Natasha Case

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body’s environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability.

Platelet-rich plasma (PRP) is an autologous blood product that contains a variety of growth factors (GFs) that are released upon platelet activation. Despite some therapeutic potential of PRP in vitro, in vivo data are not convincing. Bolus injection of PRP is cleared rapidly from the body diminishing its therapeutic efficacy. This highlights a need for a delivery vehicle for a sustained release of PRP to improve its therapeutic effect. In this study, we used microfluidics to fabricate biodegradable PRP-loaded polyethylene glycol (PEG) microspheres. PRP was incorporated into the microspheres as a lyophilized PRP powder either as is (powder PRP) or first solubilized and pre-clotted to remove clots (liquid PRP). A high PRP loading of 10% w/v was achieved for both PRP preparations. We characterized the properties of the resulting PRP-loaded PEG microspheres including swelling, modulus, degradation, and protein release as a function of PRP loading and preparation. Overall, loading powder PRP into the PEG microspheres significantly affected the properties of microspheres, with the most pronounced effect noted in degradation. We further determined that microsphere degradation in the presence of powder PRP was affected by platelet aggregation and clotting. Platelet aggregation did not prevent but prolonged sustained PRP release from the microspheres. The delivery system developed and characterized herein could be useful for the loading and releasing of PRP to promote tissue regeneration and wound healing or to suppress tissue degeneration in osteoarthritis, and intervertebral disc degeneration.

Although daily low intensity pulsed ultrasound (LIPUS) treatment has been shown to induce cellular responses supporting bone repair, in vitro studies in 3D models, such as cell-seeded scaffolds, are needed to further investigate the underlying cellular mechanisms. This requires well-controlled conditions in an US bioreactor. Computational studies are needed to investigate various effects on US wave propagation influenced by bioreactor configurations, such as reflections at interfaces and wave interference, and optimize the bioreactor design for experimental repeatability. In this study, an enclosed cylindrical sample holder that contained an inner well for placement of a scaffold immersed in culture medium was fabricated by stereolithography 3D printing and combined with an acoustic absorbent material to eliminate the presence of an air-liquid interface perpendicular to the wave propagation path. Finite element simulations conducted in the frequency domain demonstrated that weak standing waves were present within the culture medium, indicating the effects of reflections at solid-liquid interfaces within the sample holder, as expected. Focusing on the acoustic pressure at the inner well surface, it was found that the spatially-averaged pressure varied from a maximum to a minimum value as the thickness of the water layer beneath the sample holder was changed. Average pressure values at antinode positions were 2-fold higher than at node positions. A volume-averaged pressure was calculated within the culture medium corresponding to the region where a scaffold would be centrally located within the bioreactor. It was shown that the thickness of the volume analyzed had a minimal effect on the calculated average pressure. Time-dependent simulations for one complete pulse (i.e. 1 ms) showed that the acoustic pressure in volumes that would be occupied by scaffolds of two different thicknesses (diameter of 8.5 mm and thicknesses of 0.2 or 2.0 mm) reached a stable value after 45 µs, and then remained at that value until the active period of the pulse ceased. Once the active period ended, the acoustic pressure rapidly decreased to a low baseline pressure. Overall, this study showed that the proposed novel bioreactor design provided a controlled environment for the US treatment of a cell-seeded scaffold by removing the air-liquid interface using a custom-designed sample holder and an acoustic absorbent material.