Poster C2 - Peptide-Functionalized Injectable Hydrogels for Long Bone Regeneration in Critical-Sized Segmental Defects

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Yeisanai Cardona-Torres (she/her/hers)

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

Co-Author(s)

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

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

Co-Author(s)

TK

Tae Won Kim

Division Head for the Orthopaedic Oncology Center
Department of Biomedical Engineering, Rowan University, Glassboro, NJ,Bone and Joint Institute, Cooper University Health Care, Camden, NJ, and Department of Orthopedic Surgery, Cooper Medical School of Rowan University, Camden, NJ, United States

Introduction: Long-bone segmental defects are characterized by a bone discontinuity that is unable to heal on its own. These critical-sized segmental defects are caused by trauma, disease, or tumor resections, and the standard of care for these bone injuries is surgical fixation coupled with bone autografting or allografting. Bone autograft procedures harvest healthy bone from the patient to treat the segmental defect, and while efficacious at times, it involves a second surgery and there is limited bone supply. While bone allografting offsets these shortcomings, allograft bone is typically harvested from cadavers, is non-living, and does a poor job at inducing new bone formation.
Biomaterials offer a potential solution for bridging long-bone segmental defect gaps. In particular, hydrogels are soft biomaterials that can customized to feature physiochemical properties that induce bone formation. Our lab recently developed solutions that upon coming together, self-form into peptide-functionalized hydrogels with tunable gelation time and mechanics. Bone morphogenetic protein 2 (BMP-2) is an FDA-approved growth factor clinically used for spinal fusions and to treat fractures of the tibia. However, due to its delicate nature, BMP-2 has to be administered at supraphysiological doses, which leads to adverse side effects including inflammation and ectopic bone formation. The hypothesis of this work is that our injectable hydrogels, functionalized with BMP-2 mimetic peptides, can be used to bridge long-bone segmental defects while minimizing unwanted side effects of BMP-2. Our hypothesis will be tested by demonstrating the ability of our hydrogels to restore bone in a non-healing rat femur segmental defect model.

Materials and

Methods: Non-healing rat femur segmental defect model: the non-healing rat femur segmental defect model study described was reviewed and approved by Cooper University Hospital’s IACUC. To create a long-bone segmental defect gap, an incision is first made parallel to the femur, followed by the insertion of a four-pin external fixator to stabilize the bone, followed by a double osteotomy (5 mm gap in the femur midshaft) using a saw guide and a Gigli wire. (Figure 1A).
Injectable hydrogel synthesis: Norbornene-modified hyaluronic acid (HANor) and tetrazine-modified hyaluronic acid (HATet) macromers (1:1 ratio, 4 wt%) were dissolved in PBS (phosphate buffered saline), and upon combining, self-form into hydrogels via a Diels-Alder reaction (Figure 1B). To incorporate BMP-2 peptides, norbornene-modified BMP-2 peptides (DWIVA or KIPKA epitope) were formed using a solid-state peptide synthesizer (CEM, Liberty Blue), and were mixed with HATet, resulting in peptide-functionalized HATet macromer (Figure 1C).
Femur segmental defect study groups: The experimental design includes six groups (n=8 rats/group, left femur): (1) Sham Control (untreated defect), (2) Negative Control (hydrogel without peptides), (3) Positive Control (hydrogel with BMP-2, 100 ng/ml), (4) DWIVA Group (hydrogel with 2 mM peptide), (5) KIPKA Group (hydrogel with 1 mM peptide), and (6) Synergy Group (hydrogel with 2 mM DWIVA and 1 mM KIPKA peptide). RGD is an adhesive fibronectin motif peptide that significantly enhances BMP-2 peptide signaling, and it is included in all peptide groups (2 mM). Four weeks post-treatment, femurs will be harvested, and bone growth will be evaluated with microCT, histology, and biomechanical testing.

Results, Conclusions, and Discussions: Results &

Discussion: we have an approved IACUC and developed injectable hydrogels with BMP-2 peptides (DWIVA, KIPKA, and synergy) for a femur segmental defect study that will begin in August. We have also established a reproducible double osteotomy procedure with an external fixator to create a 5 mm non-healing midshaft gap in rat femurs (Figure 1A). Using this model, we formed injectable hydrogel plugs with a contrasting agent (Omnipaque) and used fluoroscopy to confirm that the external fixators maintain the midshaft gap post-suturing (Figure 1D, top) and that our hydrogels can fill this gap (Figure 1D, bottom). In a recent study published in the Journal of Orthopedic Research, we injected our hydrogels functionalized with DWIVA BMP-2 peptide (2 mM concentration), to study its effects in forming trabecular bone at the injection site. Four weeks post-injection, the femur midshafts injected with DWIVA-functionalized hydrogels (Peptide Group) exhibited significantly greater trabecular bone formation compared to the hydrogel-only group (Control Group) (Figure 1E). Based on our current progress and strong in vivo preliminary data, we anticipate bone growth across rat femur segmental defects using our BMP-2 peptide-functionalized injectable hydrogels.
Femur segmental defect healing will be evaluated by studying the presence of nascent bone ECM, bone-producing osteoblasts and bone-resorbing osteoclasts, and biomechanical strength. Bone volume and distribution will be measured via bone morphometry (microCT). Trabecular morphometry (bone volume, trabeculae count, thickness, spacing) and cortical morphometry (cortical thickness, surface area, area of the periosteal envelope, area fraction) will be compared to age-matched healthy femurs by randomly selecting contralateral femurs. Bridged femurs will undergo 3-point bending in the mediolateral direction to assess structural strength, stiffness, and energy absorption, and 3 femurs per group will be used for bone histomorphometry. Histology samples will be prepared from decalcified femurs and will be stained for H&E (to evaluate immune cell infiltration), Masson’s Trichrome (to evaluate osteoid tissue), ALP (to count osteoblasts), and TRAP (to count osteoclasts).

Conclusions: long-bone segmental defects are prevalent, the standard of care has significant limitations, and we are hopeful that our novel biomaterial-based bone-producing therapeutic will enhance efficacy and possibly someday replace bone autografting and allografting.

Acknowledgements (Optional): This project is supported by the New Jersey Health Foundation (PC 67-24) and the Foundation Venture Capital Group.