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.

Human mesenchymal stem cells (MSC), during in vitro expansion, undergo a progressive loss of proliferative potential that leads to the senescent state, associated with a reduction of their “medicinal” properties. This may hampers their efficacy in the treatment of injured tissues. Quality controls on MSC-based cell therapy products should include an assessment of the senescent state. However, a reliable and specific marker is still missing. From studies on lamin-associated disorders, has emerged the correlation between defective lamin A maturation and cellular senescence. Primary cultured hMSC lines (n = 3), were analyzed by immunostaining at different life-span stages for the accumulation of prelamin A, along with other markers of cellular senescence. During culture, cells at the last stage of their life span displayed evident signs of senescence consistent with the positivity of SA-β-gal staining. We also observed a significant increase of prelamin A positive cells. Furthermore, we verified that the cells marked by prelamin A were also positive for p21Waf1 while negative for Ki67. Overall data support that the detection of prelamin A identifies senescent MSC, providing an easy and reliable tool to be use alone or in combination with known senescence markers to screen MSC before their use in clinical applications.

In orthopedics, the use of anti-infective biomaterials is considered the most promising strategy to contrast the bacterial contamination of implant surfaces and reduce the infection rate. KSL, KSL-W, and Dadapin-1 are three antimicrobial peptides (AMPs) that possess significant antibacterial properties, making them promising candidates for producing anti-infective biomaterials not based on antibiotics. To fully assess their true potential, this study explores in detail their cytocompatibility on human osteoblast-like MG63 cells, murine fibroblastoid L929 cells, and hMSCs. To this end, the cytotoxicity of the AMPs in terms of IC₅₀ was tested over a range of concentrations of 450–0.22 µg/mL using the ATP bioluminescence assay. The tests were performed both in the presence and absence of bovine serum to assess the effects of serum components on peptide stability. IC₅₀ values obtained under the most stringent conditions were used to extrapolate the selectivity index (S.I.) toward salient bacterial species. In medium containing serum, all AMPs exhibited minimal to no cytotoxicity, with IC₅₀ values exceeding 100 µg/mL. Dadapin-1 was the peptide that exhibited the lowest cytotoxicity, KSL-W exhibited the highest stability, and KSL exhibited the highest selectivity. Overall, these findings highlight the potential of these AMPs for the future production of anti-infective materials.

In osteosarcoma (OS) and most Ewing sarcoma (EWS) patients, the primary tumor originates in the bone. Although tumor resection surgery is commonly used to treat these diseases, it frequently leaves massive bone defects that are particularly difficult to be treated. Due to the therapeutic potential of mesenchymal stem cells (MSCs), OS and EWS patients could benefit from an autologous MSCs-based bone reconstruction. However, safety concerns regarding the in vitro expansion of bone marrow-derived MSCs have been raised. To investigate the possible oncogenic potential of MSCs from OS or EWS patients (MSC-SAR) after expansion, this study focused on a biosafety assessment of MSC-SAR obtained after short- and long-term cultivation compared with MSCs from healthy donors (MSC-CTRL).

Myxofibrosarcoma (MFS) is a malignant soft tissue sarcoma (STS) that originates in the body’s connective tissues. It is characterized by the presence of myxoid (gel-like) and fibrous components and typically affects patients after the fifth decade of life. Considering the ongoing trend of increasing lifespans across many nations, MFS is likely to become the most common musculoskeletal sarcoma in the future. Although MFS patients have a lower risk of developing distant metastases compared with other STS cases, MFS is characterized by a high frequency of local recurrence. Notably, in 40–60% of the patients where the tumor recurs, it does so multiple times. Consequently, patients may undergo multiple local surgeries, removing the risk of potential amputation. Furthermore, because the tumor relapses generally have a higher grade, they exhibit a decreased response to radio and chemotherapy and an increased tendency to form metastases. Thus, a better understanding of MFS is required, and improved therapeutic options must be developed. Historically, preclinical models for other types of tumors have been instrumental in obtaining a better understanding of tumor development and in testing new therapeutic approaches. However, few MFS models are currently available. In this review, we will describe the MFS models available and will provide insights into the advantages and constraints of each model.