High-Throughput Mechanical Evaluation of Tissue Engineered Constructs During Incubation +1Lujan, T J; 1Wirtz, K M; 2Bahney, C S; 1Madey, S M; 1Johnstone, B; 2Bottlang, M

+1Legacy Research and Technology Center, Portland, OR, 2Oregon Health and Science University, Portland, OR tlujan@biomechresearch.org

INTRODUCTION: Engineered neocartilage presently lacks the mechanical integrity fundamental to the function of load-bearing tissue. However, the environmental conditions (e.g. mechano-stimulation) that effectively stimulate functional development may be rapidly identified by measuring material properties during culture.[1] Measuring transient mechanical behavior is also useful in evaluating degradable scaffolds, since volumetric scaffold degradation should coincide with matrix deposition. Unfortunately, contemporary bioreactors are unable to efficiently measure material properties during culture, and therefore little is understood about temporal growth patterns.[2] The objective of this study was to develop and validate a batch-processing mechano-stimulation bioreactor, capable of automatic and reliable material property evaluation. The bioreactor’s ability to map time-dependent mechanical properties was tested by monitoring degradable hydrogels during collagenase digestion. METHODS: A mechano-active transduction and evaluation (MATE) bioreactor was constructed to accommodate a tissue-culture laboratory environment (Fig. 1A). The culture module that houses the constructs imitates a six-well plate (Fig. 1B). Six electromagnetic voice coil motors (VCM) were housed beneath the culture wells (Fig. 1C) to apply unconfined compression by raising specimens into contact with the loading posts (Fig. 1D). To minimize system complexity and hardware, the VCM input voltage was used to predict the actual forces applied to the constructs. Calibration between voltage and force was performed in a custom casing equipped with a load cell. System accuracy was calculated during variable static and dynamic loading (amplitudes: 0.1 to 10 N; frequencies: 1 Hz, 10 Hz). To verify that viscoelastic material properties could be reliably measured in weak and strong constructs, six soft hyrdogels and six stiff bovine cartilage specimens were evaluated in the MATE and an Instron (DynaMight, Norwood MA; force accuracy = 0.01 N, displacement accuracy = 1 µm).

Fig. 1: MATE images. A) Dimensions are 15x16x22 cm. B) Six-well culture plate holds specimens. C) Electrical components housed beneath specimens. D) VCMs raise each specimen into contact with posts The MATE monitored the effect of collagenase in two groups of six cell-free hydrogels: a non-degradable group (10% w/v PEGDA) and degradable group (10% w/v of a collagenase sensitive PEGDA). Degradable hydrogels were synthesized by covalently embedding a collagenase sensitive peptide,[3] and exposing for two minutes to visible light and an initiator system.[4] Each hydrogel was placed in culture dishes with 2 ml of PBS that contained 0.005% collagenase type II. Daily mechanical tests measured material properties during ten-days of culture under standard incubation conditions (37°C, 5% CO2). After preloading to 0.10 N, specimens were quasi-statically compressed to 0.20 N, allowed one minute to creep, and sinusoidally loaded for 10 cycles (frequency = 1 Hz, amplitude = 0.1 N). Equilibrium and dynamic modulus were automatically calculated in the LabVIEW software. The effect of the test system (MATE vs Instron) and the time-dependent effects of incubation were measured with paired t-tests and repeated measures ANOVA, respectively. If significance was detected (p<0.05), LSD tests were used for pairwise comparisons. All results are reported as mean ± standard deviation.

RESULTS: The average forces delivered by the MATE strongly correlated with the target forces prescribed by the user during static (R2 = 1) and dynamic loading (R2 = 1). For static loading, there was low variability between the six MATE chambers when 0.1 N was prescribed (error = 0 ± 10%) and when 10 N was prescribed (error = 0.1 ± 0.4%). Dynamic loading was accurate at amplitudes of 0.20 N or greater (0.20 ± 0.02 N, error = 0 ± 8%). The force output at 10 Hz was on average 2.1% greater than at 1 Hz (p<0.001). On average, the equilibrium and dynamic modulus determined from the MATE’s six chambers were within 5% of Instron results for soft hydrogels (p=0.3, p=0.4, respectively; Fig. 2A), and within 8% for mature cartilage (p=0.2, p=0.3, respectively; Fig. 2B). There was over a 10x difference in modulus values between the PEGDA constructs and the bovine cartilage.

Fig. 2: Comparison of material properties acquired by the MATE and Instron for a) compliant tissue and b) stiff tissue. The MATE system detected transient structural changes in the degradable hydrogels (Fig. 3). Material properties in the degradable group were altered during culture (41% change in thickness, p<0.001; 42% change in equilibrium modulus, p=0.02; 20% change in dynamic modulus, p=0.005), but were unaltered in the non-degradable group (1% change in thickness, p=0.19; 3% change in equilibrium modulus, p=0.47; 1% change in dynamic modulus, p=0.27).

Fig. 3: Effect of collagenase digestion on A) thickness and B) modulus. DISCUSSION: The engineering of load-bearing tissue demands clear and reliable communication with regard to the functional outcome of specific culture conditions. The MATE bioreactor was developed to offer a reliable tool to facilitate this communication and expedite the potential clinical transfer of tissue engineering technology. The absence of bioreactors with the MATE’s functionality is likely related to inherent limitations in standard electromechanical instrumentation.[5] The MATE’s novel use of VCM technology permits force and displacement to be individually measured in multiple-chambers without force sensors, specimen-platen separation, or feedback mechanisms. Once validated, the MATE detected reductions in the mechanical integrity of collagenase sensitive hydrogels, indicating scaffold degradation. Since the MATE tests six specimens at a time, each daily evaluation lasted less than five minutes. This demonstrates that the MATE can quantify the functional effect of culture environments with minimal time and materials. REFERENCES: [1] Preiss-Bloom O. et al., Artif Org, 33(4), pp. 318-327 [2] Martin, I., et al., Trd Btech, 22(2), pp. 80-86. [3] Mann, B. K., et al., Biomat, 22(22), pp. 3045-3051. [4] Waldman, S. D., et al., Tiss Eng, 10(9-10), pp. 1323-1331. [5] Bikram, M., et al., Ann BioE 35, 796-807. ACKNOWLEDGEMENTS: Financial support from NIH #AR059433

Spotlight No. 78 • ORS 2011 Annual Meeting