gms | German Medical Science

Objective: Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) are important during differentiation and development. Their signalling is tightly regulated by several BMP antagonists, such as Noggin that binds to BMPs and thus inhibits the ligand receptor interaction. This inhibition is particularly undesirable when BMPs are applied during bone regeneration. Previously a Noggin resistant GDF5 mutant (N445T) was identified that causes multiple synostosis syndrome. This disease is typically caused by loss-of-function mutations in Noggin, hence a GDF5 mutant resulting in the same phenotype will exhibit altered antagonist sensitivity. We hypothesized that the healing outcome after mutant GDF5 (N445T) treatment would be better than after wild-type (wtGDF5) application.

Method: A 4 mm segmental femoral bone defect was created in female rats, stabilized by a unilateral external fixator. Within the defect a collagen scaffold was loaded with 25µl of wtGDF5 (2 mg/ml, n=6) or 30μl of N445T (1.7 mg/ml, n=7). Another group loaded with 50μl of BMP2 (1 mg/ml, n=8) and an unloaded empty collagen scaffold group (Collagen, n=4) were also included in the study. Bone healing was assessed at 2, 4, and 6 weeks post-operation by in vivo micro-computed tomography and at 6 weeks by qualitative histology. Significance was set at p<0.05. Time to consistent bony union varied between groups: Collagen: none healed; BMP2: 8/8 at 2 wks; wtGDF5: 2/6 at 4 wks, 3/6 at 6 wks; N445T: 6/7 at 4 weeks and 7/7 at 6 weeks. At 4 and 6 weeks, mineralized callus volume (BV, mm3) and bone mineral content (BMC, mg) was significantly greater (p<0.019) in defects treated with wtGDF5 compared to the Collagen group. BV and BMC in the N445T group were significantly greater at 4 and 6 weeks compared to the wtGDF5 group (p<0.008), but significantly lower at 2 and 4 weeks compared to the BMP2 group (p<0.021). Histological analysis at week 6 revealed no cartilage formation in the Collagen group, only remnants in the BMP2 group, large amounts in the wtGDF5 group and intermediate cartilage formation in the N445T group. Defects treated with BMP2 had new cortices periosteally and marrow elements, while in the wtGDF5 and N445T groups, ossification fronts were visible intercortically.

Results and conclusions: A Noggin resistant GDF5 mutant (N445T) was shown to have higher regenerative capacities than the wtGDF5 in stimulating femoral defect healing in rats. Defects treated with N445T achieved bony union at later time points compared to those treated with BMP2, which promoted healing by inducing periosteal bone formation, possibly without a prior cartilage phase, while both the wtGDF5 and the N445T promoted endochondral bone formation. The mechanism through an antagonist resistant GDF5s might be a promising new concept for bone regeneration. Its higher efficacy associated with lower doses and stimulation of the physiological process of endochondral ossification might help to minimize the risk of ectopic bone formation.