiation and supported them in culture for 14 days.

Figure 4a and 4b: a: The spermatogonial stem cells (SSCs) immunophenotyping by the flow cytometry and the SSCs are labeled with PLZF antibody. b: Cell viability of SSC on 2D and 3D groups at different times (Day 1, 3, 7) of the post culture.

Figure 5: Differentiated spermatogonial stem cells (SSCs) morphology on 2D (a, b) and 3D (c, d) groups at 7- and 14-days post culture. (scale bars: 10 μm).

  The MTT test was applied to assess the cell growth or loss rate after 1-, 3- and 7-days (Figure 4b). the cell viability in a 3D cell culture was less than 2D cell culture, but it was not significant (P > 0.05) and may happen because of hydrophobicity nature of PCL/MWCNTs scaffolds that induced weak spermatogonial attachment and discarded during medium changing. Based on MTT results, SSCs cell proliferation on 7 day was more than other times (1, 3 days) (P ≤ 0.05), it occurred due to the suitable affinity of SSCs to carbon nanotubes.

  Undifferentiated and differentiated spermatogonia‐specific genes expression and the comparison of them between 2D with 3D (PCL/ MWCNTs) groups demonstrated in Figure 6. The Id4 and PLZF gene expression pattern in two group had no significant difference. The gene expression of C‐Kit and SYCP3 in 3D group was better than 2D group.

Discussion

  Male infertility is an important failure that affects is associated with infertility of about half of infertile couples [35]. There are some techniques that give infertile men a chance to have a healthy offspring, such as microsurgical testicular sperm extraction (m-TESE), intra-cytoplasmic sperm injection (ICSI), and round spermatid injection (ROSI) which at least the round spermatids are essential for success in these processes [36]. Some of the infertile men suffer from azoospermia in which their semen have not any spermatid, one of the useful technique for them is in vitro spermatogenesis [37]. The cancer treatments and chemotherapy are gonadotoxic, the pre-pubertal patient under treatment may will sterilize in their future life [38] and sampling before chemotherapy and the culture of testicular tissue fragments is one of the way to preserve their fertility, so they are another group who take advantage of in vitro spermatogenesis [39]. The current research aims to evaluate the effect of Polycaprolactone (PCL)/multi‐walled carbon nanotube 3D scaffold on growth and differentiation of spermatogonial stem cells.

  The cells distributed uniform in 3D culture, but in 2D culture we observed the SSCs formed the colonies, moreover 3D scaffold induced better cell differentiation. The researches have revealed that the 3D culture systems could increase the stem cells differentiation to different cells such as osteoblast [40], hepatocyte [41] and neurons [42]. It may be due to several factors such as the structural similarity of scaffold to ECM which induce the suitable environment for growth and differentiation of cells [43], and another important factor can be MWCNT stimulation effect on cells differentiation [44]. The cell viability in 3D scaffold was approximately similar to 2D culture system and the 3D scaffold enhanced the SSCs proliferation, which could be due to the electrical and mechanical properties of CNTs [29], as has been demonstrated they could increase the proliferation in different cell lines [45,46]. Generally, in the first days of culture, 2D culture systems maintain the cell viability more than 3D scaffolds, but 3D scaffold increases the cell proliferation in compare to 2D culture systems [47].

  Promyelocytic leukemia zinc finger (PLZF) is essential for normal function of SSCs and indirect controls the earliest cell fate decisions in spermatogenesis [48], imbalance of PLZF gene expression impairs the self-renewal and SSCs differentiation [49]. Helix-loop-helix protein ID4 gene expression have an important role in SSCs pool maintenance [50]. Our result demonstrated that the expression of ID4 and PLZF gene in 2D and 3D culture system was similar that means none of them induce SSCs damage. Ghorbani., et al. revealed that the spermatogonial genes expression in cells that cultured in PLLA/MWCNTs scaffold were decreased [11], but another study indicated that the PCL 3D culture increased the level of PLZF gene expression but had no effect on ID4 gene expression [51]. C-kit is a marker of spermatogonial differentiation and have no direct effect on SSCs survival and proliferation [52]. Synaptonemal complex protein3 (SYCP3) is a meiosis and germ cell differentiation marker [53]. In the present study the gene expression of this two differentiation SSC markers in 3D scaffold were more than 2D culture system that our morphological assessment confirmed this data. Other studies revealed that 3D culture systems enhance the expression of differentiation SSCs genes [11,51]. This betterment may be due to scaffold affinity to ECM that could promote good environment for cells and the scaffold materials that stimulate cells to be differentiated [43,44], so the 3D culture systems improve the cells proliferation and differentiation.

Conclusion

  In this study, electrospun PCL/MWCNT was synthesized and it was shown that these scaffolds could supported SSCs attachment and proliferation also could maintain the cell survival like 2D culture system. Spermatogonial differentiation of SSCs was also demonstrated using qRT‐PCR assay indicating increase in spermatogonial genes expression which including C‐Kit and SYCP3, in 3D groups compared to 2D culture. Thus, we suggest that PCL/MWCNT scaffold can be useful as a new approach in 3D culture system especially for in vitro spermatogenesis.

Funding

No funding was received.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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Citation

Citation: Masoud Ghorbani., et al. “A 3D Engineered Scaffold Enhanced the Growth and Differentiation of Spermatogonial Stem Cells". Acta Scientific Pharmaceutical Sciences 5.8 (2020): 51-58.

Copyright

Copyright: © 2021 Masoud Ghorbani., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.