Non-invasive characterization of polyurethane-based tissue constructs in a rat abdominal repair model using high frequency ultrasound elasticity imaging

Abstract

The evaluation of candidate materials and designs for soft tissue scaffolds would benefit from the ability to monitor the mechanical remodeling of the implant site without the need for periodic animal sacrifice and explant analysis. Toward this end, the ability of non-invasive ultrasound elasticity imaging (UEI) to assess temporal mechanical property changes in three different types of porous, biodegradable polyurethane scaffolds was evaluated in a rat abdominal wall repair model. The polymers utilized were salt-leached scaffolds of poly(carbonate urethane) urea, poly(ester urethane) urea and poly(ether ester urethane) urea at 85% porosity. A total of 60 scaffolds (20 each type) were implanted in a full thickness muscle wall replacement in the abdomens of 30 rats. The constructs were ultrasonically scanned every 2 weeks and harvested at weeks 4, 8 and 12 for compression testing or histological analysis. UEI demonstrated different temporal stiffness trends among the different scaffold types, while the stiffness of the surrounding native tissue remained unchanged. The changes in average normalized strains developed in the constructs from UEI compared well with the changes of mean compliance from compression tests and histology. The average normalized strains and the compliance for the same sample exhibited a strong linear relationship. The ability of UEI to identify herniation and to characterize the distribution of local tissue in-growth with high resolution was also investigated. In summary, the reported data indicate that UEI may allow tissue engineers to sequentially evaluate the progress of tissue construct mechanical behavior in vivo and in some cases may reduce the need for interim time point animal sacrifice.

Fig. 1

Study design. 30 female 16-wk old Lewis rats under anesthesia were implanted with the 60 scaffolds (20 scaffolds per type with 2 matched type scaffolds per animal). Scaffolds were implanted in the rat’s abdomen from the midline incision, with each sample sutured into a full thickness circular abdominal wall defect surgically created in the left or right side. The week 0 scan was performed 3 days after surgery and the scans afterwards were performed bi-weekly from the first scan day until sacrifice at week 4 (n=3, one on each group), week 8 (n=3, one on each group) and week 12 (n=4, one on each group). Tissue constructs were harvested for compression testing or histological staining.

Fig. 2

Normalized strain map (in color) laid over B-mode images (morphology) of the implanted scaffolds.

Fig. 3

Average strain and compliance over time. (A) Normalized strain obtained from UEI for scaffold averaged over 8 samples (except when scaffold develops hernia) and surrounding tissue over time. Lines between two adjacent points are simple connection, not interpolation of data. (B) Mean compliance (1/elastic modulus) from compression tests at weeks 0, 4, 8, 12 (scaffolds with hernia are excluded). Lines between two adjacent points are simple connection, not interpolation of data. (C) Scatter plots of the compliance and normalized strain values based on the same samples at corresponding time points. R is the correlation coefficient and P is the p-value for the correlation between compliance and normalized strain. Samples with hernia are represented by open symbols.

Fig. 4

Histology. (A) Masson’s trichrome stained sections for PCUU, PEUU and PEEUU at weeks 4, 8, and 12. Scale bar = 1 mm. (B) Representative magnified image of Masson’s trichrome stain for PCUU, PEUU and PEEUU at weeks 4, 8 and 12 (taken from the framed area in Fig. 4A). Scale bar = 100 μm. Dimension of each image is 852 μm (width) by 639 μm (height). (C) Mean collagen area percentage within the scaffold. The values are expressed as mean +/− standard deviation.

Fig. 5

Comparison of normal and herniated implants. (A) Normalized strain map for normal and herniated PCUU sample at week 12. The dimension of each B-mode image is 14 mm (width) by 6.4 mm (depth). (B) Stress-strain curve from direct compression tests for PCUU samples at week 12, where normal samples (in blue, red, black) and samples with hernia (in yellow, green, pink) show different behavior. Blue and green curves are from the normal and herniated implants respectively shown in Fig. 5A. (C) Masson’s trichrome staining of a PCUU sample with hernia at week 12. Sectioning was performed perpendicular to the scaffold’s top and bottom surface. The trichrome stain indicates scaffold and muscle tissue as purple (center) and red/magenta (both sides) respectively. Right side is the rectus abdominis (RA) muscle, and left side are the external oblique (EO) muscle, internal oblique (IO) muscle and transversus abdominis (TA) muscle (from top to bottom). Scale bar = 1 mm.

Fig. 6

Local distribution of scaffold degradation and cell in-growth. (A) Normalized strain map for PEUU sample at week 4. The dimension of the B-mode image is 14 mm (width) by 6.4 mm (depth). For better contrast in color, the dynamic range of the color map was adjusted from 0 to 0.4. (B) Masson’s trichrome staining of the PEUU sample in Fig. 6A at week 4. Within the scaffold, the region near the center stains white which indicates insignificant cellular in-growth compared to the outer regions which stain red/purple. Scale bar = 1 mm. (C) Magnified image of the representative area (green frame) in the central white region in Fig. 6B. The light blue structure (polymer) is slightly infiltrated with cellular and extracellular material. Scale bar = 100 μm. (D) Magnified image of the representative area (blue frame) in the surrounding pink region of the scaffold. Significant collagen (blue) along with a large number of cells (red) are observed. Scale bar = 100 μm.