Raman imaging for quantification of the volume fraction of biodegradable polymers in histological preparations.

Postretrieval analysis of biodegradable polymeric constructs for degradation rates requires correct identification of the degradable polymer, de novo tissue and the confounding presence of a secondary polymer used for embedding. Similarities between the structures of many tissue engineering polymers may make them difficult to distinguish from the polymer used to embed explants prior to histological sectioning. In this study, we assessed the feasibility of a chemical imaging method, Raman microscopy, to discriminate between more than one polymer species. From the perspective of spectroscopy, this is not a straightforward process because of the emergence of multiple peaks, ubiquity of embedding medium, and presence of observations sourcing from points sampled at the interface of two phases. A multivariate K-means data clustering method was used to discriminate between different polymeric components. The method was able to classify 95% of the observations to the correct category. The remaining data displayed multiple memberships because of (1) the laser spot coinciding with the interfaces of more than one phase or (2) infiltration of histological embedding polymer. Combined with multivariate analysis methods, this technique may prove useful in the future for tissue engineering and biomaterials analysis of degradation rates of, and tissue ingrowth into, polymer scaffolds.

Biomechanical responses due to discitis infection of a juvenile thoracolumbar spine using finite element modeling.

Growth modulation changes occur in pediatric spines and lead to kyphotic deformity during discitis infection from mechanical forces. The present study was done to understand the consequences of discitis by simulating inflammatory puss at the T12/L1 disc space using a validated eight-year-old thoracolumbar spine finite element model. Changes in the biomechanical responses of the bone, disc and ligaments were determined under physiological compression and flexion loads in the intact and discitis models. During flexion, the angular-displacement increased by 3.33 times the intact spine and localized at the infected junction (IJ). The IJ became a virtual hinge. During compression loading, higher stresses occurred in the growth plate superior to the IJ. The components of the principal stresses in the growth plates at the T12/L1 junction indicated differential stresses. The strain increased by 143% during flexion loading in the posterior ligaments. The study indicates that the flexible pediatric spine increases the motion of the infected spine during physiological loadings. Understanding intrinsic responses around growth plates is important within the context of growth modulation in children. These results are clinically relevant as it might help surgeons to come up with better decisions while developing treatment protocols or performing surgeries.

Effect of fixation and embedding on Raman spectroscopic analysis of bone tissue.

Raman spectroscopy provides valuable information on the physicochemical properties of hard tissues. While the technique can analyze tissues in their native state, analysis of fixed, embedded, and sectioned specimens may be necessary on certain occasions. The information on the effects of fixatives and embedding media on Raman spectral properties is limited. We examined the effect of ethanol and glycerol as fixatives and a variety of embedding media (Araldite, Eponate, Technovit, glycol methacrylate, polymethyl methacrylate, and LR white) on Raman spectral properties (mineralization, crystallinity, and carbonation) measured from the cortical bone of mouse humeri. Humeri were fixed in ethanol or glycerol, followed by embedding in one of the media. Nonfixed, freeze-dried, and fixed but not embedded sections were also examined. Periosteal, endosteal, and midosteal regions of the intracortical envelope were analyzed. Raman spectra of fixative solutions and embedding media were also recorded separately in order to examine the specifics of overlap between spectra. We found significant effects of fixation, embedding, and anatomical location on Raman spectral properties. The interference of ethanol with tissue seemed to be relatively less pronounced than that of glycerol. However, there was no single combination of fixation and embedding that left Raman spectral parameters unaltered. We conclude that careful selection of a fixation and embedding combination should be made based on the parameter of interest and the type of tissue. It may be necessary to process multiple samples from the tissue, each using a combination appropriate for the Raman parameter in question.