SummaryPluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

Defects in the annulus fibrosus (AF) of intervertebral discs allow nucleus pulposus tissue to herniate causing painful disability. Microdiscectomy procedures remove herniated tissue fragments, but unrepaired defects remain allowing reherniation or progressive degeneration. Cell therapies show promise to enhance repair, but methods are undeveloped and carriers are required to prevent cell leakage. To address this challenge, this study developed and evaluated genipin-crosslinked fibrin (FibGen) as an adhesive cell carrier optimized for AF repair that can deliver cells, match AF material properties, and have low risk of extrusion during loading. Part 1 determined that feasibility of bovine AF cells encapsulated in high concentration FibGen (F140G6: 140 mg/mL fibrinogen; 6 mg/mL genipin) for 7 weeks could maintain high viability, but had little proliferation or matrix deposition. Part 2 screened tissue mechanics and in situ failure testing of nine FibGen formulations (fibrin: 35-140 mg/mL; genipin: 1-6 mg/mL). F140G6 formulation matched AF shear and compressive properties and significantly improved failure strength in situ. Formulations with reduced genipin also exhibited satisfactory material properties and failure behaviors warranting further biological screening. Part 3 screened AF cells encapsulated in four FibGen formulations for 1 week and found that reduced genipin concentrations increased cell viability and glycosaminoglycan production. F70G1 (70 mg/mL fibrinogen; 1 mg/mL genipin) demonstrated balanced biological and biomechanical performance warranting further testing. We conclude that FibGen has potential to serve as an adhesive cell carrier to repair AF defects with formulations that can be tuned to enhance biomechanical and biological performance; future studies are required to develop strategies to enhance matrix production.

Emergent approaches in regenerative medicine look toward the use of extracellular vesicles (EVs) as a next-generation treatment strategy for intervertebral disc (IVD) degeneration (IVDD) because of their ability to attenuate chronic inflammation, reduce apoptosis, and stimulate proliferation in a number of tissue systems. Yet, there are no Food and Drug Administration (FDA)-approved EV therapeutics in the market with an indication for IVDD, which motivates this article to review the current state of the field and provide an IVD-specific framework to assess its efficacy. In this systematic review, 29 preclinical studies that investigate EVs in relation to the IVD are identified, and additionally, the regulatory approval process is reviewed in an effort to accelerate emerging EV-based therapeutics toward FDA submission and timeline-to-market. The majority of studies focus on nucleus pulposus responses to EV treatment, where the main findings show that stem cell-derived EVs can decelerate the progression of IVDD on the molecular, cellular, and organ level. The findings also highlight the importance of the EV parent cell's pathophysiological and differentiation state, which affects downstream treatment responses and therapeutic outcomes. This systematic review substantiates the use of EVs as a promising cell-free strategy to treat IVDD and enhance endogenous repair.

Although transplantation of retinal pigment epithelial (RPE) cells has shown promise for the treatment of retinal degenerative diseases, this therapeutic approach is not without challenges. Two major challenges are RPE cell dedifferentiation and inflammatory response following transplantation. The aim of this work is to understand how the rigidity of a scaffold, a relatively unexplored design aspect in retinal tissue engineering, affects RPE cells, particularly the pathways associated with the aforementioned challenges. Poly(ethylene glycol) diacrylate (PEGDA) of varying molecular weights from 3.4 to 20 kDa were photopolymerized to fabricate scaffolds. The Young's modulus of the scaffolds varied from 60 to 1200 kPa. A cell study was then conducted to test the effects of scaffold rigidity on RPE cells. A cell adhesion peptide motif of arginine-glycine-aspartic acid-serine (RGDS) was conjugated to 60 and 1200 kPa scaffolds and ARPE-19 cells, a human RPE cell line, were seeded onto these hydrogels. Cells grown on scaffolds demonstrated qualitatively different adhesion properties, metabolic activity, and gene expression at an mRNA level. IL-6 and MCP-1, two inflammation markers known to recruit microglial into the retina, had the same expression pattern with cells having the highest expression on the high modulus scaffold and lowest expression on the control substrate. This study demonstrates that scaffold rigidity, an important design parameter in other areas of tissue engineering, affects cell adhesion, activity, and expression of RPE cells. Though more exploration is needed, this begins to lay a foundation for optimizing scaffold rigidity to promote long-term success of RPE scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1260-1266, 2017.

Hydrogels are extraordinarily versatile by design and can enhance repair in diseased and injured musculoskeletal tissues. Biological fixation of these constructs is a significant determinant factor that is critical to the clinical success and functionality of regenerative technologies for musculoskeletal repair. In the context of an intervertebral disc (IVD) herniation, nucleus pulposus tissue protrudes through the ruptured annulus fibrosus (AF), consequentially impinging on spinal nerve roots and causing debilitating pain. Discectomy is the surgical standard of care to treat symptomatic herniation; however these procedures do not repair AF defects, and these lesions are a significant risk factor for recurrent herniation. Advances in tissue engineering utilize adhesive hydrogels as AF sealants; however these repair strategies have yet to progress beyond preclinical animal models because these biomaterials are often plagued by poor integration with AF tissue and lead to large variability in repair outcomes. These critical barriers to translation motivate this article to review the material composition of hydrogels that have been evaluated in situ for AF repair, proposed mechanisms of how these biomaterials interface with AF tissue, and their functional outcomes after treatment in order to inform the development of new hydrogels for AF repair. In this systematic review, we identify 18 hydrogel formulations evaluated for AF repair, all of which demonstrate large heterogeneity in their interfacing mechanisms and reported outcome measures to assess the effectiveness of repair. Hydrogels that covalently bond to AF tissue were found to be the most successful in improving IVD biomechanical properties from the injured state, but none were able to restore properties to the intact state suggesting that new repair strategies with innovative surface chemistries are an important future direction. We additionally review biomechanical evaluation methods and recommend standardization in the field of AF tissue engineering to establish mechanical benchmarks for translation and ensure clinical feasibility.

Objective Intervertebral disk degeneration is a prevalent postoperative complication after discectomy, underscoring the need to develop preventative and bioactive treatment strategies that decelerate degeneration and seal annulus fibrosus (AF) defects. Human mesenchymal stem cell–derived exosomes (MSC-Exos) hold promise for cell-free bioactive repair; however, their ability to promote AF repair is poorly understood. The objective of this study was to evaluate the ability of MSC-Exos to promote endogenous AF repair processes and integrate MSC-Exos within a biomaterial delivery system. Design We characterize biophysical and biochemical properties of normoxic (Nx) and hypoxic (Hx) preconditioned MSC-Exos from young, healthy donors and examine their effects on AF cell proliferation, migration, and gene expression. We then integrate a poly(lactic- co-glycolic acid) microsphere (PLGA µSphere) delivery platform within an interpenetrating network hydrogel to facilitate sustained MSC-Exo delivery. Results Hx MSC-Exos led to a more robust response in AF cell proliferation and migration than Nx MSC-Exos and was selected for a downstream protection experiment. Hx MSC-Exos maintained a healthy AF cell phenotype under a TNFα challenge in vitro and attenuated catabolic responses. In all functional assays, AF cell responses were more sensitive to Hx MSC-Exos than Nx MSC-Exos. PLGA µSpheres released MSC-Exos over a clinically relevant timescale without affecting hydrogel modulus or pH upon initial embedment and µSphere degradation. Conclusions This MSC-Exo treatment strategy may offer benefits of stem cell therapy without the need for exogenous stem cell transplantation by stimulating cell proliferation, promoting cell migration, and protecting cells from the degenerative proinflammatory microenvironment.

In injured intervertebral discs, disruptions in fibre organization and in cellular contractility result in a fibrotic phenotype and progressive tissue degeneration.As essential components of every joint in the musculoskeletal system, fibrous connective tissues enable load-bearing with controlled mobility. Because these soft tissues interact with surrounding bone surfaces (which act as fixed interfaces), tissue growth and osmotic swelling cause the tissues to develop basal levels of mechanical strain (also known as 'residual strains') within their constituent fibres 1,2 . Residual strains have a marked effect on the topography of the fibrillar network and on its organization within the cellular microenvironment 3 . Along with joint loading, these strains present biophysical cues to resident cells, ultimately influencing cellular phenotype and function through mechanotransductive mechanisms 4,5 .

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