Bone Morphogenetic Protein-2 Promotes Human Mesenchymal Stem Cell Survival and Resultant Bone Formation When Entrapped in Photocrosslinked Alginate Hydrogels

Abstract

There is a substantial need to prolong cell persistence and enhance functionality in situ to enhance cell-based tissue repair. Bone morphogenetic protein-2 (BMP-2) is often used at high concentrations for osteogenic differentiation of mesenchymal stem cells (MSCs) but can induce apoptosis. Biomaterials facilitate the delivery of lower doses of BMP-2, reducing side effects and localizing materials at target sites. Photocrosslinked alginate hydrogels (PAHs) can deliver osteogenic materials to irregular-sized bone defects, providing improved control over material degradation compared to ionically cross-linked hydrogels. It is hypothesized that the delivery of MSCs and BMP-2 from a PAH increases cell persistence by reducing apoptosis, while promoting osteogenic differentiation and enhancing bone formation compared to MSCs in PAHs without BMP-2. BMP-2 significantly decreases apoptosis and enhances survival of photoencapsulated MSCs, while simultaneously promoting osteogenic differentiation in vitro. Bioluminescence imaging reveals increased MSC survival when implanted in BMP-2 PAHs. Bone defects treated with MSCs in BMP-2 PAHs demonstrate 100% union as early as 8 weeks and significantly higher bone volumes at 12 weeks, while defects with MSC-entrapped PAHs alone do not fully bridge. This study demonstrates that transplantation of MSCs with BMP-2 in PAHs achieves robust bone healing, providing a promising platform for bone repair.

Keywords: bone morphogenetic protein-2; mesenchymal stem cell; osteogenesis; photocrosslinking; survival.

Figure 1

(A) Schematic illustrating synthesis of photocrosslinked alginate hydrogels (PAHs) containing MSCs and BMP-2; (B) gross morphological image of representative hydrogel 24 hours after synthesis; (C) release kinetics of 2 µg BMP-2 from PAH over 14 days in vitro (n=4). Sustained release of rhBMP-2 was observed during the first week; (D) ALP activity in MC3T3-E1 preosteoblasts when stimulated by eluted BMP-2 as demonstration of BMP-2 bioactivity; positive control is media supplemented with 100 ng mL⁻¹ fresh BMP-2; n=4; ^***p<0.001, ^****p<0.0001 vs. −BMP-2 at Day 7; ^##p<0.01, ^###p<0.001 vs. respective Day 1 group.

Figure 2. BMP-2 promotes survival of human MSCs entrapped in PAHs under pro-apoptotic conditions

(A) Live/dead staining of MSCs entrapped in PAHs in the presence or absence of BMP-2 in serum deprivation/hypoxia (SD/H). Live cells are green; dead cells are red. Significant cell death is observed at Day 4 in the absence of BMP-2; images at 10× magnification; scale bar represents 200 µm. (B) Quantification of Live/Dead images to determine percentage of viable cells (n=4 per group; ^*p<0.05 vs. −BMP-2 at Day 4). (C) Caspase 3/7 activity of MSCs entrapped in SD/H (n=4 per group; ^**p<0.01 vs. −BMP-2 at Day 4). (D) DNA content in each condition was not significantly different (n=4 per group).

Figure 3. MSCs undergo osteogenic differentiation when entrapped in BMP-2 releasing PAHs

(A) ALP activity was higher at 1 and 7 days for MSCs in BMP-2 containing gels (***p<0.001, ****p<0.0001 vs. −BMP-2 at the same time point). (B) Secreted osteocalcin by MSCs entrapped in alginate gels (*p<0.05 vs. −BMP-2 at 7 days). (C) Calcium deposition by entrapped MSCs is significantly increased in MSCs entrapped in BMP-2 loaded PAHs (**p<0.01 vs. −BMP-2 at 14 days). All data are n=4 per group per time point.

Figure 4. Co-delivery of MSCs and BMP-2 in PAHs increased cell persistence in vivo when implanted in a femoral critical-sized defect

(A) Representative bioluminescence imaging of the same animal over 4 weeks. (B) Quantification of cell signal intensity revealed a significant increase in radiance (an indicator of live cells) in the +BMP-2 group at 2 weeks (n=5 per group; ^**p<0.01 vs. −BMP-2 at 2 weeks).

Figure 5. MSCs deployed in BMP-2-loaded PAHs enhanced defect repair compared to MSCs deployed in PAHs without BMP-2

(A) Radiographs reveal the presence of increased mineralized tissue within the defect in the +BMP-2 group at 4-, 8-, and 12-weeks post-implantation. Radiographs at 4 and 8 weeks were performed on live animals, while 12-week radiographs were performed on explanted tissue. (B) MicroCT scans reveal a significant increase in bone volume within the defect for the +BMP-2 group (bottom) at 12 weeks. (C) Bone volume within the original tissue defect is significantly increased in animals treated with BMP-2 at 12 weeks (n=6 for +BMP-2, n=4 for −BMP-2; ^***p<0.001 vs. −BMP-2).

Figure 6. Matrix deposition and bone formation are enhanced in defects containing BMP-2 loaded PAHs compared to PAHs lacking BMP-2

(A–B) Representative H&E staining of intact femur. The defect site is indicated by ellipse, representing greater tissue formation in BMP-2 treated defects; scale bar = 4 mm. (C–D) Representative Masson’s Trichrome staining at 10× magnification; scale bar = 500 µm. (E–F) Representative osteocalcin immunohistochemical staining at 10× magnification; scale bar = 500 µm. NB = native bone, A = alginate, white arrows denote matrix deposition, black arrows indicate positive osteocalcin staining.

Figure 7. Regenerated bone formed with MSCs transplanted in BMP-2-loaded PAHs exhibits improved mechanical properties compared to defects treated without BMP-2

(A) Torsional stiffness and (B) torque to failure were each significantly increased for bone defects treated with MSCs in PAHs with BMP-2 (+BMP-2) compared to those defects treated without BMP-2 (n=4 for +BMP-2, n=5 for −BMP-2; ^**p<0.01 vs. −BMP-2).