Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential.

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects' treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-ß and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.

Photobiomodulation with a 645 nm Diode Laser of Saos-2 Cells and Platelet-Rich Plasma: The Potential for a New Mechanism of Action.

Objective: The main focus of this in vitro study was to highlight possible differences between outcomes of photobiomodulation performed in the presence or absence of growth factors derived from platelet-rich plasma. Background: Photobiomodulation has garnered increasing attention, thanks to a large number of controlled clinical trials that have proven its efficacy in various oral pathologies. Nevertheless, the mechanism of action is still a matter of debate. Materials and methods: The cell model used was Saos-2ATTC HTB-85, a human osteosarcoma cell line that retains an osteogenic potential matching that of osteoblastic cells. Photobiomodulation was performed with a 645 nm diode laser; we investigated three different fluence values (2, 5, and 10 J/cm2) delivered with 3 different irradiation times (1, 2, and 4 min). The design of the study included a case-control structure. Cell viability was assessed by resazurin reduction assay before laser irradiation. We assessed cell differentiation by Alizarin-red Sigma Aldrich assay 48 h after the last laser irradiation. Results: Results show that the combination of photobiomodulation and platelet-rich plasma can lead to a statistically significant increase in both proliferation and differentiation rates. Conclusions: Only a defined amount of energy, that is, a fluence of 5 J/cm2 delivered in 2 min and of 10 J/cm2 in 4 min, was proven to be the most effective in the presence of platelet-rich plasma to induce cell proliferation and calcium deposition.

High levels of Notch intracellular cleaved domain are associated with stemness and reduced bevacizumab efficacy in patients with advanced colon cancer.

δ­like ligand 4 (DLL4)­Notch signaling is associated with tumor resistance to anti­vascular endothelial growth factor (VEGF) therapy. Furthermore, Notch signaling is critical for the maintenance of colon cancer stem cells (CSCs), which are relevant in drug resistance and tumor angiogenesis. CD44 is a transmembrane glycoprotein and is considered a putative marker of CSCs. To assess the association of Notch intracellular cleaved domain (NICD), DLL4 and CD44 expression with the efficacy of anti­angiogenic drugs, a series of samples derived from patients with advanced colon cancer enrolled in prospective clinical trials were analyzed. Histological samples from 51 primary tumors that originated from patients treated with bevacizumab­based first­line chemotherapy were analyzed by immunohistochemistry for NICD, DLL4 and CD44 expression, and CD31 for microvessel count. The expression levels of genes relevant for angiogenesis [angiopoietin (ANGPT)1, ANGPT2, fibroblast growth factor (FGF)1, FGF2, epidermal growth factor, placental growth factor, VEGFA and DLL4] were detected by reverse transcription­quantitative PCR using RNA extracted from the frozen tissues of four tumors with low and four tumors with high NICD expression. Strong NICD levels were observed in 12/51 (24%) of the patients, whereas 16/51 (31%) of the colon cancer subjects exhibited high CD44 expression. Strong CD44 staining was associated with high NICD levels compared with the CD44 expression levels noted in samples with low NICD levels (67 vs. 20%, P=0.005). No association was observed with regards to the expression levels of NICD, CD44 and the other aforementioned biomarkers. High expression levels of NICD and CD44 predicted reduced progression­free survival (P<0.001) and overall survival (P=0.002). No significant differences in the expression of angiogenesis­related genes were detected between low and high NICD­expressing tumors. In conclusion, NICD and CD44 tissue levels exhibited an association and may be related to a reduced survival rate in patients with advanced colon cancer treated with bevacizumab.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro.

Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. OBJECTIVE: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. MATERIAL AND METHODS: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. RESULTS: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. CONCLUSIONS: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.