Rapid biocompatibility analysis of materials via in vivo fluorescence imaging of mouse models

Abstract

Background: Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis.

Methodology/principal findings: Here we develop rapid, non-invasive methods for in vivo quantification of the inflammatory response to implanted biomaterials. Materials were placed subcutaneously in an array format and monitored for host responses as per ISO 10993-6: 2001. Host cell activity in response to these materials was imaged kinetically, in vivo using fluorescent whole animal imaging. Data captured using whole animal imaging displayed similar temporal trends in cellular recruitment of phagocytes to the biomaterials compared to histological analysis.

Conclusions/significance: Histological analysis similarity validates this technique as a novel, rapid approach for screening biocompatibility of implanted materials. Through this technique there exists the possibility to rapidly screen large libraries of polymers in vivo.

Figure 1. Subcutaneous Injection arrays.

Three array formats used for injecting saline and polymers subcutaneously in mice where A is 30 µl, B is 50 µl, C is 70 µl, and D is 100 µl.

Figure 2. Time evolution of cathepsin activity in response to injected materials fluorescently imaged.

In vivo fluorescence imaging using ProSense 680 for cathepsin activity at various time points for a) saline, b) polystyrene, and c) alginate. The scale bar ranges 0–6×10⁻⁴ in fluorescence efficiency. The quantified fluorescence efficiencies of cathepsin activities are shown for d) polystyrene and e) alginate as the mean with standard deviation. Symbols represent data points and lines represent linear regressions.

Figure 3. Time evolution of macrophage response to injected materials fluorescently imaged.

In vivo fluorescence imaging of F4/80 pan macrophage antibody at various time points for a) saline, b) polystyrene, and c) alginate. The scale bar ranges 0–1.5×10⁻⁴ in fluorescence efficiency. The quantified fluorescence efficiency of F4/80 pan macrophage responses are shown for d) polystyrene and e) alginate as the mean with standard deviation. Symbols represent data points and lines represent linear regressions.

Figure 4. Histological scores of materials subcutaneously injected.

Histological scores of a) neutrophils, b) macrophages, and c) fibrosis determined for tissue excised at various time points with injections of saline, polystyrene, and alginate. Values shown are means with standard deviations.

Figure 5. H&E staining of representative sections subcutaneously injected.

Representative sections stained with H&E are shown for saline, polystyrene, and alginate at various time points (1, 3, 7, 14, 21, and 28 days post-injection). (Magnification 20×, scale bar = 100 µm).