Z21 - Characterization of Extrudable Granular ECM (gECM) Biomaterials for Five Distinct Tissue-Specific Applications

Introduction: : There is a need for an extrudable biomaterial that recapitulates native tissue to create realistic in vitro models and to fill tissue defects in vivo [1]. Reliable tissue-specific in vitro models are essential to eliminate excessive animal testing and reduce failure in clinical trials of treatment candidates for a variety of applications including osteoarthritis, wound healing, and drug toxicity. A tissue-specific biomaterial could also be implanted to fill defects in tissues like cartilage and dermis [2], or could form the foundation for engineering organs for transplant. A top-down approach to biomaterial design, such as repurposing existing extracellular matrix (ECM), is an efficient way to capture the full array of ECM components. However, an intact tissue graft can be difficult to source, while solubilized ECM lacks the structural architecture of native tissue. Alternatively, processing tissue into microparticles enables preservation of both biochemical and biomechanical cues in a cellular microenvironment. These ECM particles can be packed into a hydrogel base at high density [2] to achieve a viscous yet extrudable granular biomaterial that mimics native tissue and that can be formed into any size/shape construct. In this study, we present hyaluronic acid-based granular ECM (gECM) biomaterials for porcine cartilage, bone, dermis, liver, and kidney tissue, with compositional, rheological, and mechanical characterization, and preliminary cellular responses to a subset of tissues.

Materials and

Methods: : Porcine tissue was sourced from a local abattoir (sows ~2 years old) and decellularized using viral inactivation (VI) in 0.2M HCl and 1.0M NaOH for 2 hours each [3]. Bone underwent an additional de-fatting process in 2% SDS and ethanol (50%, 75%, 95%, and 100%). Processed tissues were lyophilized, pulverized, and sorted to < 100um diameter via a micro sieve (Electron Microscopy Sciences, Hatfield PA) (Fig1A). Double-stranded DNA (dsDNA) content was quantified using a PicoGreen assay (Fig1B). Particle size distribution of particles suspended in 100% ethanol (non-swelled state) and water (swelled state) was measured in a laser diffraction particle size analyzer (Mastersizer 3000) (Fig1C). Biomaterial ink formulations consisted of 10mg/mL thiolated hyaluronic acid (HA) (~20% thiolation) packed with 0.2g/mL or 0.56g/mL (bone only) lyophilized particles. A room temperature rheological shear sweep (0.1 to 250 s-1) [4] was performed within 5 mins of mixing (MCR 702, Anton Paar) (Fig1D). Molded cylindrical biomaterial constructs were polymerized for 30 min at 37°C. For particle density analysis, constructs were stained with Ghost Dye, z-stack imaged via confocal microscopy, and thresholded in MATLAB to calculate the volume of particles to total volume (Fig1E). For mechanical testing, constructs were compressed (unconfined) to 40% strain at a quasi-static 0.1%/s strain rate (MCR 702, Anton Paar), and compressive modulus was calculated between 30-40% strain (Fig1F). Primary bovine chondrocytes were seeded into cartilage gECM biomaterial constructs and primary murine dermal fibroblasts were seeded into dermis gECM biomaterial constructs, and LIVE/DEAD stained at 14 days in culture (Fig1G).

Results, Conclusions, and Discussions:: Cartilage, bone, dermis, liver, and kidney were processed into granular extracellular matrix (gECM) particles with diameters < 100µm (Fig1A). The decellularization process effectively reduced DNA content to below the industry standard of 50 ng double-stranded DNA (dsDNA) per mg dry tissue [5] (Fig1B).

Particle size distribution analyses revealed variability between tissues and swelling states (Fig1C). In the swelled state, cartilage particles exhibited the largest peak diameter, while kidney particles exhibited the smallest. Calculated swelling ratios (swelled/non-swelled peak diameter) demonstrate that cartilage has the highest ratio (ratio = 1.58), followed by dermis (1.37), bone (1.21), kidney (1.15), and liver (1.07). This offers insight into the microenvironments created by gECM biomaterials, which can influence cellular interactions.

All gECM biomaterials were formulated with a consistent mass-to-volume ratio of particles-to-HA, except for bone, which required higher mass input to achieve comparable consistency to other gECM biomaterials, likely due to dense mineral content. Nevertheless, all gECM biomaterials demonstrated similar viscosity values and particle volume fraction. Rheological testing demonstrated that gECM biomaterials exhibited shear-thinning behavior, characterized by decreasing viscosity with increasing shear rate (Fig1D), an essential property for extrusion-based delivery methods. Volume fraction measurements of gECM particles in polymerized constructs (Fig1E) revealed that cartilage gECM, with the largest swelled peak particle diameter, resulted in the lowest volume fraction. Conversely, kidney gECM, with the smallest swelled peak particle diameter, displayed the highest volume fraction. This may suggest an inverse relationship between particle size and volume fraction, likely influenced by packing efficiency and percolation [2].

The bulk compressive modulus of bone gECM exhibited the highest stiffness and kidney the lowest (Fig1F), which is consistent with known relative stiffnesses of native tissues. Lastly, cell studies demonstrated viability of bovine chondrocytes in cartilage gECM and murine dermal fibroblasts in dermis gECM over 14 days (Fig1G), supporting the suitability of these materials for in vitro modeling and in vivo applications. Future studies include quantifying collagen and proteoglycan content in gECM particles and assessing cell-gECM interactions through gene expression analysis. These defined properties and characteristics of our gECM biomaterials demonstrate their translational potential for both in vitro and in vivo tissue engineering applications.

Acknowledgements and/or References (Optional)::

References: [1] Oliveira, J.+, Bio-Des. and Mfg., 7,237-239, 2024. [2] Barthold, J.+, Adv. Funct. Mat., 31:2103355, 2021. [3] Cameron, R.+. Pharm. Bioprocess. 2(1), 75–83, 2014. [4] Barthold, J.+, Biofab., 14:025021, 2022. [5] Crapo, P., Badylak, S., et al. Biomat., 32:12:4, 2011.

Funding Acknowledgements: NSF GRFP (JOH), NIH R01 AR083379 and U01 AR082845