Background and aimsThe recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.MethodsThe histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.ResultsThe criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively) and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively). The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively) and very high for the analysis of inflammation (ICC = 0.931, p<0.001).ConclusionsWe established a simple NAFLD scoring system with high reproducibility that is applicable for different rodent models and for all stages of NAFLD etiology.
Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simpli fied method for the quantification of the amount of degraded collagen in the collagen net work of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by a-chymotrypsin, hydrolysis in 6 M HC1 of the released fragments as well as the residual tissue, and then mea surement of the amount of hydroxyproline in both pools* Since the digestion of degraded collagen by a-chymotrypsin and measurement of hydroxyproline is not restricted to a sper cific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investi gations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy carti lage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in os-4 ■* teoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.
Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age- or OA etiology-dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA-etiology groups (age-related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor-site morbidity, typically containing>10(7) mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible. Disclosure of potential conflicts of interest is found at the end of this article.
We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.
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