A bioinspired gelatin-hyaluronic acid-based hybrid interpenetrating network for the enhancement of retinal ganglion cells replacement therapy

Biomaterial-based cell replacement approaches to regenerative medicine are emerging as promising treatments for a wide array of profound clinical problems. Here we report an interpenetrating polymer network (IPN) composed of gelatin-hydroxyphenyl propionic acid and hyaluronic acid tyramine that is able to enhance intravitreal retinal cell therapy. By tuning our bioinspired hydrogel to mimic the vitreous chemical composition and mechanical characteristics we were able to improve in vitro and in vivo viability of human retinal ganglion cells (hRGC) incorporated into the IPN. In vivo vitreal injections of cell-bearing IPN in rats showed extensive attachment to the inner limiting membrane of the retina, improving with hydrogels stiffness. Engrafted hRGC displayed signs of regenerating processes along the optic nerve. Of note was the decrease in the immune cell response to hRGC delivered in the gel. The findings compel further translation of the gelatin-hyaluronic acid IPN for intravitreal cell therapy.


Statistics
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Software and code
Policy information about availability of computer code Data collection No software was used Data analysis Graphpad Prism, MATLAB and MAKESENSE.AI For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
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2 nature portfolio | reporting summary

March 2021
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample size was mostly n=15 and experiments were set up in triplicates. We did power analysis and this was the ideal number for sample size.

Replication
All experiments in-vitro were set up in triplicates and in-vivo each group was set up in n=5. All replication were successful Randomization Animals were randomly divided in group of 5. The groups were all female and injected on the same day.

Blinding
The experiment was performed as double blind study as the surgeon was unaware of the material injected. For all data analysis with our proprietary image processing algorithms that the images were assigned to the group post analysis.

Reporting for specific materials, systems and methods
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Flow Cytometry
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A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation hRGC [(3 x 105 cells/mL in PBS or in 1 mL of Gtn-HPA, IPN75 and IPN50 hydrogels, in 6-well plates (3.5 cm diameter, polystyrene, flat bottom, sterile, fisher scientific)] were maintained in PBS for 5 days (replicating the in vivo conditions). 1000 U/mL collagenase-type IV (Invitrogen) was then added to degrade the Gtn-HPA hydrogels and 500 U/mL hyaluronidase type I-S (Sigma-Aldrich) to degrade the HA-Tyr part of the IPN; after 20 minutes of incubation, Gtn-HPA, IPN75 and IPN50 gels were fully dissolved. Samples were centrifuged, and hRGC were harvested. The phenotype of hRGC was analyzed using flow cytometry with the MACSQuant flow cytometer (Miltenyi, San Diego). hRGC, from the 4 different conditions-in PBS, in Gtn-HPA, IPN75 and IPN50 were collected and fixed with a Perm/Fix buffer (BD Biosciences) at 4 C for 15 min. Cells were then washed in a wash buffer (BD Biosciences) and incubated, at room temperature, in a blocking buffer (Pharmingen staining buffer with 2% goat serum) for 30 min. Blocked cells were seeded onto a flat bottom 96-well plate (treated, sterile, polystyrene, Thomas Scientific) and labeled overnight at room temperature with the following conjugated primary antibodies: Brn3a-FITC, RBPMS-APC, Thy1.1-APC (ganglion cell marker), Caspase9-FITC (apoptosis marker), Ki67-FITC (proliferation marker), Cmyc-FITC, Oct4-APC (stemness markers) and NeuN-APC (neuronal marker). Primary antibodies were diluted in 200 uL of antibody buffer (TBS, 0.3% Triton X-100 and 1% goat serum). After overnight incubation cells were washed three times for 15 min, and incubated in secondary antibodies and left at room temperature for 3h; secondary antibodies (goat-derived anti-rabbit and anti-mouse, DAPI-VioBlue) were diluted 1:200 in antibody buffer (Jackson Immunoresearch Laboratory) .

Software
The results were analyzed using the MACSQuantify software (https://www.miltenyibiotec.com) Cell population abundance Light scatter and fluorescence signals from each sample were measured using the MACSQuant (Miltenyi Biotech, Germany) flow cytometer (2 x 10^5 events were recorded).