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Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

Background Recurrent bleeding into joints results in a crippling arthritis, the consequence of intra-articular (IA) pathologic lysed red blood cell iron and inflammatory signals from macrophages/monocytes. Hemophilic mice that experience joint bleeding develop synovial and osteochondral changes that mimic the changes seen in human hemophilia. We previously examined in hemophilia B mice the efficacy of conventional recombinant factor IX (rFIX) compared to a half-life extended polyethyleneglycol-conjugated factor IX (N9-GP) to support wound healing when administered "on demand" after the initiation of induced joint hemorrhage. In the "on-demand" scenario, N9-GP improved wound healing when compared to rFIX, although neither treatment group demonstrated completely normal joint healing (as observed in hemostatically normal mice that experienced the same injury). We test the hypothesis that the prophylactic use of N9-GP may support wound healing in synovium, cartilage, and bone and may do so more effectively than rFIX. Method: FIX deficient mice were treated prophylactically with either rFIX or N9-GP at the dose of 250 IUnits human factor IX/kg 30 minutes, 24hr or 72hr prior to the induction of unilateral hemarthosis. Injured hemostatically normal (iWT) mice were treated with I.V. normal saline as the controls. Joint diameter was measured daily to quantify joint swelling. Plasma and tissues were harvested from cohorts of mice (n = 8-10/group) at two weeks after hemarthrosis. Pathologic synovial hyperplasia, neovascularization, iron deposition and macrophage infiltration of the synovium were quantified by histologic examination. Integrity of trabecular bone neighboring the hemarthrosis was quantified by microComputed Tomography (mCT). Result: WT mice developed no detectable joint swelling at days 1-14 after the joint bleeding challenge, whereas mice treated prophylactically with rFIX were protected from joint swelling only in the cohort treated 24 hours prior to hemarthrosis. Joint swelling did not develop in groups that received prophylactic N9-GP using any of the 3 schedules. No mouse treated with rFIX (n = 28 total) or N9-GP (n=27 total) developed FIX inhibitors or FIX-binding IgG. Synovitis was graded 0 to 10 by Valentino mouse hemophilic synovitis score, with a mean score in the iWT mice of 1.0±0.5 and in the injured untreated FIX-/- mice of 4.9±1.4. Synovitis was greatly diminished although not eliminated by prophylactic N9-GP given 30 minutes prior to injury (1.7±1.2), and this result contrasted with the relatively less good healing provided by rFIX given 30 minutes prior to injury (3.9±1.1). Synovitis, iron staining, and persistence of macrophages within the joint were each diminished by N9-GP and rFIX prophylaxis. The degree of protection diminished as the interval between dosing and injury increased. Better protection was seen using N9-GP when compared to rFIX at each timepoint. Strikingly, rFIX at this dose provided minimal or no prophylactic protection from synovial neoangiogenesis (vessels/hpf) at two weeks after hemarthrosis even when given at the shortest interval preceding the injury. Injured, untreated FIX-/- mice developed 17.6±5.1 vessels/hpf compared to rFIX given 30 minutes prior to injury (16.1±4.8), 24 hours prior (17.8±5.4) or 72 hours prior (17.1±6.3). In contrast, iWT mice displayed 7.1±3.1 at two weeks after injury, which was comparable to N9-GP given 30 minutes prior to injury (9.4±2.4) and better than N9-GP given 24 hours prior (12.6±3.3) or 72 hours prior (13.1±5.6). Consistent with previous reports, hemophilic mice when compared to iWT mice demonstrated acute losses of bone mineral density in the bone adjacent to the joint experiencing bleeding, as well as structural defects in the number, thickness, and spacing of the bone trabeculae. Prophylactic rFIX or N9-GP given at 30 minutes or 24 hours prior to injury resulted in protection that did not differ statistically from findings in iWT mice. When prophylaxis was given at 72 hours prior to injury, significant volumetric bone mineral density loss of at least 20% was evident in the rFIX group (vBMD 98 mgHA) compared to iWT (131 mgHA) and N9-GP given 72 hours prior (125 mgHA). Conclusion: The support of normal wound healing in joint and bone following hemarthrosis was significantly improved by N9-GP prophylaxis when compared to rFIX in hemophilia B mice, with neoangiogenesis particularly improved. Disclosures Knudsen: Novo Nordisk: Employment. Monroe:Novo Nordisk: Honoraria, Research Funding. Ezban:Novo Nordisk: Employment. Bateman:Novo Nordisk: Honoraria, Research Funding; Baxter: Honoraria, Research Funding. Monahan:CSL Behring: Consultancy, Honoraria; Pfizer: Honoraria; Chatham LLC: Consultancy; Baxter/Baxalta: Consultancy, Honoraria, Research Funding; Prolor: Research Funding; Bayer: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding; Asklepios BioPharmaceutical: Consultancy, Patents & Royalties: Author I.P. licensed by UNC to AskBio, Research Funding.