Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration

Abstract

Supraphysiologic doses of bone morphogenetic protein-2 (BMP-2) are used clinically to promote bone formation in fracture nonunions, large bone defects, and spinal fusion. However, abnormal bone formation (i.e., heterotopic ossification) caused by rapid BMP-2 release from conventional collagen sponge scaffolds is a serious complication. We leveraged the strong affinity interactions between heparin microparticles (HMPs) and BMP-2 to improve protein delivery to bone defects. We first developed a computational model to investigate BMP-2-HMP interactions and demonstrated improved in vivo BMP-2 retention using HMPs. We then evaluated BMP-2-loaded HMPs as a treatment strategy for healing critically sized femoral defects in a rat model that displays heterotopic ossification with clinical BMP-2 doses (0.12 mg/kg body weight). HMPs increased BMP-2 retention in vivo, improving spatial localization of bone formation in large bone defects and reducing heterotopic ossification. Thus, HMPs provide a promising opportunity to improve the safety profile of scaffold-based BMP-2 delivery.

Fig. 1. Fabrication and morphogen loading of HMPs.

HMPs were fabricated from heparin methacrylamide using the free radical initiators ammonium persulfate (APS) and N,N,N′,N′-tetramethylethane-1,2-diamine (TEMED) in a water-in-oil emulsion at 55°C. BMP-2 was loaded onto HMPs by incubating BMP-2 and HMPs together at 4°C for 16 hours. BMP-2 binds to the sulfate groups on heparin.

Fig. 2. In silico assessment of BMP-2 release from alginate/PCL tissue-engineered constructs.

(A) BMP-2 unbinds from HMPs at a rate of k_off, rebinds to HMPs at a rate of k_on, and diffuses through the alginate hydrogel and surrounding tissue at diffusion rates of D_{BMP, Alginate} and D_{BMP, Tissue}, respectively. (B) Heat map depicting the sensitivity of BMP-2 release into surrounding tissue to ±2.5-, 5-, and 10-fold changes in key parameter values. (C) Three-dimensional representations of BMP-2 release from tissue-engineered constructs into surrounding soft tissue at 14 days after injury. (D) Volume fraction analysis of BMP-2 in alginate hydrogel (red line), in surrounding tissue (blue line), and bound to HMPs (green line). Treatment groups are as follows: (i) 30 μg of BMP-2 + 1 mg of HMPs in alginate, (ii) 30 μg of BMP-2 + 0.1 of mg HMPs in alginate, (iii) 30 μg of BMP-2 + 0.01 mg of HMPs in alginate, and (iv) 30 μg of BMP-2 in alginate.

Fig. 3. In vivo tracking of BMP-2 released from alginate/PCL tissue-engineered constructs.

(A to D) Longitudinal IVIS images of subcutaneously implanted constructs containing 2.5 μg of fluorescently labeled BMP-2 loaded onto 0.1 or 1 mg of HMPs at (A) day 0, (B) day 1, (C) day 4, and (D) day 7. (E) Quantification of fluorescence within implantation sites and fit to one-phase exponential decay curves (R² = 0.88 for BMP-2, R² = 0.78 for BMP-2 + 0.1 mg of HMPs, and R² = 0.68 for BMP-2 + 1 mg of HMPs.) (F) Decay constants obtained from BMP-2 retention curves (*P < 0.05 as indicated). (G) Radiant efficiency of constructs explanted after 21 days (n = 7 to 8; *P < 0.05 as indicated).

Fig. 4. Representative radiographs and micro-CT reconstructions of femoral defects treated with alginate/PCL tissue-engineered constructs.

Constructs contained 30 μg of BMP-2, 30 μg of BMP-2 + 0.1 mg of HMPs, or 30 μg of BMP-2 + 1 mg of HMPs. Radiographs were taken at 4, 8, and 12 weeks. White arrows indicate heterotopic ossification. Mineral density evaluated by micro-CT at 12 weeks is depicted in sagittal sections.

Fig. 5. Quantification of bone volume and biomechanical properties of femoral defects treated with alginate/PCL tissue-engineered constructs.

(A) Representative micro-CT slice demonstrating that bone volume was divided into defect volume and heterotopic volume based on 6-mm circular contours defined by the PCL nanofiber mesh. (B) Total bone volume at 4, 8, and 12 weeks was divided into (C) percentage of heterotopic bone volume and (D) percentage of defect bone volume (n = 13 to 14; *P < 0.05 as indicated). Excised femurs underwent torsion testing at 12 weeks after injury to calculate (E) stiffness and (F) maximum torque of regenerated bone. Mechanical properties were compared to that of intact femurs (n = 9 to 10).

Fig. 6. Femoral bone defect histology at 12 weeks after injury.

Femurs were decalcified, paraffin processed, sectioned, and stained with Safranin O/Gill’s hematoxylin, Safranin O/Fast Green, or H&E. Black and yellow arrows indicate HMPs.