Porcine skin gelatin

Eight porcine eye balls were enucleated about 1 hour after death from 35–40 kg female pigs that were part of IACUC approved studies. The eyes were immersed in saline solution and then transported to the authors’ laboratory for experimental preparation. To support the eye ball for experiment, the eye was supported on some rubber materials and placed in a testing plastic container. A gelatin mixture was prepared from porcine skin gelatin (SIGMA-ALDRICH, Inc). The gelatin mixture was heated to 60° and then cooled down to the room temperature. The cooled gelatin mixture was poured into the plastic container to support the eye ball. The gelatin surrounded the eye ball but did not cover the surface of the eye ball completely. The gelatin mixture provided the support for the eye ball for experiment. The gelatin may also absorb some wave energy and reduce the wave reflection from hard boundaries of the testing container. The completed eye and gelatin structure was allowed to set overnight. The eye ball experiments were performed on the following day. In order to change the pressure in the eye ball, a 25 gauge butterfly needle was inserted into the vitreous humor space close to the cornea of the eye under the ultrasound imaging guidance. The needle was connected to a 10 mL syringe filled with water. The syringe was mounted on a retort stand and changed to different heights. The intraocular pressure (IOP) was obtained by the water height difference between the water level in the syringe and the water level in the testing container. Some water was put in the testing container for ultrasound measurement of the eye ball.
Intraocular pressure was maintained at a pressure between 5 and 30 mmHg and changed gradually at an interval of 5 mmHg by raising the syringe (Fig. 1). At each pressure level (5, 10 15, 20 25 and 30 mmHg), a sinusoidal vibration signal of 0.1 s duration was generated by a function generator (Model 33120A, Agilent, Santa Clara, CA). The vibration signals were used at three frequencies of 100, 150, and 200 Hz. The excitation signal at a frequency was amplified by an audio amplifier (Model D150A, Crown Audio Inc., Elkhart, IN) and then drove an electromagnetic shaker (Model: FG-142, Labworks Inc., Costa Mesa, CA 92626) mounted on a stand. The shaker applied a 0.1s harmonic vibration on the surface of the eye ball using an indenter with 3 mm diameter (Fig. 1). The propagation of the vibration wave in the ocular tissues was measured using an ultrasound probe (Verasonics, Inc, Kirkland, WA) submerged in water and mounted above the cornea on another stand. The ultrasound system was equipped with a linear array transducer (L11-4, Philips Healthcare, Andover, MA) transmitting at 6.4 MHz center frequency. The measurements were repeated three times at each frequency and each pressure level.
The wave motions were measured at eight locations in the cornea for each pressure level and for each frequency. The tissue motion at a location was measured by analyzing the ultrasound tracking beam through that location [22 (link)]. The wave speed was analyzed by the change in wave phase with distance. Using the tissue motion at the first location as a reference, the wave speed was measured using the wave phase delay of the remaining locations relative to the first location (Fig. 2). At each frequency, the wave speed in the cornea was estimated using a phase gradient method,
$$c_s(f)=2\pi f\frac{\mathrm{\Delta }r}{\mathrm{\Delta }\varphi },$$ where Δr is the distance between 2 detected locations and Δϕ is the phase change over that distance, f is the excitation frequency in Hz. Three measurements were made at each frequency. It has been shown that on gelatin phantoms the wave speed estimation has a standard error less than 10% based on 7-point regression [23 (link)]. Therefore, it is necessary to obtain multiple measurements of Δϕ and a statistical regression to get accurate estimates of wave speed. Ideally, the phase of the surface wave ϕ at a particular frequency has a linear relationship with the distance. The wave speed was measured over eight locations in the central region of the cornea and analyzed with a linear regression curve obtained using a least-squares fitting technique on multiple Δϕ measurements,
$$\mathrm{\Delta }\widehat{\varphi }=-\alpha \mathrm{\Delta }r+\beta ,$$ where Δϕ̂ denotes the linear regression value of multiple Δϕ measurements, α and β are regression parameters, and the wave speed is calculated as follows,
The quality of the measurement of wave speed was assured by the sum of squares of linear regression residuals (R²) being ≥ 0.8 [22 (link)]. R² is the coefficient of determinant ranging from 0 to 1, a statistical measure of how close the data fit the regression line. In general, the higher the R², the better the model fits the data.

Glycidal methacrylate-hyaluronic acid (GM-HA) was synthesized according to a protocol modified from previous work [25 (link)]. 1g of hyaluronic acid was first dissolved in 100ml of acetone/water (50/50) solution at room temperature overnight. 7.2ml tri-ethylamine (Sigma–Aldrich) and 7.2ml glycidyl methacrylate (Sigma–Aldrich) were added dropwise both at 20-fold excess in succession until thoroughly mixed. The solution was covered with aluminum foil and stirred overnight at room temperature. The resulting solution was then dialyzed against DI water with 3.5 kDa tubing (Spectrum Labs) at room temperature. The DI water was changed after 2 hours, 4 hours and 24 hours. The dialyzed solution was frozen overnight at −80 °C and then lyophilized for 48 hours at 0.040 mbar and −50 °C. The lyophilized GM-HA was stored at −80 °C for future use.
Gelatin methacrylate (GelMa) was synthesized according to a protocol adapted from previous work [26 (link)]. Briefly, 10% (w/v) porcine skin gelatin (Sigma–Aldrich) was dissolved into Dulbecco’s phosphate-buffered saline (DPBS) by stirring at 60 °C. Methacrylate anhydride (Sigma–Aldrich) was added to the solution at a rate of 0.5 ml/min until the final concentration of 8% (v/v) MA was reached. The reaction continued for 3 hours at 60 °C with constant stirring. After 3 hours, the resulted solution was diluted 1:1 with warm DPBS and dialyzed against DI water with 13.5 kDa tubing for 1 week at 40 °C. The dialyzed solution was then frozen at − 80 °C and lyophilized for 1 week. The lyophilized GelMa was store at − 80 °C for future use.
Photoinitiator lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) was synthesized according to previously published work [27 (link)]. Briefly, 3.2g (0.018mol) of 2,4,6-trimethylbenzoyl chloride (Sigma–Aldrich) was added dropwise to an equal molar amount of dimethyl phenylphosphonite (3g, Acros Organics) with continuous stirring at room temperature under argon. After 18 hours, 6.1g lithium bromide (Sigma–Aldrich) dissolved in 100ml of 2-butanone (Sigma–Aldrich) was added into the previous mixture at 4-fold excess. The reaction was then heated to 50 °C and a solid precipitate was formed after 10 min. The mixture was allowed to cool down to room temperature and rest for overnight before filtration. 2-butanone was used to wash the filtrate and remove the unreacted lithium bromide. After 3 times wash and filtration, the excess solvent was removed by vacuum, leaving LAP in a white solid chunk state which was pestled into powder. LAP was stored at − 80 °C under argon for future use.

GelMA was synthesized as described previously [21 (link)]. Briefly, porcine skin gelatin (Sigma Aldrich, St. Louis, MO, USA) was mixed at 10%(w/v) into phosphate buffered saline (PBS; Gibco, Billings, MT, USA) and stirred at 60 °C until fully dissolved. Methacrylic anhydride (MA; Sigma) was added to the solution at a rate of 0.5 ml/min until a concentration of 8% (v/v) of MA was obtained in the gelatin solution. The solution was then stirred for 1 h at 50 °C, followed by a 2x dilution with warm PBS and dialyzed against distilled water using 12–14 kDa cutoff dialysis tubing (Spectrum Laboratories, Rancho Dominguez, CA, USA) for one week at 40 °C to remove the unreacted groups from the solution. The GelMA solution was frozen overnight at −80 °C and lyophilized in a freeze dryer (Labonco, Kansas City, MO, USA) for one week. Freeze dried GelMA foam was stored at −80 °C until further usage.