Table 1.
Random divided animal groups.
Figure 1.
Characterization and the properties of the hydrogel.
(A) GPC analysis (in THF at 40°C); (B) 1H NMR spectrum of the block copolymer (in CDCl3); (C) Phase diagrams by invert test; (D) mass lose profiles; E, MMC release kinetics of/from PTMC15-F127-PTMC15 hydrogel (5% w/v) and F127 hydrogel (18% w/v) in PBS (pH = 7.4) at 37°C, MMC (1 mg/ml).
Figure 2.
In vitro cell toxicity of the HTFs cocultured with PTMC15-F127-PTMC15 copolymer in different concentration and with F127.
(A) primary cultured HTFs were identified with microscopy and (B) with immunofluorescence. (a) Negative control using DAPI dyed the nucleus (blue) of HTFs. (b) Vimentin antibody (red) was added. to primary HTFs (c) Fibronectin antibody (green) was added. (d) Melt. (C) LDH release assay were used to analyze cell toxicity in different concentration of PTMC15-F127-PTMC15 (3%, 5% and 10% w/v) and in 18% w/v F127 within 3 days. (D) Total apoptosis of HTFs incubated with 0.1 ml 5% w/v PTMC15-F127-PTMC15 or 0.1 ml 18% w/v F127 were detected by Annexin V/PI staining using fluorescence-activated cell sorting (FACS). (E) Apoptotic changes between 5% w/v PTMC15-F127-PTMC15 and 18% w/v F127 at 3 days. LDH = lactate dehydrogenase. ‘Control’ one is the group with no treatment.
Figure 3.
In vitro toxicity of HTFs incubated with MMC.
(A) LDH release from cells incubated with different concentrations of MMC within 48 hours. (B) Cell viability of HTFs treated with 0.1 mg/ml MMC loaded 5% w/v PTMC15-F127-PTMC15 or 18% w/v F127 were measured by LDH assay for 3 days. (C) Early stage, Late stage, and Total HTFs apoptotic cells change labeled by Annexin V/PI in different treatment groups were detected by FACS for 3 days. (D) The FACS for Annexin V/PI staining of HTFs treated with 0.5 mg/ml MMC for 5 minutes, with two type of hydrogel loaded with 0.1 mg/ml MMC and 0.1 mg/ml MMC at 3 days. LDH = lactate dehydrogenase. ‘Control’ group is the group with no treatment.
Figure 4.
Postoperative observation in rabbit glaucoma filtration surgery models.
(A) Group had filtration surgery only. (B) Group injected with 0.1 ml 5% w/v PTMC15-F127-PTMC15/MMC (0.1 mg/ml) hydrogel in glaucoma filtration surgery. (C) Treat with 0.5 mg/ml MMC for 5 minutes during glaucoma filtration surgery group. (D) Group with 0.1 ml 5% w/v PTMC15-F127-PTMC15 injected in glaucoma filtration surgery.
Figure 5.
Postoperative evaluations in rabbit models.
The rabbits were divided into four treatment groups: ‘Control group’ is the filtration surgery only group (a). Group injected with 0.1 ml 5% w/v PTMC15-F127-PTMC15/MMC (0.1 mg/ml) hydrogel in filtration surgery (b). Treat with 0.5 mg/ml MMC for 5 minutes during filtration surgery group (c). Group with 0.1 ml 5% w/v PTMC15-F127-PTMC15 injected in filtration surgery (d). (A) Survival curve of filtering blebs after glaucoma filtration surgery using Kaplan-Meier analysis. Filtering blebs in group ‘a’ (n = 4) and ‘d’ (n = 4) mostly failed within 1 weeks. Group ‘b’ (n = 4) markedly increased the bleb survival period. In group ‘c’ (n = 4), the bleb survived nearly 4 weeks. Kaplan-Meier analysis showed a significant difference in the survival distributions among the four groups. (Log Rank = 75.121, p<0.01). (B) IOP changes within postoperatively 28 days. The results were obtained and expressed as the mean± SD. There is no significant difference between each group (p>0.05). (C) Proliferating cell nuclear antigen (PCNA) was analyzed in each group at post-operative day 28.(D) Apoptotic cells were indicated by TUNEL assay in each group at day 28.
Table 2.
Corneal Endothelial Cell Count (/mm2, mean±SD).
Figure 6.
Postoperative Histological characteristics evaluations in rabbit models.
The rabbits were divided into four treatment groups: Filtration surgery only group (a). Group injected with 0.1 ml 5% w/v PTMC15-F127-PTMC15/MMC (0.1 mg/ml) hydrogel in filtration surgery (b). Treat with 0.5 mg/ml MMC for 5 minutes during filtration surgery group (c). Group with 0.1 ml 5% w/v PTMC15-F127-PTMC15 injected in filtration surgery (d). (A) In H&E staining, the subconjunctival scarring in group ‘a’ were noticed with fibrocellular scar tissue and with closed sclerotomy, group ‘b’ had loosely arranged subepithelial connective tissues, group ‘c’ had a few inflammatory cells and thin subconjunctival tissue, and group ‘d’ was similar to group ‘a’. (B) Massones Trichrome staining showed group ‘a’ and ‘d’ had more dense collagen tissues in sub-conjunctiva space than group ‘b’ and ‘c’. (C) Immunohistochemical examination of α-SMA illustrated that lesser α-SMA specific myofibroblast were differentiated in group ‘b’ and ‘c’. CE: conjunctival epithelium; TC: Tenon's Capsule;