Poster C6 - Relyophilized collagen scaffold for small molecule loading

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ashley D. Oliver

Undergraduate Research
University of Illinois Urbana-Champaign
Hoffman Estates, Illinois, United States

Co-Author(s)

Alison C. Nunes

Research Scientist
University of Illinois at Urbana Champaign
Urbana, Illinois, United States
SB

Scott D. Boden, MD

Professor of Orthopedic Surgery
Emory University, Georgia, United States
SS

Sreedhara Sangadala

Asst. Professor, Department of Orthopedics
Emory University, United States
TD

Thanh Doan

Instructor, Department of Orthopedics
Emory University, United States
NW

Nick Willett, PhD

Associate Professor
University of Oregon - Knight Campus, United States

Primary Investigator(s)

Brendan A.C Harley, Sc.D. (he/him/his)

Robert W. Schaefer Professor
University of Illinois Urbana-Champaign, United States

Introduction: Over 300,000 spinal fusion surgeries are performed every year in the United States, with 10-25% of patients suffering from non-union due to hardware failure to integrate. While it has a variety of diverse functions in orthopedic and dental industries, Bone Morphogenetic Proteins (BMPs) contribute largely to the processes of bone remodeling and homeostasis through the activation of osteogenic genes. However, BMP activity is regulated in the body by the Noggin protein, a BMP-2 antagonist, that ultimately turns off BMP-responsive genes. Historically, 40% of spinal fusion surgeries utilize BMP-2 in high concentrations due to its bone formation properties to address issues of non-union, but several adverse side effects can result from high doses of BMP-2 such as inflammation and ectopic bone growth. To combat this, we have developed a Small Molecule Inhibitor (SMI) of the Noggin protein. The use of a Noggin inhibitor should allow lower doses of BMP-2 which should decrease the local inflammatory side effects. In this work we explore a novel processing method to sequester small molecules into collagen scaffolds. We describe a process of relyophilizing scaffolds to allow for the sequestration and secretion of small molecules such as the Noggin SMI.

Materials and

Methods: Mineralized and non-mineralized Collagen type 1 bovine scaffolds formulated using chondroitin-6-sulfate (C6S) were used for all procedures. Non-mineralized scaffolds contain an acetic acid buffer, while mineralized scaffolds use a phosphoric acid buffer along with calcium hydroxide and calcium nitrate tetrahydrates. Scaffolds undergo initial lyophilization at -10 degrees Celcius. Crosslinking is performed using N-Hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) in a 5:2:1 ratio of EDAC:NHS:carboxylic acid groups within the collagen respectively. Volume absorption measurements were taken by evaluating the mass change of the scaffolds before and after being submerged in a solution of Dimethyl Sulfoxide (DMSO), seeing that it is the vehicle for delivering the SMI. Following the hydration and crosslinking steps, change in height and diameter of the scaffolds after a second lyophilization was gauged using a Johnson 6” Digital Caliper. Scaffolds were soaked in BMP-2 of varying concentrations for one hour at room temperature under mild shaking. Scaffolds were placed in PBS for 3 days with PBS replaced and collected daily. Samples were measured by running liquid chromatography-mass spectrometry (LC-MS) to evaluate release kinetics in both classifications of scaffolds. Mineralization of the scaffolds before and after relyophilization was quantified using inductively coupled plasma-mass spectrometry (ICP-MS).

Results, Conclusions, and Discussions: We expect our preliminary findings for mechanical testing to consist of similar traits seen between the lyophilized and relyophilized scaffold types. This is the target result with the final goal of using the crosslinked, relyophilized scaffolds in vivo. Figure 1 displays a slight decrease in average height and diameter for both conditions of mineralized scaffolds after relyophilization, with further statistical analysis needing to be conducted in order to determine the significance of the decrease. Figure 2 suggests that both mineralized and non-mineralized scaffolds have lower rates of absorption than the original scaffolds when hydrated and relyophilized, and the lowest values for absorption when crosslinking agents are added. The purpose of collecting this data is to find an acceptable volume for later liquid dosages of small molecules. In the future we can apply our findings by developing an assay that incorporates Mesenchymal Stem Cells (MSCs) into our scaffolds. This line of cells was chosen due to their ability to differentiate into osteoblasts: the cells that form new bones and have the ability to grown and heal existing bones. This assay could then be used to evaluate the effectiveness of the Noggin SMI by measuring the release of cytokines and inflammatory markers.

Acknowledgements (Optional):