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Functional tissue engineering involves the application of physical loads to promote the development of tissue constructs that can withstand the mechanical demands encountered in vivo [1]. Specifically, the goal of functional tissue engineering of articular cartilage is to develop an engineered cartilage construct that exhibits structure and function sufficient to replace or repair damaged articular cartilage. To accomplish this goal, bioreactors have been developed to apply mechanical stimulation to cell-laden constructs. Design strategies may impart various types of load including hydrostatic pressure, compression, or shear [2–5]. However, few bioreactors include instrumentation that allow for continuous monitoring of tissue development.  The successful in vitro development of functional tissue-engineered constructs could benefit from a method of assessment that allows for continuous evaluation of tissue while not compromising construct integrity, preserving the construct for continuous development and eventual implantation. Current methods for evaluating extracellular matrix (ECM) development and mechanical properties are time consuming and destructive to the construct, and require numerous replicates to obtain a comprehensive overview of construct quality. Nondestructive, continuous evaluation of a tissue construct during development can be useful not only for final clinical use, but also for determining appropriate bioreactor parameters to achieve sufficient structure and function.  Nondestructive measurement systems have been developed to assess construct mechanical properties as well as bulk-tissue development [6,7]. Preiss-Bloom et al. developed a bioreactor to mechanically stimulate tissue-engineered cartilage and measure real-time force response [6]. The bioreactor was outfitted with load sensors to continuously log construct resistance to deformation by the bioreactor. Such measurements give insight into the change in construct stiffness during stimulation and development in the bioreactor. Hagenmuller et al. developed a bioreactor that combines mechanical loading and online microcomputed tomography (μCT) for monitoring the development of engineered bone tissue [7]. Cartridge-like culture chambers were designed to allow for sterile mechanical stimulation and μCT monitoring of mineral deposition without removing the constructs.  Another potential method for nondestructive assessment of tissue formation is ultrasound. Ultrasonic techniques are sensitive to mechanical and biochemical properties of cartilage [8–10] and have the potential to nondestructively assess the quality of tissue-engineered cartilage during development. Ultrasonic waves are utilized to acquire acoustic images and make localized quantitative measurements of tissue properties. Propagation and scattering of ultrasonic waves depend on tissue composition and structure [11]. Specifically, the reflection coefficient, the fraction of ultrasound reflected from an interface with different acoustic impedances, is one parameter commonly used to evaluate tissue characteristics [12–16].  A number of studies have been conducted to examine the feasibility of ultrasound as a tool for diagnosis of osteoarthritis by measuring changes in ultrasonic parameters after spontaneous and selective enzymatic degradation of cartilage tissue [17–20]. Ultrasound has also been used as a tool for monitoring in vivo cartilage tissue development and repair [21–23]. However, ultrasound has only recently been used as a measurement tool for the evaluation of tissue-engineered cartilage [8,24] and has yet to be implemented for real-time evaluation of tissue development.  The objective of this work was to develop an instrumented bioreactor that could be utilized to stimulate and nondestructively evaluate tissue-engineered cartilage. Our dynamic compression bioreactor is instrumented with an ultrasonic transducer, load cells, and a video microscope for assessing ECM development and mechanical properties of tissue-engineered cartilage. Chondrocyte-laden hydrogel constructs were placed in the bioreactor and subjected to a three-part loading regime including: (1) a ramp, (2) sinusoidal compression, and (3) no load. This regime was repeated twice per day for 7 days. Constructs were nondestructively evaluated with ultrasound on days 0 and 7. Constructs were also evaluated on days 0 and 7 for cell viability, cell number, sulfated glycosaminoglycan (sGAG), and collagen content. Histological sections were stained for sGAG and collagen with safranin O and Masson's trichrome, respectively.

An Instrumented Bioreactor for Mechanical Stimulation and Real-Time, Nondestructive Evaluation of Engineered Cartilage Tissue