gms | German Medical Science

Objectives: Autologous bone graft, the gold standard in the rising number of bone graft procedures [1], comes with disadvantages (pain, morbidity). Therefore, alternative materials or material combinations are needed and Demineralised Bone Matrix (DBM) enriched with Bone Morphogenetic Protein 2 (BMP2) might be a feasible strategy.

Methods:

In vitro: 20, 40 or 80 µg of recombinant human BMP2 (Wyeth, GB) per 100 mg DBMputty (DIZG, Germany) were mixed in a special mixing syringe (Medmix Systems AG, CH) and incubated in a cell culture medium. Elution samples were taken over a time period of 56 days and the BMP-2 concentration in the samples and in the remaining DBM after 56 days (extraction: guanidine-HCl method) was quantified (ELISA kit, R&D Systems, Germany). The osteoinductivity was determined by culturing C2C12 cells with the elution samples and testing the metabolic (AlamarBlue, Biozol, Germany) and alkaline phosphatase (AP) activity. In addition, C2C12 cells were cultured on the remaining mixture samples after the 56 days and an AP staining was performed. (Statistic: Kruskal-Wallis; Mann-Whitney, Bonferroni-Holm)

In vivo: Drill holes (6 mm x 15 mm) were placed in the femur, humerus, metacarpus and -tarsus of 16 merino-mix sheep (>2.5 years) and the defects were filled with DBM or DBM enriched with BMP2 (80µg/200mg). Empty defects and defects filled with autologous bone graft were used as a control. After a healing time of 3 and 9 weeks, the defects were analysed radiographically (µCT) and histologically (Safranin O/von Kossa, Movat Pentachrom). (Statistics: Wilcoxon; ANOVA, Dunnett's T3)

Results:

In vitro: The mixing process was simple and resulted in a homogeneous mixture. The samples showed a burst release of BMP2 within the first four days, followed by a slow and incomplete release until day 56. C2C12 cells cultured with the elution samples showed a significant enhanced metabolic and AP activity. Cultivating C2C12 cells on BMP2/DBM mixture after 56 days of elution showed an intensive blue AP staining for all groups, indicating that BMP2 is still bound to the matrix.

In vivo: A centripetal bone growth could be observed in all defects. The diaphyseal samples seemed to heal better than the metaphyseal. The group treated with autologous cancellous bone showed the highest increase in newly formed bone. DBM enriched with BMP2 increased the bone healing compared to the singular use of DBM. The DBM group and the negative control showed similar healing tendencies.

Conclusion: Even after 56 days of elution, sufficient quantities of BMP2 with a high osteoinductive activity are still bound in the DBM. In vivo, augmented bone healing could be observed in the DBM/BMP2 group. This is an easy and reproducible perioperative method for enriching bone graft with BMP2. And throughout the long-term binding of BMP-2, a persisting activity in the graft is ensured, which might be beneficial for the healing of lager bone defects. [1. Greenwald AS et al. 68th AAOS 2008]