A Novel Bioreactor System Capable of Simulating the In Vivo Conditions of Synovial Joints

Abstract

Any significant in vitro evaluation of cartilage tissue engineering and cartilage repair strategies has to be performed under the harsh conditions encountered in vivo within synovial joints. To this end, we have developed a novel automated physiological robot reactor system (PRRS) that is capable of recapitulating complex physiological motions and load patterns within an environment similar to that found in the human knee. The PRRS consists of a mechanical stimulation unit (MSU) and an automatic sample changer (ASC) within an environment control box in which the humidity, temperature, and gas composition are tightly regulated. The MSU has three linear (orthogonal) axes and one rotational degree of freedom (around the z-axis). The ASC provides space for up to 24 samples, which can be allocated to individual stimulation patterns. Cell-seeded scaffolds and ex vivo tissue culture systems were established to demonstrate the applicability of the PRRS to the investigation of the effect of load and environmental conditions on engineering and maintenance of articular cartilage in vitro. The bioreactor is a flexible system that has the potential to be applied for culturing connective tissues other than cartilage, such as bone and intervertebral disc tissue, even though the mechanical and environmental parameters are very different.

Keywords: biomaterials; biomechanics; bioreactor; cartilage; tissue engineering.

FIG. 1.

Design and assembly of components of the PRRS. The PRRS is connected to a personal computer with a user-friendly interface to control the device with I/O modules via actuators and sensors (a) and is connected to gas bottles to allow for regulation of the climate (b–d). The main parts of the PRRS include an MSU (f) with four degree of freedom (three linear axes [x, y, and z] and the rotational axis [around z]), an ASC (e), and an ECB (c, e). The load is applied to the samples through an indenter on the tissues (e, g). The carousel can contain up to 24 samples (e) fixed within the sample holders (h, i). ASC, automatic sample changer; ECB, environment control box; MSU, mechanical stimulation unit; PRRS, physiological robot reactor system.

FIG. 2.

The PRRS Labview-based user interface and environmental validation feedback loops. (a) Screenshot of graphical user interface used to control the bioreactor system. The Labview interface enables user-defined communication and programming of individual parameters, including the mechanical loading pattern (force, frequency, and duration), environmental parameters (temperature, gas composition), and the sampling rate of the force and actuator position feedback. The individual parameters are recorded in CSV log files for control, analysis and validation of the mechanical properties of the samples, as well as the environmental parameters (b). CSV, comma-separated value.

FIG. 3.

Cytotoxicity assay. Bovine articular chondrocytes from three different animals run in six biological replicates (n = 3, n = 6) were cultured for 3 days in extraction medium in either sample holders (container) or tissue culture dishes (plastic). No significant difference (p = 0.092) in the mitochondrial activity of the chondrocytes was found in both conditions as assayed by XTT. XTT, sodium 3,3′-[1[(phenylamino)carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate.

FIG. 4.

Engineering of cartilage and culture of articular cartilage explants. Chondrocytes were seeded on collagen scaffolds and cultured for 14 days in a standard cell culture incubator using differentiation medium supplemented with transforming growth factor β1. The cell-seeded scaffolds were mounted on CaP substrate and cultured for further 14 days either as controls in plastic dishes (top figure) or within the PRRS in the sample holders by application of 5 N dynamic compression (bottom figure). Histological sections of the cell-seeded scaffolds were stained with Safranin-O /Fast Green to highlight the deposition of the glycosaminoglycans. (a) Histological sections of the tissue-engineered cartilage tissues in control and PRRS cultures. (b) Depicts detailed view from regions of the cell-seeded scaffolds highlighted in squares. Cartilage formation and maintenance was possible in both conditions. A cartilaginous tissue with chondrocytes located in lacunae and visually a thicker cartilage layer formed in the samples cultured in the PRRS. Dynamic compression and shear stress upregulated the levels of transcripts encoding the cartilage-specific COL2 and ACAN, whereas levels of transcripts encoding COL1 were downregulated (c). Moreover, dynamic compression or shear stress retained the tissue integrity, as observed histologically (d).

FIG. 5.

Mechanical properties of bovine articular cartilage explants. (a, b) The force was recorded over time during a 50 N dynamic compressive loading (b depicts a detailed view of a). (c) The stiffness of the cartilage was determined through consecutive calibration cycles, and (d) from the first calibration cycle of the daily stimulation. The diagrams of force over time, and force/deformation are shown from one representative articular cartilage tissue.