Application of a dense gas technique for sterilizing soft biomaterials

Abstract

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.

Figure 1

The dense CO₂ sterilization apparatus.

Figure 2

Visual characterization of the gels before and after treatment with conventional sterilization methods. The gross appearance and the microstructure of the control untreated gel is shown in (a) and (d), respectively. Corresponding images for a gel treated with gamma radiation (dosage = 0.5 Mrad at 1,527 rad/min) are shown in (b) and (e), while images for gels treated with steam sterilization (132°C, 186 kPa, 5 min) are shown in (c) and (f), respectively. Scale bar = 1 mm.

Figure 3

Visual characterization of the gels after treatment with dense CO₂. The gross appearance and the microstructure of a gel treated with dense CO₂ at 40°C and 250 bar for 1 h is shown in (a) and (c), respectively. Corresponding images for a gel treated with dense CO₂ at 70°C and 75 bar for 6 h are shown in (b) and (d). The similarity in appearance and microstructure to the control untreated gel (Fig. 2a and d) can be noted. Similar results were obtained for gels treated with dense CO₂ using the other conditions listed in Table II. Scale bar = 1 mm.

Figure 4

Elastic shear modulus G′ as a function of oscillatory frequency for PEG hydrogels treated with dense CO₂ compared to untreated control (n ≥ 3).

Figure 5

In vivo tissue response to the PEG gels in ferrets. a: H&E stained image of the untreated PEG gel implanted subcutaneously; (b) H&E stained image of the PEG gel sterilized using supercritical CO₂ (40°C, 250 bar, 1 h) implanted subcutaneously.