Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Summary

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Abstract

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

Introduction

We have designed a loading bioreactor that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. This device is primarily designed as a bioreactor for engineered replacements for articular cartilage; it could also be used for other load-bearing tissues in the human body. Our motivation in this bioreactor design stems from Drachman and Sokoloff ¹, who made the seminal observation of abnormal formation of articular cartilage in paralyzed chick embryos due to absence of motion. Similarly, physical exercise is essential for development of normal muscle and bone. In keeping with....

Protocol

1. Biaxial Loading Bioreactor Design

1. The bioreactor employs two stages manufactured by Thor-labs (Newton, MA) for precision-guided laser applications for applying uniaxial or biaxial mechanical strain to engineered tissues, with great precision of loading dose (amplitude and frequency) and application to a wide variety of tissue culture conditions from single to 24 well plates (Figure 1).
2. Bi-axial loading is accomplished by two TravelMax stages (LNR50SE). These stages are mounted orthogonally in an XZ configuration. The horizontal stage provides dynamic shearing motions by oscillating along the X-axis. The vertical stage provides....

Results

The device was tested by using agarose gels seeded with 20 million cells/ml chondrocytes and cultivated in the presence of uniaxial (compression) or biaxial (compression and shear) mechanical loading. Primary porcine chondrocytes were isolated from the articular cartilage of 2-4 month old pigs. 5 mm diameter and 1.5 mm thick samples were cultured in 2 ml of defined chondrogenic culture medium (High glucose DMEM, 1% ITS+ Premix, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 2.5 μg/ml amphotericin B, 50 μg.......

Discussion

We have designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to tissue engineered constructs fabricated for transplantation. The device can be used as a bioreactor for in vitro cultivation of engineered biocomposites or as a testing device to describe the mechanical characteristics of the native tissue or after other treatments prior to. The device subjects engineered tissue constructs to biaxial mechanical loading with great precision of loading dose (amplitude and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
DMEM, High glucose, pyruvate	Invitrogen	11995
Agarose Type II	Sigma	CAS 39346-81-1
Penicillin Streptomycin Glutamine 100X	Invitrogen	10378-016
ITS+ Premix	BD Biosciences	354352
Pen Strep Glutamine	Invitrogen	10378-016
Amphotericin B	Invitrogen	041-95780
Ascorbic Acid	Sigma	A-2218
Nonessential Amino Acid Solution 100x	Sigma	M-7145
L-proline	Sigma	P-5607
Dexamethasone	Sigma	D-2915
Recombinant Human Transforming Growth Factor β1	R&D Systems	240-B-010
EQUIPMENT
Model 31 Load Cell (1000 g)	Honeywell	AL311
Model 31 Load Cell (1000 g)	Honeywell	AL311
Single Channel Display	Honeywell	SC500
50 mm Linear Encoded Travelmax Stage with Stepper Actuator	Thorlabs	LNR50SE/M
Two Channel Stepper Motor Controller	Thorlabs	BSC102
50 mm Trapezoidal Stepper Motor Drive (2)	Thorlabs	DRV014
Adjustable Kinematic Locator (4)	Thorlabs	KL02
Precision Right Angle Plate	Thorlabs	AP90/M
Vertical Mounting Bracket	Thorlabs	LNR50P2/M
Solid Aluminum Breadboard	Thorlabs	MB3030/M
Gel Casting System with 1.5 mm and 0.75 mm spacer plates	BioRad	#1653312 and #1653310
Disposable Biopsy Punch, 5 mm	Miltex, Inc.	33-35
16 mm hollow punch	Neiko Tools
Non-Tissue Culture Treated Plates, 24 Well, Flat Bottom	BD Biosciences	351147
Ultra-Moisture-Resistant Polysulfone sheet for loading platens	McMaster-Carr	86735k19	Custom-machined