Cell death in human articular chondrocyte: a morpho-functional study in micromass model

Abstract

Chondrocyte death and loss of extracellular matrix are the central features in articular cartilage degeneration during osteoarthritis pathogenesis. Cartilage diseases and, in particular, osteoarthritis are widely correlated to apoptosis but, chondrocytes undergoing apoptosis "in vivo" more often display peculiar features that correspond to a distinct process of programmed cell death termed "chondroptosis". Programmed cell death of primary human chondrocyte has been here investigated in micromasses, a tridimensional culture model, that represents a convenient means for studying chondrocyte biology. Cell death has been induced by different physical or chemical apoptotic agents, such as UVB radiation, hyperthermia and staurosporine delivered at both 1 and 3 weeks maturation. Conventional electron microscopy was used to analyse morphological changes. Occurrence of DNA fragmentation and caspase involvement were also investigated. At Transmission Electron Microscopy, control cells appear rounding or slightly elongated with plurilobated nucleus and diffusely dispersed chromatin. Typically UVB radiation and staurosporine induce chromatin apoptotic features, while hyperthermia triggers the "chondroptotic" phenotype. A weak TUNEL positivity appears in control, correlated to the well known cell death patterns occurring along cartilage differentiation. UVB radiation produces a strong positivity, mostly localized at the micromass periphery. After hyperthermia a higher number of fluorescent nuclei appears, in particular at 3 weeks. Staurosporine evidences a diffuse, but reduced, positivity. Therefore, DNA fragmentation is a common pattern in dying chondrocytes, both in apoptotic and "chondroptotic" cells. Moreover, all triggers induce caspase pathway activation, even if to a different extent, suggesting a fundamental role of apoptotic features, in chondrocyte cell death.

Chondroptotic chondrocytes in the loaded area of chondrocalcinotic cartilage: A clinical proposal?

“…This type of cell death has some features in common with classical apoptosis such as cell shrinkage, chromatin condensation, and the probable involvement of caspases. In addition, chondroptosis shows some peculiar characteristics, including cytoplasmic vacuolization, autophagic vacuoles, extrusion of cellular material into the extracellular space, and a final disruption of cell remnants (Battistelli et al, 2014). To date, no evidence has revealed chondroptosis in patients affected by chondrocalcinosis.…”

“…Chondrocytes, which showed chondroptotic features, have been found in the cartilage middle layer from the area particularly subjected to mechanical loading, suggesting a strong relationship between compressive stress and chondroptosis, as well as for the other cell death types and osteoarthritis (OA) (Levin et al, 2001). Chondroptotic chondrocytes, already showed in osteoarthritis and alkaptonuria, are characterized by a specific morphology, in the absence of apoptotic patterns, like chromatin clumping into large solid masses at nuclear periphery (known as cupshaped masses) and presence of micronuclei, allowing to clearly distinguish between them (Battistelli et al, 2014;Hwang and Kim, 2015;Millucci et al, 2015). One of the most interesting feature of chondroptosis is its ability to reduce the cellular components without producing residual material, which could induce an inflammation process.…”

Tyrosol prevents apoptosis in irradiated keratinocytes

“…In addititon, molecular analyses revealed that the antioxidant compound induced a caspase activation decrease. UVB radiation can be considered a powerful apoptotic trigger [23,25], able to activate both the intrinsic and extrinsic apoptotitc pathway. In particular, Tyr administration resulted in decreasing the intrinsic and extrinsic pathway activation, with a reduced expression of caspase-8, -9 and -3.…”

“…All the specimens were post-fixed in 1% OsO 4 for 1 h, alcohol dehydrated and embedded in araldite [25]. Thin sections were stained with uranyl acetate and lead citrate and analyzed with a Philips CM10 transmission electron microscope.…”

In vitro cytotoxicity evaluation of cadmium by label‐free holographic microscopy

“…The conventional light microscopy provides a quite facile and rapid view of cell morphological changes and plasma membrane integrity by using trypan blue dye in cell culture dishes but only qualitatively, that is, without any quantitative information about others biophysical parameters . The electron microscopy provides high spatial resolution and is usually considered the ‘gold standard’ in cell death research. However, it is very expensive, time consuming and requires cell fixing, thus suspending any biological process.…”