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The application of a novel biomaterial based on the secreted products of human mesenchymal stem cells and collagen for spermatogenesis restoration in the model of experimental cryptorchidism.
Abstract and Figures
Male infertility is a widespread problem, and there is lack of effective methods for its treatment. The authors have developed a novel biomaterial for the stimulation of regenerative processes based on the secreted products of human adipose-derived mesenchymal stem/stromal cells (MSC). MSC conditioned medium (MSC-CM), containing bioactive factors produced by the cells, has a great regenerative potential, stimulates vascularization and innervation recovery of the tissue after injury, contributes to the activation of endogenous repair processes due to the additional attraction of stem and progenitor cells to the damaged area. This study investigated the ability of a novel biomaterial, consisted of MSC-CM combined with type I collagen, to stimulate spermatogenesis restoration. Experimental cryptorchidism (2 weeks) was used as a model of infertility. The results of this study clearly show that the administration of MSC suspension or MSC- CM combined with a collagen gel under the testis tunica albuginea effectively stimulates the recovery of spermatogenesis. It was found that biomaterial, containing concentrated MSC-CM, attenuated the hypotrophy of cryptorchid testes and stimulated the recovery of spermatogenesis by enhancing both the total number of spermatozoa and their motile fraction. Using this type of biomaterial, the authors observed 3-times more prominent effect on spermatogenesis compared to non-concentrated MSC conditioned medium. Importantly, this effect was comparable with the results obtained for local administration of MSCs themselves. Use of MSC- CM, containing MSC secretions, including specific growth factors to maintain vitality of spermatogonial stem cells (GDNF, FGF-2, etc.), in combination with collagen gel may be a therapeutic advantage to stimulate spermatogenesis and treat male infertility. This approach is considered to be even more preferable than administration of the cells themselves, due to the various clinical and ethical problems associated with transplantation of cellular material.
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... The advantage of this model is combined damage to key cell components involved in spermatogenesis (both SSC and supporting cells -Sertoli and Leydig cells), which reflects the pathogenesis of testicular deficiency. It is important to note that spontaneous recovery of fertility, at least partial, after the descent of testicles remains possible indicating the retention of some SSC [10]. In addition, this model is feasible to set different degrees of damage by adjusting the duration of exposure of testicles in the abdominal cavity. ...
... The present study was focused on the feasibility of the rat abdominal cryptorchidism model for the evaluation of regenerative drug potency to treat the testicular deficiency. Earlier we showed that concentrated MSC CM stimulated recovery of spermatogenesis in cryptorhid testes with the comparable or even superior effectiveness than MSC themselves [10]. Six experimental groups were included in the study; rats were injected by the following formulations: unconcentrated and concentrated MSC CM within collagen gel, MSC from the same patients both in suspension or combined with collagen gel, and basal DMEM-LG in the collagen gel ("medium control group") along with rats without therapy as a negative control group. ...
... In the group of animals treated by MSC within collagen gel injections we obtained some promising results one month after testes moving into scrotum. But histological analysis of the testes revealed histiocytic granuloma, a severe pathologic factor led to testicular mass decrease, in a half of animals three months after testicle descention [10]. Only the groups of animals treated by concentrated MSC CM or MSC suspension demonstrated the significantly positive results of spermatogenesis restoration compared to the negative control group; therefore, data obtained in these groups were predominantly used to explore the feasibility of the abdominal cryptorchid model for the evaluation the drug-driven regenerative effects on spermatogenesis restoration after the injury. ...
Male infertility represents a severe social and medical challenge. In recent years the progress in regenerative medicine promoted the development of novel options to overcome this medical condition. We are elaborating a promising approach to restore spermatogenesis using mesenchymal stromal cell (MSC) secretome components as a novel class of cell-free cell therapy medicinal products for regenerative medicine. However, the choice of the representative in vivo model of spermatogenesis failure to evaluate the effectiveness of regenerative drugs remains challenging. Using the rat model of bilateral abdominal cryptorchidism, we studied the contribution of MSC conditioned medium contained bioactive cell secreted products to the spermatogenesis recovery. The feasibility of this model to evaluate the drug-driven regenerative effects on spermatogenesis restoration after the injury was demonstrated. We revealed significant correlations between the extent of spermatogonial stem cell niche recovery, spermatozoa count and serum concentration of androgens as an indicator of Leydig cell function. The obtained results can be applied in preclinical studies to choose the proper criteria to appraise the specific activity of novel regenerative drugs developed for the treatment of non-obstructive spermatogenesis disorders.
... Viscoll is also used to alleviate spinal cord injury by injecting it into the trunk of the spinal cord in a rat model (Ryabov et al., 2018). Moreover, hydrogel based on Viscoll is a biocompatible carrier for prolonged release of incorporated growth factors as a treatment for cryptorchidism in a rat model (Kamalov et al., 2017). ...
... consistent with other results obtained in various in vivo studies using Viscoll collagen biomaterials (Kamalov et al., 2017;Kirpatovckii et al., 2016;Ryabov et al., 2018). ...
Although collagen based materials are widely used in corneal tissue engineering with promising results. The usage of such materials for the improvement of corneal biomechanical properties is still unclear. In this study, we aimed to investigate a new Viscoll collagen-based membrane for the improvement of corneal biomechanical characteristics.The right eyes of 15 Chinchilla rabbits were implanted with the membrane via an intrastromal pocket, with the contralateral intact eyes as controls. At 7, 30, 90, and 180 days post-surgery, the rabbits underwent anterior segment optical coherence topography, clinical examination, and slit-lamp microscopy. Additionally, the corneal samples also underwent histological examination followed by the assessment of the biomechanical characteristics of four treated and non-treated corneas at 30, 90, and 180 days, including keratometry at 180 days, post operation. Data are presented as means ± confidence intervals with a 95% confidence level. All the operated corneas retained their transparency throughout the study. Implantation approximately doubled the central corneal thickness. Corneas became stronger by approximately 87% between 1 and 6 months after surgery (maximum fracture load, 13.3 ± 0.8 and 24.9 ± 1.4 N, respectively), and their elasticity increased by approximately 27% over the same time frame (maximum slope of the elastic region of the stress-strain curve, 11.5 ± 0.2 and 14.6 ± 1.4 N/mm respectively). We have thus proposed a new method to increase corneal thickness and strengthen the corneal tissues while preserving their transparency and demonstrated its safety and efficacy in a rabbit model over 6 months. This may be a suitable alternative to the existing corneal collagen crosslinking procedure.
... In the present study, numbers of primary spermatocytes as well as numbers of Leydig cells were also comparable in secretome-, MSC-, and vehicle- LG to support high metabolic demands of Sertoli and germ cells at initial stages of recovery [29]. Proliferation of Leydig cells may have been inhibited by germ cells [30] in which numbers were reportedly increased in vehicletreated rats [31]. However, spermatogenesis remained dysfunctional in the vehicle group. ...
Background:
Injury of stem cell niches may disturb tissue homeostasis and regeneration coordinated by specific niche components. Yet, the mechanisms of stem cell niche restoration remain poorly understood. Herein, we examined the role of mesenchymal stromal cells (MSCs) as pivotal regulators of stem cell niche recovery focusing on the effects of their secretome.
Methods:
The spermatogonial stem cell (SSC) niche was selected as a model. SSC niches were injured by inducing abdominal cryptorchidism in rats. Briefly, testes of anesthetized rats were elevated into the abdominal cavity through the inguinal canal for 14 days. After descent of testes, MSC or MSC secretome treatment was applied to the animals by local subtunical injections.
Results:
Local administration of MSC or MSC secretome was sufficient to recover spermatogenesis and production of functional germ cells. The effects of MSC and their secreted components were comparable, leading to restoration of Sertoli cell pools and recovery of Leydig cell secretory functions.
Conclusion:
Our data suggest that MSCs mimic the functions of lost supportive cells within the stem cell niche, transiently providing paracrine stimuli for target cells and triggering tissue regenerative processes after damage.
... Due to their prominent viscoelastic properties, Viscoll solutions serve as carriers for growth factors. The controlled release of growth factors from the hydrogel formed from injected Viscoll was effectively shown in the model of cryptorchism consequences treatment in rats [21]. Viscoll was also used as a template for injection of stem cells into the damaged spinal cord in the model of the spinal trauma [22]. ...
Collagen is one of the most promising materials for 3D bioprinting because of its distinguished biocompatibility. Cell-laden constructs made of pure collagen with or without incorporated growth supplements support engineered constructs persistence in culture and are perfectly suitable for grafting. The limiting factor for direct 3D collagen printing was poor printability of collagen solutions, especially admixed with cells or tissue spheroids. In our study, we showed that concentrated solutions of native collagen branded Viscoll were effective as bioinks with high fidelity performance. Viscoll containing 20, 30, or 40 mg/ml collagen were used for direct extrusion 3D bioprinting to form scaffolds appropriate to support spatial arrangement of tissue spheroids into rigid patterns with resolution of 0.5 mm in details. Incorporated cells demonstrated sufficient viability. Associated rheological study showed that good printability of the collagen solutions correlates with their increased storage modulus value, notably exceeding the loss modulus value. The proper combination of these physical parameters could become technological criteria for manufacturing various collagen bioinks for 3D bioprinting.
Background and Objectives
The dura mater is a barrier between the brain and the surrounding environment. Injuries to the dura can lead to serious complications, therefore, ensuring a hermetic closure of the dura is a primary task for a neurosurgeon. The aim of the study is to compare the effectiveness of applying the newly developed ViscollDURA collagen membrane (VDCM), with the commercially available Durepair (xenogeneic collagen) in animal model.
Methods
A dural tear model was utilized in rats with membrane implantation using an application method. The sample size consisted of 24 rats. Group I underwent VDCM implantation, while Group II underwent Durepair implantation. Results were evaluated at 30, 60, and 90 days. The study was assessed using magnetic resonance imaging, histology, electron scanning microscopy, and immunohistochemistry. The obtained results underwent statistical analysis.
Results
In the clinical presentation, there were no difference between groups. Histologically, Group 1 showed comparable results to Group 2. The integration process of the membrane statistically differed between the groups. In Group 1, neovascularization and tissue replacement showed better results than in Group 2. Magnetic resonance imaging differences were observed at later stages, with group 2 showing adhesion and brain deformation in the implantation area.
Conclusions
Both membranes showed safety and compatibility. The collagen membrane produced under sterile conditions demonstrated better regeneration with minimal inflammatory reaction. The study suggests that VDCM exhibits biocompatibility comparable to Durepair, providing prospects for potential applications in neurosurgery.
In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pig. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3 and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.
Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells.
BACKGROUND Extracellular vesicles (EVs) are membrane-bound vesicles, found in biofluids, that carry and transfer regulatory molecules,
such as microRNAs (miRNAs) and proteins, and may mediate intercellular communication between cells and tissues. EVs have been
isolated from a wide variety of biofluids, including plasma, urine, and, relevant to this review, seminal, follicular and
uterine luminal fluid. We conducted a systematic search of the literature to review and present the currently available evidence
on the possible roles of EVs in follicular growth, resumption of oocyte development and maturation (meiosis), sperm maturation,
fertilization and embryo implantation.
Factors affecting the blood-testis barrier function may be involved in testicular damage and male infertility. Two cytokines play an important role in the barrier regulation, namely transforming growth factor beta 3 (TGF-β3) and tumor necrosis factor (TNF-α). The aim of this study was to investigate the potential association between TGF-β3 (TGFB3) and TNF-α (TNF) gene polymorphisms and male infertility. A total of 846 subjects, 423 diagnosed with male infertility and 423 fertile men were enrolled. TGFB3 (rs2268626:T > C, rs3917158:C > T, rs2284792:A > G, rs2268625:T > C, rs3917187:C > T) and TNF (rs1800629:-308G > A) gene polymorphisms were genotyped. No association between TNF genotype and infertility was observed. As for TGFB3, the genotypes distribution was similar in infertile and fertile men. However, rs2284792 minor allele frequency was significantly higher among infertile subjects. Heterozygous rs2284792 AG genotype was associated with increased odds for infertility [OR = 1.40 (95% CI 1.05-1.86), p = 0.021] and similar results were observed for G allele carrier status [OR = 1.40 (95% CI 1.06-1.84), p = 0.017]. Heterozygosity in TGFB3 rs3917158 was also associated with the infertility [OR = 1.37 (95% CI 1.01-1.87), p = 0.041]. The TGFB3 variant genotypes were associated with lower spermatozoa motility parameters in fertile men. The results indicate that variants in TGFB3 gene may be associated with male infertility. However, the findings require further replication and validation.
This study was aimed at deciphering the secretome of adipose-derived mesenchymal stromal cells (ADSCs) cultured in standard and hypoxic conditions to reveal proteins, which may be responsible for regenerative action of these cells.
Human ADSCs were isolated from 10 healthy donors and cultured for 3–4 passages. Cells were serum deprived and cell purity was assessed using multiple cell surface markers. Conditioned media was collected and analyzed using LC-MS with a focus on characterizing secreted proteins.
Purity of the ADSC assessed as CD90+/CD73+/CD105+/CD45-/CD31- cells was greater than 99 % and viability was greater than 97 %. More than 600 secreted proteins were detected in conditioned media of ADSCs. Of these 100 proteins were common to all cultures and included key molecules involved in tissue regeneration such as collagens and collagen maturation enzymes, matrix metalloproteases, matricellular proteins, macrophage-colony stimulating factor and pigment epithelium derived factor. Common set of proteins also included molecules, which contribute to regenerative processes but were not previously associated with ADSCs. These included olfactomedin-like 3, follistatin-like 1 and prosaposin. In addition, ADSCs from the different subjects secreted proteins, which were variable between different cultures. These included proteins with neurotrophic activities, which were not previously associated with ADSCs, such as mesencephalic astrocyte-derived neurotrophic factor, meteorin and neuron derived neurotrophic factor. Hypoxia resulted in secretion of 6 proteins, the most prominent included EGF-like repeats and discoidin I-like domains 3, adrenomedullin and ribonuclease 4 of RNase A family. It also caused the disappearance of 8 proteins, including regulator of osteogenic differentiation cartilage-associated protein.
Human ADSCs with CD90+/CD73+/CD105+/CD45-/CD31-/PDGFRβ+/NG2+/CD146+(−) immunophenotype secrete a large array of proteins, the most represented group is comprised of extracellular matrix components. Number of secreted proteins is largely unaffected by prolonged hypoxia. Variability in the secretion of several proteins from cultured ADSCs of individual subjects suggests that these cells exist as a heterogeneous population containing functionally distinct subtypes, which differ in numbers between donors.
Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems.
Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.
De novo formation of testis tissue from single cell suspensions allows manipulation of different testicular compartments before grafting to study testicular development and the spermatogonial stem cell niche. However, the low percentages of newly formed seminiferous tubules supporting complete spermatogenesis and lack of a defined protocol have limited use of this bioassay. Low spermatogenic efficiency in de novo formed tissue could result from the scarcity of germ cells in the donor cell suspension, cell damage caused by handling or from hypoxia during tissue formation in the host environment. Here, we compared different proportions of spermatogonia in the donor cell suspension and the use of Matrigel as scaffold to support de novo tissue formation and spermatogenesis. Then, we utilized the system to investigate the role of Vascular Endothelial Growth Factor-165 during testicular morphogenesis on blood vessel and seminiferous tubule formation, and on presence of germ cells in the de novo developed tubules. Our results show that donor cell pellets with 10x106 porcine neonatal testicular cells in Matrigel efficiently formed testis tissue de novo. Contrary to what was expected, the enrichment of the cell suspension with germ cells did not result in higher numbers of tubules supporting spermatogenesis. The addition of VEGF-165 did not improve blood vessel or tubule formation but it enhanced the number of tubules containing spermatogonia. These results indicate that spermatogenic efficiency was improved by the addition of Matrigel, and that VEGF-165 may have a protective role supporting germ cell establishment in their niche.
Quality of cartilaginous tissue derived from bone marrow mesenchymal stromal stem cell (BMSC) transplantation has been correlated with clinical outcome. Therefore, culture conditions capable of modulating tissue phenotype, such as oxygen tension and scaffold composition, are under investigation. The objective of this study was to assess the effect of hypoxia on in vitro BMSC chondrogenesis within clinically approved porous scaffolds composed of collagen and hyaluronic acid (HA). It was hypothesized that hypoxic isolation/expansion and differentiation would improve BMSC chondrogenesis in each construct.
Ovine BMSCs were isolated and expanded to passage two under hypoxia (3% oxygen) or normoxia (21% oxygen). Cell proliferation and colony forming characteristics were assessed. BMSCs were seeded at 10 million cells/cm(3) on cylindrical scaffolds composed of either collagen I sponge or esterified hyaluronic acid (HA) non-woven mesh. Chondrogenic differentiation was performed in a defined medium under hypoxia or normoxia for 14 days. Cultured constructs were assessed for gene expression, proteoglycan staining, glycosaminoglycan (GAG) quantity, and diameter change.
Isolation/expansion under hypoxia resulted in faster BMSC population doublings per day (p < 0.05), while cell and colony counts were not significantly different (p = 0.60 and 0.30, respectively). Collagen and HA scaffolds seeded with BMSCs that were isolated, expanded and differentiated under hypoxia exhibited superior aggrecan and collagen II mRNA expressions (p < 0.05), GAG quantity (p < 0.05) and proteoglycan staining in comparison to normoxia. GAG/DNA was augmented with hypoxic isolation/expansion in all constructs (p < 0.01). Comparison by scaffold composition indicated increased mRNA expressions of hyaline cartilage-associated collagen II, aggrecan and SOX9 in collagen scaffolds, although expression of collagen X, which is related to hypertrophic cartilage, was also elevated (p < 0.05). Proteoglycan deposition was not significantly improved in collagen scaffolds unless culture involved normoxic isolation/expansion followed by hypoxic differentiation. During chondrogenesis, collagen-based constructs progressively contracted to 60.1 ± 8.9% of the initial diameter after 14 days, while HA-based construct size was maintained (109.7 ± 4.2%).
Hypoxic isolation/expansion and differentiation enhance in vitro BMSC chondrogenesis within porous scaffolds. Although both collagen I and HA scaffolds support the creation of hyaline-like cartilaginous tissue, variations in gene expression, extracellular matrix formation and construct size occur during chondrogenesis.
Spermatogenesis is a dynamic and complex process that involves endocrine and testicular factors. During xenotransplantation of testicular tissue fragments into immunodecifient mouse mice a functional communication between host brain and donor testis is established. This interaction allows for the progression of a normal spermatogenesis and recovery of fertilization-competent spermatozoa from a broad range of mammalian species. In the last years, significant progression have has been achieved in testis tissue xenografting that improves our knowledge about factors determining the success of grafting. The goal of this review is to provide up to date information about the role of factors such as donor age, donor species, testis tissue preservation or type of recipient mouse on the efficiency of this technique. Applications will be are described and compared with other techniques with similar purposes. Recent works demonstrates that testicular tissue xenografting is being used as a model to study gonadotoxicity of drugs and to obtain sperm from valuable young males.