Assistant Professor Rowan University, United States
Introduction:: While bone fractures typically heal with current medical interventions including splints, braces, and orthopedic hardware, about 10% of long bone fractures fail to heal properly. Biomaterials such as hydrogels can be synthesized with biophysical and biochemical properties that augment the healing process, thereby reducing the incidence of nonunion. Mesenchymal stem cells (MSCs) are somatic cells that can be expanded in vitro and readily differentiate into bone-producing osteoblasts based on cues from their extracellular environment. For instance, hydrogel properties can be tailored to provide encapsulated MSCs with osteogenic signals and MSC-laden osteogenic hydrogels can be photopolymerized at the fracture site to promote healing. Bone morphogenetic protein-2 (BMP-2) is a powerful inducer of osteogenesis, and our lab and others have shown that peptides within the wrist (DWIVA) and knuckle (KIPKA) epitopes of BMP-2 promote MSC bone differentiation and nascent bone formation in vivo. MSC spreading is also conducive to osteogenic differentiation, and MSC spreading in 3D hydrogels can be regulated by incorporating enzymatically degradable moieties within the hydrogel. The hypothesis of this work is that BMP-2 peptide functionalized hydrogels formed with enzymatically degradable crosslinkers will enhance 3D spreading and osteogenic differentiation of encapsulated MSCs. To test this hypothesis, MSCs were encapsulated in HANor (norbornene-modified hyaluronic acid) hydrogels with tunable biophysical (degradable crosslinkers) and biochemical (BMP-2 peptides) properties. Spreading was evaluated through the morphology of encapsulated MSCs and osteogenic differentiation was assessed via 3D confocal microscopy of MSCs stained with alkaline phosphatase (ALP), a well-established biomarker of osteogenesis.
Materials and Methods:: Hydrogel formation was the result of a thiol-ene click chemistry reaction between hyaluronic acid macromers modified with norbornene (HANor) and thiolated crosslinkers in the presence of a photoinitiator and ultraviolet light. Hydrogel solutions were created by combining the HANor macromer (3 wt%), thiolated crosslinker, photoinitiator (Irgacure 2959, 0.05 wt%), thiolated cell adhesion peptide RGD (1 mM), and MSC culture medium (αMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin). Thiolated (cysteine “C” containing) DWIVA (sequence: GCGGGDWIVAG) and KIPKA (sequence: GCGGGKIPKASSVPTELSAISTLYLG) peptides sequences were also included in some hydrogel solutions at a 2mM concentration. To encapsulate MSCs (Lonza, passage 4), MSC pellets (1E6 cells/ml) were resuspended in hydrogel solution, added to clear cylindrical molds (8 mm diameter, 2 mm height), and irradiated under UV light (320-390 nm) at 10 mW/cm2 for 10 minutes. The hydrogels were removed from the mold, placed in a well plate containing MSC culture medium, and incubated at 37 ℃. To evaluate viability, a Live/Dead staining assay was performed 1-, 3-, and 7-days post-encapsulation. To evaluate morphology, the cytoskeleton and nuclei were stained with phalloidin (1:100) and Hoescht (1:1,000), respectively after 7 days in culture. To evaluate osteogenic differentiation, MSC samples were stained for ALP (alkaline phosphatase) using a VectorBlue assay per manufacturer’s instructions after 7 days post-encapsulation. A Nikon A1 confocal microscope was used to acquire immunofluorescence images of all stained samples. Viability (% live cells), morphology (Volume, Sphericity), and osteogenic differentiation (ALP fluorescence) were measured using ImageJ software (National Institutes of Health, Bethesda, MD).
Results, Conclusions, and Discussions:: Incorporation of three different crosslinkers (enzymatically degradable Deg and nondegradable Nondeg peptides, and dithiothreitol DTT) and BMP-2 peptides DWIVA and KIPKA were both achieved using thiol-norbornene click chemistry and photopolymerization (Figure 1A). Encapsulated MSCs demonstrated high cell viability in hydrogels functionalized with DWIVA and KIPKA peptides and were comparable to the no peptide control group. The percentage of live cells remained above 97% for 1-, 3-, and 7-days post-encapsulation across all groups (Figure 1B). The impact of the degradable crosslinker Deg on encapsulated MSC 3D spreading and osteogenic differentiation was investigated against the control DTT and Nondeg crosslinker groups. MSCs in hydrogels formed with crosslinkers that prevent cell spreading remained round, whereas MSCs in Deg hydrogels were significantly more spread, as evidenced by average Sphericity values for MSCs in Deg (0.361), DTT (0.591), and Nondeg (0.525) hydrogels after 7 days in culture (Figure 1C). Cell volume was also significantly highest in the Deg group when compared to the DTT and Nondeg crosslinker groups (Figure 1D). The Deg crosslinker’s susceptibility to proteolysis resulted in local hydrogel remodeling from MMPs (matrix metalloproteinases) released by encapsulated MSCs. MSC spreading favors 3D osteogenesis, and this suggests that MSCs in the Deg group should exhibit increased osteogenic differentiation. MSCs encapsulated in the Deg crosslinker group exhibited the highest levels of ALP (alkaline phosphatase) (Figure 1E). These findings suggest that MSC spreading enhances osteogenesis of encapsulated MSCs. We are currently investigating the role of DWIVA and KIPKA peptides on 3D osteogenic differentiation within Deg hydrogels, in addition to potential synergistic effects of exposing MSCs to both peptides simultaneously. Once optimal BMP-2 peptide dosing is established, we will test the best performing hydrogel groups (as evidenced by the presence of osteogenic biomarkers) on accelerating the healing time of long bone fractures using a preclinical femur fracture rat model.
Acknowledgements (Optional): : This research was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) (T34 GM136492), the NIH National Institute of Deafness and other Communicative Diseases (NIDCD) (R21 DC018818), and a National Science Foundation (NSF) CAREER award (2239922).
