Perlecan knockdown significantly alters extracellular matrix composition and organization during cartilage development

Functional repair of diseased or injured tissues remains a significant challenge for regenerative medicine. Extracellular matrix (ECM) composition during tissue assembly (e.g., development) is dramatically different from that of the homeostatic adult and may aid the design of engineered therapeutics that will promote regrowth and functionality of damaged tissues. Implementing a top-down approach, we evaluated perlecan (HSPG2), a pericellular ECM protein critical for proper cartilage development. When HSPG2 is knocked down, the non-lethal phenotype mimics the musculoskeletal defects observed in human Schwartz-Jampel syndrome. We previously demonstrated that HSPG2 knockdown significantly decreased the stiffness of the interstitial matrix and chondrocytes in developing cartilage (Xu et al., 2016b). However, it is not clear what changes occur in ECM structure and organization to cause the observed decrease in stiffness when HSPG2 is knocked down. Therefore, we performed proteomic analysis using mass spectrometry to test the hypothesis that ECM components that contribute to the developing structural integrity of cartilage will be in lower abundance in Hspg2^C1532Y-Neo mutants (Neo/Neo) than in wildtype littermates (+/+). Relative increases in the abundance of ECM and associated proteins highlighted the expected developmental changes in ECM composition between embryonic day (E)16.5 and postnatal day (P)3 timepoints. The relative abundance of multiple proteins was significantly higher in Neo/Neo vs. +/+ P3 mice, contrasting our original hypothesis. Further investigation confirmed that the total collagen content increased with HSPG2 knockdown. However, similar collagen fibril diameter and ECM volume fractions between P3 Neo/Neo and +/+ littermates suggested HSPG2 knockdown did not affect matrix protein organization and assembly. Sulfated glycosaminoglycan (GAG) abundance showed no difference between groups, but safranin O staining indicated atypical GAG deposition. This, and increased hyaluronic acid binding protein expression, suggested increased hyaluronic acid deposition leads to decreased mechanical stiffness in Neo/Neo cartilage. Chondrocytes in perlecan-deficient cartilage may upregulate the synthesis of key ECM to compensate for decreased matrix stiffness; however, without HSPG2, GAGs accumulate and the matrix assembles into a structure with less mechanical integrity. Overall, the study of perlecan-deficient mice will provide insight into the influence of HSPG2 on chondrogenesis, matrix secretion, and functional cartilage assembly to enhance our design of engineering scaffolds that mimic cartilage to promote restoration of tissue function.