Growth factor-mediated augmentation of long bones: evaluation of a BMP-7 loaded thermoresponsive hydrogel in a murine femoral intramedullary injection model

Abstract

Background: Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible.

Methods: In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 μl saline, 100 μl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 μl hydrogel loaded with 1 μg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination.

Results: Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples.

Conclusions: In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation.

Keywords: BDI-hydrogel; Bone morphogenetic protein 7 (BMP-7); Murine femoral intramedullary injection model; Osteoporosis.

Fig. 1

Schematic representation of the VOIs selected for analysis of cortical and trabecular bone microarchitecture (a), the distal growth plate as an example of an anatomical landmark (b), representative images of the manually drawn ROIs for analysis of cortical (c), and trabecular (d) bone. The distal growth plate and the division into greater trochanter and femoral neck served as internal references, i.e., positioning of the VOIs for analysis of the distal and proximal femora as well as the femoral neck. For examination of the distal femur (1), VOI started 50 slices apart from the growth plate (b) and consisted of 100 slices. The VOI in the region diaphysis (2) started 400 slices apart from the growth plate and consisted of 100 slices. In case of the proximal femur (3), trab and cort VOIs consisted of 80 slices each and started 50 slices distal from the division into greater trochanter and femoral neck. For analysis of the femoral neck (4), cort VOI and trab VOI included 20 slices each and an offset of 15 slices from the division into greater trochanter and femoral neck was used

Fig. 2

Significant association of normalized TbN (a) and normalized BMD (b) with normalized BV/TV determined at the femoral neck (red triangle), proximal (blue circle) and distal femur (white circle), respectively. Spearman’s rank coefficients of correlation: femoral neck, R = 0.94 (a) and R = 0.79 (b); proximal femur, R = 0.88 (a) and R = 0.90 (b); distal femur, R = 0.95 (a) and R = 0.88 (b); each p < 0.001

Fig. 3

Hematoxylin-Eosin (HE)-stained sagittal sections of demineralized femora obtained 4 and 12 weeks after surgery. Femora on the right side were treated with either BMP-7 loaded BDI-hydrogel, intramedullary application of plain BDI hydrogel or saline (SHAM), the left sides serve as untreated controls. (Scale bar, 100 μm)

Fig. 4

Immunohistochemical detection of BMP-7 in mineralized femora obtained 4 weeks (left column) and 12 weeks (right column) after intramedullary application of BMP-7 loaded BDI-hydrogel, plain BDI-hydrogel, or saline (SHAM) (Scale bar, 100 μm)