Application of a three-dimensional graft of autologous osteodifferentiated adipose stem cells in patients undergoing minimally invasive transforaminal lumbar interbody fusion: clinical proof of concept.

BACKGROUND: The authors applied a scaffold-free osteogenic three-dimensional (3D) graft made of adipose-derived mesenchymal stem cells (AMSCs) in patients undergoing minimally invasive transforaminal lumbar interbody fusion (MI-TLIF). METHODS: Three patients (two patients and one patient with 1 and 2 levels, respectively) with degenerative spondylolisthesis underwent MI-TLIF with 3D graft made of AMSCs. To obtain the AMSCs, fatty tissue was collected from the abdomen by lipoaspiration and differentiated afterwards in our Cell/Tissue bank. Clinical outcomes, including the Oswestry Disability Index (ODI) and visual analog scale (VAS) as well as fusion status were assessed preoperatively and up to 12 months postoperatively. RESULTS: At 12 months, all four operated AMSC levels could be assessed (n = 4). Grade 3 fusion could be confirmed at two levels out of four. Mean VAS score improved from 8.3 to 2 and ODI also improved from 47 to 31%. No donor site complication was observed. The final AMSC osteogenic product was stable, did not rupture with forceps manipulation, and was easily implanted directly into the cage with no marked modification of operating time. CONCLUSIONS: A scaffold-free 3D graft made of AMSCs can be manufactured and used as a promising alternative for spinal fusion procedures. Nevertheless, further studies of a larger series of patients are needed to confirm its effectiveness.

Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells.

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm x 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25-125 ng/ml of cell extract by a 1/x(2) weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of +/-15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n=48) or with Stocrin (600 mg once a day, n=50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus (500 mg twice a day). The patients treated by Kaletra showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin showed mean CC of 2388.11 ng/ml while both patients under Aptivus showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.