Background: The clinical application of freshly isolated connective-tissue progenitors, as well as the potential preparation of culture-expanded mesenchymal stem cell populations for therapeutic applications, will benefit from clinical methods that maximize the yield of the starting population. We compared the number of cells, concentration, and prevalence of colony-founding connective-tissue progenitors from the anterior and posterior iliac crest. In addition, we compared the expansion kinetics and multilineage differentiation potential of their culture-expanded progeny when processed to form mesenchymal stem cells. Methods: Marrow aspirate was collected from both the anterior and posterior iliac crest of twenty-two patients. The concentration and prevalence of colony-founding connective-tissue progenitors were estimated with use of a colony formation assay. The expansion kinetics and multilineage differentiation potential of the culture-expanded mesenchymal stem cell populations derived from these starting samples were compared. Results: The yield of colony-founding connective-tissue progenitors was 1.6 times greater in the posterior compared with the anterior iliac crest. No differences were found with respect to the viability, phenotype, expansion kinetics, or multilineage differentiation potential of mesenchymal stem cell populations derived from these two sites. Conclusions: The concentration and yield of colony-founding connective-tissue progenitors were greater when aspirate was obtained from the posterior compared with the anterior iliac crest, whereas the biological potential of the cells derived from these sites appeared comparable. Clinical Relevance: The harvesting of bone marrow from the posterior iliac crest appears to be preferred, as it provided a modestly higher concentration of colony-founding connective-tissue progenitors than comparable aspirate from the anterior iliac crest.

Background: Osteosarcoma (OS) is an aggressive malignant neoplasm that still suffers from poor prognosis in the case of distal metastases or occurrence of multi-drug resistance. It is therefore crucial to find novel therapeutic options able to go beyond these limitations and improve patients’ survival. The objective of this study is to exploit the intrinsic properties of mesenchymal stromal cells (MSCs) to migrate and infiltrate the tumor stroma to specifically deliver therapeutic agents directly to cancer cells. In particular, we aimed to test the efficacy of the photoactivation of MSCs loaded with nanoparticles in vitro and in a murine in vivo ectopic osteosarcoma model. Methods: AlPcS₄@FNPs were produced by adding tetra-sulfonated aluminum phthalocyanine (AlPcS₄) to an aqueous solution of positively charged poly-methyl methacrylate core-shell fluorescent nanoparticles (FNPs). The photodynamic therapy (PDT) effect is achieved by activation of the photosensitizer AlPcS₄ in the near-infrared light with an LED source. Human MSCs were isolated from the bone marrow of five donors to account for inter-patients variability and used in this study after being evaluated for their clonogenicity, multipotency and immunophenotypic profile. MSC lines were then tested for the ability to internalize and retain the nanoparticles, along with their migratory properties in vitro. Photoactivation effect was evaluated both in a monolayer (2D) co-culture of AlPcS₄@FNPs loaded MSCs with human OS cells (SaOS-2) and in tridimensional (3D) multicellular spheroids (AlPcS₄@FNPs loaded MSCs with human OS cells, MG-63). Cell death was assessed by AnnexinV/PI and Live&Dead CalceinAM/EthD staining in 2D, while in the 3D co-culture, the cell killing effect was measured through ATP content, CalceinAM/EthD staining and TEM imaging. We also evaluated the effectiveness of AlPcS₄@FNPs loaded MSCs as delivery systems and the ability of the photodynamic treatment to kill cancer cells in a subcutaneous mouse model of OS by bioluminescence imaging (BLI) and histology. Results: MSCs internalized AlPcS₄@FNPs without losing or altering their motility and viability in vitro. Photoactivation of AlPcS₄@FNPs loaded MSCs induced high level of OS cells death in the 2D co-culture. Similarly, in the 3D co-culture (MSCs:OS ratios 1:1 or 1:3), a substantial decrease of both MSCs and OS cells viability was observed. Notably, when increasing the MSCs:OS ratio to 1:7, photoactivation still caused more than 40% cells death. When tested in an in vivo ectopic OS model, AlPcS4@FNPs loaded MSCs were able to decrease OS growth by 68% after two cycles of photoactivation. Conclusions: Our findings demonstrate that MSCs can deliver functional photosensitizer-decorated nanoparticles in vitro and in vivo and inhibit OS tumor growth. MSCs may be an effective platform for the targeted delivery of therapeutic nanodrugs in a clinical scenario, alone or in combination with other osteosarcoma treatment modalities.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma.

The aim of this study was to exploit silk fibroin’s properties to develop innovative composite microcarriers for mesenchymal stem cell (MSCs) adhesion and proliferation. Alginate microcarriers were prepared, added to silk fibroin solution, and then treated with ethanol to induce silk conformational transition. Microcarriers were characterized for size distribution, coating stability and homogeneity. Finally, in vitro cytocompatibility and suitability as delivery systems for MSCs were investigated. Results indicated that our manufacturing process is consistent and reproducible: silk/alginate microcarriers were stable, with spherical geometry, about 400 μm in average diameter, and fibroin homogeneously coated the surface. MSCs were able to adhere rapidly onto the microcarrier surface and to cover the surface of the microcarrier within three days of culture; moreover, on this innovative 3D culture system, stem cells preserved their metabolic activity and their multi-lineage differentiation potential. In conclusion, silk/alginate microcarriers represent a suitable support for MSCs culture and expansion. Since it is able to preserve MSCs multipotency, the developed 3D system can be intended for cell delivery, for advanced therapy and regenerative medicine applications.

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α₅ integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α₅β₁ integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α₅β₁-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion.