Rapid Detection of Shear-Induced Damage in Tissue-Engineered Cartilage Using Ultrasound

Abstract

Previous investigations have shown that tissue-engineered articular cartilage can be damaged under a combination of compression and sliding shear. In these cases, damage was identified in histological sections after a test was completed. This approach is limited, in that it does not identify when damage occurred. This especially limits the utility of an assay for evaluating damage when comparing modifications to a tissue-engineering protocol. In this investigation, the feasibility of using ultrasound (US) to detect damage as it occurs was investigated. US signals were acquired before, during, and after sliding shear, as were stereomicroscope images of the cartilage surface. Histology was used as the standard for showing if a sample was damaged. We showed that US reflections from the surface of the cartilage were attenuated due to roughening following sliding shear. Furthermore, it was shown that by scanning the transducer across a sample, surface roughness and erosion following sliding shear could be identified. Internal delamination could be identified by the appearance of new echoes between those from the front and back of the sample. Thus, it is feasible to detect damage in engineered cartilage using US.

Keywords: acoustic analysis; articular cartilage; nondestructive testing; tissue engineering; tissue failure identification; ultrasonography.

FIG. 1.

Flowchart of the experiments in this article. Left column was done using the sliding shear device described previously and a separate US measurement system. Right column used a newer integrated device described in Figure 4. Other than ease-of-use, there are no fundamental differences between the two devices with respect to sliding shear loading. US, ultrasound.

FIG. 2.

(A) Device used to characterize damage in cartilage samples using US. A sample is attached to a magnetic stainless steel holder and is immersed in PBS, which couples the focused US beam and sample. Reflections from the front surface of the sample and from the interface between the sample and the sample-holder are recorded and analyzed to identify signs of damage. The X-Y stage is used to scan the transducer across the sample. (B) A schematic of the testing device in (A). PBS, phosphate-buffered saline. Color images available online at www.liebertpub.com/tec

FIG. 3.

Samples were loaded in compression and sliding shear to induce damage. Compression was applied by deadweights placed on a platform above a sample, and the oscillating movement of the table to which the lubricant wells are attached applied shear. Glass microscope slides were used for the sliding counterface. This is a close-up of one loading station, the complete device consists of four of these. Color images available online at www.liebertpub.com/tec

FIG. 4.

Combined sliding shear and US apparatus. A single-axis linear slide driven by a stepper motor produces oscillating motion of a sliding stage carrying a PBS-filled well with a glass bottom. The sample is fixed in space above the stage; deadweight is used to provide normal force. A single element focused US transducer is attached to the linear slide below the fluid-filled dish and can be moved into position under the sample using the slide. Fine positioning of the sample relative to the transducer is accomplished using the z-axis adjustment micrometer. Color images available online at www.liebertpub.com/tec

FIG. 5.

Relaxation of the sample centerpoint after sliding shear, expressed as TOF to front of cartilage (normal bovine cartilage). The parallel but shifted curves suggest a small, but permanent loss of thickness between 100 and 1100 cycles. Before = reference line of sample thickness before loading. TOF, time of flight. Color images available online at www.liebertpub.com/tec

FIG. 6.

(A) Stereomicroscope image of a sample before sliding shear. The surface is relatively smooth and continuous. The colored dots show the size and position of the focused US beam. (B) Stereomicroscope image of a sample after sliding shear. After shear, cracks have opened, which in some cases, extend through the full thickness of the sample (red arrows). These cracks would not necessarily be identified when the sample is interrogated using a single fixed-position element US transducer (green dots) or by histology. When a line-scan across the sample was taken (blue dots), more area is covered, but some damage can be missed. Sample 31 in Table 1. Color images available online at www.liebertpub.com/tec

FIG. 7.

Histology of (A) normal bovine cartilage test sample and (B) tissue-engineered cartilage samples. Toluidine blue staining, scale bar is 1 mm. Color images available online at www.liebertpub.com/tec

FIG. 8.

(A) US signals before and after sliding shear. Note the attenuation of the amplitude of the reflection from the sample's surface (double-headed blue arrow). There is also a relative shift of the “After” signal to the left of the “Before” signal, suggesting that the sample was thicker after damage (green arrow). A new area of attenuated signal appears in the “After” signal (blue arrow), close to the return from the metal holder (magenta arrow). (B) Histology clearly showing that the sample was damaged. Where the sample has cracked and separated into two legs, it is thicker than the bulk material, which is consistent with the thickening shown by US. Sample 35 in Table 1. Scale bar is 500 μm. Color images available online at www.liebertpub.com/tec

FIG. 9.

Stereomicroscope images of the surface before (A) and after (B) sliding. Damage is clearly evident in (B). (C) Histology of the damaged sample. (D) Single point (150 μm diameter spot) US traces before and after sliding shear. The salient feature is the loss of an easily identifiable front face reflection in the “After” (red) trace due to destruction of the surface and scattering of the US wave. (E) When multiple thickness measurements were taken at 1 mm increments across the sample's surface, the roughness of the surface (red line) can be clearly determined from US TOF. The black line shows the original thickness of the sample. Histology scale bar is 500 μm. Sample 29 in Table 1. Color images available online at www.liebertpub.com/tec

FIG. 10.

Surface profiles of two TE and one native bovine cartilage samples taken before and after the indicated number of cycles showing different types of surface changes. (A) Native bovine cartilage before and after 100 and 1100 cycles showing no significant changes (sample 1117). (B) TEC showing slight compaction at the center of the sample, slight swelling elsewhere (sample 1110). (C) Sample showing marked increase in thickness. Histology shows tissue to be broken up with multiple cracks (sample 1108). Color images available online at www.liebertpub.com/tec