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Neural Engineering

Neural Engineering - Poster Session D

Poster Z15 - Accelerated Biofabrication of Micro Tissue Engineered Neural Networks for Brain Pathway Reconstruction

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Parkinson’s Disease (PD) arises from progressive degeneration of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc). As greater than 50% of DA neurons are often lost before clinical presentation of PD symptoms, direct circuit-level pathway replacement remains a critical avenue toward future PD therapeutics.[1] We thus previously conceived Tissue-Engineered Nigrostriatal Pathways (TE-NSPs) as implantable neural pathways intended to replace lost dopaminergic fibers between the SNpc and Striatum.[2] TE-NSPs belong to a category of micro-tissue engineered neural networks (micro-TENNs) consisting of discrete dopaminergic neuronal somata with long-distance axonal tracts housed within a bio-compatible hydrogel microcolumn. In terms of design criteria, the human nigrostriatal pathway can exceed 30mm in length, and faithful reconstruction may demand more than 100,000 DA neurons.[3] However, typical TE-NSP axonal proliferation rates rapidly taper to < 500µm / day, and canonical fabrication methods achieve low yield beyond 20mm casing length, making clinical-grade implants difficult to produce.[3] To address these challenges, this study assesses the impact of hydrogel casing composition on micro-TENN axonal proliferation rates and maximum single-unit fabrication length. We synthesized micro-TENN casings from Methacrylated Hyaluronic Acid (HA-MA or MeHA) with varying degrees of chemical backbone modification and varying solution concentrations (weight/volume) to achieve varying polymeric network densities. As a proof-of-concept, micro-TENNs were fabricated using primary cortical neurons to analyze the growth of axon tracts.

Materials and

Methods:  Methacrylated Hyaluronic Acid (MeHA) was synthesized by esterification of Hyaluronic Acid (HA, 60kDa, Lifecore Biomedical) and Methacrylic Anhydride (MA, Sigma). Backbone modification was closely controlled through stoichiometric addition of MA.[4] Briefly, 1g HA was dissolved in 140mL DI H2O, and MA was added dropwise. pH was then maintained between 7.5-8.5 over 3h via automated dropwise delivery of 1N sodium hydroxide (Fisher). After 5 days of dialysis against DI H2O (SnakeSkin Dialysis Tubing, 10kDa MWCO, Thermo Fisher) and lyophilization over 4 days, backbone modification was quantified by 1H NMR. A range of backbone methacrylation from 12-55% was thus achieved. Hollow cylindrical micro-TENN casings were then coaxially molded in 20+ mm long, 1049 x 500µm OD/ID units. For each degree of methacrylation (DoM), casings were fabricated by photopolymerization of 1-5% w/v MeHA solution under collimated UV light (10mw/cm2, 365nm). In parallel, E18 Sprague Dawley rat cortical neurons were isolated, dissociated, and re-aggregated (~110,000 neurons/aggregate) via custom inverted pyramidal wells.[2] Micro-TENN casings were then seeded with an extracellular matrix-like (ECM) mixture of collagen (Type I, Rat, Corning) and Laminin (Mouse, Corning). This mixture was allowed to polymerize for 12h, and a single neuronal aggregate was then installed at one end of each casing. Micro-TENNs were then cultured for 14+ days in neurobasal medium supplemented with 2% B-27, 0.2% L-glutamine, and 2% Penicillin-Streptomycin antibiotic (v/v, Figure 1A). Axonal outgrowth was quantified via FIJI after Calcein AM/Ethidium Homodimer staining and subsequent confocal microscopy 14-15 days after cell seeding.

Results, Conclusions, and Discussions: 

Results: Based on previously determined clinically inspired design criteria, MeHA DoM was titrated downward in 5% increments until stable micro-TENN casings did not reliably form ( < 20% yield) at 1-5% w/v solution. 17% DoM was determined as an approximate floor for micro-TENN casing fabrication (Fig 1B). To assess single-unit fabrication length, casing mold length was increased from 20 mm in 10 mm increments until < 20% yield was achieved. Compared to canonical 55% modified MeHA, 17-25% modification enabled production of 6-7cm long casings for the first time (Fig 1C). To assess initial outgrowth, micro-TENN casings were divided into 12mm units and cultured as described above. After 14-15 days, axonal outgrowth was measured via confocal microscopy (Fig 2A). Aggregate length did not differ significantly among the various groups, suggesting similar achieved cell mass (Fig 2B). At each DoM, 3% w/v casings exhibited significantly faster axonal proliferation than their respective 5% counterparts, while 23% modification yielded the fastest overall axonal proliferation (Fig 2C-D). Finally, the difference between leader axon and dense front outgrowth was quantified for each construct. No significant differences were observed among all groups, suggesting similar growth patterns in which leader axons were ~1mm ahead of the dense front within each construct (Fig 2E).

Conclusions: MeHA with 17-25% methacrylation enabled the fabrication of substantially longer micro-TENN casings (7 cm vs. 3 cm), while significantly increasing axonal outgrowth, relative to 55% modified controls. We demonstrate the preferential growth of E18 rodent cortical axons at median (23%) MeHA modification and minimum (3%) solution concentration. The present study suggests a critical balance between casing stability and nutrient transport which will be explored in future works. Follow up studies will involve validation of the selected MeHA micro-columns for TE-NSP development using dopaminergic neurons to reconstruct the nigrostriatal dopaminergic pathway, which can be implanted in the brain via minimally invasive techniques.

Acknowledgements (Optional):  

References:
1. Cheng, H.-C., Ulane, C.M. and Burke, R.E. (2010), Clinical progression in Parkinson disease and the neurobiology of axons. Ann Neurol., 67: 715-725. https://doi.org/10.1002/ana.21995

2. Struzyna LA, Browne KD, Brodnik ZD, et al. Tissue engineered nigrostriatal pathway for treatment of Parkinson's disease. J Tissue Eng Regen Med. 2018; 12: 1702–1716. https://doi.org/10.1002/term.2698

3. Gordián-Vélez WJ, Chouhan D, España RA, et al. Restoring lost nigrostriatal fibers in Parkinson's disease based on clinically-inspired design criteria. Brain Research Bulletin. 2021 Oct;175:168-185. DOI: 10.1016/j.brainresbull.2021.07.016. PMID: 34332016; PMCID: PMC8404430.

4. Burdick JA, Chung C, Jia X, Randolph MA, Langer R. Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules. 2005 Jan-Feb;6(1):386-91. doi: 10.1021/bm049508a. PMID: 15638543; PMCID: PMC2678566.