Chondrocytes extract from patients with osteoarthritis induces chondrogenesis in infrapatellar fat pad-derived stem cells.

OBJECTIVE: Infrapatellar fat pad of patients with osteoarthritis (OA) contains multipotent and highly clonogenic adipose-derived stem cells that can be isolated by low invasive methods. Moreover, nuclear and cytoplasmic cellular extracts have been showed to be effective in induction of cell differentiation and reprogramming. The aim of this study was to induce chondrogenic differentiation of autologous mesenchymal stem cells (MSCs) obtained from infrapatellar fat pad (IFPSCs) of patients with OA using cellular extracts-based transdifferentiation method. DESIGN: IFPSCs and chondrocytes were isolated and characterized by flow cytometry. IFPSCs were permeabilized with Streptolysin O and then exposed to a cell extract obtained from chondrocytes. Then, IFPSCs were cultured for 2 weeks and chondrogenesis was evaluated by morphologic and ultrastructural observations, immunologic detection, gene expression analysis and growth on 3-D poly (dl-lactic-co-glycolic acid) (PLGA) scaffolds. RESULTS: After isolation, both chondrocytes and IFPSCs displayed similar expression of MSCs surface makers. Collagen II was highly expressed in chondrocytes and showed a basal expression in IFPSCs. Cells exposed to chondrocyte extracts acquired a characteristic morphological and ultrastructural chondrocyte phenotype that was confirmed by the increased proteoglycan formation and enhanced collagen II immunostaining. Moreover, chondrocyte extracts induced an increase in mRNA expression of chondrogenic genes such as Sox9, L-Sox5, Sox6 and Col2a1. Interestingly, chondrocytes, IFPSCs and transdifferentiated IFPSCs were able to grow, expand and produce extracellular matrix (ECM) on 3D PLGA scaffolds. CONCLUSIONS: We demonstrate for the first time that extracts obtained from chondrocytes of osteoarthritic knees promote chondrogenic differentiation of autologous IFPSCs. Moreover, combination of transdifferentiated IFPSCs with biodegradable PLGA 3D scaffolds can serve as an efficient system for the maintenance and maturation of cartilage tissue. These findings suggest its usefulness to repair articular surface in OA.

[Ability of leukocyte reduction filters to remove fat particles from blood in experimental models simulating blood salvage in orthopedic surgery]. / Capacidad de los filtros leucorreductores para la eliminación de partículas de grasa en sangre. Modelo experimental de simulación de la sangre recuperada en cirugía ortopédica.

BACKGROUND: Salvaged autologous blood in orthopedic surgery may contain tissular debris such as fat particles (FP), possibly increasing the risk of fat embolism after bone surgery. Therefore, this study was initiated to ascertain the capacity of leukocyte filters to remove FP using in vitro models. METHODS: All experiments were performed in triplicate using donor blood bags within 15 days of their donation. Five different olive oil volumes were added to blood to obtain 5 oil concentrations (1% to 5%), and blood was subsequently filtered through a PureCell (Pall Biomedical, Portsmouth, UK) leukocyte-reduction filter. In another set of experiments, 5 different oil volumes (1, 2.5, 5, 7.5 or 10 mL) were injected into the line during filtration of oil-free blood. In addition, 3 preparations of blood supplemented with 5% oil were processed in the autotransfusion device OrthoPAT (Haemonetics Corp, Braintree, MA, USA), and the obtained red cell concentrate was subsequently filtered through PureCell. We collected samples for cell counting and analysis and FP detection with a Pentra 120 Retic (ABX, Montpellier, France) flow cytometer. RESULTS: Specific signals corresponding to FP were clearly detected in the white blood cell scattergrams yielded by the cytometer for oil supplemented blood. PureCell removed FP up to an oil concentration of 3% or up to an injected oil volume of less than 10 mL. Addition of a filtration step through a PureCell filter after blood washing by the OrthoPAT device completely removed FP. CONCLUSIONS: Leukocyte filters seem to be useful for removing FP from unprocessed blood with a low degree of fat contamination (less than 10 mL) and to complete FP removal from processed blood. Therefore, using a leukocyte filter in the patient's line should contribute to improving the safety of perioperative autologous blood salvage.

[Determination of serum ferritin: ideas for avoiding induced ferropenia in blood donors]. / Determinación de ferritina sérica: consideraciones para evitar ferropenia inducida en donantes de sangre.

PURPOSE: To evaluate blood donation as a cause of iron deficiency. MATERIAL AND METHODS: Serum ferritin levels were determined by enzymoimmunoassay with an SRItm autoanalyser in 500 blood donors of both sexes chosen at random and in 200 suitors for blood donation, used as control group. Iron deficiency was defined by ferritin values below 15 ng/dL. Age, sex, total number of blood donations and those carried out in the last year were all correlated for the statistical analysis, performed with the SPSS/PC+ pack. RESULTS: The mean ferritin value in men was 86.0 ng/dL, and in women this was 27.1 ng/dL. With respect to the control group, blood donors showed increased iron deficiency, 7.4% for men and 11.8% for women. Highly significant direct correlation was found in male donors between total donations, last-year donations and age, and between total number of donations and age in female donors; highly significant inverse correlation was found between total number of donations, last-year donations and ferritin levels among the male donors, while these correlations lacked significance in the female donors. When correlating last-year donations with mean ferritin levels in women, low, although constant, ferritin values were seen, whereas a marked descent was found in men. Iron deficiency was strikingly spread among women, ranging between 21% of those with one blood donation to 46% in those with 4 donations during the last year; in men, iron deficiency was present in 14% of those with 4 or more blood donations in the last year. With respect to total number of blood donations and mean ferritin values, iron deficiency was found in 50% of the women with 8 donations and in 12.8% of men with 14 donations. Ferritin levels decrease in blood donors with aging beyond two blood donations in both sexes. CONCLUSIONS: 1st) Iron deficiency related to blood donation is demonstrated. This deficiency is clearly seen in men after the first blood donations and is more intense in women, as their previous reserves are lower. 2nd) Ferritin is the best marker for estimating iron deposits, and enzymoimmunoassay is the technique of choice as it seems easy to perform and is automatic. 3rd) Determining ferritin levels in the first blood donation seems advisable in order to assess previous deposits and to evaluate yearly the state or iron reserves. 4th) Iron supplement is advisable during the 4 first donations in regular blood donors and in those with iron deficiency, with ferrous sulphate at a dose of 100 mg/day for 10 days.