Volume 27, Issue 4 (6-2020)
RJMS 2020, 27(4): 37-48 |
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Majidi Gharenaz N, Movahedin M, Mazaheri Z. Production of biocompatible testis scaffold for use in tissue engineering. RJMS 2020; 27 (4) :37-48
URL: http://rjms.iums.ac.ir/article-1-5988-en.html
URL: http://rjms.iums.ac.ir/article-1-5988-en.html
Tarbiat Modares University, Tehran, Iran , movahed.m@modares.ac.ir
Abstract: (2932 Views)
Background: Cryopreservation of immature testicular tissue before chemo/ radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cell, induction of in vitro spermatogenesis could be considered. Induction of in vitro spermatogenesis using spermatogonial cells requires a suitable platform for cell growth and proliferation. The extracellular matrix of the testis could be used for adhesion, proliferation, migration and differentiation of spermatogonial cells. The extracellular matrix of the testis consists of glycosaminoglycans (GAGs), fibronectin, collagen and laminin. It can mimic specific microenvironment of testis. The extracellular matrix as a biological scaffold provided an appropriate platform for proliferation and differentiation of spermatogonial cells. Biological scaffolds were developed using decellularization of tissues and organs. Decellularization is a process that removes the cells, their nuclei and debris from tissues and organs without sever damage to structure and biochemical component of the tissues. The aim of our study was decellularization of whole testis for preparation of scaffold and evaluation of spermatogonia cells homing after injection into the scaffold
Methods: In order to prepare the scaffolds, adult mouse testes and different concentrations of detergents were used. Initially, the adult mice were scarified using chloroform and their testes were removed and washed with PBS, then decellularization was performed using different concentrations of detergents according following protocols.
Protocol 1: The testes were immersed in 0.1% SDS solution for 24 hours
Protocol 2: The testes were immersed in 0.5% SDS solution for 24 hours.
Protocol 3: The testes were immersed in 1% SDS solution for 24 hours.
Protocol 4: The testes were immersed in 0.5% SDS solution for 18 hours, then washed with PBS and immersed in 0.5% Triton solution for 18 hours.
In order to remove detergents, scaffolds were washed using PBS and disinfected by 70% ethanol. All protocols of decellularization and washing were done at room temperature on orbital shaker with 50 rpm speed. The efficiency of the decellularization process was determined by hematoxylin-eosin staining and DNA quantification. To evaluate the preservation of collagen and GAGs, Masson's trichrome staining and alcian blue staining were done respectively. Confirmation of fibronectin, collagen 4 and laminin presence in decellularized scaffolds was done using immunohistochemistry (IHC). The quantity of totlal collagen and GAGs in scaffolds was evaluated using Sicrol assay kit and Blyscan assy kit respectively. The biocompatibility of testicular scaffolds was evaluated using MTT test. Initially, mouse embryonic fibroblast cells were cultured on testicular scaffold for 24 hours and 72 hours. Then, the culture medium was removed and 200 μl of MTT reagent with a concentration of 0.5 mg / ml was added to the cells and incubated at 37 ° C for 4 h. Finally, 200 micrometers of DMSO was added and the samples were transferred to the 96 well plates and located in ELISA reader. In order to evaluation of spermatogonial cells support by scaffolds, the isolated cells from neonatal testes were injected to scaffolds via efferent ductile and then cultured on agarose gel for two weeks. Histological studies were carried out at the end of culture.
Results: The results of hematoxylin-eosin staining showed that immersion testis in 0.1% SDS solution and 0.5% SDS solution couldn’t decellularize the testes. On the other hand, immersion testis in 1% SDS solution led to destruction of seminiferous base membrane. Immersion testis in 0.5% SDS and 0.5% Triton resulted in complete decellularization of the testes without severe damage to seminiferous base membrane. In order to further evaluation of methods efficiency, the amount of DNA residue in the scaffolds was extracted using kit and examined by nanodrop. Spectrophotometric analysis showed 50% and 70% of DNA were removed in first and second methods respectively, while more than 98% of DNA was removed in third and forth methods. The first and second methods were discarded due to inefficiency in DNA removal from the testes and third method due to destruction of the basement membrane of the tubes. So, the scaffolds that prepared by forth method were selected for further evaluation. The result of alcian blue staining indicated the good preservation of the GAGs in decellularized testes scaffolds .The result of thrichrom staining confirmed the preservation of collagen decellularized testes scaffolds. Presence of blue fibers in the scaffold (representing collagen fibers) and the lack of red dots (representing the cell nuclei) indicate that the prepared scaffolds are cell-free and Collagen strands are well preserved. Examination of fluorescent microscopic images showed that extracellular testicular matrix proteins including fibronectin, collagen type 4 and laminin were expressed in testicular scaffolds, indicating preservation of these proteins in scaffolds. Quantified evaluation of GAGs and collagen content of decellularized scaffolds showed that there was no significant reduction in GAGs and collagen level in scaffolds compared to testes. In order to evaluation of the cytotoxicity of testicular scaffolds, MTT test was done. The results of the MTT test showed that the survival rate of mouse embryo fibroblastic cells didn’t show significant difference after 24 and 72 hours of culture in the presence of testicular scaffolds compared to culture without scaffolds, so the scaffolds were biocompatible and did not negative effect on cell survival. Mouse embryo fibroblastic cells could metabolize MTT in the presence of scaffolds, so mitochondria of the cells were active in the presence of scaffolds and led to the survival and proliferation of cells. Examination of hematoxylin-eosin images showed that the injected cells were located on basement membrane of seminiferous tubules and in the interstitial space and created colonies that resemble organoid structures. The tubes were completely collapsed in control group, and no cells were seen in the scaffolds.
Conclusion: Immersion of adult mouse testes in 0.5% SDS solution and 0.5% triton solution was an effective method for decellularization of whole testes without severs damage to seminiferous tubules. Our decellularization method could preserve important proteins of extra cellular matrix including fibronectin, collagen type 4 and laminin in testicular scaffolds. Decellularized testicular scaffolds were biocompatible and did not have a harmful effect on MEF and spermatogonial cells viability. Also prepared scaffolds could support the proliferation of spermatogonial cells during two weeks culture
Methods: In order to prepare the scaffolds, adult mouse testes and different concentrations of detergents were used. Initially, the adult mice were scarified using chloroform and their testes were removed and washed with PBS, then decellularization was performed using different concentrations of detergents according following protocols.
Protocol 1: The testes were immersed in 0.1% SDS solution for 24 hours
Protocol 2: The testes were immersed in 0.5% SDS solution for 24 hours.
Protocol 3: The testes were immersed in 1% SDS solution for 24 hours.
Protocol 4: The testes were immersed in 0.5% SDS solution for 18 hours, then washed with PBS and immersed in 0.5% Triton solution for 18 hours.
In order to remove detergents, scaffolds were washed using PBS and disinfected by 70% ethanol. All protocols of decellularization and washing were done at room temperature on orbital shaker with 50 rpm speed. The efficiency of the decellularization process was determined by hematoxylin-eosin staining and DNA quantification. To evaluate the preservation of collagen and GAGs, Masson's trichrome staining and alcian blue staining were done respectively. Confirmation of fibronectin, collagen 4 and laminin presence in decellularized scaffolds was done using immunohistochemistry (IHC). The quantity of totlal collagen and GAGs in scaffolds was evaluated using Sicrol assay kit and Blyscan assy kit respectively. The biocompatibility of testicular scaffolds was evaluated using MTT test. Initially, mouse embryonic fibroblast cells were cultured on testicular scaffold for 24 hours and 72 hours. Then, the culture medium was removed and 200 μl of MTT reagent with a concentration of 0.5 mg / ml was added to the cells and incubated at 37 ° C for 4 h. Finally, 200 micrometers of DMSO was added and the samples were transferred to the 96 well plates and located in ELISA reader. In order to evaluation of spermatogonial cells support by scaffolds, the isolated cells from neonatal testes were injected to scaffolds via efferent ductile and then cultured on agarose gel for two weeks. Histological studies were carried out at the end of culture.
Results: The results of hematoxylin-eosin staining showed that immersion testis in 0.1% SDS solution and 0.5% SDS solution couldn’t decellularize the testes. On the other hand, immersion testis in 1% SDS solution led to destruction of seminiferous base membrane. Immersion testis in 0.5% SDS and 0.5% Triton resulted in complete decellularization of the testes without severe damage to seminiferous base membrane. In order to further evaluation of methods efficiency, the amount of DNA residue in the scaffolds was extracted using kit and examined by nanodrop. Spectrophotometric analysis showed 50% and 70% of DNA were removed in first and second methods respectively, while more than 98% of DNA was removed in third and forth methods. The first and second methods were discarded due to inefficiency in DNA removal from the testes and third method due to destruction of the basement membrane of the tubes. So, the scaffolds that prepared by forth method were selected for further evaluation. The result of alcian blue staining indicated the good preservation of the GAGs in decellularized testes scaffolds .The result of thrichrom staining confirmed the preservation of collagen decellularized testes scaffolds. Presence of blue fibers in the scaffold (representing collagen fibers) and the lack of red dots (representing the cell nuclei) indicate that the prepared scaffolds are cell-free and Collagen strands are well preserved. Examination of fluorescent microscopic images showed that extracellular testicular matrix proteins including fibronectin, collagen type 4 and laminin were expressed in testicular scaffolds, indicating preservation of these proteins in scaffolds. Quantified evaluation of GAGs and collagen content of decellularized scaffolds showed that there was no significant reduction in GAGs and collagen level in scaffolds compared to testes. In order to evaluation of the cytotoxicity of testicular scaffolds, MTT test was done. The results of the MTT test showed that the survival rate of mouse embryo fibroblastic cells didn’t show significant difference after 24 and 72 hours of culture in the presence of testicular scaffolds compared to culture without scaffolds, so the scaffolds were biocompatible and did not negative effect on cell survival. Mouse embryo fibroblastic cells could metabolize MTT in the presence of scaffolds, so mitochondria of the cells were active in the presence of scaffolds and led to the survival and proliferation of cells. Examination of hematoxylin-eosin images showed that the injected cells were located on basement membrane of seminiferous tubules and in the interstitial space and created colonies that resemble organoid structures. The tubes were completely collapsed in control group, and no cells were seen in the scaffolds.
Conclusion: Immersion of adult mouse testes in 0.5% SDS solution and 0.5% triton solution was an effective method for decellularization of whole testes without severs damage to seminiferous tubules. Our decellularization method could preserve important proteins of extra cellular matrix including fibronectin, collagen type 4 and laminin in testicular scaffolds. Decellularized testicular scaffolds were biocompatible and did not have a harmful effect on MEF and spermatogonial cells viability. Also prepared scaffolds could support the proliferation of spermatogonial cells during two weeks culture
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