研究目的
To investigate the role of pH in wound healing by detecting non-invasive pH changes in tissue-engineered human skin models in response to wounding, inflammation, and bacterial infection using Raman confocal spectroscopy.
研究成果
The study successfully demonstrated that confocal Raman spectroscopy can non-invasively detect pH changes in TE-skin models, revealing the loss of acid mantle in wounded and inflamed skin and heterogeneous pH microenvironments in bacterially infected skin. These findings highlight the potential role of pH in wound healing and infection treatment, warranting further investigation into the implications for antimicrobial therapies and wound management.
研究不足
The study is limited to in vitro TE-skin models, which may not fully replicate in vivo conditions. The Raman spectroscopy method requires specific equipment and expertise, and the detection of pH microenvironments might be influenced by sample preparation and bacterial strain variability. Further optimization is needed for real-time in vivo applications.
1：Experimental Design and Method Selection:
A confocal Raman microspectroscopy method was employed to measure pH non-invasively in 3D tissue-engineered human skin (TE-skin) models. The method utilized the protonation state of phosphate groups, with pH calculated using the Henderson-Hasselbalch equation based on Raman peak intensities at 968 cm?1 and 1074 cm?1. Principal component analysis (PCA) was used for data analysis.
2：Sample Selection and Data Sources:
TE-skin models were produced using de-epidermised dermis (DED) from human skin, seeded with keratinocytes and fibroblasts. Models included controls, wounded models (created by incisional cuts or epithelial removal), inflamed models (treated with IL-17), and infected models (inoculated with Staphylococcus aureus or Pseudomonas aeruginosa).
3：List of Experimental Equipment and Materials:
Equipment included a DXRxi Raman imaging microscope system (Thermo Scientific), Zeiss Axiovert 200M inverted fluorescence microscope, and various reagents from Sigma-Aldrich and Fisher Scientific. Materials included de-epidermised dermis, keratinocytes, fibroblasts, bacteria cultures, and buffers for pH calibration.
4：Experimental Procedures and Operational Workflow:
TE-skin models were cultured for 14 days at an air-liquid interface. For infection studies, bacteria were applied to wound models and cultured for 48 hours. Raman spectra were collected every 15 μm in the Y-axis and every 50 μm in the Z-axis up to 600 μm depth using a 780 nm laser. Samples were fixed and processed for histology (H&E staining, Gram staining, immunohistochemistry) post-analysis.
5：Data Analysis Methods:
pH was calculated from the ratio of Raman peak intensities at 968 cm?1 and 1074 cm?1. PCA was performed over the spectral range 950–1080 cm?1 to distinguish pH variations. Data were processed using OMNICxi software.
独家科研数据包，助您复现前沿成果，加速创新突破
获取完整内容
