Poster 51 - Design and Evaluation of DWIVA-Functionalized Hydrogels for Enhanced Stem Cell Bone Differentiation in 3D Environments

Saturday, October 26, 2024

10:00 AM – 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Aabha Pokar

High School Student
Rowan University, New Jersey, United States

Co-Author(s)

Niva Shah

Student
Rowan University
Mount Laurel, New Jersey, United States
Umu S. Jalloh (she/her/hers)

Graduate Student at Rowan University
Rowan University
Glassboro, New Jersey, United States

Co-Author(s)

Daniella N. Concha-Ortiz (she/her/hers)

High School Student at Clearview Regional High School
Rowan University
Mullica Hill, New Jersey, United States
MS

Myranda Sims

Graduate Student at Rowan University
Rowan University, United States

Primary Investigator(s)

Sebastian Vega, PhD

Associate Professor
Department of Biomedical Engineering, Rowan University, Glassboro, NJ and Department of Orthopedic Surgery, Cooper Medical School of Rowan University, Camden, NJ, New Jersey, United States

Introduction: Bone fractures pose significant complications, with 10% leading to nonunion fractures and a malunion rate of 23.5%. Current treatments, including bone grafts and fixations, often fail due to alignment issues, infections, and poor blood supply. Innovative solutions, like osteoinductive biomaterials, offer promising alternatives. The goal of this study is to design an osteoinductive hydrogel that reduces fracture healing time and helps prevent nonunion fractures. Photopolymerizable hydrogels can be inserted into fracture sites during surgery and polymerized under UV light, providing a scaffold that degrades while allowing bone regrowth as the hydrogel dissipates. Successful implementation of such a material is crucial, as millions of bone fractures occur annually in the U.S, with a significant portion affecting osteoporotic individuals.

Bone morphogenetic protein (BMP-2), an FDA-approved protein used for spine fusions and to treat non-unions, has limitations, such as inflammation and ectopic bone formation. Researchers found that BMP-2 mimetic peptides, KIPKA and DWIVA, promote bone formation via Smad-independent and Smad-dependent signaling, respectively and can potentially offset the negative effects of the BMP-2 protein. Our lab and others found that the DWIVA peptide induces bone differentiation of human mesenchymal stem cells (MSCs) in vitro and promotes bone growth in the femur of rats when injected through a self-forming hydrogel in vivo. In this study, we redesigned our former DWIVA peptides with a new GCEAAAK spacer sequence to improve solubility, and studied the effects of our improved DWIVA peptide design on BMP-2 mediated changes in 3D MSC morphology and bone differentiation.

Materials and

Methods: Hydrogel synthesis and MSC encapsulation: Hydrogel formation involved a thiol-ene click chemistry reaction between hyaluronic acid macromers modified with norbornene (HANor) and di-thiolated degradable (Deg) crosslinkers (sequence: GCNSVPMS↓MRGGSNCG) in the presence of a photoinitiator (I2959, 0.05 wt%) and UV light (10 mW/cm², 5 minutes) (Figure 1A). Hydrogel solutions were prepared by combining HANor (3 wt%), degradable crosslinker (2.5 mM), photoinitiator, thiolated cell adhesion peptides (RGD, 2 mM, sequence: GCGYGRGDSPG), and BMP-2 mimetic peptides (DWIVA, 0.01 to 2 mM, sequence: GCEAAAKDWIVAG) in MSC growth medium (GM, αMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin). To encapsulate MSCs, MSC pellets were resuspended in the hydrogel solution (1E6 cells/ml), added to clear cylindrical molds (3.5 mm diameter, 1 mm height), and irradiated under UV light. The hydrogels were removed from the molds, placed in a well plate containing GM, and incubated at 37°C. Cell morphology was evaluated by staining the MSC cytoskeleton (phalloidin, 1:100, 1 hour) and nuclei (Hoechst, 1:1000, 15 minutes).
MSC viability, morphology, and osteogenic differentiation assessment in 3D HANor hydrogels: Samples were then imaged with a Nikon A1 confocal microscope, and ImageJ was used to quantify the percentage of live cells using a live/dead viability kit (living cells green, dead cell nuclei red), cell morphology (volume, sphericity), and osteogenic differentiation. Alkaline phosphatase (ALP) is a bone-specific biomarker, and population-based ALP activity was determined using Vector Blue and a Quantichrome ALP kit.

Results, Conclusions, and Discussions: Results &

Discussion: The study demonstrated that MSCs maintained high cell viability (> 86%) across all DWIVA concentrations (0.01 to 2 mM), and these values were comparable to the control groups using GM (growth medium) and OM (osteogenic induction medium) in the absence of peptides (Figure 1B). These results indicate that MSC viability remains robust throughout encapsulation and photopolymerization, with no adverse effects from the peptide concentrations tested.

ALP activity in MSCs encapsulated within hydrogels containing DWIVA peptides showed a positive correlation with increasing DWIVA concentration. The highest ALP activity was observed at 2 mM (20.8 U/L), surpassing other groups (GM: 3.3 U/L; 0.01 mM: 3.5 U/L; 0.1 mM: 4.8 U/L; 1 mM: 5.4 U/L; and OM: 16.9 U/L) (Figure 1C). These findings suggest that ALP activity may continue to increase at higher DWIVA concentrations. The improved DWIVA peptide design is fully soluble at 10 mM, which is 5 times the highest concentration tested, which allows for DWIVA peptide dosing studies at higher concentrations.

Cell morphology analysis revealed that after 7 days, cell spreading occurred across all groups, with the OM group exhibiting the largest cell volume (33,244 µm³ ± 7,561 µm³). Cell volume also increased in a DWIVA concentration-dependent manner (0.01 mM: 13,600 µm³ ± 6708 µm³ vs. 2 mM: 20,833 µm³ ± 15,001 µm³); however, this was not statistically significant (Figure 1D). There was also no statistically significant change in sphericity across the groups, and this is due to the fact that MSCs spread in all groups (Figure 1E). Increased cell volumes in OM and high concentration DWIVA groups suggest enhanced cellular matrix mechanosensing, which is an important contributor towards osteogenic differentiation. While a positive correlation between cell volume and ALP activity was seen, further studies are needed to confirm this relationship.

Conclusions: The study successfully developed biocompatible HANor hydrogels for 3D cell culture and BMP-2 peptide functionalization. MSCs in 2 mM DWIVA-functionalized hydrogels exhibited larger cell volumes and enhanced ALP activity. Future studies will explore the effects of higher DWIVA and KIPKA concentrations on early 3D matrix mechanosensing and long-term osteogenic differentiation.

Acknowledgements (Optional): I would like to express my gratitude towards Rowan University, specifically Dr. Sebastián Vega, who runs the RISER Program for high school students, for sponsoring and contributing to the research shown in this paper.
I extend my sincere thanks to my project mentor Umu Jalloh, as well as Myranda Sims, Niva Shah, and my fellow rising high school seniors in the RISER Program, who provided me with invaluable guidance and aided me with writing my first biomedical research paper.
Finally, I am deeply grateful to Dr. Sebastián Vega for unwavering patience, and dedicated leadership as head of the RISER program, which were indispensable throughout this research project.
This work was funded by an NSF CAREER (2239922) and a New Jersey Health Foundation (PC 67-24) grant.

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Stacy A. Love and Kirstene A. Gultian. S.A.L. and K.A.G. Mesenchymal stem cells enhance targeted bone growth. 7 February 2024. 25798. 1-8.

Sebastián L. Vega. S.L.V. Injectable hydrogel with immobilized BMP-2 mimetic peptide for local bone regeneration. 22 July 2022. 1-948493.

Kirstene L. Gultian. K.L.G. Self-Forming Norbornene-Tetrazine Hydrogels. 09 December 2022. 202200425.

Asghar Elmi A.E. Treatment of neglected malunion. 17 February 2014. 28:7 Medical Journal of the Islamic Republic of Iran. Introduction.

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