Wound healing is a complex and dynamic process with different distinct and overlapping
phases from homeostasis, inflammation and proliferation to remodelling. Monitoring the
healing response of injured tissue is of high importance for basic research and clinical prac-
tice. In traditional application, biological markers characterize normal and abnormal wound
healing. Understanding functional relationships of these biological processes is essential for
developing new treatment strategies. However, most of the present techniques (in vitro or in
vivo) include invasive microscopic or analytical tissue sampling. In the present study, a non-
invasive alternative for monitoring processes during wound healing is introduced. Within this
context, hyperspectral imaging (HSI) is an emerging and innovative non-invasive imaging
technique with different opportunities in medical applications. HSI acquires the spectral
reflectance of an object, depending on its biochemical and structural characteristics. An in-
vitro 3-dimensional (3-D) wound model was established and incubated without and with
acute and chronic wound fluid (AWF, CWF), respectively. Hyperspectral images of each
individual specimen of this 3-D wound model were assessed at day 0/5/10 in vitro, and
reflectance spectra were evaluated. For analysing the complex hyperspectral data, an effi-
cient unsupervised approach for clustering massive hyperspectral data was designed,
based on efficient hierarchical decomposition of spectral information according to archetypal
data points. It represents, to the best of our knowledge, the first application of an advanced
Data Mining approach in context of non-invasive analysis of wounds using hyperspectral
imagery. By this, temporal and spatial pattern of hyperspectral clusters were determined
within the tissue discs and among the different treatments. Results from non-invasive imag-
ing were compared to the number of cells in the various clusters, assessed by Hematoxylin/
Eosin (H/E) staining. It was possible to correlate cell quantity and spectral reflectance during
wound closure in a 3-D wound model in vitro.