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Osteosarcoma is a primary bone tumor characterized by a dismal prognosis, especially in the case of recurrent disease or metastases. Therefore, tools to understand in-depth osteosarcoma progression and ultimately develop new therapeutics are urgently required. 3D in vitro models can provide an optimal option, as they are highly reproducible, yet sufficiently complex, thus reliable alternatives to 2D in vitro and in vivo models. Here, we describe 3D in vitro osteosarcoma models prepared by printing polyurethane (PU) by fused deposition modeling, further enriched with human mesenchymal stromal cell (hMSC)-secreted biomolecules. We printed scaffolds with different morphologies by changing their design (i.e., the distance between printed filaments and printed patterns) to obtain different pore geometry, size, and distribution. The printed PU scaffolds were stable during in vitro cultures, showed adequate porosity (55–67%) and tunable mechanical properties (Young’s modulus ranging in 0.5–4.0 MPa), and resulted in cytocompatible. We developed the in vitro model by seeding SAOS-2 cells on the optimal PU scaffold (i.e., 0.7 mm inter-filament distance, 60° pattern), by testing different pre-conditioning factors: none, undifferentiated hMSC-secreted, and osteo-differentiated hMSC-secreted extracellular matrix (ECM), which were obtained by cell lysis before SAOS-2 seeding. Scaffolds pre-cultured with osteo-differentiated hMSCs, subsequently lysed, and seeded with SAOS-2 cells showed optimal colonization, thus disclosing a suitable biomimetic microenvironment for osteosarcoma cells, which can be useful both in tumor biology study and, possibly, treatment.

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 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.

The preparation and selection of biobased materials compatible with skin is essential for producing innovative and highly eco-friendly beauty masks. The use of a commercial elastomeric poly(hydroxyalkanoate) and starch was fundamental to select materials for bioplastic films with the necessary resistance in wet conditions, skin compatibility and capacity for a fast release of polysaccharides and similar active and functional molecules. Micrometric calcium carbonate was also used to control the stickiness of film during moulding. Starch release in water was investigated by gravimetric and infrared analyses. The compatibility with skin was investigated via two different in vitro tests based on human keratinocytes and human mesenchymal stromal cells. The materials were highly cytocompatible with skin, enabled immune modulation by keratinocytes and starch release in water up to 49% by weight in 30 min. These outcomes are a good starting point for boosting the production of biobased and biodegradable beauty masks, thus decreasing the impact onto environment of cosmetic products that are currently still mainly produced using petrol-based substrates.

Biodegradable polymers are promising materials for films and sheets used in many widely diffused applications like packaging, personal care products and sanitary products, where the synergy of high biocompatibility and reduced environmental impact can be particularly significant. Plasticized poly(lactic acid) (PLA)/poly(butylene succinate) (PBS) blend-based films, showing high cytocompatibility and improved flexibility than pure PLA, were prepared by laboratory extrusion and their processability was controlled by the use of a few percent of a commercial melt strength enhancer, based on acrylic copolymers and micro-calcium carbonate. The melt strength enhancer was also found effective in reducing the crystallinity of the films. The process was upscaled by producing flat die extruded films in which elongation at break and tear resistance were improved than pure PLA. The in vitro biocompatibility, investigated through the contact of flat die extruded films with cells, namely, keratinocytes and mesenchymal stromal cells, resulted improved with respect to low density polyethylene (LDPE). Moreover, the PLA-based materials were able to affect immunomodulatory behavior of cells and showed a slight indirect anti-microbial effect. These properties could be exploited in several applications, where the contact with skin and body is relevant.

The external auditory canal (EAC) is an osseocartilaginous structure extending from the auricle to the eardrum, which can be affected by congenital, inflammatory, and neoplastic diseases, thus reconstructive materials are needed. Current biomaterial-based approaches for the surgical reconstruction of EAC posterior wall still suffer from resorption (biological) and extrusion (synthetic). In this study, 3D fiber deposited scaffolds based on poly(ethylene oxide terephthalate)/poly(butylene terephthalate) were designed and fabricated to replace the EAC wall. Fiber diameter and scaffold porosity were optimized, leading to 200 ± 33 µm and 55% ± 5%, respectively. The mechanical properties were evaluated, resulting in a Young's modulus of 25.1 ± 7.0 MPa. Finally, the EAC scaffolds were tested in vitro with osteo-differentiated human mesenchymal stromal cells (hMSCs) with different seeding methods to produce homogeneously colonized replacements of interest for otologic surgery. This study demonstrated the fabrication feasibility of EAC wall scaffolds aimed to match several important requirements for biomaterial application to the ear under the Tissue Engineering paradigm, including shape, porosity, surface area, mechanical properties and favorable in vitro interaction with osteoinduced hMSCs. This study demonstrated the fabrication feasibility of outer ear canal wall scaffolds via additive manufacturing. Aimed to match several important requirements for biomaterial application to ear replacements under the Tissue Engineering paradigm, including shape, porosity and pore size, surface area, mechanical properties and favorable in vitro interaction with osteo-differentiated mesenchymal stromal cells.

Background Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System. Methods Bone marrow aspirates were obtained from healthy adult individuals undergoing routine total hip replacement surgery and used to generate primary cultures in the bioreactor. HPL and ABS were tested as supplements to culture medium. Morphological observations, cytofluorimetric analysis, lactate and glucose level assessment were performed. Results ABS gave rise to both heterogeneous MSC and MPC population. About 95% of cells cultured in HPL showed a fibroblast-like morphology and typical mesenchymal surface markers, but MPCs were scarcely represented. Discussion The use of ABS appeared to sustain a large scale MSC production, as well as the recovery of a subset of MPCs, and resulted a suitable alternative to HPL in the cell generation based on the Quantum System.

Sensorineural hearing loss, primed by dysfunction or death of hair cells in the cochlea, is the main cause of severe or profound deafness. Piezoelectric materials work similarly to hair cells, namely, as mechano-electrical transducers. Polyvinylidene fluoride (PVDF) films have demonstrated potential to replace the hair cell function, but the obtained piezoresponse was insufficient to stimulate effectively the auditory neurons. In this study, we reported on piezoelectric nanocomposites based on ultrafine PVDF fibers and barium titanate nanoparticles (BTNPs), as a strategy to improve the PVDF performance for this application. BTNP/PVDF fiber meshes were produced via rotating-disk electrospinning, up to 20/80 weight composition. The BTNP/PVDF fibers showed diameters ranging in 0.160–1.325 μm. Increasing collector velocity to 3000 rpm improved fiber alignment. The piezoelectric β phase of PVDF was well expressed following fabrication and the piezoelectric coefficients increased according to the BTNP weight ratio. The BTNP/PVDF fibers were not cytotoxic towards cochlear epithelial cells. Neural-like cells adhered to the composite fibers and, upon mechanical stimulation, showed enhanced viability. Using BTNP filler for PVDF matrices, in the form of aligned ultrafine fibers, increased the piezoresponse of PVDF transducers and favored neural cell contact. Piezoelectric nanostructured composites might find application in next generation cochlear implants. Keywords: Electrospinning, Barium titanate, Polyvinylidene fluoride, Aligned fibers, Neural cells, Transducer

This study aims at identifying compositional and architectural (pore size and distribution) parameters of biocompatible scaffolds, which can be best suitable for both osteoblasts and endothelial cells to produce optimized 3D cocultured constructs. Spongy scaffolds are prepared using poly(vinyl alcohol) (PVA) and gelatin (G) at different weight compositions (PVA/G range: 100/0–50/50, w/w) via emulsion and freeze-drying. The higher the gelatin content, the larger is the volume occupied by higher size pores. Human umbilical vein endothelial cells and human mesenchymal stromal cells are independently differentiated on the scaffolds to select the best candidate for the coculture. The results of metabolic activity and histology on single platforms show both cell- and material-type dependent outcomes. PVA/G 80/20 scaffolds are finally selected and allow the formation of mineralized matrix containing organized endothelial-like structures. This study highlights the need for systematic investigations on multifactorial parameters of scaffolds to improve vascularized bone substitutes.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions — namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Tissue engineering scaffolds have shown an intrinsic ability to provide cellular stimulation, thus behaving as physically active microenvironments. This study reports on the interaction between human mesenchymal stromal cells (hMSCs) and dry-wet spun polymer microfiber meshes. The following scaffolding parameters were tested: i) polymer type: poly-L-lactide (PLLA) vs poly-ε-caprolactone (PCL); ii) non-solvent type: ethanol (Et-OH) vs isopropanol/ gelatin; iii) scaffold layout: patterned vs random microfiber fabrics. After two culture weeks, the effects on metabolic activity, scaffold colonization and function of undifferentiated hMSCs were assayed. In our study, the polymer type affected the hMSC metabolic activity timeline, and the metabolic picks occurred earlier in PLLA (day 6) than in PCL (day 9) scaffolds. Instead, PLLA vs PCL had no endpoint effect on alkaline phosphatase (ALP) activity expression. On average, the hMSCs grown on all the random microfiber fabrics showed an ALP activity statistically superior to that detected on patterned microfiber fabrics, with the highest in Et-OH random subtypes. Such findings are suggestive of enhanced osteogenic potential. The understanding of scaffold-driven stimulation could enable environmental hMSC commitment, paving the way for new regenerative strategies.

Introduction Due to their properties and characteristics, human mesenchymal stem cells (MSCs) appear to have great therapeutic potential, because they can contribute to the tissue regeneration. Many different populations of MSCs that differ for the embryonic origin and the anatomical localization have been described and to understand whether they have equivalent biological properties is a critical issue for their therapeutic application. Spine fusion is frequently used to treat traumatic, degenerative, and oncological spine diseases. Autologous bone graft has been considered the gold standard for spine fusion procedures because of its osteogenic, osteoinductive, and osteoconductive ability. However, its use is associated with significant disadvantages, including donor site pain, increased operative time, insufficient availability, and nonunion postlumbar fusion. Various bone substitutes have been developed to promote spinal fusion. Also, MSCs have been successfully tested for spinal fusion in several small and large animal models. Material and Methods To define the properties of MSCs for the use in clinical applications, we proposed to analyze the cellular and molecular characteristics of human adult MSCs derived from different body locations, such as bone marrow from the iliac crest (Ic-MSCs), sternum (St-MSCs), and vertebrae (vMSCs), as well as colon (Co-MSCs) and dental pulp (DPSCs), to identify the cell population showing better biological properties for spine fusion clinical application. Results MSCs from different sources presented very different growth kinetics. While iliac crest and sternum-derived MSCs could be maintained in culture for about 3 months, undergoing a limited expansion, mesenchymal cultures derived from dental pulp and colon grew much more rapidly, but could not be maintained longer. Strikingly, vMSCs continued to propagate at high rate even after 3.5 months ex vivo. MSCs from different sources were also induced to osteogenic, adipogenic, and chondrogenic differentiation following exposure to specific inducing agents. We observed that vMSCs generate mature cells of all mesenchymal lineages with greater efficiency, resulting into the best population both in terms of expansion and differentiation. Conclusion This finding could be very interesting and open new perspectives for the improvement of spine fusion, which is a mandatory step for the success of spinal surgical procedures. In the course of a surgical procedure for spinal fusion, vertebral bone marrow can be harvested simultaneously with the preparation of the site for pedicle screw insertion. Then multipotent MSCs can be isolated from vertebral bone marrow during the progress of the surgical procedure and reintroduced in the fusion site (using homologous or autologous bone tissue or biomaterials as scaffold), without any additional surgical time or any other donor site involvement.