Patterning expression of regenerative growth factors using high intensity focused ultrasound

Abstract

Temporal and spatial control of growth factor gradients is critical for tissue patterning and differentiation. Reinitiation of this developmental program is also required for regeneration of tissues during wound healing and tissue regeneration. Devising methods for reconstituting growth factor gradients remains a central challenge in regenerative medicine. In the current study we develop a novel gene therapy approach for temporal and spatial control of two important growth factors in bone regeneration, vascular endothelial growth factor, and bone morphogenetic protein 2, which involves application of high intensity focused ultrasound to cells engineered with a heat-activated- and ligand-inducible gene switch. Induction of transgene expression was tightly localized within cell-scaffold constructs to subvolumes of ∼30 mm³, and the amplitude and projected area of transgene expression was tuned by the intensity and duration of ultrasound exposure. Conditions for ultrasound-activated transgene expression resulted in minimal cytotoxicity and scaffold damage. Localized regions of growth factor expression also established gradients in signaling activity, suggesting that patterns of growth factor expression generated by this method will have utility in basic and applied studies on tissue development and regeneration.

FIG. 1.

Development of a heat shock protein (HSP)-based and rapamycin-dependent gene switch for control of BMP2 expression. (a) Gene switch design. The first component of the gene switch comprises a human HSP70B promoter that drives expression of a bimodular synthetic transactivator (TA). One module contains the FRB rapamycin-binding domain (derived from human FRAP) and transcriptional activation domains from the p65 subunit of NF-κB and heat shock factor 1 (HSF1). The second module contains FKBP rapamycin-binding domains and zinc finger homeodomain 1 (ZFHD1) DNA-binding domains. In the presence of a dimerizer, the TA autoactivates its own expression through ZFHD1 elements and also drives expression of a transgene of interest [firefly luciferase (fLuc), human VEGF₁₆₅, BMP2] via the second component of the gene switch. (b) BMP2 induction requires heat shock and rapamycin/rapalog treatment. C3H10T½-BMP2 cells were suspended in a fibrin scaffold and exposed to vehicle, 10 nM rapamycin (Rap), and/or a 30 min 45°C hyperthermic stimulus as indicated. BMP2 was measured in the conditioned medium (“medium”) and lysates (“construct”) of the cell-scaffold constructs. ^#p<0.05 versus vehicle and 37°C controls. Data are mean±standard deviation with n=4. (c) BMP Responsive Element (BRE) fLuc reporter cell assay. Cells were activated as in b except that the rapalog, AP21967 (AP), was used in place of Rap. Conditioned media were obtained from each condition and added to the BRE luciferase BMP reporter cell line. ^#p<0.05 versus vehicle and 37°C controls. Data are mean±standard deviation with n=4. Color images available online at www.liebertpub.com/tec

FIG. 2.

Application of focused ultrasound to cell-fibrin constructs and induction of localized, mild hyperthermia. (a) Apparatus for delivering focused ultrasound to cell-fibrin constructs. Circuitry for driving the ultrasound transducer is shown on the left, including a function generator (Fxn Gen), an RF amplifier (RF Amp), and an impedance matching network (Z Network). A digital oscilloscope (O-scope) was used to monitor the quality of the driving signal. On the right is the tank filled with degassed 37°C water and a six-well Bioflex dish with fibrin scaffolds or cell-fibrin constructs positioned near the focus of the transducer. An absorber limits reflection of ultrasound energy into the samples. (b) Time course of temperature rises within a fibrin scaffold upon exposure to continuous wave ultrasound of I_SPTA=658 W/cm². Temperature measurements were made once per second at the indicated distances from the ultrasound focus using a thermocouple. Color images available online at www.liebertpub.com/tec

FIG. 3.

Spatially restricted activation of gene switch activity using focused ultrasound. (a) Time-dependence of induction. Triplicate fibrin scaffolds containing C3H10T½-fLuc cells were exposed to focused ultrasound (I_SPTA=754 W/cm²) for the indicated times before measurement of bioluminescence. Scale bar=5 mm. (b) Quantification of the bioluminescence signals (average radiance and total flux) in nonirradiated and ultrasound-treated regions of the constructs. ^#p<0.05 versus nonirradiated. Data are mean±standard deviation, n=3. (c) Ultrasound intensity-dependent activation of C3H10T½-fLuc cells. Cell-scaffold constructs were exposed to increasing ultrasound intensities for 5 min (I_SPTA=0–850 W/cm²) and the bioluminescence of treated and nonirradiated regions was quantified 24 h later. ^#p<0.05 versus nonirradiated. Data are mean±standard deviation with n=6. (d) Ultrasound intensity-dependent increases in the lateral width of fLuc-expressing region. Mean best-fit Gaussian profiles are shown. $ p<0.05 for width of 658 W/cm² versus 850 W/cm². Dashed line indicates the geometry of the acoustic beam at the focus. Inset shows typical quality of fit between the measured distribution of fLuc activity and the best-fit Gaussian profile. (e) A cross section of a cell-fibrin construct treated with three ultrasound exposures (denoted by three yellow arrowheads; 10 min at I_SPTA=754 W/cm² for each exposure) focused at different depths (separated longitudinally by 1.5 mm, laterally by 2 mm) within the construct. The incident surface is on the bottom. Scale bar=5 mm.

FIG. 4.

Ultrasound-induced expression of bioactive BMP2 in cell-fibrin constructs. (a) Accumulation of BMP2 in conditioned media (“medium”) and lysates (“construct”) from cell-fibrin constructs exposed to focused ultrasound (I_SPTA=0–850 W/cm²). Irradiated and nonirradiated regions of the constructs were extracted following HIFU exposure and cultured separately. ^#p<0.05 versus nonirradiated controls. Data are mean±standard error of the mean with n=12 for controls and n=9/group for ultrasound-treated samples. (b) Schematic describing experimental approach to localizing expression of bioactive BMP2 following HIFU treatment of C3H10T½-BMP2 cells in a fibrin scaffold. (c) After localization of BRE reporter activity in cells plated on an ultrasound-treated cell-fibrin construct. Asterisks indicate nominal locations of ultrasound exposure and the pink dashed line denotes line scan region for quantification. Scale bar=5 mm. Lower panels indicate quantification of BRE reporter cell fLuc expression in a line scan across the center of the cell-fibrin construct and a calibration curve showing the relationship between BRE reporter cell fLuc expression and treatment with known concentrations of rhBMP2.

FIG. 5.

Viability and morphology of cell-fibrin constructs exposed to focused ultrasound. (a) Metabolic activity of portions of cell-fibrin constructs treated with focused ultrasound for increasing times (0–15 min., I_SPTA=754 W/cm²). No differences in metabolic activity measured using an alamarBlue dye reduction assay were detected. Data are mean±standard deviation with n=9 for controls and n=3/group for ultrasound-treated samples and are expressed as percent control fluorescence of cells not exposed to ultrasound. (b) Metabolic activity of cell-fibrin constructs treated for 5 min with increasing ultrasound intensities (I_SPTA=0–850 W/cm²). ^#p<0.05 versus nonirradiated controls. Data are mean±standard deviation with n=9 for controls and n=6/group for ultrasound-treated samples. (c) Confocal microscopic images of viable cells (labeled with CMFDA, green) and fibrin scaffold morphology (labeled with AlexaFluor647-fibrinogen, AF647-Fbgn, red) in control and ultrasound-treated regions (10 min, I_SPTA=0–850 W/cm²) of cell-fibrin constructs. Left images captured at the surface of the construct incident to the acoustic beam. Right images captured 80 μm distal from the incident surface. Scale bar=250 μm. Color images available online at www.liebertpub.com/tec

FIG. 6.

In vivo activation of heat shock- and ligand-inducible gene switch activity with focused ultrasound. (a) Bioluminescence imaging of a representative animal with control (encircled by pink dashed line) and ultrasound-irradiated (I_SATA=658 W/cm² for 10 min; asterisk) cell-fibrin subcutaneous implants. Image was captured 24 h following ultrasound exposure. Scale bar=10 mm. (b) Hematoxylin and eosin staining of histologic sections of subcutaneous implants following ultrasound exposure. Dashed line indicates implant–skin interface. Scale bars=50 μm.