Surface functionalization of microscaffolds produced by high-resolution 3D printing: A new layer of freedom

Abstract

Scaffolded-spheroids represent novel building blocks for bottom-up tissue assembly, allowing to produce constructs with high initial cell density. Previously, we demonstrated the successful differentiation of such building blocks, produced from immortalized human adipose-derived stem cells, towards different phenotypes, and the possibility of creating macro-sized tissue-like constructs in vitro. The culture of cells in vitro depends on the supply of various nutrients and biomolecules, such as growth factors, usually supplemented in the culture medium. Another means for growth factor delivery (in vitro and in vivo) is the release from the scaffold to alter the biological response of surrounding cells (e.g. by release of VEGF).¹ As a proof of concept for this approach, we sought to biofunctionalize the surface of the microscaffolds with heparin as a "universal linker" that would allow binding a variety of growth factors/biomolecules. An aminolysis step in an organic solvent made it possible to generate a hydrophilic and charged surface. The backbone of the amine, as well as reaction conditions, led to an adjustable surface modification. The amount of heparin on the surface was increased with an ethylene glycol-based diamine backbone and varied between 8 and 40 ng per microscaffold. Choosing a suitable linker allows easy adjustment of the loading of VEGF and other heparin-binding proteins. Initial results indicated that up to 5 ng VEGF could be loaded per microscaffold, generating a steady VEGF release for 16 days. We report an easy-to-perform, scalable surface modification approach of polyester-based resin that leads to adjustable surface concentrations of heparin. The successful surface aminolysis opens the route to various modifications and broadens the spectrum of biomolecules which can be delivered.

Keywords: Growth factors; High-resolution 3D printing; Microscaffolds; Scaffolded spheroids; Surface modification; Tissue engineering; VEGF.

Graphical abstract

Scheme 1

Two reaction pathways are possible to introduce amino groups under the investigated conditions. A) Aza-Michael addition of the primary amine on the α,β-unsaturated carbonyl group provided by unreacted acrylate end groups of the prepolymer. B) Aminolysis of the ester group on the backbone of the prepolymer leading to an alcohol and an amide. The stability of the formed amide hinders the backreaction.

Fig. 1

A) FT-ATR-IR spectra from the material control (black) and after the surface modification performed in 1-propanol (red) and THF (blue). The peak at 1567 cm-1 indicates the presence of primary amides formed during the aminolysis reaction. Due to the distinct presence of this peak in THF, this solvent was chosen for the modification of MS; B) Preliminary study on the surface modification on thin films using TTDA and DBN as reactive component in 1-propanol and THF as well as a material control. Before (top), during (middle) staining and after washing (bottom). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

A) Staining of heparin on material discs for preliminary tests. The blue areas indicate the binding of the dye to the heparin, which was bound to the material's surface. B) ATR-IR spectrum of the surface after heparin conjugation. It can be seen that the transmittance at wavenumber above 3000 cm⁻¹ is further decreased, indicating more available -OH and -NH₂ groups introduced during the heparin-binding. The increase in signals in the fingerprint area (below 1000 cm⁻¹) further suggests the introduction of heparin, which is distinctly different from the material. C) The change of contact angle of water on a material film. Although already hydrophilic as raw material, introducing amino groups on the surface further increases surface hydrophilicity. The binding of heparin did not lead to a further change in contact angle. D) Apparent Young's modulus was calculated before and after surface modification. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Quantification of heparin per MS as (A) function of TTDA concentration during the aminolysis and (B) function of heparin concentration in the conjugation step. C) 100 MS in a 1.5 Eppendorf tube after staining with 1,1-dimethylene blue (top) and after the release of bound dye (bottom) with increasing surface concentration of heparin (left to right). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Cumulative VEGF release (A) and measured VEGF concentration per time point (right) of 10 MS. The measured concentration at a given time can be interpreted as the global concentration of VEGF in solution. The concentration of VEGF increased steadily until day 6 before the release was reduced almost but a steady concentration of 1 ng ml⁻¹ was obtained until the end of the release study.

Fig. 5

Spider chart depicting the amount of VEGF bound to scaffold in [ng mg⁻¹], VEGF biding of microscaffolds outperforms, previous published results of VEGF-releasing heparinzed scaffolds.

Fig. 6

Effects of basal DMEM preconditioned using MS loaded with/without VEGF (VEGF+/−). A) Graphical representation of the image analysis using ImageJ; B) metabolic activity measured during serum-starvation (normalized to the first timepoint = 48 h after starting of serum-starvation/culture = dotted line); C) Representative microscopic images depicting the tube formation progression from 4 to 24 h after seeding on the Matrigel surface; For both tested groups, D) Quantification of the number of master junctions, total tube length, and total mesh area covered by the tubular network.