Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds

https://doi.org/10.1016/j.jbiomech.2003.10.005Get rights and content

Abstract

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.

Introduction

Articular cartilage defects may occur as a result of traumatic injury, or from degenerative diseases such as osteoarthritis. Such defects have a limited capacity for repair, and may require novel regenerative approaches to restore biological and mechanical functionality of damaged or diseased tissue. Tissue engineering strategies target the use of cell populations in order to regenerate functional repair tissue. One approach is to deliver isolated chondrocytes (Brittberg et al., 1994) or chondro-progenitor cells to voids created by the removal of dysfunctional tissue. To facilitate delivery, cells may be encapsulated in or attached to a biocompatible scaffold. A variety of materials are under investigation for cartilage tissue engineering, including biologically derived and synthetic polymers and hydrogels (Glowacki, 2000). Such scaffolds provide a three-dimensional template in which cells can initiate and mediate tissue repair.

The ultimate success of cell-seeded constructs for cartilage repair depends on appropriate long-term maturation of functional repair tissue in vivo, and integration with surrounding cartilage and bone. To achieve these goals, constructs may benefit from a period of in vitro culture prior to implantation to initiate a cellular repair response (Schaefer et al., 2002; Lee et al (2003a), Lee et al (2003b)). Culture conditions may be optimized to enhance extracellular matrix (ECM) synthesis, or stimulate proliferation and chondrogenesis of progenitor cells. ECM deposition in vitro may increase the mechanical functionality of the cell-seeded construct to withstand loading forces encountered after implantation (Butler et al., 2000). This could be critical for scaffolds that are significantly weaker than native cartilage (Lee et al., 2001; Rotter et al., 2002). Recent studies have incorporated physical stimulation of cell-seeded constructs during culture. Bioreactors have been designed to convect culture medium around (Vunjak-Novakovic et al., 1999) or perfuse directly through scaffolds (Pazzano et al., 2000), or impart hydrostatic pressure on the constructs (Carver and Heath, 1999; Mizuno et al., 2002; Angele et al., 2003). For each physical factor, conditions have been identified in which ECM synthesis and/or cell division was accelerated over static controls.

The effects of dynamic compression on chondrocyte biosynthesis have been well-characterized in cartilage explants and chondrocyte-seeded scaffolds. In explants, continuously applied dynamic compression (Sah et al., 1989) and dynamic tissue shear (Jin et al., 2001) have been found to increase synthesis of proteins and proteoglycans (PGs) over 24 h of loading. In chondrocyte-seeded agarose hydrogels, continuous dynamic compression increased chondrocyte biosynthesis during short-term loading (Buschmann et al., 1995; Lee and Bader, 1997; Hunter and Levenston, 2002). Intermittent compressive loading increased PG synthesis during short-term loading (Chowdhury et al., 2003) and material properties and GAG content over several weeks of culture (Mauck et al (2000), Mauck et al (2002)). Additional studies showed that dynamic compression (Davisson et al., 2002; Lee et al (2003a), Lee et al (2003b)) and shear (Waldman et al., 2003) could affect chondrocyte biosynthesis in tissue engineering scaffolds. These studies suggest that loading may enhance the long-term deposition of ECM in cell-seeded constructs during in vitro culture.

In this study, we investigated the effects of long-term dynamic compression on cellular biosynthesis and ECM retention in a chondrocyte-seeded peptide hydrogel, a scaffold actively under investigation for applications to cartilage repair (Kisiday et al., 2002). Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. Thus, our first objective was to identify an appropriate protocol that would increase PG synthesis over control cultures maintained in free-swelling conditions, using a well-established chondrocyte culture scaffold (Benya and Shaffer, 1982; Buschmann et al., 1992). Combinations of periods of loading and free-swelling culture were explored to maximize retention of newly synthesized ECM. Based on these results, an optimized loading protocol was applied to cell-seeded peptide hydrogels with the second objective of quantifying the effects of dynamic compression on synthesis and accumulation of ECM, viable cell density, and mechanical properties for up to 39 days of in vitro loading.

Section snippets

Isolation of chondrocytes and casting of cell-seeded peptide and agarose hydrogels

Chondrocytes were isolated from femoral condyles of 1–2-week-old bovine calves as previously described (Ragan et al., 2000). Chondrocytes were seeded in a self-assembling peptide hydrogel of sequence AcN-KLDLKLDLKLDL-CNH2 at 0.4% (w/v) or 2% low melting temperature agarose (Gibco) hydrogels in 1.6-mm-thick slab structures as previously described (Kisiday et al., 2002). Cells were seeded at a concentration of 15–30×106 cells ml, similar to seeding densities previously found to be responsive to

Normalization of radiolabel incorporation and retention in the scaffold

In our previous studies, incorporation of 35S-sulfate and 3H-proline in free-swelling chondrocyte-seeded peptide and agarose hydrogels reached stable levels from days 15 to 28 after casting (Kisiday et al., 2002). Given that radiolabel incorporation in this study was performed between 15 and 30 days after casting, steady-state radiolabel incorporation was expected for each free-swelling time course. This was, indeed, found to occur in all experiments (data not shown): for any given time course,

Discussion

In this study, we found that application of dynamic compression on alternate days was optimal for long-term stimulation of PG synthesis in hydrogel cultures. The extent to which periods of free-swelling culture were necessary to intersperse between periods of loading was unexpected, given that continuous loading over short-term culture (24–48 h) was previously found to stimulate PG synthesis (Buschmann et al., 1995; Lee and Bader, 1997). In the present study, the significant inhibition of PG

Acknowledgements

This research was supported by NIH Grant AR33236 and Biotechnology Training Grant, the DuPont-MIT Alliance, and the Cambridge-MIT Institute.

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