Osteogenic differentiation modulates the cytokine, chemokine, and growth factor profile of ASCs and SHED

Great efforts have been made to improve bone regeneration techniques owing to a growing variety of sources of stem cells suitable for autologous transplants. Specifically, adipose-derived stem cells (ASCs) and stems cells from human exfoliated deciduous teeth (SHED) hold great potential for bone tissue engineering and cell therapy. After a preliminary characterization of the main biomolecules ASCs and SHED released in their conditioned media, cells were kept both in normal and osteo-inducing conditions. Conventional assays were performed to prove their osteogenic potential such as quantitative real-time polymerase chain reaction (qRT-PCR) (for RUNX-2, collagen type I, osteopontin and osteonectin), alkaline phosphatase activity, osteocalcin production, and von Kossa staining. Conditioned media were tested again after the osteogenic induction and compared to maintaining condition both at base line and after 14 days of culture. The osteogenic condition inhibited the release of all the biomolecules, with the exception, concerning SHED, of growth-regulated alpha protein precursor (GRO\u3b1), and, to a lesser extent, interleukin (IL)-8. In conclusion, our data support that undifferentiated ASCs and SHED may be preferable to committed ones for general cell therapy approaches, due to their higher paracrine activity. Osteoinduction significantly affects the cytokine, chemokine, and growth factor profile in a differential way, as SHED kept a more pronounced pro-angiogenic signature than ASCs

Isolation and characterization of buccal fat pad and dental pulp mscs from the same donor

Mesenchymal stem cells (MSCs) can be harvested from different sites in the oral cavity, representing a reservoir of cells useful for regenerative purposes. As direct comparisons between at least two types of MSCs deriving from the same patient are surprisingly rare in scientific literature, we isolated and investigated the osteoinductive potential of dental pulp stem cells (DPSCs) and buccal fat pad stem cells (BFPSCs). MSCs were isolated from the third molar dental pulp and buccal fat pads of 12 patients. The number of viable cells was quantified through manual count. Proliferation and osteodifferentiation assays, flow cytometry analysis of cell phenotypes, and osteocalcin release in vitro were performed. The isolation of BFPSCs and DPSCs was successful in 7 out of 12 (58%) and 3 out of 12 (25%) of retrieved samples, respectively. The yield of cells expressing typical stem cell markers and the level of proliferation were higher in BFPSCs than in DPSCs. Both BFP-SCs and DPSCs differentiated into osteoblast-like cells and were able to release a mineralized matrix. The release of osteocalcin, albeit greater for BFPSCs, did not show any significant difference between BFPSCs and DPSCs. The yield of MSCs depends on their site of origin as well as on the protocol adopted for their isolation. Our data show that BFP is a valuable source for the derivation of MSCs that can be used for regenerative treatments

A novel method to optimize autologous adipose tissue recovery with extracellular matrix preservation

This work aims to characterize a new method to recover low-manipulated human adipose tissue, enriched with adipose tissue-derived mesenchymal stem cells (ATD-MSCs) for autologous use in regenerative medicine applications. Lipoaspirated fat collected from patients was processed through Lipocell, a Class II-a medical device for dialysis of adipose tissue, by varying filter sizes and washing solutions. ATD-MSC yield was measured with flow cytometry after stromal vascular fraction (SVF) isolation in fresh and cultured samples. Purification from oil and blood was measured after centrifugation with spectrophotometer analysis. Extracellular matrix preservation was assessed through hematoxylin and eosin (H&E) staining and biochemical assay for total collagen, type-2 collagen, and glycosaminoglycans (GAGs) quantification. Flow cytometry showed a two-fold increase of ATD-MSC yield in treated samples in comparison with untreated lipoaspirate; no differences where reported when varying filter size. The association of dialysis and washing thoroughly removed blood and oil from samples. Tissue architecture and extracellular matrix integrity were unaltered after Lipocell processing. Dialysis procedure associated with Ringer’s lactate preserves the proliferation ability of ATD-MSCs in cell culture. The characterization of the product showed that Lipocell is an efficient method for purifying the tissue from undesired byproducts and preserving ATD-MSC vitality and extracellular matrix (ECM) integrity, resulting in a promising tool for regenerative medicine applications

A novel method to optimize autologous adipose tissue recovery with extracellular matrix preservation

This work aims to characterize a new method to recover low-manipulated human adipose tissue, enriched with adipose tissue-derived mesenchymal stem cells (ATD-MSCs) for autologous use in regenerative medicine applications. Lipoaspirated fat collected from patients was processed through Lipocell, a Class II-a medical device for dialysis of adipose tissue, by varying filter sizes and washing solutions. ATD-MSC yield was measured with flow cytometry after stromal vascular fraction (SVF) isolation in fresh and cultured samples. Purification from oil and blood was measured after centrifugationwith spectrophotometer analysis. Extracellularmatrix preservationwas assessed through hematoxylin and eosin (H&E) staining and biochemical assay for total collagen, type-2 collagen, and glycosaminoglycans (GAGs) quantification. Flow cytometry showed a two-fold increase of ATD-MSC yield in treated samples in comparisonwith untreated lipoaspirate; no differenceswhere reportedwhen varying filter size. The association of dialysis and washing thoroughly removed blood and oil from samples. Tissue architecture and extracellular matrix integrity were unaltered after Lipocell processing. Dialysis procedure associated with Ringer's lactate preserves the proliferation ability of ATD-MSCs in cell culture. The characterization of the product showed that Lipocell is an efficient method for purifying the tissue from undesired byproducts and preserving ATD-MSC vitality and extracellular matrix (ECM) integrity, resulting in a promising tool for regenerative medicine applications

Evaluation of the immune state activation in patients affected by ONJ: preliminary data

Bisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) is a serious adverse event characterized by non-healing necrotic bone tissue of mandible or maxilla. BPs target osteoclasts, inhibiting their action and blocking bone resorption, nonetheless an increased bone resorption in the oral cavity is associated to ONJ. One of the causes of ONJ is the dysregulation of the immune system, in particular the strong reduction of gamma/delta T cells circulating in peripheral blood (PB). The rate of circulating osteoclast precursors (OCPs) is altered in bone metastatic patients, suggesting a systemic alteration of osteoclast compartment, but we do not know wether it is altered in cancer patients affected by active lesion of ONJ induced by Zoledronic acid treatment. By flow cytometric analysis of patients' and controls' PB cells, we studied the presence of different T cell subsets, according to the expression of gamma/delta chains, CD4, CD8, CD25 and CD69 and of OCPs. We also evaluated the capability of PBMCs to spontaneously differentiate into osteoclasts in vitro, which is an index of pathological bone resorption. Our preliminary results confirmed in ONJ patients a marked reduction in gamma/delta T cells compared to their level befor patients started BP treatment. The subsets of activated T cells and OCPs were not significantly modified. In ONJ patients, in vitro osteoclastogenesis was comparable to the one of healthy control, while before patients started BP treatment osteoclastogenesis was significantly increased. This result suggests that BPs correctly act in reducing the systemic activation of osteoclasts, associated to bone metastatic patients, as expected, but BPs fail to block osteoclast activity locally

Electron-Beam-Induced Grafting Of Chitosan Onto HDPE/ATZ Composites for Biomedical Applications

HDPE and HDPE/ATZ surfaces were functionalised with chitosan Via electron-beam irradiation technique in order to prepare materials suitable for biomedical purposes. ATR–FTIR and wettability measurements were employed for monitoring the surface changes after both irradiation and chitosan grafting reaction. The presence of ATZ influenced both the EB irradiation process and the surface functionalisation. Mechanical properties of irradiated materials were not remarkably affected by irradiation processing. Biological assays indicated that electrostatic interactions between the negative charges of the surface of cell membranes and the –NH3+ sites on chitosan chains promoted cell adhesion, while some oxidized species produced during the irradiation process were thought to cause a detrimental effect on the cell Viability