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. 2017 Aug;23(15-16):847-858.
doi: 10.1089/ten.TEA.2016.0467. Epub 2017 Mar 27.

* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability

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Free PMC article

* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability

Robert J Nims et al. Tissue Eng Part A. .
Free PMC article

Abstract

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-β before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

Keywords: cartilage tissue engineering; collagen; constrained culture; proteoglycans; pyridinoline.

Conflict of interest statement

No competing financial interest exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
(A) Study 1 (agarose) and Study 2 (CDMH) overview including channeling, transforming growth factor-β3 supplementation, caging, and construct time points. (B) Cage assembly diagram (left) and with construct (right).
<b>FIG. 2.</b>
FIG. 2.
Study 1, agarose constructs: (A) Image of construct differences from FS (left) and cage (right; front faceplate removed for construct viewing) on day 112. (B) Morphological differences in cage growth from top and side perspectives (FS side view only shows middle of construct). (C) Swelling ratio differences between FS and caged constructs. *Denotes significant (p < 0.05) differences between factor levels. Denotes significant (p < 0.05) differences between individual groups at the same time point. X-axis spacing is not temporally representative. (D) Histological images (side view) of constructs (Safranin-O displays GAG, Picrosirius displays collagen). Dashed lines represent construct midlines and separates Safranin-O and Picrosirius Red images; dotted lines represent construct channels or preexisting channels; * are voids or tears in construct. GAG, glycosaminoglycan; FS, free swelling.
<b>FIG. 3.</b>
FIG. 3.
Study 1, agarose constructs: (A) equilibrium compressive modulus, EY, (B) dynamic modulus, G*, (C) GAG (%ww), (D) collagen (%ww), (E) GAG (%D0-ww), (F) collagen (%D0-ww), (G) cell density, (H) total cell content, (I) PYD (nmol/mL), (J) PYD/collagen (mol/mol), (K) collagen solubility. *Denotes significant (p < 0.05) differences between factor levels. Denotes significant (p < 0.05) differences in individual groups at the same time point. X-axis spacing is not temporally representative. PYD, pyridinoline; ww, wet weight.
<b>FIG. 4.</b>
FIG. 4.
Study 2, CDMH constructs: (A) Morphological differences (top and side profiles) of FS and cage groups on day 85. (B) Swelling ratio differences between FS and caged constructs. *Denotes significant (p < 0.05) differences between factor levels. Denotes significant (p < 0.05) differences between individual groups at the same time point. X-axis spacing is not temporally representative. (C) Histological (top view) images of constructs (Safranin-O displays GAG, Picrosirius displays collagen) on day 56. + represents channel location, * represent voids or tears in construct. CDMH, cartilage-derived matrix hydrogel.
<b>FIG. 5.</b>
FIG. 5.
Study 2, CDMH constructs: (A) equilibrium compressive modulus, EY, (B) dynamic modulus, G*, (C) GAG (%ww), (D) collagen (%ww), (E) GAG (%D0-ww), (F) collagen (%D0-ww), (G) cell density, (H) total cell content, (I) PYD (nmol/mL), (J) PYD/collagen (mol/mol), (K) collagen solubility. *Denotes significant (p < 0.05) differences between factor levels. Denotes significant (p < 0.05) differences between individual groups at same time point. X-axis spacing is not temporally representative.

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