Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles

Figures

• 

  Figure 1. In vitro release of vascular growth factors from NPs incorporated alone or in combination with other NPs, in an alginate hydrogel. (A) VEGF-loaded PLGA:PLGA-PEG NPS and (B) PDGF-loaded PLGA:PLGA-PEG NPs were embedded in a 1% alginate matrix and VEGF and PDGF were quantified in the release buffer (40 mM CaCl2 PBS) at 34 °C for 21 days (n = 4).

• 

  Figure 2. Impact of NP supplementation on seminiferous tubule integrity. Mouse testicular tissues were encapsulated in alginate supplemented with five different combinations in addition to a negative control (Alginate, V, V+P, N, V+N and V+P+N) for 21 days. Seminiferous tubule integrity was (A) visualized by hematoxylin–eosin-staining 5 and 21 days after orthotopic auto-transplantation and (B) quantified after 21 days. Results are expressed as the mean percentage of intact (Score 1) STs ± standard deviation. a: p = 0.02; b: p = 0.02; c: p = 0.04; d: p = 0.04; e: p = 0.03, f: p = 0.03; g: p = 0.04 (n = 3).

• 

  Figure 3. Impact of NP supplementation on germ cell survival 21 days after grafting. Mouse testicular tissue fragments were encapsulated in alginate supplemented with five different combinations in addition to a negative control (Alginate, V, V+P, N, V+N and V+P+N). (A) Immunostaining against PLZF was used to identify undifferentiated spermatogonia, (Red arrows highlight positive cells, scale bar = 60 μm) and (B) to quantify them. Results are expressed as the mean PLZF positive cells/STs (Alginate 1.77 ± 0.17; 1.79 ± 0.46; V+P: 1.87 ± 0.31; N: 2.4 ± 0.29; V+N. 1.76 ± 0.16; and V+P+N: 2.23 ± 0.08) (n = 3). * p = 0.03.

• 

  Figure 4. Impact of NP supplementation on the vascularization of mice testicular tissue auto-transplanted for 5 and 21 days. Mouse testicular tissue fragments were encapsulated in alginate supplemented with five different combinations in addition to a negative control (Alginate, V, V+P, N, V+N and V+P+N) and the grafts were stained after 5 and 21 days for α-SMA and CD31. Red arrows highlight positive vascular structures. Scale bar = 60 μm. n = 3.

• 

  Figure 5. Impact of NP supplementation on the vascularization of mice testicular tissue auto-transplanted for 5 and 21 days. Mouse testicular tissue fragments were encapsulated in alginate supplemented with five different combinations in addition to a negative control (Alginate, V, V+P, N, V+N and V+P+N) and the positive surface for α-SMA (A and C, 5 and 21 days, respectively) and CD31 (B and D, 5 and 21 days, respectively) was quantified using VisioPharm (n = 36). (A) IHC for α-SMA, 5 days. Encapsulation with a matrix containing VEGF-NPs (alone or combined with PDGF-NPs or NECINH-NPs) increased vascular surfaces. Results are expressed as the mean positive vascular surface per total graft surface ± standard deviation: Alginate (1.74 × 10−3 ± 1.81 × 10−3); V (1.47 × 10−2 ± 4.21 × 10−3); V+P (1.09 × 10−2 ± 3.29 × 10−4); N (4.63 × 10−3± 6.85 × 10−4); V+N (1.23 × 10−2 ± 1.80 × 10−3); V+P+N (1.05 × 10−2 ± 2.64 × 10−3). a: p < 0.01; b: p < 0.01; c: p < 0.01; d: p < 0.01; e: p < 0.01; f: p = 0.01; g: p = 0.05; h: p < 0.01. (B) IHC for α-SMA, 21 days. Encapsulation with a matrix containing VEGF-NPs and PDGF-NPs increased vascular surface: alginate (4.13 × 10−3 ± 2.69 × 10−3); V (5.79 × 10−3 ± 3.92…

• 

  Figure 6. Impact of NP supplementation on the blood vessels’ evolution between 5 and 21 days in mice testicular tissue auto-transplants. (A) α-SMA- and (B) CD31-positive surfaces in the grafts were compared after 5 and 21 days of transplantation. a: p = 0.03; b: p = 0.04; c: p < 0.01; d: p = 0.01; e: p = 0.02; f: p = 0.03. n = 3.

• 

  Figure 7. Impact of NP supplementation on neo-vascularization stabilization. The CD-31/α-SMA ratio was calculated for grafts retrieved after 21 days a: p = 0.04; b: p = 0.02; c: p = 0.02. n = 18.