(L-471) Granular Biomaterial Inks Using Ground Tissue Matrix Enable 3D Bioprinting for In Vitro Models

Introduction:: High-throughput screening of disease pathogenesis and new therapeutics is 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. While there has been recent progress in the development of in vitro models, such as tissue-on-chip technology, there is still a need for reliable models that recapitulate native tissues. Extracellular matrix (ECM) tissue particles can be utilized (Fig 1A), as they preserve both the biochemical and structural micro-architecture of ECM. These particles can be packed into a hydrogel base to achieve a viscous yet extrudable granular biomaterial ink conducive to 3D extrusion bioprinting [1]. The particle packing density and hydrogel base material enables tuning of biomaterial properties for a variety of tissue types. In this study, we present hyaluronic acid-based granular tissue particle biomaterial inks for bone, cartilage, kidney, and dermis tissue, with rheological and mechanical characterization and proof-of-concept 3D printed in vitro models.


Materials and Methods:: Bone tissue was sourced from humans (Allosource, Centennial CO), and all other tissues were sourced from porcine animals via a local abattoir. Tissues were pulverized via cryo-mill and sorted to < 100um diameter via a micro sieve (Electron Microscopy Sciences, Hatfield PA). Porcine tissue was decellularized in 2% sodium dodecyl sulfate (SDS). Biomaterial ink formulations consisted of 10mg/mL hyaluronic acid (HA) (~20% thiolation) packed with either 0.72g/mL bone or 0.2g/mL cartilage, dermis, or kidney lyophilized particles to standardize volume ratio of particles to HA (determined via ImageJ analysis of confocal microscopy images). Molded cylindrical ink constructs were polymerized for 30 min at 37°C and compressed to 40% strain (0.1% per second) for quasi-static loading (Bose ElectroForce 5500 system). A room temperature rheological shear sweep (0.01 to 250 s^-1) was performed within 5 mins of mixing (AR-G2, TA Instruments). Biomaterial inks were 3D printed on a pneumatic extrusion printer with 20 G or 18 G nozzles (BioX, Cell Ink Life Sciences) and varying pressure and flow rate parameters.


Results, Conclusions, and Discussions:: This work provides macro-scale rheological and mechanical characterization and 3D printing demonstrations of novel bone, cartilage, kidney, and dermis granular ECM biomaterial inks. The higher bone particle mass required to achieve consistent volume fraction compared to cartilage, kidney, and dermis inks is likely due to lower swelling capabilities of the bone particles. Further studies will be done to characterize swelling of particles and assess relationship to mass and volume ratio of the biomaterial ink.



After disulfide bond crosslinking between HA and particles [2], unconfined compression testing demonstrated a compressive modulus of 741 kPa for bone, 39 kPa for cartilage, 30 kPa for kidney, and 39 kPa for dermis (Fig 1B). Previous work showed that as the tissue matrix packing density increased in the microparticle cartilage ink, compressive modulus rapidly increased up to 300 kPa [1]. As such, it is expected that biomaterial ink using cartilage matrix could reach physiologically-relevant stiffness under load. Furthermore, we have seen a similar relationship between packing density and compressive modulus of the bone ink (not shown). This relationship enables us to tune granular biomaterial inks to achieve desired properties for other tissues.



Rheological measurements indicate shear-thinning behavior of granular biomaterial inks (Fig 1C). Bone, cartilage, and kidney inks were easily extruded through pneumatic-based bioprinting (Fig 1D). Based on the shear-thinning rheological behavior and successful extrusion from a syringe (not shown) seen with the dermis ink, we anticipate smooth extrusion bioprinting of this ink as well. Proof-of-concept osteochondral (Fig 1E) and kidney (Fig 1F) scaffolds and PDMS “on chip” models were successfully printed with bone/cartilage and kidney inks, respectively, to demonstrate potential in vitro models.



We have previously shown that biomaterial inks using cartilage matrix preserve ECM micro-architecture and biochemical factors, and promote infiltration of chondrocytes and a chondrogenic phenotype [1]. Additional studies support particle-guided cell differentiation [3]. Follow-up studies will assess cellular response to bone, kidney, and dermis biomaterial inks. Ultimately, we aim to recapitulate the structure and function of various tissues for realistic in vitro models.



Acknowledgements (Optional): : This work was supported by grant CDMRP W81XWH-20-1-0268, Lab Venture Challenge, and State of Colorado (OEDIT).

References (Optional): : [1] Barthold, J.+ , Adv. Funct. Mat., 31:2103355, 2021. [2] Barthold, J.+, Biofab., 14:025021, 2022. [3] Yin, H.+, Acta Biomat., 77:127-141, 2018.