(165aa) Degradable Oxidized Alginate Microbeads Promote Cell Viability and Extracellular Matrix Synthesis within Genipin-Crosslinked Fibrin Composite Hydrogel Constructs

Introduction: Back pain is a pressing public health concern, accounting for $134.5 billion in healthcare spending in 2016.1 It is estimated that the intervertebral disc (IVD) is involved in 39-42% of adult back pain cases, making the IVD a prime therapeutic target for treating back pain.2 Cell therapy has shown efficacy for slowing progressive IVD degeneration in numerous clinical and preclinical studies, making it a promising treatment option.3 Injectable cell delivery biomaterials may enhance the regenerative potential of IVD cell therapies by retaining injected cells at the injury site and providing an instructive microenvironment that supports cell-mediated healing.4,5 Engineering cell delivery biomaterials for the annulus fibrosus (AF) region of the IVD is challenging because biomaterials capable of withstanding high-magnitude spinal loads require a high degree of crosslinking, which is cytotoxic to cells and inhibits cell-biomaterial binding.6 To overcome this challenge, this study developed a novel composite cell delivery biomaterial construct that uses oxidized alginate (OxAlg) microbeads(MBs) to safely deliver AF cells within high-modulus genipin-crosslinked fibrin (FibGen). The conceptual model is that OxAlg MBs will initially protect cells from the cytotoxic genipin crosslinking, then degrade to promote extracellular matrix (ECM) synthesis and cell-mediated repair within FibGen. Part 1 of this study assessed whether OxAlg MBs protect AF cells from genipin crosslinker cytotoxicity in FibGen+MB constructs. Part 2 characterized the rate that acellular OxAlg MBs degrade within FibGen+MB constructs and measured whether constructs were stable over time. Part 3 measured the ECM synthesis of AF cells within FibGen+MB constructs over time.

Methods: 2% (w/v) OxAlg solutions, with or without bovine AF cells at 20 M cells/mL, were passed through a Nisco VarJ1 microencapsulation system to generate MBs. Resultant MBs were suspended in the FibGen pre-polymer solution, then cast to form cellular and acellular FibGen+MB composite constructs. FibGen+MB-RGD composites utilized MBs generated from OxAlg functionalized with RGD peptides. FibGen composites were cultured up to 6 weeks in growth medium (high-glucose DMEM, 10% fetal bovine serum, 1% penicillin streptomycin and 0.2% ascorbic acid). Fibrin and FibGen hydrogels were used for comparison because they promote strong biological performance and biomechanical competence, respectively. OxAlg hydrogels were not used as controls because of their rapid degradation; however, we have previously demonstrated that AF cells are highly viable throughout OxAlg microencapsulation and release.7 At culture day (D) 7, cellular constructs were harvested for cleaved caspase-3 immunohistochemistry to measure apoptosis. Acellular constructs were harvested at D1, D21 and D42, then scanning electron microscopy (SEM), wet weight and parallel plate rheometry were used to characterize the degradation kinetics of OxAlg MBs within FibGen+MB composites and whether constructs were stable over time. At D21 and D42, cellular constructs were harvested to assess ECM synthesis. Alcian blue and picrosirius red staining were used to visualize the deposition of glycosaminoglycans (GAGs) and collagens, respectively. The specific deposition of collagen I was measured with immunohistochemistry. Significant differences (p < 0.05) in cleaved caspase-3 immunopositivity were determined using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. Two-way ANOVA with Bonferroni correction was used to determine significant differences in wet weight and complex shear modulus (G*) over time.

Results: Microencapsulation within OxAlg MBs was able to protect AF cells from genipin cytotoxicity. Compared to direct cell seeding into FibGen hydrogels, FibGen+MB constructs showed significantly reduced levels of apoptosis (Fig 1A). However, apoptosis levels in FibGen+MB composites were greater than fibrin hydrogel controls. AF cells within FibGen+MB-RGD composites, which had RGD peptides conjugated to the OxAlg polymer backbone for enhanced cell-biomaterial binding, displayed significantly lower levels of apoptosis than FibGen and FibGen+MB constructs. Moreover, there were no significant differences in apoptosis levels between FibGen+MB-RGD composites and fibrin controls. These results indicate that microencapsulation in OxAlg MBs effectively protects AF cells from the cytotoxic crosslinking reaction of FibGen hydrogels.

Acellular OxAlg MBs degraded within FibGen+MB constructs throughout culture. OxAlg MB degradation was macroscopically visible by D21 in FibGen+MB composites (Fig 1B). Degradation of OxAlg MBs was more apparent by D42; whereby, degraded OxAlg MBs became incorporated into the FibGen matrix and changed the construct from dark blue to a purple/gray. SEM confirmed these macroscopic signs of MB degradation at the microscale. At D1, OxAlg was localized to discrete pockets within FibGen+MB composites, indicating MBs were intact and dispersed throughout the FibGen matrix (Fig 1C). MBs began to degrade by D21, as demonstrated by the lack of discrete OxAlg pockets and the presence of OxAlg pieces throughout FibGen+MB composites. By D42, there were no visible OxAlg pieces in FibGen+MB composites, verifying that OxAlg MBs had degraded and began diffusing into the FibGen matrix. This degradation supports the conceptual model that OxAlg MBs will be able to degrade to release regenerative AF cells into FibGen constructs.

Since OxAlg MBs degrade to form pores within FibGen+MB constructs, it was important to assess whether these constructs were stable over time. FibGen+MB composites displayed an increased normalized wet mass by D42 and constant G*, indicating that these constructs were stable over time (Fig 1D & E). Fibrin hydrogels were less stable over time, as shown by macroscopic signs of degradation at D42, microscopic signs of degradation at D21 and decreased normalized wet mass at D42. Furthermore, FibGen+MB composites were more stable than FibGen constructs, which showed microscopic signs of degradation at D42 and decreasing G* over time. Mechanically, FibGen+MB and FibGen constructs had significantly greater G* than fibrin hydrogels at all timepoints. OxAlg has a G* of 5.82 ± 2.69 kPa, which was significantly lower than FibGen and FibGen+MB constructs. Incorporation of low-modulus OxAlg MBs into FibGen caused G* to decrease for FibGen+MB composites compared to FibGen hydrogels at D1 and D21. However, there were no significant differences between G* at D42 for FibGen vs. FibGen+MB.

AF cells within FibGen+MB-RGD constructs showed greater ECM synthesis than FibGen constructs. At D21, AF cells within FibGen+MB constructs showed positivity for GAGs and collagens (Fig 1F). By D42, synthesized GAGs and collagens accumulated within the pericellular space. FibGen+MB-RGD constructs exhibited more extensive ECM synthesis; whereby, GAGs and collagens were visualized beyond the pericellular space in the interstitial space between cells. AF cells within FibGen hydrogels showed limited positivity for ECM markers and negligible staining for ECM proteins at the pericellular space or beyond. Cellular fibrin hydrogels underwent significant degradation before D21, so they could not be assessed at long-term timepoints.

Discussion: Cellular microencapsulation with OxAlg was an effective strategy to improve AF cell viability and ECM synthesis within high-modulus FibGen hydrogels. OxAlg MBs likely reduced AF cell apoptosis by forming a diffusive barrier,8 which allowed genipin to be consumed by the fibrin crosslinking reaction before directly interacting with AF cells. Previous studies investigating the survival of AF cells in FibGen hydrogels demonstrated that AF cells undergo apoptosis because of genipin cytotoxicity, and because genipin crosslinking inhibits cell-biomaterial binding.9 Therefore, conjugating RGD peptides to OxAlg, which has no integrin recognition sites, enhanced cell-biomaterial binding and reduced apoptosis induced by cell-matrix detachment, or anoikis. Since OxAlg has lower biomechanical moduli than FibGen, the presence of OxAlg MBs within FibGen+MB composites initially reduced G* by the rule of mixtures. It remains notable that FibGen+MB and FibGen constructs had similar G* at D42 and FibGen+MB composites have greater biomechanical moduli than most published IVD cell delivery biomaterials.6 Furthermore, cellular FibGen+MB and FibGen+MB-RGD composites were more stable over time and showed enhanced ECM synthesis, compared to fibrin and FibGen hydrogels.

Overall, the FibGen+MB-RGD composite biomaterial strategy is a step towards next-generation regenerative AF repairs that provide immediate biomechanical stabilization and cell delivery for long-term healing. This strategy may be broadly applicable for engineering musculoskeletal tissues that exhibit limited healing capacity and experience high mechanical demands.

References: [1] Dieleman JAMA 2020, [2] Ohtori+ Spine J 2015, [3] Sakai+ Nat Rheum Rev 2015, [4] Vadalà+ J Tissue Eng Regen Med 2012, [5] Burdick+ Cell Stem Cell 2016, [6] Panebianco, DiStefano+ Eur Cell Mater 2020a, [7] Panebianco+ Trans ORS 2020, [8] Gasperini+ J Roy Soc Interface 2014, [9] Panebianco, Meyers+ Eur Cell Mater 2020b