Low shear, continuous flow biofilm reactor for testing anti-infective wound care materials: recent prototype validation with chlorhexidine nanoparticles in alginate films

摘要

Introduction: There is demand among the clinical community for innovative materials and devices to prevent or treat infected wounds. However, antimicrobial validation is often difficult at an early stage of material design. Simplistic experimental models are frequently used, and these neglect many important features of a real wound environment. Here, a novel 3D printed low shear continuous flow biofilm reactor which allows access for materials testing whilst nutrients are continuously supplied is described and validated against alginate films containing chlorhexidine nanoparticles (CHX-NPs).The device simulates the low shear flow environment of an exudating wound and provides a continuous supply of nutrients to a biofilm from beneath, similar to how a biofilm would appear and be accessed during treatment of a real colonised wound. Materials and methods CHX-NPs synthesis 10 mM aqueous solutions of CHX digluconate and sodium hexametaphosphate were combined (1:1 ratio). CHX-NPs aloinate films Powdered alginate was dissolved in diluted CHX-NPs to give doping of 0,3 and 6 wt%. These solutions were cast (55g alginate per m~2), and the water allowed to evaporate (RT, 3d). These thin films were cross-linked in CaCl_2_((aq)) (0.18 M) for 25 min, washed with distilled water and air dried. Disks (ø=10 mm) were cut from this material and used in subsequent work. Biofilm reactor validation A novel low shear, continuous flow biofilm reactor was designed and additively manufactured. Agar disks were placed within flowing nutrient channels (0.083 mL min~(-1)). A S. aureus, P. aervginosa biofilm was grown on a nitrocellulose disk sat atop the agar over 24 or 48h. A single alginate disk was placed on 9 of the biofilms, with 3 as controls. After 24h, any films were removed, the agar and nitrocellulose disk were vortexed into PBS. Serial dilutions were made, plated and counted. Results and discussions A low shear continuous flow biofilm reactor has been designed and manufactured. The device contains 12 wells split into 4 channels, in which biofilms can be grown using a single peristatlc pump and has been validated by the production of consistent polymicrobial biofilms over 24h across all 12 chambers. Add Figure 1 Add Figure 2 To validate the model, alginate films containing 0,3 and 6 wt% CHX-NPs were loaded onto biofilms grown for 48h and the bacterial load recorded after 24h in situ (Fig 3). Add Figure 3 The control channel biofilms yielded bacterial loads of 10.10 ± 0.17 and 8.51 ± 0.52 CFU ml~(-1) for S. aureus and P. aervginosa respectively. Alginate films containing 0 wt% CHX-NPs resulted in slight changes in these bacterial loads to 9.53 ± 0.40 and 8.85 ± 0.22. Reductions of two orders of magnitude were observed with 3 wt% CHX-NPs: to 7.78 ± 0.26 and 6.45 ± 0.54 and a further, albeit smaller, decrease with 6 wt% CHX-NPs: to 7.02 ±0.17 and 5.86 ± 0.34 CFU ml~(-1) respectively. Conclusions: The novel multi-well 3D printed low shear, continuous flow biofilm reactor presented here allows top access to biofilms whilst their nutrient supply is maintained -creating a much more relevant biofilm model on which novel anti-infective materials can rapidly be screened. The large number of channels within the single device allows one peristatic pump to be used and means it can be incubated within an appropriate environment. The device has been validated here using CHX-NPs embedded within an alginate film and shown them to be effective at disrupting a polymicrobial biofilm over 24 h.