(G-277) Evaluating the Effects of Extrusion Parameters on Injectable Hydrogel Dispersion within Osteoporotic Analogs

Sub-Track: Musculoskeletal Tissue Engineering

Osteoporosis, a medical condition characterized by weakened bone density, can significantly increase the risk of fragility fractures in weight-bearing bones, which, especially in the elderly, may lead to long-term hospitalizations, lower quality of life, or even death. Therefore, creating novel methods for preventing osteoporotic fragility fractures is imperative for the safety and improved well-being of people living with osteoporosis. Our lab recently developed injectable hydrogels that can locally grow trabecular bone in the femur, which is a long bone prone to osteoporotic fragility fractures. In these studies we functionalized the hydrogels with BMP-2 mimetic peptides and injected them in cored femur shafts, and a study of hydrogel dispersion within regions containing trabeculae remains unknown. Therefore, a study of flow dynamics is important in achieving an optimal dispersion of the treated hydrogels throughout bones, leading to the most effective therapeutic outcomes. Bone analogs of various densities representing models of osteoporosis,  osteopenia, and healthy bones can be used to explore flow kinetics when injected with a hydrogel solution. Other parameters such as needle gauge and flow rate are also likely to affect hydrogel dispersion patterns within the bone analogs. This information will lead to a deeper understanding of the factors influencing hydrogel dispersion within bone analogs and provide crucial insights for optimizing the delivery of therapeutic agents in strengthening bones in the trabecular space of varying bone mineral density.

Experimental Setup: A syringe pump was used to inject 1 mL of hydrogel solution made from 1% or 2% wt./vol. hyaluronic acid modified with tetrazine (HATet) and norbornene (HANor) motifs, which upon mixing self-polymerize into a hydrogel. HATet was dissolved in PBS, and HANor was dissolved in Omnipaque solution so the formed hydrogel contrasts under X-ray imaging. 45 seconds after mixing, hydrogel solutions were injected into one of three different bone analogs (Sawbones), representing High (12.5, #1522-11), Medium (7.5, #1522-507), and Low (5.5, #1522-505) bone densities of "healthy bone," "osteopenia," and "osteoporosis," respectively. Hydrogels were extruded from different syringe needle sizes (18, 22, or 25 gauge) at a flow rate of 3 mL/min or 6 mL/min, and X-ray images were captured every 20 s until completion. An n=3 was used for all groups. Experiment Design: The experiment aimed to investigate the effects of varying hydrogel composition, flow rates, and needle gauges on hydrogel dispersion within bone analogs of different densities. The three bone analogs were chosen to simulate distinct bone health conditions. X-ray imaging allowed real-time observation of hydrogel dispersion kinetics. Experimental Analysis: Following X-ray imaging, hydrogel dispersion was evaluated using ImageJ analysis of X-ray images captured in the x-y direction (top-down view). The data obtained from the analysis were plotted as the average ± standard deviation over time, from 0 to 200 s, and at post-gelation at 350 s. This analysis facilitated a comprehensive understanding of the kinetics of hydrogel dispersion across different bone analogs and experimental conditions.

Analysis of preliminary trials revealed that the extrusion properties of the hydrogel injections displayed distinct behaviors in bone analogs of varying porosity. X-ray images were taken to reveal hydrogel dispersion under various conditions (Fig. 1a). High density analogs representing “healthy bone” were observed to exhibit larger steady state dispersive patterns, reported as hydrogel surface area. No significant differences were observed in hydrogel dispersion between Medium and Low density analogs, which represented osteopenia and osteoporosis, respectively (Fig. 1b).

Flow rate played a small role in hydrogel dispersion as well, where larger surface areas of the hydrogel solution were observed when the flow rate increased from 3 mL/min (Fig. 1c) to 6 mL/min (Fig. 1d). This is likely because the higher force of the injection facilitated dispersion directionality, pushing the solution further out prior to gelation. This effect was observed in all samples, regardless of the analog's density.

The injected solution exhibited wider dispersive patterns along the x-y axis in the High density analogs, as it encountered smaller pores that necessitated a broader spread. Conversely, in more porous bone analogs, the injected hydrogels penetrated deeper in the z-direction where the solution could accumulate in its larger pores (Fig. 1e).

These initial findings hold immense significance for our lab's future work, particularly as we plan to employ these experimental hydrogels in live animal studies. Subsequent research will focus on tracking the hydrogel's spreading patterns under more conditions, such as different hydrogel weight percentages and needle gauge sizes. Understanding the extent of hydrogel dispersion within the bone post-injection is crucial to verify its potential for promoting bone regeneration.

In conclusion, our investigation demonstrates that the behavior of injected hydrogels varies depending on the porosity of the bone analogs. The two-dimensional distribution observed in denser bone analogs could be attributed to limited pore accessibility. These results underscore the need for further exploration to optimize hydrogel spreading in different bone conditions. Thus, contributing to the development of effective therapies for bone regeneration and paving the way for innovative treatments using these hydrogels to enhance bone tissue regeneration in vivo.

This research was supported by the National Institutes of Health (NIH) National Institute of Deafness and other Communicative Diseases (NIDCD) (R21 DC018818) and a National Science Foundation (NSF) CAREER award (2239922).