Thermosensitive hydrogels deliver bioactive protein to the vaginal wall - Fig 4

<p><b>Effect of HG ± fibulin-5 on MMP-9 activity in vaginal stromal cells from WT (A) or Fbln5</b>^<b>-/-</b><b>(B) mice. HG,</b> hydrogel; <b>Fib-5</b>_<b>5,</b> recombinant fibulin-5 5 μg/ml; <b>Fib-5</b>_<b>10,</b> recombinant fibulin-5 10 μg/ml. <b>-Ctl,</b> vaginal tissues from MMP-9 KO mice; <b>+Ctl,</b> vaginal tissues from Fbln5^-/- mice.</p

Long-term effects of HG ± fibulin-5 on vaginal MMP-9 in <i>Fbln5</i> KO mice treated for 14–28 d.

<p><b>A.</b> Cumulative results of MMP-9 activity in vaginal tissues from 38 mice treated with HG ± fibulin-5 (10 μg/ml) for 2–4 weeks. *P < 0.05 compared with HG alone, ANOVA. <b>B.</b> Immunoblot analysis of urea-extracted protein from vaginal tissues of Fbln5^-/- mice treated with HG or HG + FBLN5 (10 μg/ml) x 2 weeks. The left blot was incubated with <b>anti- His Tag</b> antibody whereas the right blot was incubated with anti-fibulin-5. Positive controls on each blot are recombinant fibulin-5 (rFBLN5, 200 ng). <b>Bl</b>, blank.</p

Trichrome-stained sections of the vaginal wall of <i>Fbln5</i>^<i>-/-</i> mice treated with HG (A) or HG+fibulin-5 (10 μg/ml).

<p>Note increased thickness of epithelial and stromal layers in animals treated with fibulin-5. Bar = 20 μm.</p

Thermosensitive property of the polymer solution.

<p><b>A</b>. Transmittance of the polymer aqueous solution (0.5 wt%) as a function of temperature. <b>B</b>. Thermally-induced gelation of the HG aqueous solution (25 wt%).</p

Effect of HG ± fibulin-5 on vaginal MMP-9 and MMP-2 in <i>Fbln5</i> KO mice treated for 7 d.

<p><b>A.</b> Gelatin zymography with protein extracts (10 μg/lane) from adult <i>Fbln5</i>^<i>-/-</i> mice injected with hydrogel alone (HG), HG incorporated with fibulin-5 (5 ug/ml), or fibulin-5 alone x 7 d. <i>Mmp9</i> KO was used as a neg ctl. <b>B.</b> Cumulative results of 34 mice treated with HG ± various doses of fibulin-5 x 7 d. *P < 0.05 compared with HG alone, ANOVA.</p

<i>In vitro</i> cell compatibility and <i>in vivo</i> tissue compatibility of HG was assessed.

<p>(A) HG was incubated with DMEM media for different periods of time (1, 3, 5 and 7 days) to generate conditioned media. The cell compatibility of the conditioned media was then evaluated using 3T3 fibroblasts and MTT assay. (B) Balb/c mice were subcutaneously implanted with HG or saline as the control. After implantation for 7 days, the animals were sacrificed and the implant-surrounding tissues were recovered for histological analyses. H&E stain and implant-associated cell numbers support that HG implant exerted minimal tissue response similar to saline control.</p

Recovery of fibulin-5 in vaginal tissues from <i>Fbln5</i>^<i>-/-</i> mice injected with hydrogel (HG) ± recombinant fibulin-5 (FN5).

<p>Urea extracts (10 μg/lane, <b>A</b>) revealed incorporation of fibulin-5 in matrix fractions of KO animals 7 d after injection with HG+FN-5 (equivalent to that in WT animals). WT urea extracts were used as the + control in the soluble fractions (<b>B</b>).</p

Release kinetics from PEG hydrogels.

<p>Upper panels are representative images of BSA-NIR fluorescence intensities at intravaginal injections as a function of time. Release kinetics from hydrogels (BSA-HG) are compared with that of BSA without HG. The percent BSA released was calculated based on the following formula. Release % = 100X [(Fluorescent intensity at time 0)–(fluorescent intensity at time T)] / (Fluorescent intensity at time 0). Note that release rates are delayed with HG compared with BSA alone. *P < 0.05.</p