The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy

Liming Bian, Chieh Hou, Elena Tous, Reena Rai, Robert L. Mauck, Jason A. Burdick

Research output: Contribution to journalArticlepeer-review

241 Scopus citations


Hyaluronic acid (HA) hydrogels formed via photocrosslinking provide stable 3D hydrogel environments that support the chondrogenesis of mesenchymal stem cells (MSCs). Crosslinking density has a significant impact on the physical properties of hydrogels, including their mechanical stiffness and macromolecular diffusivity. Variations in the HA hydrogel crosslinking density can be obtained by either changes in the HA macromer concentration (1, 3, or 5% w/v at 15 min exposure) or the extent of reaction through light exposure time (5% w/v at 5, 10, or 15 min). In this work, increased crosslinking by either method resulted in an overall decrease in cartilage matrix content and more restricted matrix distribution. Increased crosslinking also promoted hypertrophic differentiation of the chondrogenically induced MSCs, resulting in more matrix calcification in vitro. For example, type X collagen expression in the high crosslinking density 5% 15 min group was ∼156 and 285% higher when compared to the low crosslinking density 1% 15 min and 5% 5 min groups on day 42, respectively. Supplementation with inhibitors of the small GTPase pathway involved in cytoskeletal tension or myosin II had no effect on hypertrophic differentiation and matrix calcification, indicating that the differential response is unlikely to be related to force-sensing mechanotransduction mechanisms. When implanted subcutaneously in nude mice, higher crosslinking density again resulted in reduced cartilage matrix content, restricted matrix distribution, and increased matrix calcification. This study demonstrates that hydrogel properties mediated through alterations in crosslinking density must be considered in the context of the hypertrophic differentiation of chondrogenically induced MSCs.

Original languageEnglish (US)
Pages (from-to)413-421
Number of pages9
Issue number2
StatePublished - Jan 2013
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health grant R01EB008722 . The authors thank Ms. Emily Zhang for her assistance in the experimental work.


  • Chondrogenesis
  • Crosslinking
  • Hyaluronic acid
  • Hypertrophy
  • Mesenchymal stem cells
  • Mineralization


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