A reduction in tumor volume exceeding 65% predicts a good histological response to neoadjuvant chemotherapy in patients with Ewing sarcoma.

OBJECTIVE: No consensus exists for tumor volume response criteria in patients with Ewing sarcoma. This study aimed to identify an optimal cutoff for predicting a good histological response by analyzing tumor volume changes and tumor necrosis after neoadjuvant chemotherapy. MATERIALS AND METHODS: We performed a retrospective analysis of 184 Ewing sarcoma patients, analyzing tumor volume changes before and after neoadjuvant chemotherapy. Patients were divided into two groups based on histological response: good (tumor necrosis ≥ 95%) and poor (tumor necrosis < 95%) responders. The receiver operating characteristic (ROC) area under the curve (AUC) method was used to determine the optimal thresholds for predicting the histological response. Additionally, the prognostic value of this cutoff for relapse-free survival was assessed. RESULTS: Out of 184 patients, 83 (45%) had tumor necrosis ≥ 95%, while 101 (55%) had tumor necrosis < 95%. ROC analysis identified the optimal cutoff for a good histological response as over 65% tumor volume reduction (AUC = 0.69; p < 0.001). Patients with volume reduction of ≥ 65% had a higher likelihood of a good histological response than those with lesser reductions (p = 0.004; odds ratio = 2.61). Multivariable analysis indicated a correlation between poor histological response and reduced relapse-free survival (hazard ratio = 2.17; p = 0.01), while tumor volume reduction itself did not impact survival. CONCLUSION: We reported that a tumor volume reduction of ≥ 65% was able to predict a good histological response in Ewing sarcoma patients. We recommend preoperative tumor volume assessment to identify patients at greater risk for poor histological response who could benefit from more intensive chemotherapy protocols or additional radiotherapy.

Histological and imaging features of myoepithelial carcinoma of the bone and soft tissue.

OBJECTIVE: To depict histological and imaging features of myoepithelial carcinoma of the bone and soft tissue. MATERIALS AND METHODS: We retrospectively examined histological features in 22 patients with myoepithelial carcinoma of the bone (4 patients) and soft tissue (18 patients) at a single institution. Imaging analysis of 15 patients (bone, 3 patients; soft tissue, 12 patients;) with preoperative images involved classifying lytic bone lesions via the modified Lodwick-Madewell classification; the growth patterns of soft tissue lesions were classified as well-defined, focally invasive, or diffusely invasive. RESULTS: Local recurrence occurred in eight out of 22 patients (36.3%). Four of 22 patients (18.2%) had metastasis at presentation, whereas 11 of 22 patients (50.0%) had distant metastasis during follow-up. Severe cytological pleomorphism was observed in 14 of 22 patients (63.6%), and 10 of 22 tumors (45.5%) showed ≥ 10 mitoses/10 high-power fields. Vascular invasion was observed in 10 of 22 patients (45.5%). Extracapsular/extraskeletal infiltration into the surrounding tissues was assessed in 20 patients, with 14 of them (70%) showing infiltration beyond the tumor border. Regarding imaging of bone lesions, two patients had Ludwick type IIIB, whereas one patient had type II. The growth pattern of soft tissue lesions was well-defined in two patients (16.7%), focally invasive in seven patients (58.3%), and diffusely invasive in three (25.0%) out of 12 patients. CONCLUSION: Myoepithelial carcinoma of the bone and soft tissue presents high risk of local recurrence and distant metastasis. Histological and imaging features might be important to understand the aggressive behavior of the tumor.

A novel patient-derived immortalised cell line of myxofibrosarcoma: a tool for preclinical drugs testing and the generation of near-patient models.

BACKGROUND: Myxofibrosarcoma is a rare malignant soft tissue sarcoma characterised by multiple local recurrence and can become of higher grade with each recurrence. Consequently, myxofibrosarcoma represents a burden for patients, a challenge for clinicians, and an interesting disease to study tumour progression. Currently, few myxofibrosarcoma preclinical models are available. METHODS: In this paper, we present a spontaneously immortalised myxofibrosarcoma patient-derived cell line (MF-R 3). We performed phenotypic characterization through multiple biological assays and analyses: proliferation, clonogenic potential, anchorage-independent growth and colony formation, migration, invasion, AgNOR staining, and ultrastructural evaluation. RESULTS: MF-R 3 cells match morphologic and phenotypic characteristics of the original tumour as 2D cultures, 3D aggregates, and on the chorioallantoic membrane of chick embryos. Overall results show a clear neoplastic potential of this cell line. Finally, we tested MF-R 3 sensitivity to anthracyclines in 2D and 3D conditions finding a good response to these drugs. CONCLUSIONS: In conclusion, we established a novel patient-derived myxofibrosarcoma cell line that, together with the few others available, could serve as an important model for studying the molecular pathogenesis of myxofibrosarcoma and for testing new drugs and therapeutic strategies in diverse experimental settings.

Lengthening Patients Previously Treated for Massive Lower Limb Reconstruction for Bone Tumors with the PRECICE 2 Nail.

The objective of this study was to determine the efficacy of the PRECICE 2® nail in the treatment of lower limb length discrepancy in patients with a history of bone tumors. This study reports on outcomes, complications, and the safety of the PRECICE 2 limb lengthening nail in a cohort of pediatric patients with limb length discrepancy after surgery for bone tumors. Seventeen patients were treated with intramedullary magnetic nails. The average patient age at the time of surgery was 19 (range 11-32). The PRECICE 2 nail was used on 14 femurs (6 retrograde and 8 anterograde) and 3 tibias. The average consolidation time was 141 days (range 50-360) with a mean CI of 31 ± 12 days/cm. The ASAMI bone score showed 14 (82%) excellent results, 1 (6%) good result, and 2 (12%) poor results. The ASAMI functional score showed 13 (84.6%) excellent results, 3 (11.5%) good results, and 1 (3.8%) fair result. Patients treated with chemotherapy for bone cancer did not show any increase in distraction time or consolidation time. A total of 3 (17%) problems, 1 obstacle (5.5%), and 1 complication (5.5%) were encountered in our case series. The PRECICE 2 nail allows for effective and accurate lengthening preserving the range of motion in patients treated for bone tumors.

Aneurysmal bone cyst: Is selective arterial embolization effective as curettage and bone grafting?

INTRODUCTION: Aneurysmal bone cyst (ABC) is a lytic benign bone lesion representing about 1% of all primary bone tumors. Method to treat ABC's have developed over time. The standard of care cure for ABC has been curettage with or without bone grafting of the defect but is burdened by recurrence rates of approximately 25%-31%. Based on the assumption that ABCs usually supplied by one or more pathological feeding arteries, selective arterial embolization has been described as an adjuvant preoperative procedure to reduce intra-operative hemorrhage, and as primary treatment for lesions in difficult surgical access. In the current study, we therefore asked whether (1) a single or a repeat selective arterial embolization (SAE) for treating ABCs would produce comparable healing rates compared with curettage and bone grafting; (2) evaluated the relationship of recurrence in relation to the site of the cyst, the age, and gender of the patients; and (3) the two techniques differ in term of long-term complication. MATERIAL AND METHODS: We retrospectively reviewed 265 patients who underwent curettage and bone grafting or SAE performed at our institute from 1994 to 2018. The diagnosis of ABC was always established with percutaneous CT-guided biopsy or open biopsy. Patients were followed clinically with plain radiographs or CT scan at 3, 6, 9, and 12 months then annually in the absence of symptoms. Treatment success was determined evaluating pre- and postprocedural imaging according to Chang classification. RESULTS: Two hundred and nineteen were treated with curettage and bone grafting (curettage group), and 46 with SAE Group. Of the 219 patients treated with Curettage and bone grafting (curettage group), 165 out of 219 (75.3%) experienced bone healing, while local recurrence was observed in 54 cases (24.7%) after 12 months on average (range: 3-120 months) from surgery. After the first SAE, bone ossification was seen in 27 (58.7%), without needing any further treatment. Eleven recurred patients were treated with SAE (four patients need two while seven need three SAE to heal), and eight patients with curettage and bone grafting. Thirty-eight out of 46 (82%) patients experienced bone ossification regardless the number of SAE. The overall rate of local recurrence for all patients was 26.7%. SAE group presented a lower complication rate (6%) where two patients experienced skin necrosis, and one limb-length discrepancies (2% of all cohort). DISCUSSION: The use of SAE is an attractive option to treat ABC as it combines on one hand a lower complication rate than curettage and bone grafting, on the other it can be carried out in case of nonresectable ABCs, significantly reducing the size of viable ABC lesions, fostering bone remodeling and mineralization, and most importantly, significantly improving the patient's quality of life.

Is the association of electrochemotherapy and bone fixation rational in patients with bone metastasis?

BACKGROUND: Bone metastases are frequent in patients with cancer. Electrochemotherapy (ECT) is a minimally invasive treatment based on a high-voltage electric pulse combined with an anticancer drug. Preclinical and clinical studies supported the use of ECT in patients with metastatic bone disease, demonstrating that it does not damage the mineral structure of the bone and its regenerative capacity, and that is feasible and efficient for the treatment of bone metastases. Year 2014 saw the start of a registry of patients with bone metastases treated with ECT, whose data are recorded in a shared database. QUESTIONS/PURPOSES: (1) Among patients who underwent ECT and internal fixation for bone metastasis, how many experienced a reduction of pain? (2) How many cases showed a radiological response? (3) How many patients presented local or systemic complication after ECT and fixation? PATIENTS AND METHODS: Patients were treated in Bologna at Rizzoli Orthopaedic Institute between March 2014 and February 2022 and recorded in the REINBONE registry (a shared database protected by security passwords): clinical and radiological information, ECT session, adverse events, response, quality of life indicators, and duration of follow-up were registered. We consider only cases treated with ECT and intramedullary nail during the same surgical session. Patients included in the analysis were 32: 15 males and 17 females, mean age 65 ± 13 years (median 66, range 38-88 years), mean time since diagnosis of primary tumor 6.2 ± 7.0 years (median 2.9, range 0-22 years). Nail was indicated in 13 cases for a pathological fracture in, 19 for an impending fracture. Follow-up was available for 29 patients, as 2 patients were lost to follow-up and 1 was unable to return to controls. Mean follow-up time was 7.7 ± 6.5 months (median 5, range 1-24), and 16 patients (50%) had a follow-up longer than 6 months. RESULTS: A significant decrease in pain intensity was observed at the mean Visual Numeric Scale after treatment. Bone recovery was observed in 13 patients. The other 16 patients remained without changes, and one presented disease progression. One patient presented a fracture occurrence during the ECT procedure. Among all patients, bone recovery was observed in 13 patients: complete recovery in 1 patient (3%) and partial recovery in 12 patients (41%). The other 16 patients remained without changes, and one presented disease progression. One patient presented a fracture occurrence during the ECT procedure. However, healing was possible with normal fracture callus quality and healing time. No other local or systemic complications were observed. CONCLUSION: We found that pain levels decreased after treatment in 23 of the 29 cases for a pain relief rate of 79% at final follow-up. Pain is one of the most important indicators of quality of life in patients that undergo palliative treatments. Even if conventional external body radiotherapy is considered a noninvasive treatment, it presents a dose-dependent toxicity. ECT provides a chemical necrosis preserving osteogenic activity and structural integrity of bone trabeculae; this is a crucial difference with other local treatments and allows bone healing in case of pathological fracture. The risk of local progression in our patient population was small, and 44% experienced bone recovery while 53% of the cases remained unchanged. We observe intraoperative fracture in one case. This technique, in selected patients, improves outcome in bone metastatic patients combing both the efficacy of the ECT in the local control of the disease and the mechanical stability with the bone fixation to synergize their benefits. Moreover, the risk of complication is very low. Although encouraging data, comparative studies are required to quantify the real efficacy of the technique. Level of Evidence Level I, therapeutic study.

Two Beats One: Osteosarcoma Therapy with Light-Activated and Chemo-Releasing Keratin Nanoformulation in a Preclinical Mouse Model.

Osteosarcoma treatment is moving towards more effective combination therapies. Nevertheless, these approaches present distinctive challenges that can complicate the clinical translation, such as increased toxicity and multi-drug resistance. Drug co-encapsulation within a nanoparticle formulation can overcome these challenges and improve the therapeutic index. We previously synthetized keratin nanoparticles functionalized with Chlorin-e6 (Ce6) and paclitaxel (PTX) to combine photo (PDT) and chemotherapy (PTX) regimens, and the inhibition of osteosarcoma cells growth in vitro was demonstrated. In the current study, we generated an orthotopic osteosarcoma murine model for the preclinical evaluation of our combination therapy. To achieve maximum reproducibility, we systematically established key parameters, such as the number of cells to generate the tumor, the nanoparticles dose, the design of the light-delivery device, the treatment schedule, and the irradiation settings. A 60% engrafting rate was obtained using 10 million OS cells inoculated intratibial, with the tumor model recapitulating the histological hallmarks of the human counterpart. By scheduling the treatment as two cycles of injections, a 32% tumor reduction was obtained with PTX mono-therapy and a 78% reduction with the combined PTX-PDT therapy. Our findings provide the in vivo proof of concept for the subsequent clinical development of a combination therapy to fight osteosarcoma.

Mesenchymal stromal cells mediated delivery of photoactive nanoparticles inhibits osteosarcoma growth in vitro and in a murine in vivo ectopic model.

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.

CXCR4 in human osteosarcoma malignant progression. The response of osteosarcoma cell lines to the fully human CXCR4 antibody MDX1338.

Osteosarcoma (OS) is the most frequent primary malignant tumour of bone and metastases occur in 30% of cases, the 5-year survival rate is 25-30%. Although pre- and post-operative chemotherapy has improved prognosis in osteosarcoma (OS), high toxicity and natural and acquired drug-resistance are the first cause of treatment failure. The identification of new predictive and therapeutic biomarkers may increase drug sensitivity and better control localized and metastatic disease. By the evidence that CXCR4 receptor by binding its ligand CXCL12 activates downstream critical endpoints for tumour malignancy, we first studied human OS progression correlating CXCR4 expression in OS biopsy with patient clinical data. By Real-time PCR and immunoistochemistry we found that high levels of CXCR4 gene and protein expression significantly correlated with OS progression, emphasizing the role of CXCR4/CXCL12 axis in tumour prognosis. This was supported by univariate analyses that showed a higher probability of local and/or systemic relapse in OS patients with a high CXCR4 gene expression and a significant increase of metastasis risk associated with an increasing score of CXCR4 protein staining intensity. Secondarily, to study the role of CXCR4 as a target for new therapeutic strategies, we evaluated the response of OS cells to the fully human CXCR4 antibody, MDX1338. In the study we also included AMD3100, the most studied CXCR4 antagonist. In CXCR4-positive OS cells cultured in CXCL12-rich BM-MCS-CM (bone marrow-derived mesenchymal stem conditioned medium), a decrease of cell proliferation up to 30%-40% of control was seen after drug exposure. However, an increase of apoptosis was seen in p53-positive U2OS and 143B after CXCR4 inhibitor incubation, while no changes were seen in treated SAOS-2 cells which also present a different labeling profile. The role of p53 in apoptotic response to CXCR4 inhibitors was confirmed by p53 silencing in U2OS cell line. Our data suggest that the response to anti-CXCR4 agents could be influenced by the genetic background and labeling profile which induces a different cross-talk between tumour cells and environment. The delay in cell cycle progression associated with increased apoptosis could sensitize p53-positive cells to conventional therapy and in vivo preclinical experiments are on going with the aim to suggest new combined target therapies in human OS.

Nonunion fracture healing: Evaluation of effectiveness of demineralized bone matrix and mesenchymal stem cells in a novel sheep bone nonunion model.

Nonunion treatment has a high rate of success, although recalcitrant nonunion may determine the need for amputation. Therefore, new treatment options are continuously investigated in order to further reduce the risk of nonunion recurrence. This study aimed to (a) develop a new large animal model for bone atrophic nonunion and (b) compare the efficacy of demineralized bone matrix (DBM) and DBM in combination with mesenchymal stem cells (MSC) in the new nonunion model. The new model consists of a noncritical, full-thickness segmental defect created in the sheep tibia, stabilized by an intramedullary nail, and involves the creation of a locally impaired blood supply achieved through periosteum excision and electrocauterization of the stump ends. Six weeks after defect creation, lack of hard tissue callus and established nonunion was observed in all operated tibiae both by radiographic and clinical evaluation. Nonunion was treated with allogeneic DBM or autologous MSC cultivated on DBM particles (DBM + MSC) for 1 day before implantation. Twelve weeks after treatment, radiographic, microtomographic, histologic, and histomorphometric analysis showed the formation of bone callus in DBM group, whereas the fracture healing appeared at an early stage in DBM + MSC group. Torsional strength and stiffness of the DBM group appeared higher than those of DBM + MSC group, although the differences were not statistically significant. In conclusion, a new sheep bone nonunion model resembling the complexity of the clinical condition was developed. DBM is an effective option for nonunion treatment, whereas MSC do not improve the healing process when cultivated on DBM particles before implantation.

Selective sensitiveness of mesenchymal stem cells to shock waves leads to anticancer effect in human cancer cell co-cultures.

AIM: Mesenchymal stem cells (MSC) possess the distinctive feature of homing in on and engrafting into the tumor stroma making their therapeutic applications in cancer treatment very promising. Research into new effectors and external stimuli, which can selectively trigger the release of cytotoxic species from MSC toward the cancer cells, significantly raises their potential. MAIN METHODS: Shock waves (SW) have recently gained recognition for their ability to induce specific biological effects, such as the local generation of cytotoxic reactive oxygen species (ROS) in a non-invasive and tunable manner. We thus investigate whether MSC are able to generate ROS and, in turn, affect cancer cell growth when in co-culture with human glioblastoma (U87) or osteosarcoma (U2OS) cells and exposed to SW. KEY FINDINGS: MSC were found to be the cell line that was most sensitive to SW treatment as shown by SW-induced ROS production and cytotoxicity. Notably, U87 and U2OS cancer cell growth was unaffected by SW exposure. However, significant decreases in cancer cell growth, 1.8 fold for U87 and 2.3 fold for U2OS, were observed 24h after the SW treatment of MSC co-cultures with cancer cells. The ROS production induced in MSC by SW exposure was then responsible for lipid peroxidation and cell death in U87 and U2OS cells co-cultured with MSC. SIGNIFICANCE: This experiment highlights the unique ability of MSC to generate ROS upon SW treatment and induce the cell death of co-cultured cancer cells. SW might therefore be proposed as an innovative tool for MSC-mediated cancer treatment.

Development of near-infrared photoactivable phthalocyanine-loaded nanoparticles to kill tumor cells: An improved tool for photodynamic therapy of solid cancers.

Conventional photodynamic therapy has shown to be beneficial in the treatment of a variety of tumors. However, one of its major limitations is the inadequate penetration depth of visible light. In order to overcome this constraint, we developed 80nm poly-methylmethacrylate core-shell fluorescent nanoparticles (FNP) loaded with the photosensitizer tetrasulfonated aluminum phthalocyanine (Ptl). To demonstrate the efficacy of our Ptl@FNP we performed in vitro and in vivo studies using a human prostate tumor model. Our data reveal that Ptl@FNP are internalized by tumor cells, favour Ptl intracellular accumulation, and efficiently trigger cell death through the generation of ROS upon irradiation with 680nm light. When directly injected into tumors intramuscularly induced in SCID mice, Ptl@FNP upon irradiation significantly reduce tumor growth with higher efficiency than the bare Ptl. Collectively, these results demonstrate that the newly developed nanoparticles may be utilized as a delivery system for antitumor phototherapy in solid cancers.

Secreted adiponectin as a marker to evaluate in vitro the adipogenic differentiation of human mesenchymal stromal cells.

BACKGROUND AIMS: Multipotency is one of the hallmarks of mesenchymal stromal cells (MSCs). Given the widespread adoption of MSC-based clinical applications, the need for rapid and reliable methods to estimate MSC multipotency is demanding. Adipogenic potential is commonly evaluated by staining cell lipid droplets with oil red O. This cytochemical assay is performed at the terminal stage of adipogenic induction (21-28 days) and necessitates the destruction of the specimen. In this study, we investigated whether it is possible to assess MSC adipogenic differentiation in a more efficient, timely and non-destructive manner, while monitoring in vitro secretion of adiponectin, a hormone specifically secreted by adipose tissue. METHODS: A commercially available enzyme-linked immunosorbent assay kit was used to quantify adiponectin secreted in the culture medium of adipo-induced human bone marrow-derived MSCs. Oil red O staining was used as a reference method. RESULTS: Adiponectin is detectable after 10 days of induction at a median concentration of 5.13 ng/mL. The secretion of adiponectin steadily increases as adipogenesis proceeds. Adiponectin is undetectable when adipogenic induction is pharmacologically blocked, inefficient or when human MSCs are induced to differentiate toward the osteogenic lineage, proving the specificity of the assay. Furthermore, the results of adiponectin secretion strongly correlate with oil red O quantification at the end of induction treatment. CONCLUSIONS: Our results demonstrate that quantification of secreted adiponectin can be used as a reliable and robust method to evaluate adipogenic potential without destroying samples. This method provides a useful tool for quality control in the laboratory and in clinical applications of human MSCs.

CD99 drives terminal differentiation of osteosarcoma cells by acting as a spatial regulator of ERK 1/2.

Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99-transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane-bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21(WAF1/CIP1) , leading to G0 /G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate.

A rapid method for obtaining mesenchymal stem cells and platelets from bone marrow aspirate.

Mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) are currently used alone or in combination for therapeutic applications especially for bone repair. We tested whether MSCs can be isolated from bone marrow (BM) aspirate using a commercially available kit commonly used to obtain PRP from peripheral blood (PB). Results revealed that mononuclear cells and platelets from both PB and BM could be efficiently isolated by obtaining a mononuclear and platelet rich fraction (PB-MPRF and BM-MPRF, respectively). Starting with comparable volumes, the number of platelets increased 1.5-fold in BM-MPRF compared to PB-MPRF. The number of clonogenic cells in BM-MPRF samples was significantly higher than whole BM samples as revealed by CFU-F assay (54.92 ± 8.55 CFU-F/1.5 x 10(5) nucleated cells and 32.50 ± 12.43 CFU-F/1.5 x 10(5) nucleated cells, respectively). Cells isolated from BM-MPRF after in vitro expansion fulfilled the definition of MSCs by phenotypic criteria, and differentiated along osteogenic, adipogenic and chondrogenic lineages following induction. Results showed that the kit isolated MSCs and platelets from BM aspirate. Isolated MSCs were further expanded in a laboratory and BM-MPRF was used clinically following BM withdrawal for rapid intra-operative cell therapy for the treatment of bone defects.

Physiological formation of fluorescent and conductive protein microfibers in live fibroblasts upon spontaneous uptake of biocompatible fluorophores.

We have recently reported initial results concerning an original approach to introduce additional properties into fibrillar proteins produced by live fibroblasts and extruded into the ECM. The key to such an approach was biocompatible, fluorescent and semiconducting synthetic molecules which penetrated spontaneously the cells and were progressively encompassed via non-bonding interactions during the self-assembly process of the proteins, without altering cell viability and reproducibility. In this paper we demonstrate that the intracellular secretion of fluorescent microfibers can be generalized to living primary and immortalized human/mouse fibroblasts. By means of real-time single-cell confocal microscopy we show that the fluorescent microfibers, most of which display helical morphology, are generated by intracellular coding of the synthetic molecules. We also describe co-localization experiments on the fluorescent microfibers isolated from the cell milieu demonstrating that they are mainly made of type-I collagen. Finally, we report experimental data indicating that the embedded synthetic molecules cause the proteins not only to be fluorescent but also capable of electrical conductivity.

The posterior iliac crest outperforms the anterior iliac crest when obtaining mesenchymal stem cells from bone marrow.

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.

Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells.

This study was designed to investigate the migratory behavior of adult human mesenchymal stem cells (MSC) and the underlying mechanism. Cell migration was assessed by transwell, wound healing and time-lapse in vivo motility assays. Pharmacological inhibitors were used to determine the potential mechanism responsible for cell migration and invasion. The tests that were implemented revealed that MSC were fairly migratory. Protein kinase B (AKT) was strongly activated at the basal level. Through our analyses we demonstrated that pharmacological inactivation of AKT2 but not AKT1 significantly decreased cell migration and invasion. Although preliminary, collectively our results indicate that AKT2 activation plays a critical role in enabling MSC migration.

Efficient isolation and enrichment of mesenchymal stem cells from bone marrow.

BACKGROUND AIMS: Bone marrow (BM) mesenchymal stromal cells (MSC) have been identified as a source of pluripotent stem cells used in clinical practice to regenerate damaged tissues. BM MSC are commonly isolated from BM by density-gradient centrifugation. This process is an open system that increases the risk of sample contamination. It is also time consuming and requires technical expertise that may result in variability regarding cellular recovery. The BD Vacutainer® Cell Preparation Tube™ (CPT) was conceived to separate mononuclear cells from peripheral blood. The main goal of this study was to verify whether MSC could be isolated from BM using the CPT. METHODS: BM was harvested, divided into two equal aliquots and processed using either CPT or a Ficoll-Paque™ PREMIUM density gradient. Both methods were compared regarding cell recovery, viability, proliferation, differentiation capacities and the presence of MSC progenitors. RESULTS: Similar numbers of mononuclear cells were isolated from BM when comparing the two methods under study. No differences were found in terms of phenotypic characterization, viability, kinetics and lineage differentiation potential of MSC derived by CPT or Ficoll. Surprisingly, a fibroblast-colony-forming unit (CFU-F) assay indicated that, with CPT, the number of MSC progenitors was 1.8 times higher compared with the Ficoll gradient separation. CONCLUSIONS: The CPT method is able to isolate MSC efficiently from BM, allowing the enrichment of MSC precursors.

Cell growth inhibition and apoptotic effect of the rexinoid 6-OH-11-O-hydroxyphenantrene on human osteosarcoma and mesenchymal stem cells.

Natural derivatives of vitamin A, including all-trans-retinoic acid (ATRA), commonly known as retinoids, currently produce favorable results in the treatment of many types of tumors. The rexinoid 6-OH-11-O-hydroxyphenantrene (IIF) is a synthetic derivative of ATRA. Previous in vitro and in vivo studies demonstrated that IIF is able to induce growth inhibition of various cancer cells and is a potent apoptosis-inducing agent with clinical potential. Osteosarcoma (OS) is the most common type of bone cancer, characterized by a rising aggressiveness. Recent evidences suggest that mesenchymal stem cells (MSC) may favour tumor growth and progression. Thus, it is important to investigate whether a compound with potential anti-tumoral properties such as IIF affects not only tumor cells but also MSC. The current study is an attempt to understand the mode of the potential cytotoxicity of IIF on OS cells and MSC. The response to IIF treatment of osteosarcoma SaOS-2, MG63, and U2OS cells and of bone marrow-derived MSC was the subject of investigation. The results showed that IIF significantly inhibited cell growth in OS cell lines and MSC in both a time- and dose-dependent manner, as evaluated by methylene blue assay. This was also associated with altered cell morphology and an increase in cell death with the involvement of apoptosis as demonstrated by NucleoCounter, Hoechst 33342 staining and FACS analysis. No cell death and apoptosis was found in U2OS cells. Analysis of cells treated with 20 and 40µM IIF for 24h by western blot suggests the activation of initiator caspase 9, indicating the involvement of caspases in inducing apoptosis. Furthermore, IIF upregulated the expression of the pro-apoptotic protein Bax and downregulated the anti-apoptotic protein Bcl2. For the first time, our results collectively provide an evidence for cell growth inhibition and activation of apoptosis in human OS cells and MSC by IIF. These results confirm that IIF may be an effective compound for anticancer treatment, including that of OS.