Purpose: Joint damage upon bleeding causes significant morbidity in patients with hemophilia, and adds to joint degeneration after trauma and major joint surgery. Even a single bleed damages cartilage, synovium and bone, but the pathophysiology is not completely understood. A large part of joint degeneration research uses smaller rodent models with histology as the primary outcome measure. However, histological changes take time to develop and are subject to interpretation despite initiatives to harmonize semi-quantitative scores. Determining proteoglycan synthesis rate, by incorporation of radioactive 35Sulphate (35SO42-) in cartilage, is a sensitive method to detect early cellular changes in cartilage previously applied in human and larger animal models. Isolating cartilage of small animals can be challenging, so a technique to shave off rat’s cartilage was developed and the 35Sulphate incorporation assay applied to study cartilage degradation following blood exposure.

Methods: A total of 13 factor VIII knock out (F8 KO) rats were bred and housed at Novo Nordisk A/S, Maaloev, Denmark. After euthanasia, the legs were removed and transported to UMC Utrecht, The Netherlands.

In vitro: within 24 hours after euthanasia the cartilage of six healthy F8 KO rats was obtained by shaving off cartilage fragments of the tibia plateau by use of a scalpel. All cartilage explants were then cultured for four days; in addition to culture medium, half of the cartilage samples were cultured with 50% v/v whole blood. After four days proteoglycan synthesis rate was determined by adding 35SO42- to the cultures for four hours. The 35SO42- becomes incorporated in new synthesized proteoglycans. After digesting the cartilage pieces, cetylpyridinium chloride was added to the samples to precipitate the proteoglycans and the amount of radioactivity was measured by liquid scintillation analysis. The 35Sulphate incorporation rate was normalized to the specific activity of the pulse medium, labeling time, and weight of cartilage.

In vivo: in seven F8 KO rats a unilateral joint bleed was induced by needle puncture and in the following four days until euthanasia, the animals received analgesia. At UMC Utrecht, the tibial cartilage was removed and proteoglycan synthesis activity determined according to the previously described method.

All animal experiments were performed under anesthesia and analgesia in accordance with and approved by the Danish Animal Experiments Council, Ministry of Food, Agriculture and Fisheries, Denmark.

Results: On average, a total of 1.6mg (0.8–3.1mg) cartilage per tibia could be obtained. The proteoglycan synthesis rate of healthy cartilage determined after four days of culturing in vitro was on average 49.5 nmol/h.g. This could be modulated by in vitro exposure to blood, resulting in a diminished synthesis: 7.7 nmol/h.g (p=0.0191), corresponding to an 84% decrease comparable to previously published human experiments. Moreover, when a joint bleed was induced in vivo a diminished proteoglycan synthesis rate could also be demonstrated by this assay (13.5 vs 7.5 nmol/h.g, p=0.0151), although more variation was seen.