Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

[Abstract] Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-0040Ministerio Ciencia e Innovacion; PLE2009-0144Ministerio Ciencia e Innovación; PI 08/202

Nastal čas prodloužit dobu expirace kryoprezervovaných alograftů srdečních chlopní

V současné době existuje velké množství umělých komerčních náhrad chlopní. Přesto jsou stále žádané a úspěšně transplantované kryoprezervované semilunární alografty srdečních chlopní (C-AHV). U těchto náhrad zatím není přesně definována doba expirace.Většina tkáňových bank používá pět let. Z fyziologického, funkčního a operačního pohledu představuje morfologie a mechanické vlastnosti aortálních a pulmonárních kořenů hlavní limitaci doby expirace C-AHV. Cílem této práce je podat přehled metod strukturní a mechanické analýzy tkání AHV, které jsou vhodné pro stanovení doby expirace. alograftů. Pro stanovení mikrostruktury je vhodná kvantitativní morfologie za použití stereologické testovací mřížky. Touto metodou lze snadno, efektivně a opakovatelně stanovit možství buněk a mezibuněčných komponent. Pro stanovení mechanických parametrů, jako Youngův modul pružnosti, mezní napětí a deformace, lze využít tahovou zkoušku. C-AHV jsou v různých tkáňových laboratořích připravovány podle různých protokolů. Je tedy nutné, aby každá laboratoř stanovila dobu expirace samostatně.Despite the wide choice of commercial heart valve prostheses, cryopreserved semilunar allograft heart valves (C-AHV) are required, and successfully transplanted in selected groups of patients. The expiration limit (EL) criteria have not been defined yet. Most Tissue Establishments (TE) use the EL of 5 years. From physiological, functional, and surgical point of view, the morphology and mechanical properties of aortic and pulmonary roots represent basic features limiting the EL of C-AHV. The aim of this work was to review methods of AHV tissue structural analysis and mechanical testing from the perspective of suitability for EL validation studies. Microscopic structure analysis of great arterial wall and semilunar leaflets tissue should clearly demonstrate cells as well as the extracellular matrix components by highly reproducible and specific histological staining procedures. Quantitative morphometry using stereological grids has proved to be effective, as the exact statistics was feasible. From mechanical testing methods, tensile test was the most suitable. Young’s moduli of elasticity, ultimate stress and strain were shown to represent most important AHV tissue mechanical characteristics, suitable for exact statistical analysis. C-AHV are prepared by many different protocols, so as each TE has to work out own EL for C-AHV