Loss of spatial organization and destruction of the pericellular matrix in early osteoarthritis in vivo and in a novel in vitro methodology

Abstract

Objectives Current repair procedures for articular cartilage cannot restore the tissue’s original form and function because neither changes in its architectural blueprint throughout life nor the respective biological understanding is fully available. We asked whether two unique elements of human cartilage architecture, the chondrocyte-surrounding pericellular matrix (PCM) and the superficial chondrocyte spatial organization (SCSO) beneath the articular surface are congenital, stable or dynamic throughout life. We hypothesized that inducing chondrocyte proliferation in vitro impairs organization and PCM and induces an advanced OA-like structural phenotype of human cartilage. Methods We recorded propidium-iodine-stained fetal and adult cartilage explants, arranged stages of organization into a sequence, and created a lifetime-summarizing SCSO model. To replicate the OA-associated dynamics revealed by our model, and to test our hypothesis, we transduced specifically early OA-explants with hFGF-2 for inducing proliferation. The PCM was examined using immuno- and auto-fluorescence, multiphoton second-harmonic-generation, and scanning electron microscopy. Results Spatial organization evolved from fetal homogeneity, peaked with adult string-like arrangements, but was completely lost in OA. Loss of organization included PCM perforation (local micro-fibrillar collagen intensity decrease) and destruction (regional collagen type VI signal weakness or absence). Importantly, both loss of organization and PCM destruction were successfully recapitulated in FGF-2-transduced explants. Conclusion Induced proliferation of spatially characterized early OA-chondrocytes within standardized explants recapitulated the full range of loss of SCSO and PCM destruction, introducing a novel in vitro methodology. This methodology induces a structural phenotype of human cartilage that is similar to advanced OA and potentially of significance and utility.

Proof-of-concept for the detection of early osteoarthritis pathology by endomicroscopy

“…In this study, we utilized only AC samples that unambiguously displayed one pattern of cellular organization. However, this was not the case for the double string pattern, as strings are known to be interspersed with double strings, indicating an early OA pathology (24,26). The local elastic moduli of the AC surfaces (superficial zone) significantly correlate with the distinct stages of spatial chondrocyte organization (r S = −0.68; p ≤ 2•10 -7 ; number of force vs indentation measurements n = 17402; see Note S2 for details).…”

“…Prior to AFM measurements the AC samples were fluorescent-stained in order to categorize the samples and use the chondrocyte spatial organization as image-based biomarker. Categorization is based on our lifetime-summarizing model of the cellular spatial organization of a hypothetical human individual (26), with the intention to use distinct stages of cellular spatial organization as a marker of OA severity. Fig.…”

“…To discuss the here presented data in a mechanistic context, we speculate that repetitive mechanical trauma to the articular surface, known to lead to microstructural damage, might have contributed to inducing the here observed loss of elastic modulus of AC surface. In addition, this damage might release a proliferation-inducing growth factor such as FGF-2 which is released upon mechanical injury (31) to induce changes in the cellular spatial organization of human AC, as demonstrated in (26).…”

“…Two-dimensional images capturing the entire cartilage disc were digitally recorded (Axio Observer, Axiocam 506, ZEISS, Germany; magnifications 10x, 20x) and displayed using the ZEN software (ZEISS, Germany). Visual classification was performed according to the sample's predominant type of chondrocyte organization in the superficial zone by two experienced researchers (23,26). Samples displaying the most unambiguous type of organization were selected for AFM measurements and stored at 4 °C.…”

“…Fluorescence microscopy has been used in studies to characterize the spatial organization of superficial zone chondrocytes and for detecting early, preclinical OA (22)(23)(24)(25)(26). However, so far it has not been known whether the differences in chondrocyte organization within the superficial zone of AC are related to the nanomechanical changes observed in early OA.…”