This study shows that, using the current tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage that is suitable for in vivo testing in laryngotracheal reconstruction in rabbits. In vitro biomechanical testing designed around the functional requirements of the cartilage graft indicated that tissue-engineered cartilage generated from auricular chondrocytes, but not articular or nasal chondrocytes, had the mechanical properties necessary for in vivo testing as a graft. Consistent with the biomechanical results, biochemical characterization showed that, compared to the articular and nasal samples, auricular samples generally had more cell-produced extracellular matrix, more extensive immunostaining for collagen II, significantly higher GAG content and concentration, and significantly higher collagen content. The dGEMRIC method, a recently developed MRI method to assess cartilage GAG concentration and spatial distribution, revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples.
Biomechanical testing was performed to determine the equilibrium unconfined compression modulus of the tissue-engineered cartilage samples. The in vitro conditions were chosen to simulate the in vivo loading conditions and the maximum applied load the engineered cartilage is expected to experience when implanted into the rabbit trachea, as determined by in situ measurements. For this reason, in vitro biomechanical tests were performed under load control, rather than displacement control, to ensure that samples were tested up to the maximum expected in vivo load.
Biomechanical testing revealed a profound difference in mechanical integrity and stiffness of the auricular samples when compared to the articular and nasal samples. Five of six auricular samples successfully completed biomechanical testing and had measured equilibrium unconfined compression moduli approaching those of native hyaline cartilages tested under similar conditions of unconfined compression (210 ± 93 kPa at 3 weeks and 100 ± 65 kPa at 6 weeks in the present study, compared to 270 kPa for trochlear groove cartilage of 6-month-old cows,27
677 ± 223 kPa for humeral head cartilage of 1- to 2-year-old cows,28
and 310 ± 180, 570 ± 170, and 800 ± 330 kPa for femoral, patellar, and humeral cartilage from cows of unreported age29
). The one auricular sample that did not complete testing exceeded the range of the linear variable displacement transducer during the highest load level (215.8 mN). In contrast, all tissue-engineered cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness for completion of the biomechanical testing. These samples were either damaged during handling or exceeded the range of the linear variable displacement transducer during the first experimental load level (68.7 mN), preventing estimation of equilibrium unconfined compression modulus. Based on these findings, it is clear that the articular and nasal samples were unsuitable for in vivo
testing in laryngotracheal reconstruction in rabbits, because handling less demanding than that present during implantation damaged some samples, and small loads (less than one-third of that expected to exist in vivo
) compressed the remaining samples by 50% or more.
Determining whether the tissue-engineered cartilage generated by auricular chondrocytes in the present study is truly suitable for laryngotracheal reconstruction in rabbits will ultimately require in vivo testing, particularly since relatively little is known about the biomechanics of laryngotracheal reconstruction. The equilibrium unconfined compression modulus of human costal cartilage, the current gold standard for laryngotracheal reconstruction in children, has not, to our knowledge, been reported. In the present study, it proved infeasible to harvest a sample of rabbit costal cartilage of the appropriate size and shape for biomechanical testing. In addition, the equilibrium unconfined compression modulus of cricoid cartilage or tracheal rings have also not been reported, and would not represent a standard with which to compare the properties of engineered cartilage because of the composite nature of the trachea.
The differences in quality of cartilage generated in the present study likely indicate that the baseline conditions used in this study are more favorable to auricular chondrocytes than to articular or nasal chondrocytes, not that articular and nasal chondrocytes are fundamentally unsuitable for use in this tissue engineering application. It has previously been shown that a number of factors can affect the behavior of chondrocytes during tissue engineering, and these factors can affect chondrocytes from different anatomic locations in different ways. Exposure to growth factors during expansion 16,30–32
can improve the chondrogenic capacity of chondrocytes, and chondrocytes harvested from different anatomic locations have been found to respond to the same growth factors in different ways or to different extents.16
Similarly, scaffold composition and architecture can have a significant effect on chondrocyte behavior 35
(and, in purely mechanical terms, use of a stiffer scaffold in the present application might allow more samples to withstand the loads the engineered cartilage is expected to experience when implanted). In addition, dynamic mechanical loading of agarose cartilage constructs has been shown to stimulate accumulation of GAGs and increase stiffness.36,37
Although the responses to mechanical loading of chondrocytes from different anatomic locations have not been compared, it is reasonable to expect that chondrocytes conditioned by different mechanical environments in vivo
, such as the mechanical environment of the shoulder versus the mechanical environment of the ear, would respond differently to mechanical signals applied during culture in vitro
. Experiments to optimize the present tissue engineering approach, with respect to growth factors, scaffold composition and architecture, and mechanical loading, are ongoing. Such optimization may result in chondrocytes from articular, nasal, or other cartilages being viable, if not preferred, alternatives to the use of auricular chondrocytes.
It is potentially very useful to use chondrocytes from different anatomic locations to produce engineered cartilage. The present results suggest that chondrocytes harvested from one anatomic location, the ear, are capable of producing functional tissue-engineered cartilage for use in another anatomic location, the laryngotracheal segment. The results also suggest that chondrocytes from different anatomic locations will require different isolation and culture conditions to permit their use in this specific tissue engineering application for laryngotracheal reconstruction. Other cartilage tissue engineering applications that employ comparable approaches can be expected to require similar optimization with respect to anatomic location of chondrocyte harvest.
Viewing these findings in the context of other tissue engineering studies,13–16,38,39
we suggest that optimization for specific sources of chondrocytes, whether anatomic location or even articular cartilage zone, is emerging as an important consideration when developing cartilage tissue engineering approaches that use autologous chondrocytes. The results demonstrate that the rabbit is a valuable model for continued development of the present cartilage tissue engineering approach for pediatric laryngotracheal reconstruction. The rabbit also provides a useful model for investigating the different ways in which chondrocytes from different anatomic locations behave when applied in tissue engineering approaches. Continued development of the approach presented here will allow further investigation of differences in chondrocytes from different anatomic locations while pursuing the long-term goal of tissue engineering autologous cartilage grafts for pediatric laryngotracheal reconstruction.