Poster 67 - Enhancing Bone Healing in Rat Femur Segmental Defects with BMP-2 Epitope-Loaded Hydrogels

Saturday, October 26, 2024

10:00 AM – 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Divya Prajapati

High School Student
Shawnee High School, Medford, NJ and Department of Biomedical Engineering, Rowan University, Glassboro, NJ
Medford, New Jersey, United States

Co-Author(s)

Yeisanai Cardona-Torres (she/her/hers)

Undergraduate Student
Department of Biomedical Engineering, Rowan University, Glassboro, NJ
Bridgeton, New Jersey, United States
SL

Stacy Love

Post Doc Researcher
Department of Biomedical Engineering, Rowan University, Glassboro, NJ and Bone and Joint Institute, Cooper University Health Care, Camden, NJ, United States
ED

Eduard I. Dedkov, MD, PhD (he/him/his)

Associate Professor
Cooper Medical School of Rowan University
Camden, New Jersey, United States
TK

Tae Kim, MD (he/him/his)

Associate Professor
Department of Orthopaedic Surgery, Cooper Medical School of Rowan University, Camden, NJ, 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: A non-union fracture disrupts natural bone regeneration, necessitating surgery and diminishing quality of life. Current research focuses on expediting effective fracture treatments through hydrogel technology. Hydrogels are three-dimensional networks of hydrophilic polymers, which retain large amounts of water. Previously, our lab introduced self-polymerizing hydrogels with osteogenic properties into rat femurs to replace hollowed-out trabecular bone. The osteo-functionalized hydrogels formed more trabecular bone than non-functionalized hydrogel controls. Expanding on this work, we will create a femoral shaft segmental defect to regenerate cortical and trabecular bone via hydrogel treatment, enhancing clinical relevance. Recombinant human bone morphogenetic protein 2 (rhBMP-2), a vital growth factor in the TGF-β family, is used in bone regeneration treatments. However, despite FDA approval, rhBMP-2 requires high concentrations due to its short half-life, leading to side effects such as inflammation and ectopic bone formation. Research into rhBMP-2 epitopes aims to mitigate these effects. This study investigates new hydrogels incorporating DWIVA and KIPKA (KIPKASSVPTELSAISTLYLG) epitopes of rhBMP-2, exploring various concentrations and potential synergies between them. Additionally, the peptide RGD (GCGYGRGDSPG), essential for cell adhesion, facilitates cell-to-cell interaction, thereby enhancing the effects of osteogenesis. These hydrogels show promise in areas where accelerating bone healing, preventing non-unions, and improving recovery outcomes would empower patients to reclaim control over their lives after long bone fractures and potential complications.

Materials and

Methods: This clinically relevant study involves using a surgical procedure to create a five-millimeter segmental defect on rat femurs to implant an injectable hydrogel to study bone regeneration. Briefly, this IACUC-approved procedure begins with a longitudinal incision along the femur to expose the femoral shaft (refer to Fig.1A). Then, four mounting pins are attached to an external fixator and securely fixed to the bone before adding the saw guide and Gigli wire (Fig.1B). Utilizing the saw guide and Gigli wire, the defect is generated by precisely excising a five-millimeter segment of the femoral shaft (Fig.1C). The injectable hydrogels, synthesized through crosslinking of norbornene-modified hyaluronic acid (HANor) and tetrazine-modified hyaluronic acid (HATet) via an inverse Diels Alder reaction (Fig.1D), are subsequently introduced into the defect gap (Fig.1E). All hydrogels incorporate RGD peptides to enhance interaction with mesenchymal stem cells (MSCs) within the bone microenvironment, promoting osteogenesis. DWIVA and KIPKA are peptides derived from the osteogenic domains of rhBMP-2 (Fig.1F). These peptides will be added to hydrogels to test their effectiveness in inducing osteogenesis. The segmental rat femur defect will be treated with one of the following groups: (1) sham (defect without hydrogel), (2) negative control (hydrogel alone), (3) positive control (hydrogel with rhBMP-2), or (4) experimental groups consisting of hydrogels with different concentrations of DWIVA, KIPKA, or both peptides. Following an eight-week recovery period post-surgery, the femurs will be harvested and subjected to comprehensive analysis using micro-CT scanning, histological examination, and biomechanical testing.

Results, Conclusions, and Discussions: This segmental defect study is currently ongoing. The Journal of Orthopaedic Research published a study from this lab where either a hydrogel (G) or a hydrogel containing DWIVA peptide (GP) replaced bone marrow in the intramedullary canal of rat femurs (Love+ JOR 2024). The GP hydrogel effectively stimulated trabecular mineralized bone growth at the injection site, indicated by representative micro-CT axial views (Fig.1G). To evaluate osteoid tissue formation, femurs were stained with Masson’s Trichrome. Representative histological staining shows increased osteoid tissue (dark blue) in the trabecular space in the GP group when compared to the G group (Fig.1H). Building upon these findings, our current study aims to take a more clinically relevant approach by evaluating the effectiveness of BMP-2 peptides administered via an injectable hydrogel in bridging segmental defects within long bones.

Based on prior results, we expect significant bone growth in the experimental groups receiving BMP-2 peptides compared to those without. DWIVA and KIPKA peptides are anticipated to enhance osteogenic differentiation of MSCs, thereby promoting healthy bone growth and mitigating the adverse side effects of rhBMP-2, such as inflammation and ectopic bone formation.

We will use micro-CT images to quantify new trabecular and cortical bone by assessing changes in bone volume fraction, trabecular and cortical count and thickness, cortical surface area, and cortical region of the periosteal envelope. Additionally, we will use histological staining (H&E, TRAP, Masson’s Trichrome, and ALP) and immunohistochemistry (Runx2 and RANK) to evaluate bone histomorphometry by analyzing unmineralized bone extracellular matrix and studying the presence and distribution of osteoblasts and osteoclasts. Furthermore, we will use biomechanical testing to ensure that restored bone strength matches normal, native levels, validating the regenerated bone’s structural integrity.

In conclusion, this clinically relevant study aims to deepen our understanding of peptide-enhanced bone regeneration in segmental defects using advanced imaging, histological analysis, and biomechanical testing to analyze results. These findings will contribute to developing effective therapies for long bone non-union fractures, possibly leading to clinical trials. Successful outcomes can improve patient quality of life by reducing the need for surgeries and hospitalizations, thus allowing more focus on recovery and overall well-being.

Acknowledgements (Optional): The presenting author would like to thank the RISER (Research Immersion in Biomedical Science and Engineering at Rowan) program for providing them with an opportunity to participate in a guided yet independent research project that led to the data submitted in this abstract. This project is supported by the Cooper Foundation (402860), the New Jersey Health Foundation (PC 67-24), and the Foundation Venture Capital Group.

References (Optional): Love SA, Gultian KA, Jalloh US, Stevens A, Kim TWB, Vega SL. Mesenchymal stem cells enhance targeted bone growth from injectable hydrogels with bmp‐2 peptides. J Orthop Res. 2024;42:1599‐1607. doi:10.1002/jor.25798