Deficiency of hyaluronan synthase 1 (Has1) results in chronic joint inflammation and widespread intra-articular fibrosis in a murine model of knee joint cartilage damage

Abstract

Objective Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis, we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1−/−). Design Femoral groove cartilage of WT and Has1−/− mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. Results Acute responses (1–2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1−/− mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. Conclusions We conclude that the ineffective repair of injured cartilage in Has1−/− joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.

Pirfenidone reduces subchondral bone loss and fibrosis after murine knee cartilage injury

“…Mice were housed in groups of 4–5 males, which were treated with no surgery (naïve), sham operation, or cartilage injury, without or with PFD; individual mice were then assigned towards experimental outcomes as needed. With a mouse under anesthesia, cartilage injury to the patellar groove of the right knee was performed as previously described 42 . Briefly, after medial parapatellar arthrotomy, femoral groove cartilage was debrided from a rectangular surface (~2-mm wide × 4-mm long) without penetrating the subchondral bone.…”

“…Using an estimated absorption efficiency of ~20% 43 and average weights for each genotype, the daily dosage of PFD absorbed into the bloodstream upon ingestion of PFD chow was estimated by cage. For each group (naïve control with normal chow (NN), naïve with PFD chow (NP), sham (SN), sham with PFD (SP), injured (IN), injured with PFD (IP)), the minimal number of mice needed for each experimental outcome was determined based on previous studies 42; 44 , and mouse numbers in experimental groups are given (Table S-1) with experiment timelines (Figure S-1). Treatments were the same within a cage, and mice within each cage were randomly assigned to experimental outcomes as described below.…”

“…Joint wide pathology ( n = 3) was assessed in naïve and operated joints, without and with PFD treatment, as previously described 42; 44 for all experimental groups. These joints were then reserved for subsequent μCT, as described further in the Methods below.…”

“…For histology ( n = 3), skin and muscle were removed, joints decalcified, processed and embedded as described 42 . Specimens were sectioned (6-μm thick) with sagittal sections 1–60, 61–120, and 121–190 spanning the medial, central, and lateral regions respectively 44 .…”

“…Accordingly, in this study we use gene expression, histology, gross pathology, and micro-computed tomography (μCT) to evaluate the effects of PFD after cartilage injury in the murine joint. Additionally, among the numerous genes linked to the tissue injury response, hyaluronan synthase 1 (Has1) has been linked to abnormal wound healing and hypertrophy in the skin, muscle, and lungs 8; 38–40 , as well as the synovial membranes of OA patients 41 and murine joints after cartilage injury 42 . Therefore, we also use homozygous Has1 knockout (Has1 −/− ) mice, which show abnormal wound healing 38 and chronic inflammation and fibrosis with joint injury 42 , to examine whether PFD can alter the outcome in the absence of Has1.…”