Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles
Abstract
:1. Introduction
2. Results
2.1. Nanoparticle Characterization
2.2. Tissue Integrity
2.3. Spermatogonial Survival
2.4. Vascularization
3. Discussion
4. Materials and Methods
4.1. Encapsulation of Bioactive Factors in Polymeric Nanoparticles
4.1.1. Nanoparticle Characterization
4.1.2. In Vitro Drug Release Profile
4.2. Testicular Tissue Collection
4.3. Encapsulation of Tissue and NPs
4.4. Testicular Tissue Transplantation
4.5. Tissue Analyses
4.6. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
- Wyns, C.; Curaba, M.; Vanabelle, B.; Van Langendonckt, A.; Donnez, J. Options for fertility preservation in prepubertal boys. Hum. Reprod. Updat. 2010, 16, 312–328. [Google Scholar] [CrossRef] [Green Version]
- Stukenborg, J.-B.; Jahnukainen, K.; Hutka, M.; Mitchell, R.T. Cancer treatment in childhood and testicular function: The importance of the somatic environment. Endocr. Connect. 2018, 7, R69–R87. [Google Scholar] [CrossRef] [Green Version]
- Pampanini, V.; Wagner, M.; Asadi-Azarbaijani, B.; Oskam, I.C.; Sheikhi, M.; Sjödin, M.O.D.; Lindberg, J.; Hovatta, O.; Sahlin, L.; Björvang, R.D.; et al. Impact of first-line cancer treatment on the follicle quality in cryopreserved ovarian samples from girls and young women. Hum. Reprod. 2019, 34, 1674–1685. [Google Scholar] [CrossRef] [PubMed]
- Wyns, C.; Kanbar, M.; Giudice, M.G.; Poels, J. Fertility preservation for prepubertal boys: Lessons learned from the past and update on remaining challenges towards clinical translation. Hum. Reprod. Updat. 2021, 27, 433–459. [Google Scholar] [CrossRef] [PubMed]
- Kanbar, M.; De Michele, F.; Giudice, M.G.; Desmet, L.; Poels, J.; Wyns, C. Long-term follow-up of boys who have undergone a testicular biopsy for fertility preservation. Hum. Reprod. 2020, 36, 26–39. [Google Scholar]
- Thomson, A.B.; Campbell, A.J.; Irvine, D.C.; Anderson, R.A.; Kelnar, C.J.H.; Wallace, W.H.B. Semen quality and spermatozoal DNA integrity in survivors of childhood cancer: A case-control study. Lancet 2002, 360, 361–367. [Google Scholar] [CrossRef]
- Picton, H.M.; Wyns, C.; Anderson, R.A.; Goossens, E.; Jahnukainen, K.; Kliesch, S.; Mitchell, R.T.; Pennings, G.; Rives, N.; Tournaye, H.; et al. A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum. Reprod. 2015, 30, 2463–2475. [Google Scholar] [CrossRef]
- Ming, J.M.; Chua, M.E.; Lopes, R.I.; Maloney, A.M.; Gupta, A.A.; Lorenzo, A.J. Cryopreservation of testicular tissue in pre-pubertal and adolescent boys at risk for infertility: A low risk procedure. J. Pediatr. Urol. 2018, 14, 274.e1–274.e5. [Google Scholar] [CrossRef] [PubMed]
- Valli-Pulaski, H.; Peters, K.A.; Gassei, K.; Steimer, S.R.; Sukhwani, M.; Hermann, B.P.; Dwomor, L.; David, S.; Fayomi, A.P.; Munyoki, S.; et al. Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers. Hum. Reprod. 2019, 34, 966–977. [Google Scholar] [CrossRef] [PubMed]
- Goossens, E.; Jahnukainen, K.; Mitchell, R.T.; van Pelt, A.; Pennings, G.; Rives, N.; Poels, J.; Wyns, C.; Lane, S.; Rodriguez-Wallberg, K.A.; et al. Fertility Preservation in Boys: Recent Developments and New Insights (Dagger). Hum. Reprod. Open 2020, 2020, hoaa016. [Google Scholar] [CrossRef]
- Demeestere, I.; Simon, P.; Dedeken, L.; Moffa, F.; Tsépélidis, S.; Brachet, C.; Delbaere, A.; Devreker, F.; Ferster, A. Live birth after autograft of ovarian tissue cryopreserved during childhood. Hum. Reprod. 2015, 30, 2107–2109. [Google Scholar] [CrossRef] [Green Version]
- Jadoul, P.; Guilmain, A.; Squifflet, J.; Luyckx, M.; Votino, R.; Wyns, C.; Dolmans, M. Efficacy of ovarian tissue cryopreservation for fertility preservation: Lessons learned from 545 cases. Hum. Reprod. 2017, 32, 1046–1054. [Google Scholar] [CrossRef]
- Lambertini, M.; Peccatori, F.A.; Demeestere, I.; Amant, F.; Wyns, C.; Stukenborg, J.B.; Paluch-Shimon, S.; Halaska, M.J.; Uzan, C.; Meissner, J.; et al. Fertility Preservation and Post-Treatment Pregnancies in Post-Pubertal Cancer Patients: Esmo Clinical Practice Guidelines(Dagger). Ann. Oncol. 2020, 31, 1664–1678. [Google Scholar] [CrossRef] [PubMed]
- Clermont, Y. Kinetics of spermatogenesis in mammals. Arch. D’anatomie Microsc. Et De Morphol. Exp. 1967, 56, 7–60. [Google Scholar]
- Wyns, C.; Curaba, M.; Martinez-Madrid, B.; van Langendonckt, A.; François-Xavier, W.; Donnez, J. Spermatogonial Survival after Cryopreservation and Short-Term Orthotopic Immature Human Cryptorchid Testicular Tissue Grafting to Immunodeficient Mice. Hum. Reprod. 2007, 22, 1603–1611. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Poels, J.; Van Langendonckt, A.; Many, M.-C.; Wese, F.-X.; Wyns, C. Vitrification preserves proliferation capacity in human spermatogonia. Hum. Reprod. 2013, 28, 578–589. [Google Scholar] [CrossRef] [Green Version]
- Del Vento, F.; Vermeulen, M.; De Michele, F.; Giudice, M.G.; Poels, J.; Rieux, A.D.; Wyns, C. Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments. Int. J. Mol. Sci. 2018, 19, 286. [Google Scholar] [CrossRef] [Green Version]
- De Michele, F.; Vermeulen, M.; Wyns, C. Fertility restoration with spermatogonial stem cells. Curr. Opin. Endocrinol. Diabetes Obes. 2017, 24, 424–431. [Google Scholar] [CrossRef]
- De Michele, F.; Poels, J.; Vermeulen, M.; Ambroise, J.; Gruson, D.; Guiot, Y.; Wyns, C. Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys. Front. Physiol. 2018, 9, 1413. [Google Scholar] [CrossRef]
- Vermeulen, M.; Del Vento, F.; Kanbar, M.; Ruys, S.P.D.; Vertommen, D.; Poels, J.; Wyns, C. Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix. Int. J. Mol. Sci. 2019, 20, 5476. [Google Scholar] [CrossRef] [Green Version]
- Liu, Z.; Nie, Y.H.; Zhang, C.C.; Cai, Y.J.; Wang, Y.; Lu, H.P.; Li, Y.Z.; Cheng, C.; Qiu, Z.L.; Sun, Q. Generation of Macaques with Sperm Derived from Juvenile Monkey Testicular Xenografts. Cell Res. 2016, 26, 139–142. [Google Scholar] [CrossRef] [Green Version]
- Ntemou, E.; Kadam, P.; Van Saen, D.; Wistuba, J.; Mitchell, R.T.; Schlatt, S.; Goossens, E. Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate. Hum. Reprod. 2019, 34, 403–4133. [Google Scholar] [CrossRef]
- Fayomi, A.P.; Peters, K.; Sukhwani, M.; Valli-Pulaski, H.; Shetty, G.; Meistrich, M.L.; Houser, L.; Robertson, N.; Roberts, V.; Ramsey, C.; et al. Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring. Science 2019, 363, 1314–1319. [Google Scholar] [CrossRef] [PubMed]
- Wyns, C.; Van Langendonckt, A.; Wese, F.-X.; Donnez, J.; Curaba, M. Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue. Hum. Reprod. 2008, 23, 2402–2414. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Goossens, E.; Geens, M.; De Block, G.; Tournaye, H. Spermatogonial survival in long-term human prepubertal xenografts. Fertil. Steril. 2008, 90, 2019–2022. [Google Scholar] [CrossRef] [PubMed]
- Poels, J.; Abou-Ghannam, G.; Herman, S.; Van Langendonckt, A.; Wese, F.-X.; Wyns, C.; Abou-Ghannam, G.; Wese, F.-X. In Search of Better Spermatogonial Preservation by Supplementation of Cryopreserved Human Immature Testicular Tissue Xenografts with N-acetylcysteine and Testosterone. Front. Surg. 2014, 1, 47. [Google Scholar] [CrossRef] [Green Version]
- Schlatt, S.; Westernströer, B.; Gassei, K.; Ehmcke, J. Donor-Host Involvement in Immature Rat Testis Xenografting into Nude Mouse Hosts1. Biol. Reprod. 2010, 82, 888–895. [Google Scholar] [CrossRef] [Green Version]
- Van Eyck, A.-S.; Jordan, B.F.; Gallez, B.; Heilier, J.-F.; Van Langendonckt, A.; Donnez, J. Electron paramagnetic resonance as a tool to evaluate human ovarian tissue reoxygenation after xenografting. Fertil. Steril. 2009, 92, 374–381. [Google Scholar] [CrossRef]
- Poels, J.; Abou-Ghannam, G.; Decamps, A.; Leyman, M.; Rieux, A.D.; Wyns, C. Transplantation of testicular tissue in alginate hydrogel loaded with VEGF nanoparticles improves spermatogonial recovery. J. Control. Release 2016, 234, 79–89. [Google Scholar] [CrossRef]
- Ntemou, E.; Kadam, P.; Van Laere, S.; Van Saen, D.; Vicini, E.; Goossens, E. Effect of recombinant human vascular endothelial growth factor on testis tissue xenotransplants from prepubertal boys: A three-case study. Reprod. Biomed. Online 2019, 39, 119–133. [Google Scholar] [CrossRef] [Green Version]
- Ferrara, N.; Davis-Smyth, T. The Biology of Vascular Endothelial Growth Factor. Endocr. Rev. 1997, 18, 4–25. [Google Scholar] [CrossRef]
- Yancopoulos, G.D.; Davis, S.; Gale, N.W.; Rudge, J.S.; Wiegand, S.J.; Holash, J. Vascular-specific growth factors and blood vessel formation. Nat. Cell Biol. 2000, 407, 242–248. [Google Scholar] [CrossRef] [PubMed]
- Schmidt, J.A.; De Avila, J.M.; McLean, D.J. Effect of Vascular Endothelial Growth Factor and Testis Tissue Culture on Spermatogenesis in Bovine Ectopic Testis Tissue Xenografts. Biol. Reprod. 2006, 75, 167–175. [Google Scholar] [CrossRef] [PubMed]
- Hariawala, M.D.; Horowitz, J.R.; Esakof, D.; Sheriff, D.D.; Walter, D.H.; Keyt, B.; Isner, J.M.; Symes, J.F. VEGF Improves Myocardial Blood Flow but Produces EDRF-Mediated Hypotension in Porcine Hearts. J. Surg. Res. 1996, 63, 77–82. [Google Scholar] [CrossRef]
- Folkman, J. Role of Angiogenesis in Tumor Growth and Metastasis. Semin. Oncol. 2002, 29 (Suppl. 16), 15–18. [Google Scholar] [CrossRef]
- Eiselt, P.; Kim, B.-S.; Chacko, B.; Isenberg, B.; Peters, M.; Greene, K.; Roland, W.; Loebsack, A.; Burg, K.; Culberson, C.; et al. Development of Technologies Aiding Large-Tissue Engineering. Biotechnol. Prog. 1998, 14, 134–140. [Google Scholar] [CrossRef] [PubMed]
- Strobel, H.A.; Qendro, E.I.; Alsberg, E.; Rolle, M.W. Targeted Delivery of Bioactive Molecules for Vascular Intervention and Tissue Engineering. Front. Pharmacol. 2018, 9, 1329. [Google Scholar] [CrossRef]
- Del Vento, F.; Vermeulen, M.; Ucakar, B.; Poels, J.; Rieux, A.D.; Wyns, C. Significant Benefits of Nanoparticles Containing a Necrosis Inhibitor on Mice Testicular Tissue Autografts Outcomes. Int. J. Mol. Sci. 2019, 20, 5833. [Google Scholar] [CrossRef] [Green Version]
- Folkman, J.; Klagsbrun, M. Angiogenic factors. Science 1987, 235, 442–447. [Google Scholar] [CrossRef] [PubMed]
- Lindahl, P.; Johansson, B.R.; Leveen, P.; Betsholtz, C. Pericyte Loss and Microaneurysm Formation in Pdgf-B-Deficient Mice. Science 1997, 277, 242–245. [Google Scholar] [CrossRef]
- Benjamin, L.; Hemo, I.; Keshet, E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 1998, 125, 1591–1598. [Google Scholar] [CrossRef] [PubMed]
- Israely, T.; Nevo, N.; Harmelin, A.; Neeman, M.; Tsafriri, A. Reducing ischaemic damage in rodent ovarian xenografts transplanted into granulation tissue. Hum. Reprod. 2006, 21, 1368–1379. [Google Scholar] [CrossRef] [PubMed]
- Nam, S.-Y.; Shin, B.-H.; Lee, M.; Lee, S.; Heo, C.Y. NecroX-5 ameliorates inflammation by skewing macrophages to the M2 phenotype. Int. Immunopharmacol. 2019, 66, 139–145. [Google Scholar] [CrossRef] [PubMed]
- Wang, C.; Liu, Y.; He, D. Diverse effects of platelet-derived growth factor-BB on cell signaling pathways. Cytokine 2019, 113, 13–20. [Google Scholar] [CrossRef] [PubMed]
- Soriano, P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 1994, 8, 1888–1896. [Google Scholar] [CrossRef] [Green Version]
- Wick, G.; Grundtman, C.; Mayerl, C.; Wimpissinger, T.-F.; Feichtinger, J.; Zelger, B.; Sgonc, R.; Wolfram, D. The Immunology of Fibrosis. Annu. Rev. Immunol. 2013, 31, 107–135. [Google Scholar] [CrossRef] [Green Version]
- Basciani, S.; Mariani, S.; Spera, G.; Gnessi, L. Role of Platelet-Derived Growth Factors in the Testis. Endocr. Rev. 2010, 31, 916–939. [Google Scholar] [CrossRef] [Green Version]
- Ergün, S.; Kiliç, N.; Fiedler, W.; Mukhopadhyay, A. Vascular endothelial growth factor and its receptors in normal human testicular tissue. Mol. Cell. Endocrinol. 1997, 131, 9–20. [Google Scholar] [CrossRef]
- Caires, K.C.; De Avila, J.M.; Cupp, A.S.; McLean, D.J. VEGFA Family Isoforms Regulate Spermatogonial Stem Cell Homeostasis in Vivo. Endocrinology 2012, 153, 887–900. [Google Scholar] [CrossRef] [Green Version]
- Baltes-Breitwisch, M.M.; Artac, R.A.; Bott, R.C.; McFee, R.M.; Kerl, J.G.; Clopton, D.T.; Cupp, A.S. Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis. Reproduction 2010, 140, 319–329. [Google Scholar] [CrossRef] [Green Version]
- Caires, K.C.; De Avila, J.; McLean, D.J. Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis. Reproduction 2009, 138, 667–677. [Google Scholar] [CrossRef] [Green Version]
- Simon-Yarza, T.; Formiga, F.R.; Tamayo, E.; Pelacho, B.; Prosper, F.; Blanco-Prieto, M.J. Vascular Endothelial Growth Factor-Delivery Systems for Cardiac Repair: An Overview. Theranostics 2012, 2, 541–552. [Google Scholar] [CrossRef]
- Zhang, Z.D.; Xu, Y.Q.; Chen, F.; Luo, J.F.; Liu, C.D. Sustained delivery of vascular endothelial growth factor using a dextran/poly(lactic-co-glycolic acid)-combined microsphere system for therapeutic neovascularization. Hear. Vessel. 2019, 34, 167–176. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Q.; Hubenak, J.; Iyyanki, T.; Alred, E.; Turza, K.C.; Davis, G.; Chang, E.I.; Branch-Brooks, C.D.; Beahm, E.K.; Butler, C.E. Engineering vascularized soft tissue flaps in an animal model using human adipose–derived stem cells and VEGF+PLGA/PEG microspheres on a collagen-chitosan scaffold with a flow-through vascular pedicle. Biomaterials 2015, 73, 198–213. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Matsuki, K.; Sugaya, H.; Takahashi, N.; Kawasaki, T.; Yoshimura, H.; Kenmoku, T. Degradation of Cylindrical Poly-Lactic Co-Glycolide/Beta-Tricalcium Phosphate Biocomposite Anchors After Arthroscopic Bankart Repair: A Prospective Study. Orthopedics 2018, 41, e348–e353. [Google Scholar] [CrossRef] [PubMed]
- Jiang, R.; Lin, C.; Jiang, C.; Huang, Z.; Gao, W.; Lin, D. Nobiletin enhances the survival of random pattern skin flaps: Involvement of enhancing angiogenesis and inhibiting oxidative stress. Int. Immunopharmacol. 2020, 78, 106010. [Google Scholar] [CrossRef] [PubMed]
- Kong, H.S.; Lee, J.; Youm, H.W.; Kim, S.K.; Lee, J.R.; Suh, C.S.; Kim, S.H. Effect of Treatment with Angiopoietin-2 and Vascular Endothelial Growth Factor on the Quality of Xenografted Bovine Ovarian Tissue in Mice. PLoS ONE 2017, 12, e0184546. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rieux, A.D.; Ucakar, B.; Mupendwa, B.P.K.; Colau, D.; Feron, O.; Carmeliet, P.; Préat, V. 3D systems delivering VEGF to promote angiogenesis for tissue engineering. J. Control. Release 2011, 150, 272–278. [Google Scholar] [CrossRef] [PubMed]
- Rieux, A.D.; De Berdt, P.; Ansorena, E.; Ucakar, B.; Damien, J.; Schakman, O.; Audouard, E.; Bouzin, C.; Auhl, D.; Simón-Yarza, T.; et al. Vascular endothelial growth factor-loaded injectable hydrogel enhances plasticity in the injured spinal cord. J. Biomed. Mater. Res. Part A 2013, 102, 2345–2355. [Google Scholar] [CrossRef]
- Silva, E.A.; Mooney, D.J. Effects of VEGF temporal and spatial presentation on angiogenesis. Biomaterials 2010, 31, 1235–1241. [Google Scholar] [CrossRef] [Green Version]
- Sun, Q.; Silva, E.A.; Wang, A.; Fritton, J.C.; Mooney, D.; Schaffler, M.B.; Grossman, P.M.; Rajagopalan, S. Sustained Release of Multiple Growth Factors from Injectable Polymeric System as a Novel Therapeutic Approach Towards Angiogenesis. Pharm. Res. 2009, 27, 264–271. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Costoya, J.A.; Hobbs, R.M.; Barna, M.; Cattoretti, G.; Manova, K.; Sukhwani, M.; Orwig, K.E.; Wolgemuth, D.J.; Pandolfi, P.P. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat. Genet. 2004, 36, 653–659. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bai, H.; Wang, Z.; Li, M.; Sun, P.; Wei, S.; Wang, Z.; Xing, Y.; Dardik, A. Adult Human Vein Grafts Retain Plasticity of Vessel Identity. Ann. Vasc. Surg. 2020, 68, 468–475. [Google Scholar] [CrossRef]
- Onufer, E.J.; Aladegbami, B.; Imai, T.; Seiler, K.; Bajinting, A.; Courtney, C.; Sutton, S.; Bustos, A.; Yao, J.; Yeh, C.-H.; et al. EGFR in enterocytes & endothelium and HIF1α in enterocytes are dispensable for massive small bowel resection induced angiogenesis. PLoS ONE 2020, 15, e0236964. [Google Scholar]
Size (nm) | PDI | Z-Potential (mV) | Encapsulation Efficiency | |
---|---|---|---|---|
VEGF-NPs | 199 ± 18 | 0.18 | −37 ± 7 | 90% ± 4% |
PDGF-NPs | 191 ± 8 | 0.14 | −42 ± 9 | 94% ± 2% |
NECINH-NPs | 310 ± 13 | 0.26 | −27 ± 5 | 65% ± 5% |
Condition | 1% Alginate (Control) | V | V+P | N | V+N | V+P+N | |
---|---|---|---|---|---|---|---|
Intact (Score 1) | 0.65% ± 0.71% | 0.10% ± 0.17% | 0.58% ± 0.61% | 0.14% ± 0.25% | 0.13% ± 0.23% | 0% | |
Satisfactory (Score 2) | 59.51% ± 15.91 | 50.70% ± 10.4% | 67.21% ± 6.00% | 62.60% ± 11.10 | 68.31% ± 3.30% | 62.11% ± 3.81% | |
Damaged (Score 3) | 38.70% ± 16.52% | 48.79% ± 10.43% | 32.11% ± 5.71% | 37.19% ± 10.91 | 31.53% ± 3.60% | 37.90% ± 3.81% |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Del Vento, F.; Poels, J.; Vermeulen, M.; Ucakar, B.; Giudice, M.G.; Kanbar, M.; des Rieux, A.; Wyns, C. Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles. Int. J. Mol. Sci. 2021, 22, 5779. https://doi.org/10.3390/ijms22115779
Del Vento F, Poels J, Vermeulen M, Ucakar B, Giudice MG, Kanbar M, des Rieux A, Wyns C. Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles. International Journal of Molecular Sciences. 2021; 22(11):5779. https://doi.org/10.3390/ijms22115779
Chicago/Turabian StyleDel Vento, Federico, Jonathan Poels, Maxime Vermeulen, Bernard Ucakar, Maria Grazia Giudice, Marc Kanbar, Anne des Rieux, and Christine Wyns. 2021. "Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles" International Journal of Molecular Sciences 22, no. 11: 5779. https://doi.org/10.3390/ijms22115779