Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Summary

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Abstract

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue^1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz^1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue^4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

Protocol

1. Isolation of Primary Articular Chondrocytes

Harvest 10-15 full thickness cartilage slices from the articular surfaces of animal joints (e.g. the metacarpal-phalangeal joint of skeletally mature cows obtained from a local abbatoir).

1. Place cartilage slices in a 100 mm Petri dish and Incubate in 20 ml of 0.5% protease in Ham's F-12 (w/v) for 2 hr at 37 °C. Rinse three times in Ham's F-12 culture media, and incubate with 20 ml of 0.15% Collagenase A in Ham's F-12 culture media overnight at 37 °C.
2. Filter the cell suspension through a 200 mesh screen filter into a clean dish. Wash the filter with 5 ml of Ham's F-....

Discussion

The described method for applying controlled mechanical stimuli to cell-seeded agarose hydrogels allows for the direct investigation into the effects of dynamic compressive forces on chondrocyte metabolism. The use of the custom-testing rig in conjunction with the retaining rings provided lateral constraint for the constructs to avoid potential problems of sample tipping. The use of dead-weighted loading platens secured by sets crews ensures direct contact with constructs despite potential differences in sample height. R.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ham's F-12	Thermo Fisher Scientific	SH3001002
Collagenase A	Sigma Aldrich Ltd.	C0130
Protease	Sigma Aldrich Ltd.	P5147
Fetal Bovine Serum	Sigma Aldrich Ltd.	F1051
Ascorbate	Sigma Aldrich Ltd.	A4034
Antibiotics/antimycotics	Sigma Aldrich Ltd.	A5955
HEPES	Bioshop Canada Ltd.	HEP001
Trypan blue	Sigma Aldrich Ltd.	93595
Reichert Bright-Line Hemacytometer	Hausser Scientific	1490
Quant-iT PicoGreen	Invitrogen	P7589
Papain from papaya latex	Sigma Aldrich Ltd.	P3125
Ammonium Acetate	Sigma Aldrich Ltd.	A1542
Ethyldiaminetetraacetic Acid	Sigma Aldrich Ltd.	E9884
DL-Dithiothreitol	Sigma Aldrich Ltd.	43819
Low Melting Point Agarose, Type VII	Sigma Aldrich Ltd.	A9045
Mesh Screen (200) Filter	Sigma Aldrich Ltd.	S4145
Mach-1 Micromechanical Tester	Biomomentum Inc.	V500cs
Compression Loading Jig	Custom-built		Similar product could be supplied by Biomomentum Inc.
Falcon 24 Well Culture Plate	Thermo Fisher Scientific	B353047
β-Liquid Scintillation Counter	Beckman Coulter	LS6500
[3H] Proline	Perkin-Elmer	NET323005MC
[35S] Sulfur	Perkin-Elmer	NEX041005MC