Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone

Abstract

We studied articular calcified cartilage (ACC) and the immediately subchondral bone (SCB) in normal and osteoarthritic human femoral heads. Thick slices of human normal reference post mortem (PM) and osteoarthritic (OA) femoral heads (age 55-89 years) were embedded in PMMA, micromilled, carbon coated and studied using quantitative backscattered electron (qBSE) imaging to determine variations in degree of mineralization. With exact microanatomical location, nanoindentation was performed on the same block faces in representative superior (more highly loaded) and medial regions of the joint surface. Using a partial unloading method, elastic modulus as a function of indenter penetration depth was determined using a spherical tipped diamond indenter. A pointed indenter was used to determine the tissue hardness in selected locations. The relationship between mineralization and indentation modulus was more distinct in ACC than in SCB, the latter having a higher matrix concentration with variable collagen orientation. In OA, the bulk of the measurements were coincident with those in the PM samples, although there was a greater range in the levels of mineralization and modulus in ACC. In OA, extremely hypermineralized ACC was found in ACC proper, especially in superior regions, and translocated into SCB and hyaline cartilage. The very highly mineralized cartilage fragments may function as a hard grinding abrasive, accelerating wear rates whether attached to or fragmented from the eburnated surfaces of OA ACC. Highly mineralized regions would also alter loading patterns and thereby contribute to further destruction of the joint tissues.

Fig. 1

Montage of qBSE images of the mineralized tissues closest to the joint in an OA femoral head, 74-year-old female, showing eburnation of ACC and SCB on the superior, highly loaded surface and gross hypermineralization in medial SCB. Each square image has a 2.7-mm field width (1024² pixels). qBSE montages were used to select areas for nanoindentation testing.

Fig. 2

qBSE (20 kV) image of PM field, 67-year-old male. (a) Grey levels were scaled using the halogenated dimethacrylate standards and ranked into eight classes: the colour look-up table allows visual inspection of bone mineralization density from the qBSE–SEM images. (b) Histogram of this image field using the same colour look-up table. (c) Topographic BSE image of the same field to permit location of indents (here, in two adjacent arrays): five high load indents at end of each array simplify the search process. (d) Indent array distribution overlain on a qBSE image to extract BSE grey-level measurements at the (−220 pixels) indent sites (white patches: grey patches rejected). Labels: C = ACC, B = SCB, H = hyaline articular cartilage, M = marrow space in bone domain. Fieldwidths (a, c, d) = 594 μm.

Fig. 3

qBSE (20 kV) images of representative regions. (a) Superior, 81-year-old male PM. (b) Medial, 85-year-old male PM. (c) Superior, 89-year-old female OA. (d) Medial, 55-year-old female OA. Overlays show indent array sites (white patches: grey patches rejected). Labels: C = ACC, B = SCB, H = hyaline articular cartilage, M = marrow, O = osteonal canal space in bone domain, JS = joint space where cartilage is destroyed in (b). Fieldwidths = 594 μm.

Fig. 4

qBSE (20 kV) images. (a) 89-year-old female OA, medial layered ACC, with overlay showing indentation array locations: Spherical indenter = white patches: grey patches rejected: Berkovich indenter = white circles: grey circles rejected. (b) 55-year-old female OA, showing impacted and eburnated superior ACC. (c) 59-year-old female OA, medial, with projection of hypermineralized ACC into HAC domain. Labels C = ACC, B = SCB, H = hyaline articular cartilage, M = marrow, O = osteonal canal space in bone domain, JS = joint space. Asterisk indicates contaminant debris. Fieldwidths = 594 μm.

Fig. 5

Mean ±95% CI values for (a) mineralization and (b) indentation modulus in PM samples, matched set size data. Significantly greater values for mineralization in PM ACC occur with no change in the corresponding indentation modulus or in vivo loading (medial vs. superior: one-way anova, α= 0.05: asterisk denotes significant differences from SCB values).

Fig. 6

Mean ±95% CI values for mineralization and indentation modulus in PM and OA samples, matched set size data. In OA SCB, mineralization is lower and indentation modulus tends to decrease from PM levels, with no effect of in vivo loading conditions (medial vs. superior). In OA, mineralization and indentation modulus are lower in medial ACC and greater in superior ACC. (One-way anova, α= 0.05: asterisk denotes significant differences from PM values.)

Fig. 7

Indentation modulus vs. mineralization for all data, showing that ACC (black) exhibits a greater range of values than SCB (grey), where indentation modulus increases in large amounts with small increases in mineralization.

Fig. 8

Indentation modulus vs. mineralization for all indents in SCB and ACC. Grey symbols indicate the medial and superior matched set size data. Black shows data from regions of special interest. Note the greater range of mineralization levels in both tissues in OA.

Fig. 9

Hardness vs. mineralization in areas adjacent to previously indented arrays seen in Fig. 4(a–c), for three OA samples.

Fig. 10

Values for hardness (top), and indentation modulus (bottom, ACC = dark blue; SCB = green symbols) from Berkovich array shown in Fig. 4a (image at centre, ACC = dark blue; SCB = mid blue; rejected = white). Note increase in both measured parameters in the deeper, more mineralized ACC. The apparent modulus matching between ACC and SCB may facilitate bonding and load transmission between ACC and SCB.

Fig. 11

(a) Indentation modulus vs. mineralization for 74-year-old female in medial OA SCB: poorly mineralized woven bone and highly mineralized bone. Grey points derive from three indent arrays in three similar, neighbouring qBSE image fields, one of which is shown in (b), colour-coded qBSE image. Indent sites classified as ACC or SCB are shown as white patches. Labels: C = ACC, B = SCB, M = marrow space, JS = joint space. Fieldwidth = 594 µm.

Fig. 12

71-year-old male, PM. (a) Indentation modulus vs. mineralization for highly mineralized deposit within medial HAC, not attached to the mineralizing front of the ACC, which had greater modulus values than medial PM ACC. (b) qBSE (20 kV) image. Indent sites shown as white patches are those used in (a), unused patches on HAC, edges or cracks are grey. Fieldwidth = 594 µm.

Fig. 13

Indentation modulus vs. mineralization for 59-year-old female medial OA ACC from indent locations shown as white patches in Fig. 4(c).