Introduction

Due to the avascular nature of articular cartilage tissue, once injured it has a limited healing capacity and significant damage can result in reduced quality of life and osteoarthritis. Collagen arrangement and mechanical anisotropy is widely reported to vary throughout each cartilage zone, which may serve to distribute joint loads. Indeed, biochemical gradients and strain variations within articular cartilage have been well described [1,2], and inverse methods have been used to predict properties. However, property measurements of individual articular cartilage zones are largely imprecise due to testing excised samples ex situ in a non-native stress environment and are complicated by imperfect sample geometries and the inability to obtain large enough samples to evaluate anisotropy. Moreover, such measures are influenced by the complex behaviors characteristic of cartilage tissue including time dependence and anisotropy [3], both of which vary with distance from the articular surface. As new treatment modalities and tissue engineered replacements are developed, it is critical to elucidate contributors to the relationships between structure and function throughout the full cartilage thickness in all directions. Moreover, it is critical to analyze these relationships at length scales that are relevant to the cells that maintain, remodel, and degrade surrounding tissue.

Content

In a series of studies, we evaluated human zonal articular knee cartilage in situ for evidence of mild osteoarthritis via material properties, structural organization, and chemical composition along three orthogonal directions (i.e., parallel and perpendicular to the gliding surface). Cartilage zones were identified as superficial (surface), middle, and deep. Intact human cadaver knees were assessed for OA status via morphologic and quantitative 7T MRI [4]. The medial femoral condyles showed T1ρ and T2 values within ranges characteristic of healthy cartilage with evidence of moderate cartilage degeneration in two of the lateral condyles. Histological assessment (Safranin-O) and Secondary Harmonic Generation (SHG) imaging showed GAG distributions and collagen fiber orientations characteristic of moderately healthy zonal articular cartilage, with minor GAG depletion in the superficial zone from all four donors.

Microindentation [5] was used to produce large maps of spatial property and behavior variation spanning all three cartilage zones; SHG imaging facilitated visualization of collagen orientation; histology was performed to assess glycosaminoglycans; and hyperspectral Raman spectroscopy permitted mapping of tissue biochemistry [6]. Through a biphasic, contact-based analysis of indentation responses [8], the equilibrium tensile and compressive moduli and permeability were computed for superficial, middle, and deep zones. As expected, modulus values were higher in the deep zone as compared to the surface zone, a trend which was most obvious when comparing compressive moduli. Regardless of orientation, tensile modulus values exceeded compressive moduli. These modulus values were also directionally dependent, where indents placed normal to the articulating surface produced lower moduli than those in a parallel orientation. Permeability was greatest in the superficial zone and was greater when indenting in directions perpendicular, rather than parallel, to the articular surface. These behaviors compare well with observations of articular cartilage compressive strain [2] and results from finite element modeling [7].

Histology and SHG revealed GAG distributions and collagen fiber orientations characteristic of moderately healthy zonal articular cartilage, with minor GAG depletion in the superficial zone in all donors. The collagen fiber orientation from SHG increased with depth relative to the articulating surface. When correlating collagen fiber orientation to modulus, the equilibrium compressive modulus showed a stronger correlation as compared to the tensile modulus. GAG distributions from histological assessment compared well to Raman spectral maps showing a zonal gradient in GAG and collagen prevalence. The ratio of chondroitin sulfate to collagen (CHS:Col) increased significantly with increasing distance from the articular surface across all three zones. Further, a positive and significant relationship existed between CHS:Col and collagen orientation and also with tissue material properties (i.e., compressive and tensile modulus). These results indicate that in articular cartilage, the modulus and permeability variations are modulated by the biochemical composition of the tissue.

These outcomes provide new insight into relationships between properties, organization and biochemistry of cartilage that are assessed in three orthogonal directions and, critically, at length scales relevant to cells. Moreover, multimodal data sets that define a range of unique metrics across multiple spatial scales are sensitive to even small variations of organization and chemistry within articular cartilage – and thus hold potential to describe early degenerative changes. This study demonstrates micro-meter length scale variations in key mechanical property measures within human zonal articular cartilage, and maps how these changes correlate with the underlying tissue structure (e.g. collagen orientation, quantitative MRI) and biochemistry (i.e. from Raman spectroscopy) with potential to detect early osteoarthritic changes. This work also provides important and novel targets for tissue engineers and modelers as they seek to recreate the complex mechanical, chemical, and structural environment in articular cartilage.

References

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