| Background:Blood vessel formation is a crucial process in the development,homeostasis,and regeneration of most types of tissue.Vessel formation is regulated by the concentration,spatiotemporal presentation,and sequence of pro-angiogenic growth factors?GFs?.Methods to stimulate vessel formation could impact the treatment of ischemic diseases as well as improve the viability of transplanted organs and tissue substitutes.The challenge of translating pro-angiogenic growth factors for therapeutic purposes has stimulated a myriad of biomaterials-based,delivery approaches.Many techniques rely on incorporating a growth factor into a hydrogel.The kinetics of release can be tuned basedonthe physiochemical properties of the growth factor and scaffold.An ARS consists of a fibrin matrix doped with a growth factor-loaded,sonosensitive emulsion.We have previously shown that focused ultrasound?FUS?can be used to control the delivery of bioactive payloads from composite scaffolds,termed acoustically-responsive scaffolds?ARSs?.Objectives:1.To assess the dose response of basic fibroblast growth factor?bFGF?concentration on angiogenic sprouting in a co-culture model consisted of endothelial cell-coated microbeads and dispersed fibroblasts.2.To assess the bFGF release from the ARS in response to the emulsion volume as well as acoustic pressure of US and the distribution of bFGF in this in vitro system.3.To evaluate endothelial sprouting in response to the volume fraction of the bFGF-loaded emulsion or blank emulsion and acoustic pressure used for bFGF release.4.To investigate the impact of temporal presentation of bFGF from gel-in gel constructs on tubule length and fibroblast density.Materials and Methods:1.Experimental apparatus:?1?A calibrated,single-element high intensity focused US transducer?2.5?MHz,H-108,f-number?=?0.83,focal length?=?50?mm,Sonic Concepts Inc.,Bothell,WA,USA?was used to generate acoustic droplet vaporization?ADV?within the ARSs.?2?Coulter counter,Multisizer 4,Beckman Coulter,USA.?3?Fluorescence microscopy,Eclipse TiE,Nikon,USA.2.Experimental reagents:?1?VascuLife VEGF endothelial cell culture medium,Lifeline Cell Technology,USA.?2?Dulbecco's modified eagle medium,DMEM,Gibco,USA.?3?DMEM/F12,Gibco,USA.?4?Cytodex microcarrier beads,C3275,Sigma,USA.?5?Bovine fibrinogen,Sigma,USA.?6?Basic fibroblast growth factor?bFGF?,EMD Millipore,USA.?7?Bovine thrombin,King Pharmaceuticals,USA.?8?Ulex europaeus agglutin I,UEA-I,Vector Laboratories,USA.?9?4',6-diamidino-2-phenylindole,DAPI,Thermo Fisher Scientific,USA.?10?Perfluoroheptane,PFHep,Sigma,USA.?11?Enzyme-linked immunosorbent assay,ELISA,R&D System,USA.?12?Quartz microfluidic chip,Dolomite,Royston United Kingdom.?13?6-well HT Bioflex plates,Flexcell International Co.,USA.3.Cells:?1?Human umbilical vein endothelial cells,HUVECs,Lonza,USA.?2?Normal human dermal fibroblasts,NHDFs,Lonza,USA.4.Study protocol:?1?Dose response of bFGF concentration on angiogenic sprouting?1?Construct fabrication:The gel consisted of 2.5 mg/mL fibrin,2 U/mL thrombin,2.5×104/mL normal human dermal fibroblasts and 50/mL microcarrier beads coated with HUVECs.The day before construct assembly,104 beads were combined with 4×106HUVECs.?2?Overlying medium:The gels were covered with complete media following polymerization.The next day,the overlying media was replaced with starvation media with the desired concentration?0.1 ng/mL,1 ng/mL,2 ng/mL,5 ng/mL,10 ng/mL or 100 ng/mL?of bFGF.?3?Imaging and analysis of sprouting and NHDF density:Constructs were fixed on day7 or day 14.Constructs were imaged via fluorescence microscopy using MetaMorph software after UEA-I and DAPI staining.Total tubule length has been used to quantify the magnitude of vascular morphogenesis and NHDF density was counted using ImageJ.?2?bFGF release from the ARS in response to ADV?1?Preparation of the emulsions:The water-in-perfluorocarbon-in water?W1/PFC/W2?double emulsion was prepared on a microfluidic device.The W1 phase consisted of 10mg/mL bovine serum albumin,0.4 U/mL heparin,and 0.5 mg/mL bFGF in PBS whereas the PFC phase was perfluoroheptane(C7F16).?2?Construct fabrication and US exposure:The inner gel ARS with either 0.25%or 1%?v/v?bFGF-loaded emulsion,or fibrin containing an equivalent loading of unencapsulated bFGF as in the two ARS conditions.The outer gel?1.5 mL volume?consisted of 2.5 mg/mL fibrin,2 U/mL thrombin and DMEM.The gels were covered with complete media following polymerization.The next day,the overlying media was replaced with starvation media and the ARSs were exposed to pulsed US?3.3 MPa or 8.8 MPa peak rarefactional pressure?via a computer-controlled raster pattern.?3?Collecting and mearsurement of bFGF release:The overlying media were sampled daily by collecting 0.1 mL of the media for 8 days,including immediately after US exposure.On day 2 or day 8,the outer gels wer biopsied??:6 mm?and digested by adding0.05 U of plasmin.The concentration of bFGF was measured using an ELISA.?3?Endothelial sprouting in response to ADV?1?Preparation of the emulsions:The water-in-perfluorocarbon-in water?W1/PFC/W2?double emulsion was prepared on a microfluidic device.The W1 phase consisted of 10mg/mL bovine serum albumin,0.4 U/mL heparin,and 0.5 mg/mL bFGF in PBS whereas the PFC phase was perfluoroheptane(C7F16).Blank emulsion was prepared as described above with only PBS as the W1 phase.?2?Construct fabrication and US exposure:The inner gel consisted of an ARS?0.4 mL volume?containing 2.5 mg/mL fibrin,2 U/mL thrombin,and either 0.25%or 1%?v/v?double emulsion with bFGF.The outer gel?1.5 mL volume?consisted of 2.5 mg/mL fibrin,2 U/mL thrombin,2.5×104/mL normal human dermal fibroblasts and 75 microcarrier beads coated with HUVECs.The gels were covered with complete media following polymerization.The next day,the overlying media was replaced with starvation media and the ARSs were exposed to pulsed US?3.3 MPa or 8.8 MPa peak rarefactional pressure?via a computer-controlled raster pattern.?3?Imaging and analysis of sprouting:Constructs were fixed on day 7.Constructs were imaged via fluorescence microscopy using MetaMorph software after UEA-I and DAPI staining.Total tubule length has been used to quantify the magnitude of vascular morphogenesis.?4?Impact of temporal presentation of bFGF on endothelial network formation?1?Preparation of the emulsions:The water-in-perfluorocarbon-in water?W1/PFC/W2?double emulsion was prepared on a microfluidic device.The W1 phase consisted of 10mg/mL bovine serum albumin,0.4 U/mL heparin,and 0.5 mg/mL bFGF in PBS whereas the PFC phase was perfluoroheptane(C7F16).Blank emulsion was prepared as described above with only PBS as the W1 phase.?2?Construct fabrication and US exposure:The inner gel consisted of an ARS?0.4 mL volume?containing 2.5 mg/mL fibrin,2 U/mL thrombin,and either 0.25%or 1%?v/v?double emulsion with bFGF.The outer gel?1.5 mL volume?consisted of 2.5 mg/mL fibrin,2 U/mL thrombin,2.5×104/mL normal human dermal fibroblasts and 75 microcarrier beads coated with HUVECs.The gels were covered with complete media following polymerization.The next day,the overlying media was replaced with starvation media.On day 1,different subvolumes of the ARSs?i.e.,none,half,or complete?were exposed to US?8.8 MPa peak rarefactional pressure?.At a later time?i.e.,day 4 or day 7?,the remaining half of the ARS was exposed to US.?3?Imaging and analysis of sprouting:Constructs were fixed on day 7,day 10 or day13.Constructs were imaged via fluorescence microscopy using MetaMorph software.Total tubule length has been used to quantify the magnitude of vascular morphogenesis and NHDF density was counted using ImageJ.Results:1.bFGF stimulates endothelial sprouting?1?Quantification of the UEA-I stained images revealed a sigmoidal relationship between total tubule length and bFGF concentration.The C50 on day 14,5.9?ng/mL[4.7,7.3],was significantly higher than on day 7,2.7?ng/mL[2.3,3.2]?P?0.05?.Analogously,Lmax on day 14,8008??m[7358,8710],was significantly higher than on day 7,4303??m[4051,4567]?P?0.05?.?2?On day 14,bFGF concentration of 5 ng/mL and higher yielder NHDF densities that were significantly greater than 0.1 ng/mL?P?0.05?.2.ADV enhanced bFGF release?1?The fraction of bFGF released into the overlying media increased significantly following US exposure at 3.3 MPa or 8.8 MPa?P?0.05?.Negligible release?i.e.,<0.02%?was measured for the-US conditions.bFGF release correlated with acoustic pressure for each emulsion concentration.In response to exposure at 8.8 MPa,greater bFGF release was measured from the ARS containing 1%?v/v?bFGF-loaded emulsion versus 0.25%?v/v??P?0.05?;no difference was observed with the 3.3 MPa exposure?P>0.05?.?2?For the inner gel did not contain any emulsion,the masses of bFGF incorporated were equivalent to the 0.25%?v/v??i.e.,167 ng?and 1%?v/v??i.e.,668 ng?ARS conditions.For both mass loadings,values of Cmax and K were not statistically different.Cmax was23.5%[22.4,24.7]and 22.1%[21.2,23.1]for constructs with 167 ng and 668 ng of bFGF,respectively?P?0.05?.K was 1.1 day-1[0.8,1.6]and 0.9 day-1[0.7,1.2]for constructs with167 ng and 668 ng of bFGF,respectively?P>0.05?.?3?The majority of the added bFGF remained in the inner gel for all conditions.For each experimental condition,the fraction of the bFGF in the inner gel or media increased when comparing day 2 versus day 8.3.ADV enhanced sprout formation?1?The PFHep emulsion had a mean diameter of 6.2±0.13?m and a coefficient of variance of 13.4%±0.6%.?2?For constructs containing blank?i.e.,without bFGF?emulsion,tubule formation in the absence of bFGF delivery was lower but yielded significantly sprouting?P?0.05?.The total tubule length for constructs cultured in fully supplemented media?4235±1816?m?was 16-fold higher compared to constructs cultured in starvation media?258±77?m??P?0.05?.?3?Both-US conditions as well as the 3.3 MPa exposure with 0.25%?v/v?emulsion yielded sprouting that was not different than the starvation condition?P?0.05?.Significantly greater sprouting was measured following exposure of the 1%?v/v?ARS at3.3 MPa,0.25%?v/v?ARS at 8.8 MPa,and 1%?v/v?ARS at 8.8 MPa?P?0.05?.Overall,sprouting correlated with the volume fraction of bFGF-loaded emulsion in the ARS and the acoustic pressure used for bFGF release.4.Temporal presentation of bFGF?1?Tubule length increased with incubation time within a given group of conditions?i.e.,C,D,E?.All+US conditions yielded significantly greater sprouting than the-US groups.However,there were no observed differences in sprouting when comparing the different US exposure patterns with the same incubation period?i.e.,C1 vs D1 vs E1;C2 vs D2;C3 vs E2??P>0.05?.Similar trends were observed with NHDF density?P>0.05?.?2?An analysis of tubule length versus distance from the ARS revealed no correlation based on a slope analysis?P>0.05?.Conclusions:1.Quantification of the UEA-I stained images revealed a sigmoidal relationship between total tubule length and bFGF concentration.The density of NHDFs tended to increase with bFGF concentration.2.When directly incorporated into fibrin,a burst release of bFGF occurs.bFGF release correlated with acoustic pressure for each emulsion concentration.When comparing bFGF release into the media following exposure at 8.8 MPa,the rate of release qualitatively correlated inversely with the volume fraction of emulsion in the ARS,which supports the hypothesis regarding an increased path length.3.Bubble formation and sprouting correlated with the volume fraction of bFGF-loaded emulsion in the ARS and the acoustic pressure used for bFGF release.A slight increase was also seen with blank ARS constructs.4.Staggering the release of bFGF did not impact tubule lengths.It is hypothesized that the concentration of released bFGF may not have been high enough to observe a difference in sprouting using the different temporal patterns of bFGF release. |
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