Raman spectroscopy of osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor. In the last years, several studies have demonstrated that the increase of Hydroxyapatite (HA) and Interleukin-6 (IL-6) syntheses compared to those expressed by normal osteoblasts could be used to detect the degree of malignancy of osteosarcoma cells. Conventional biochemical methods widely employed to evaluate bone cell differentiation, including normal and cancerous phenotypes, are time consuming and may require a large amount of cells. HA is a mineral form of calcium phosphate whose presence increases with maturation of osteosarcoma cells. Analogously, IL-6 is a fundamental cytokine whose production is highly increased in osteosarcoma cells. In this study, we employ Raman spectroscopy to the identification and discrimination of osteosarcoma cells from osteo-differentiated mesenchymal stromal cells (MSCs) by detecting the presence of HA and IL-6. However, while the identification of HA is facilitated by the characteristic peak at 960 cm-1, corresponding to symmetric stretching (P-O) mode, the quantification of IL-6 it is much more elusive, being its Raman signal characterized by cysteine, but also by phenylalanine, amide I II and III whose signals are common to other proteins. Supported by an accurate multivariate analysis, the results show that Raman spectroscopy is a high sensitivity technique dealing out a direct and quantitative measurement of specific mineralization levels of osteosarcoma cells. In turn, by exploiting the Surface-Enhanced Raman Scattering stimulated by internalized Gold Nanoshells (AuNSs) and combined with scanning probe microscopies, we were able to employ Raman spectroscopy to study subcellular components locally

Polysaccharide hydrogels for multiscale 3D printing of pullulan scaffolds

Structurally and mechanically similar to the extracellular matrix (ECM), biomimetic hydrogels offer a number of opportunities in medical applications. However, the generation of synthetic microenvironments that simulate the effects of natural tissue niches on cell growth and differentiation requires new methods to control hydrogel feature resolution, biofunctionalization and mechanical properties. Here we show how these goals can be achieved by using a pullulan-based hydrogel, engineered in composition and server as cell-adhesive hydrogel, 3D photo-printable in dimension, ranging from the macro- to the micro-scale dimensions, and of tunable mechanical properties. For this, we used absorbers that limit light penetration, achieving 3D patterning through stereolithography with feature vertical resolution of 200 μm and with overall dimension up to several millimeters. Furthermore, we report the fabrication of 3D pullulan-modified hydrogels by two-photon lithography, with sub-millimetric dimensions and minimum feature sizes down to some microns. These materials open the possibility to produce multiscale printed scaffolds that here we demonstrate to be inert for cell adhesion, but biologically compatible and easily functionalizable with cell adhesive proteins. Under these conditions, successful cell cultures were established in 2D and 3D. Keywords: Hydrogel, Biomaterials, Polysaccharide, Pullulan, 3D printing, Two photon laser lithography, Mesenchymal stromal cell

Nanomaterials-based strategies for piezoelectric cochlear implants

Nanomaterials-based strategies for piezoelectric cochlear implant

Growing bone tissue-engineered niches with graded osteogenicity: an in vitro method for biomimetic construct assembly

The traditional bone tissue-engineering approach exploits mesenchymal stem cells ( MSCs) to be seeded once only on three-dimensional (3D) scaffolds, hence, differentiated for a certain period of time and resulting in a homogeneous osteoblast population at the endpoint. However, after achieving terminal osteodifferentiation, cell viability is usually markedly compromised. On the other hand, naturally occurring osteogenesis results from the coexistence of MSC progenies at distinct differentiative stages in the same microenvironment. This diversification also enables long-term viability of the mature tissue. We report an easy and tunable in vitro method to engineer simple osteogenic cell niches in a biomimetic fashion. The niches were grown via periodic reseeding of undifferentiated MSCs on MSC/scaffold constructs, the latter undergoing osteogenic commitment. Timefractioning of the seeded cell number during differentiation time of the constructs allowed graded osteogenic cell populations to be grown together on the same scaffolds (i.e., not only terminally differentiated osteoblasts). In such cell-dynamic systems, the overall differentiative stage of the constructs could also be tuned by varying the cell density seeded at each inoculation. In this way, we generated two different biomimetic niche models able to host good reservoirs of preosteoblasts and other osteoprogenitors after 21 culture days. At that time, the niche type resulting in 40.8% of immature osteogenic progenies and only 59.2% of mature osteoblasts showed a calcium content comparable to the constructs obtained with the traditional culture method (i.e., 100.03 – 29.30 vs. 78.51 – 28.50 pg/cell, respectively; p = not significant), the latter colonized only by fully differentiated osteoblasts showing exhausted viability. This assembly method for tissue-engineered constructs enabled a set of important parameters, such as viability, colonization, and osteogenic yield of the MSCs to be balanced on 3D scaffolds, thus achieving biomimetic in vitro models with graded osteogenicity, which are more complex and reliable than those currently used by tissue engineers

Assessing cytotoxicity of boron nitride nanotubes: Interference with the MTT assay

Thanks to a non-covalent wrapping with glycol-chitosan, highly biocompatible and highly concentrated dispersions of boron nitride nanotubes were obtained and tested on human neuroblastoma cells. A systematic investigation of the cytotoxicity of these nanovectors with several complementary qualitative and quantitative assays allowed a strong interference with the MTT metabolic assay to be highlighted, similar to a phenomenon already observed for carbon nanotubes, that would wrongly suggest toxicity of boron nitride nanotubes. These results confirm the high complexity of these new nanomaterials, and the needing of extensive investigations on their exciting potential applications in the biomedical field

Development of tissue-engineered substitutes of the ear ossicles: PORP-shaped poly(propylene fumarate)-based scaffolds cultured with human mesenchymal stromal cells.

This is a novel study aimed at exploring possible tissue engineering (TE) options for fabricating middle ear ossicle replacements. Alternatives to prosthetic replacements currently used in ossiculoplasty are desirable, considering that current devices are known to suffer from a persistent rejection phenomenon, known as extrusion. In this study a biocompatible and biodegradable polymer, poly(propylene fumarate)/poly(propylene fumarate)-diacrylate (PPF/PPF-DA), was chosen to assess the fabrication feasibility of highly porous devices shaped as Partial Ossicular Replacement Prostheses (PORPs). PORP-like scaffolds were produced, and their poral features (porosity and pore interconnectivity) were evaluated via micro-CT. In addition, their capability to support human mesenchymal stromal cell (hMSC) colonization and osteoblastic differentiation in vitro was investigated with both quantitative and qualitative analyses. This report summarizes and discusses all the fundamental issues associated with ossicle prosthetization as well as the challenging opportunities potentially offered to middle ear reconstruction by TE; moreover it demonstrates that PPF/PPF-DA PORP-like scaffolds can be appropriately fabricated to allow both the colonization of hMSCs and their osteoblastic maturation in vitro. Specifically, the expression patterns of the main osteogenic markers (alkaline phosphatase, calcium) and of various matrix biomolecules (glycoproteins, glycosaminoglycans, collagen I) were studied. These preliminarily obtained outcomes may launch a new trend in otology dedicated to TE ossicle development to improve on the performance of current prosthetic replacements