(A-12) Peptide-modified, hydrogel-coated melt electrowritten scaffolds with planar and tubular morphology

Introduction:: Melt electrowriting (MEW) is a high-resolution 3D printing technique that enables the development of customized scaffolds for tissue engineering (TE) applications, mostly with poly(ε-caprolactone) (PCL). One distinctive property of MEW is the high porosity scaffolds that are produced, compared to other techniques. The porosity of MEW scaffolds is typically between 85% and 98%, and their 3D printing design is known to have well-defined mechanical properties. This research aims to alter the cell-scaffold interface between MEW scaffolds and cells/tissue using a hydrogel coating. We report the discovery of a fast surface modification process based on phase separation with a biomimetic yet synthetic hydrogel that is readily functionalized with peptides. This research enables peptide-modified hydrogel coatings onto MEW reinforcement scaffolds by quenching the poly(2-hydroxymethyl methacrylate (HEMA)) (pHEMA) phase separation process with peptides that allow Michael-Type addition reactions. Overall, we aim to combine MEW tubular scaffolds with enhanced surface coatings to construct hybrid fabricated tubes for applications in neural TE.


Materials and Methods:: Flat scaffolds

MEW scaffolds were printed with 20 layers of 10 µm diameter fibers into boxed or triangular pores, printed into circles of 14 mm diameter. Medical grade PCL was heated to 75 °C and extruded from a 25 G nozzle onto a glass slide resting on a metal substrate. Processing conditions used were pressure of 2.0 bar, voltage difference 4.8 kV and printing speed 500 mm/min.

Tubular scaffolds

MEW tubular scaffolds were printed onto a 3 mm mandrel. Medical grade PCL was heated to 80 °C and printing conditions were optimized for 10 µm diameter fibers, otherwise conditions were similar to the flat scaffold. Printing designs include 60 turns on either side of the tubular scaffold.

Coating of flat PCL scaffolds with pHEMA and peptide functionalization

PCL scaffolds were sterilized and etched with 0.1 M sodium hydroxide (NaOH) for 30 minutes. Phase separated pHEMA hydrogels were formed using 80:20, dH2O:HEMA and a redox initiator combination of 2 wt.% ammonium persulfate (APS) and 0.2 wt.% tetramethylethylenediamine (TEMED). At 30 seconds post phase separation, scaffolds were placed in the solution for 1:20 mins and then immediately placed in water to remove non-crosslinked monomer. The pHEMA coated scaffolds were functionalized with 8 pmol of RGD peptide per scaffold for 30 mins at pH 8. Each scaffold was seeded with 20,000 3T3-GFP cells. Cells were incubated 24, 48, or 72 hours before analysis of metabolic activity using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). Cells were stained for nuclei and actin filament for confocal imaging.


Results, Conclusions, and Discussions:: Results

A hydrogel coating was achieved by capturing droplets from a phase-separated solution of pHEMA onto PCL microfibers by scaffold addition and removal. When polymerized in excess water, phase separated HEMA droplets are still liquids and wet the PCL microfibers and stretch across them, creating a webbing structure (Figure 1A-B). Excess pHEMA droplets are removed and phase separation is quenched by washing in water, and then functionalized by dipping the scaffold into a buffer with the peptide. The surface area of the scaffold is increased, when compared to the PCL scaffold. It was discovered that the agitation of the pHEMA solution while the scaffold is in place also affected the coating morphology. We demonstrate that a cysteine-terminated RGD-peptide conjugated to the pHEMA coating promoted cell adhesion on the hydrogel-coated MEW scaffolds over unmodified pHEMA. Applied within intricate MEW tubes, these peptides may serve as cell instructive signals which are promising for several fields of TE, for example, nerve grafts. For high resolution MEW printing of tubular structures, we achieved an inter-fiber spacing of 100 µm for 10 layers with 10 µm fiber diameter for scaffolds with inner diameter of 3 mm (Figure 1C). As expected, the unmodified pHEMA coatings show less cell attachment than non-coated PCL scaffolds (over 50% lower metabolic activity for fibroblasts cultured for 72h), while RGD-functionalized pHEMA scaffolds achieved higher cell attachment when compared to non-functionalized pHEMA.

Conclusions

We discovered that a phase-separated pHEMA solution could be used to quickly alter the surface of a MEW PCL scaffold, to increase both surface area and provide functional signals. This functionalized hydrogel coating that has the potential to be tailored with cell-specific peptides, while agitation of the liquid during scaffold immersion altered the resulting hydrogel morphology. This coating is applied to highly resolved MEW tubes so as to impart new properties on tubular scaffolds.

Discussions

The use of phase-separated solutions to coat and increase the surface area of MEW scaffolds was investigated. This research is remarkably generic, as the process can be applied to different scaffolds while the peptide-modification may be directed towards a spectrum of different tissues.


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