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.

Introduction

In the tissue engineering paradigm scaffolds are designed from biodegradable materials to support tissue in-growth, extracellular matrix (ECM) elaboration, and eventual replacement of the implant site with native tissue. In many instances the scaffolds provide important mechanical function that must eventually be transferred to the native tissue without dropping below some critical value that would precipitate mechanical failure. The remodeling process is dynamic and complex, dependent not only on the scaffold chemistry and morphology, but on the host implant site, inflammatory response, mechanical environment, and disease state to name but a few important parameters. Despite this complex situation, scaffold design principles provide some general options in terms of chemistry and processing to tune degradation provided sufficient in vivo data are available to guide such design modifications. The collection of such in vivo data, particularly mechanical parameters at the implant site, often requires destructive analysis and substantial animal use to allow the construct to be explanted and characterized histologically and mechanically [1], [2], [3], [4]. Ideally, non-invasive methods would allow the monitoring of the scaffold site with the provision of some of the desired mechanical parameters in situ and temporally [5], [6].

In considering some of the non-invasive methods that have been reported, Dhollander et al. employed magnetic resonance imaging (MRI) to evaluate the implantation of alginate-based scaffolds containing human allogenic chondrocytes for the treatment of knee cartilage defects [7]. Despite the cartilage-like appearance the repair tissue exhibited in MRI, the study observed a poor correlation between clinical outcome and MRI findings. This finding agreed with the findings by Tins et al., where it was shown that the morphological appearance of cartilage implants on MRI did not correlate with histological findings of tissue development [8]. Similar to MRI, computed tomography (CT) [9], [10], [11] provides only morphological information, and scanning and image reconstruction procedures are quite extensive [9], [10], [11]. MR-based elastography (MRE) can measure the stiffness of a scaffold, but is limited by relatively poor spatial resolution (>a few mm) and extensive scanning and image reconstruction procedures [12], [13]. Sinkus et al. applied MRE to a polyvinyl alcohol breast phantom and demonstrated that MRE utilizing an advanced reconstruction algorithm was capable of depicting 6 mm objects size at a minimum [13]. Most current MRE is limited with spatial resolution near 5 mm [14], [15]. Rogowska et al. [16], [17] evaluated optical coherence elastography (OCE) as a method for assessing the mechanical properties of atherosclerotic arterial samples and different tissue-mimicking phantoms, measuring their elastic modulus with high resolutions of 18 μm [16] and 5 μm [17]. OCE provides superior spatial resolution, but the imaging depth is only 2–3 mm, which currently limits its broad application in vivo.

Ultrasound elasticity imaging (UEI) or ultrasound (US) elastography has the potential to become a valuable tool for characterizing the mechanical and structural changes of the implanted engineered tissues at reasonably high resolution with substantial imaging depth. Since it was introduced in early 1990s as a non-invasive tool to investigate mechanical properties of biological tissues [18], [19], [20], UEI or US elastography has been applied in a wide spectrum of applications for native biological tissues and organs in vitro and in vivo [21], [22], [23], [24]. With some uniqueness in signal processing has been adopted in each approach, the most commonly used elasticity imaging techniques are based on 2-D correlation-based speckle tracking methods [25], [26], [27], [28]. This approach uses US radio frequency (RF) signal to estimate tissue motion, tracking US speckles between consecutive frames during tissue deformation. Speckle displacements are estimated from correlation lags corresponding to the maximum correlation coefficient between the frames. Displacement can be accumulated over frames throughout the entire deformation procedure, and strain can be derived from accumulated displacement. In the past several years, US elastography or UEI based on speckle tracking has been shown to have great potential for clinical use, but mostly in applications involving native tissues [29], [30], [31], [32].

In our previous study [5], UEI was applied in vivo to detect the degradation of the poly (1,8-octanedio-co-citrate) pre-polymer (POC) scaffolds subcutaneously implanted in the backs of mice. With limited sample numbers (n = 3) and time points (days 1 and 7), the results supported the feasibility of UEI as a non-invasive monitoring tool for mechanical property changes of tissue scaffolds in vivo. The change in strains from UEI due to scaffold degradation compared well with direct mechanical measurements; however, any tissue in-growth was not included in the investigation. To investigate systematically the correlation of the dynamic, adaptive mechanical and structural property changes with varying rates of scaffold degradation and tissue in-growth, porous scaffolds made from three biodegradable elastomers with varying degradation rates were used in this study: poly(ether ester urethane) urea (PEEUU) for a fast degradation rate, poly(ester urethane) urea (PEUU) for a moderate degradation rate and poly(carbonate urethane) urea (PCUU) for a slow degradation rate.

Tissue constructs made of biodegradable elastomers offer attractive mechanical properties for many soft-tissue engineering applications, which can be fabricated by electro-spinning natural polymers, synthetic polymers or polymer blends. In previous work [33], [34], [35], [36], [37], [38], biodegradable polyurethanes were developed and processed into three-dimensional scaffolds for a variety of mechanical support applications in vivo. For this study, we implanted scaffolds made from three types of polyurethanes (PEEUU, PEUU and PCUU) as full thickness replacements of the rat muscular abdominal wall, and then systematically applied UEI using a high frequency US scanner at time points for up to 12 weeks. Compression testing in vitro was chosen to compare with strains from UEI to provide similar conditions for tissue deformation [5]. Histological assessments were performed to monitor scaffold infiltration with tissue and morphological remodeling. The objective of this study was to demonstrate the ability of non-destructive US methodology to provide an alternative method for the assessment of mechanical behavior as three different types of elastic, biodegradable scaffolds remodeled in a mechanically loaded environment in situ.