Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs

Abstract

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (Ø10 mm, 2.34 mm thick), with zero or three nutrient channels (Ø1 mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct EY, glycosaminoglycan (GAG), and collagen contents, whereas rocking had detrimental effects on GAG and collagen versus static control. Nutrient channels increased EY as well as GAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of EY (~400 kPa) and glycosaminoglycans (GAG; ~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs.

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement

“…[41][42][43] Thus, the in vitro culture conditions for molded constructs will need to be optimized to produce improved cell viability, better ECM deposition, and higher mechanical stiffness at the center. This may be accomplished by dynamic loading, 20 culture in hydrodynamic environments that encourage nutrient exchange, 24,44 or even the ordered introduction of channels within the construct to facilitate more consistent nutrient exchange to the central regions of these large constructs. 45,46 A limitation in this study is the use of juvenile bovine MSCs to develop engineered cartilage constructs that clinically will be applied to elderly patients.…”

Matrix Production in Large Engineered Cartilage Constructs Is Enhanced by Nutrient Channels and Excess Media Supply

“…For biomechanical assessment, construct dimensions were measured before an unconfined compression (10% specimen height) and relaxation (*11,000 s); E Y was calculated from the relaxed tissue load and the undeformed construct dimensions. 14,28 Photographs of constructs were taken to assess the degree of channel filling and images were imported into NIH ImageJ (http://imagej.nih.gov/ij/) for data analysis. Day 56 channel area was normalized to day 0 area (based on B1 mm channel) to measure percentage channel filling.…”

“…This reduction in nutrient path length within large constructs produced tissues with higher compressive mechanical properties and improved matrix distribution over channel-free constructs, although differences in the total matrix content were insignificant. [12][13][14] Despite these advances, our limited understanding of the specific nutrient requirements for construct synthesis compromises the application of culture strategies to larger constructs. This notion is supported by the variability of media supply rates found in the literature (Table 1).…”

Nutrient Channels Aid the Growth of Articular Surface-Sized Engineered Cartilage Constructs

“…36,37 The system was scaled up such that each Ø40 mm construct was cast in a three-dimensional (3D) printed mold and held by a culture rack within a 473 mL polypropylene container with a polyethylene lid (Fig. 1).…”

“…10,35 In our recent studies, the combination of suspension culture and sufficient glucose supply permitted Ø10 mm constructs with 3 or 12 evenlyspaced Ø1 mm channels (CH3 or CH12, respectively) to attain functional properties on par with those of Ø4 mm constructs, including native E Y and GAG content. 36,37 Many tissue engineering studies using bovine chondrocytes opt for the use of primary cells. 19,[38][39][40] In this approach, numerous calf joints may be harvested and pooled together to obtain enough chondrocytes for large-scale studies.…”