Optimizing the osteo-immunomodulatory balance: threshold-saturation effects of BMP-2-loaded allografts in the...

The Masquelet induced membrane technique (MIMT) is an established strategy for reconstructing critical-sized bone defects (CSDs). However, the limited osteoinductivity of allografts often necessitates supplementation with bone morphogenetic protein-2 (BMP-2), whose supra-physiological doses may cause excessive inflammation. This study aimed to identify a BMP-2 concentration range that balances osteogenic efficacy with systemic safety in the MIMT setting.

A 1.5-cm critical-sized radial defect was created in 36 New Zealand white rabbits. In Stage I, a polymethyl methacrylate spacer was implanted to induce membrane formation. In Stage II, performed 4 weeks later, the spacer was removed and the defect was filled with freeze-dried allograft standardized to the nominal defect volume, calculated by the cylindrical formula (V = πr²L). Using a radius of 0.2 cm and a length of 1.5 cm, the nominal defect volume was 0.188 cm³ (≈0.188 mL). BMP-L, BMP-M, and BMP-H allografts were loaded with rhBMP-2 at 5, 10, and 20 μg mL^-1, corresponding to nominal doses of 0.94, 1.89, and 3.77 µg per defect; the Allograft group received unloaded allograft. Bone repair was assessed by serial X-ray at 2, 4, 8, 12, and 16 weeks, by micro-CT at 2, 8, and 16 weeks, and by histological and immunohistochemical analyses up to 16 weeks. Systemic inflammatory and reparative dynamics were evaluated at 12 timepoints by ELISA.

At 16 weeks, radiographic and micro-CT analyses showed that BMP-M and BMP-H achieved significantly higher bone mineral density and BV/TV than BMP-L and allograft alone (P < 0.05). Osteogenic marker expression increased dose-dependently from 0 to 10 μg mL^-1. However, increasing the dose from 10 to 20 μg mL^-1 did not yield meaningful additional benefit in cortical bridging or trabecular maturation. The BMP-H group also showed an early post-Stage II inflammatory rebound in serum CRP and IL-6, whereas BMP-M maintained a more stable systemic immune profile with a higher IL-10/IL-6 ratio.

Within the tested conditions, 10 μg mL^-1 BMP-2 appeared to provide the most favorable balance between bone regeneration and systemic inflammatory stability in MIMT.