In vivo non-invasive monitoring of tissue development in 3D printed subcutaneous bone scaffolds using fibre-optic Raman spectroscopy.

The development of novel biomaterials for regenerative therapy relies on the ability to assess tissue development, quality, and similarity with native tissue types in in vivo experiments. Non-invasive imaging modalities such as X-ray computed tomography offer high spatial resolution but limited biochemical information while histology and biochemical assays are destructive. Raman spectroscopy is a non-invasive, label-free and non-destructive technique widely applied for biochemical characterization. Here we demonstrate the use of fibre-optic Raman spectroscopy for in vivo quantitative monitoring of tissue development in subcutaneous calcium phosphate scaffolds in mice over 16 weeks. Raman spectroscopy was able to quantify the time dependency of different tissue components related to the presence, absence, and quantity of mesenchymal stem cells. Scaffolds seeded with stem cells produced 3-5 times higher amount of collagen-rich extracellular matrix after 16 weeks implantation compared to scaffolds without. These however, showed a 2.5 times higher amount of lipid-rich tissue compared to implants with stem cells. Ex vivo micro-computed tomography and histology showed stem cell mediated collagen and bone development. Histological measures of collagen correlated well with Raman derived quantifications (correlation coefficient in vivo 0.74, ex vivo 0.93). In the absence of stem cells, the scaffolds were largely occupied by adipocytes. The technique developed here could potentially be adapted for a range of small animal experiments for assessing tissue engineering strategies at the biochemical level.

Adhesive Hydrogels for Maxillofacial Tissue Regeneration Using Minimally Invasive Procedures.

Minimally invasive surgical procedures aiming to repair damaged maxillofacial tissues are hampered by its small, complex structures and difficult surgical access. Indeed, while arthroscopic procedures that deliver regenerative materials and/or cells are common in articulating joints such as the knee, there are currently no treatments that surgically place cells, regenerative factors or materials into maxillofacial tissues to foster bone, cartilage or muscle repair. Here, hyaluronic acid (HA)-based hydrogels are developed, which are suitable for use in minimally invasive procedures, that can adhere to the surrounding tissue, and deliver cells and potentially drugs. By modifying HA with both methacrylate (MA) and 3,4-dihydroxyphenylalanine (Dopa) groups using a completely aqueous synthesis route, it is shown that MA-HA-Dopa hydrogels can be applied under aqueous conditions, gel quickly using a standard surgical light, and adhere to tissue. Moreover, upon oxidation of the Dopa, human marrow stromal cells attach to hydrogels and survive when encapsulated within them. These observations show that when incorporated into HA-based hydrogels, Dopa moieties can foster cell and tissue interactions, ensuring surgical placement and potentially enabling delivery/recruitment of regenerative cells. The findings suggest that MA-HA-Dopa hydrogels may find use in minimally invasive procedures to foster maxillofacial tissue repair.

Simple Defocused Fiber Optic Volume Probe for Subsurface Raman Spectroscopy in Turbid Media.

We investigated the ability to perform deep subsurface Raman spectroscopy in turbid media using a simple fiber optic volume probe. Being able to collect Raman signals from regions deep within a biological sample provides the ability to noninvasively study underlying living tissue and tissue engineered constructs with high chemical specificity. Spatially offset Raman spectroscopy has shown great potential for obtaining subsurface Raman signals in biological samples. The applicability of the method for in vivo studies depends on the system complexity and small size probes are often desirable. Most real-time studies on human patients utilizing Raman spectroscopy have been performed with easy-to-handle miniaturized probes. Here we show both experimentally and theoretically that the sampling depth from a simple volume probe can be controlled by changing the probe to sample distance effectively suppressing Raman and fluorescence contributions from shallow sample layers while favoring the collection of signals from deeper layers. Relative spectral intensities as function of probe to sample distance were investigated for layered phantoms of poly(methyl methacrylate) and trans-stilbene and compared with theory. The volume probe was then utilized for the collection of spectra from phantoms mimicking in vivo transcutaneous measurement configurations of bone and engineered scaffold as well as from an ex vivo sample of bone and soft tissue. Together the results show that Raman fiber optic volume probes can be utilized for subsurface Raman spectroscopy in turbid media, providing a simple alternative to spatially offset Raman systems for, e.g., noninvasive monitoring and studying mineralized tissue and implanted scaffolds in vivo.