Influence of Intermittent Pressure, Fluid Flow, and Mixing on the Regenerative Properties of Articular Chondrocytes

Scott E. Carver, Carole A. Heath
Department of Chemical Engineering, Iowa State University, Ames, Iowa
50011-2230; telephone (515) 294-4828; fax: (515) 294-2689; e-mail:
cheath@iastate.edu
Received 1 July 1998; accepted 13 May 1999

Summary:
This paper examines the effects of physical stimuli on growing cartilaginous tissue constructs from chondrocytes embedded in a polyglycolic acid matrix. In order to develop an articular cartilage replacement suitable in vivo, the tissue construct must have an ECM with native levels of type II collagen and sulfated glycosaminoglycans (GAG) and a compressive modulus comparable to healthy tissues. Previous studies have shown that a three dimensional environment is needed for type II collagen production and dynamic pressure increases collagen concentration. It also been demonstrated that either intermittent pressure or fluid flow alone can increase GAG level. Convection helps to overcome mass transfer limitations, which results in larger tissue constructs than in an unmixed system.

The authors created a system that combined mixed and pressurized environments with fluid flow and studied their effects on foal articular chondrocyte cultures over a 6-week period. Mixing was provided by growing cells in spinner flasks. For applying intermittent pressure (5 seconds on/15 seconds off), a reactor that applied 3.44 MPa for 20 min every four hours was used. Medium perfusion took place during the rest of the time the cultures were in the reactor. They performed a series of five experiments: (1) 1 week in spinner, 5 weeks in reactor with no pressure, (2) 1 week in spinner, 5 weeks in reactor with pressure, (3) 2 weeks in spinner, 4 weeks in reactor with pressure, (4) 4 weeks in spinner, 2 weeks in reactor with pressure, and (5) 6 weeks in spinner. The cultures were assayed for cell concentration, GAG and collagen concentrations and compressive moduli.

Assays for the concentration of GAG and type II collagen demonstrated that a combination of pressure and flow led to higher GAG and collagen concentration than from just applying pressure or fluid flow alone. This suggested a synergistic relationship of the physical stimuli and that the optimal culture environment was one that had both pressure and fluid flow. From SEM images, cells that benefitted from convective mass transfer had earlier ECM development, resulting in a denser ECM by week 6. Mixing also promoted cellular attachment and distribution, which helped the tissue grow larger in size.

The compressive modulus determined from measurement of the strength of the material and was shown to be directly proportional to GAG level, which meant that those cells in mixing and pressurized environments were more resistive to compression. However, the compressive modulus in the tissue construct was not nearly high as that in native tissues. The authors suggested that it may be due to GAG not aggregating to form a similar structure found in vivo. Another possible explanation was that the low levels of collagen limited the tissue’s resistance to compression as collagen normally serves to aggregate proteoglycan of the ECM. The paper finishes by indicating the importance of physical stimuli on composition and structural properties that can be used to produce functional tissues.

Relevance
Osteoarthritis is the most common form of arthritis in the United States and can normally be reversed if detected early by injections. The studies discussed in this paper are useful for developing tissue grafts for patients in later stages of osteoarthritis, when the defect is large. By studying the stimuli that affect the regenerative properties of articular chondrocytes, the tissue construct can be grown in such a way that it would act as healthy cartilage tissue.

This paper demonstrates the importance of applying physical stimuli that cells normally experience in vivo for engineering functional tissues. These conditions will affect the structural properties and protein composition of the tissue. This is an important lesson for tissue engineering as mimicking the environment in vivo will likely lead to tissues that are structurally and compositionally similar to those found in the body.