Elsevier

Biomaterials

Volume 31, Issue 26, September 2010, Pages 6756-6763
Biomaterials

Effect of media mixing on ECM assembly and mechanical properties of anatomically-shaped tissue engineered meniscus

https://doi.org/10.1016/j.biomaterials.2010.05.039Get rights and content

Abstract

This study investigated the hypothesis that controlled media mixing will enhance tissue formation and increase mechanical properties of anatomically-shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2% w/v alginate, cross-linked with 0.02 g/mL CaSO4, and injected into molds of menisci. Engineered menisci were incubated for up to 6 weeks. A mixing media bioreactor was designed to ensure proper mixing of culture medium while protecting constructs from the spinning impeller. Impeller speeds were calibrated to produce Reynolds number (Re) of 0.5, 2.9, 5.8, 10.2, and 21.8. Constructs were divided a tested in confined compression and in tension to determine the equilibrium and tensile moduli, respectively. Media stimulation resulted in a 2–5 fold increase in mechanical properties and a 2–3 fold increase in matrix accumulation in constructs over 6 weeks in culture. Benefits from mixing stimulation for collagen accumulation and compressive modulus appeared to peak near Re 2.9, and decreased with increased mixing intensity. This study suggests that fluid mixing can be optimized to enhance mechanical properties of anatomically-shaped engineered constructs.

Introduction

Meniscal lesions are frequent injuries that lead to degeneration of knee articular cartilage [1]. Cadaveric meniscal allografts remain a preferred method of treatment [2], but this approach suffers from the scarcity of donor tissue and the risk of disease transmission. Moreover, size matching is of high importance because geometry is crucial for proper functional performance [3], [4]. Collectively, these factors have spurred interest in meniscal tissue engineering (TE).

Current meniscal TE efforts have focused on the repair of focal defects through the use of stem cells [5], [6] and scaffold materials [7], [8]. There have also been efforts to characterize the behavior of meniscal fibrochondrocytes and how these cells generate extracellular matrix (ECM) in scaffolds with simple geometry, including agarose [9], alginate [10], chitosan-graft-poly(N-isopropylacrylamide) [11], ploy (ɛ-caprolactone) [12], polyglycolic acid (PGA) [9], polyethylene terephthalate [13], and poly(l-lactic acid) [14]. Few studies have attempted to engineer whole menisci [15], [16], [17], due to the large size and the challenges associated with replicating the complex geometry.

Many biomaterials have been used to engineer small tissue samples, few can be formed into a prescribed geometry, especially an anatomical one such as the meniscus. Fewer still, can be combined with cells and formed into a desired shape while maintaining cell viability. Recently, the generation of anatomically-shaped engineered menisci based on MRI and μCT images was made possible using alginate combined with tissue injection molding [15], [18]. These studies were encouraging, but the implants had heterogeneous matrix distribution and mechanical properties that were significantly worse than those of native tissue. Producing constructs that mimic the mechanical properties of native tissue still remains a challenge.

Controlled media mixing is widely used to stimulate TE constructs seeded with articular chondrocytes (AC) [19], [20], [21], [22] and has been used with meniscal fibrochondrocytes [13], [23]. Several studies demonstrated that mixing bioreactors increase the amount of extracellular matrix (ECM) 3–9 fold and mechanical properties 3–4 fold for TE cartilage [9], [13], [20], [21], [22]. Media mixing stimulation is relatively simple to implement compared to direct compressive or tensile stimulation.

Based on the large size and unusual geometry of anatomically-shaped TE menisci, significant gradients in nutrient transport and ECM composition likely exist [15]. We hypothesized that media mixing will enhance transport of nutrients and ECM around and inside of TE constructs, improving the amount and homogeneity of ECM assembly. To test this hypothesis, we developed a bioreactor to control the extent of media mixing and determined how mixing affected the spatial pattern of ECM assembly and mechanical properties in anatomically-shaped TE menisci.

Section snippets

Molded constructs

Molds for generating anatomically-shaped TE menisci were made as previously described [15], [24]. Briefly, bovine meniscal fibrochondrocytes were isolated from freshly slaughtered 1–3 day old calves by 0.3% collagenase digestion. Cells were then seeded into sterile 2% w/v low viscosity high G-content alginate at 50 × 106 cells/mL. The alginate-cell suspension was combined with 0.02 g/mL CaSO4 and injected into either silastic impression molds of bovine menisci or μCT/MRI-based ABS plastic molds

Construct appearance and composition

All groups of engineered constructs retained shape for the duration of culture (Fig. 2, column 1). Large volume static samples at 6 weeks exhibited a dense region of tissue formation at the center of the cross-sections (Fig. 2, columns 1 and 2). Visual inspection of cross-sections at 6 weeks showed an increase in tissue homogeneity with increased mixing intensity. As mixing intensity increased, the constructs contained a darker and more opaque center compared to static samples (Fig. 2, column

Discussion

We investigated the hypothesis that media mixing will enhance transport of nutrients and ECM around and inside of TE meniscal constructs, improving the amount and homogeneity of ECM assembly. We found that the process of ECM assembly in injection molded TE menisci was significantly altered by culture in a mixing bioreactor (Fig. 4, Fig. 5, Fig. 6). The primary effects were to redistribute ECM, forming a tissue that was more spatially homogeneous than static controls, with the exception of the

Conclusions

This study began with the hypothesis that mixing media stimulation would enhance construct development. We found that mixing does affect ECM accumulation in large volume anatomically-shaped TE menisci. The largest effect of mixing was the redistribution of ECM and the enhanced homogeneity in large volume constructs. However, mixing stimulation was not uniformly beneficial as indicated by DNA and ECM loss to the media and increased rate of scaffold degradation. But intermediate intensities

Acknowledgments

The authors would like to thank the following funding sources: The Alfred P. Sloan Foundation, AO Research Foundation F-08-10B, and Cornell BME NSF GK-12 program.

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