Fig 1.
Fabrication of biomimetic tissue-engineered blood vessels (bTEBV).
(A) The scaffold is composed of the negatively charged inert polymers, N,O-carboxymethyl chitosan (N,O-CMC) and alginate. This scaffold is enriched with the bioactive polyanionic polyP, as well as gelatin. The hydrogel is pressed through an extruder and immediately submersed in a solution, containing the cation Ca2+, followed by hardening of the material. The Ca2+ can be partially substituted by polycations, e.g. poly(l-Lys), or the peptide His/Gly-tagged RGD. After addition of poly(l-Lys), especially together with the His/Gly-tagged RGD peptide, the endothelial cells densely cover the surface of the hydrogel. The outer cell layer, composed of mural cells, is only sketched for completeness, but not studied here. (B to D) Human, umbilical vein/vascular endothelial cells, EA.HY926 cells, grown for 2 weeks on the following scaffold matrices; (B) basic scaffold alone, (C) basic scaffold, supplemented with poly(d-Lys), or (C) basic scaffold, supplemented with His/Gly-tagged RGD. The cells are stained, after fixation, with DRAQ5 (blue fluorescence) and labeled antibodies against actin (red), as described under “Material and methods”.
Fig 2.
Technical drawing of the extruder used for the fabrication of the bTEBV.
The N,O-CMC-based material is filled into a syringe (s) which is hooked to the extruder (e). (A and B) By pressing the material with the plunger (p) into the syringe and through the aperture disc (ad), hooked within the molder (m) via the aperture disc in the molder, into the hardening tube, a tube around the central stab is formed. The tube, the bTEBV, undergoes hardening in the hardening tube, a process driven by Ca2+.
Fig 3.
Sequential addition of the polymers to prepare the hydrogel.
(A and B) Suspending of N,O-CMC and silica in saline, and preparation of a solution; (C and D) addition of Na-polyP (polyP) and preparation of a homogeneous solution, again by stirring; (E and F) supplementation of solid sodium alginate (alg), together with low-melting gelatin (gel), resulting in a viscous gel. (G) Filling of the hydrogel, the scaffold to be used for fabrication of the bTEBV, in the extruder or for bioprinting, into a syringe. (H) Printed hydrogel discs, prior (left) or after (right) hardening with CaCl2.
Fig 4.
Experimental fabrication of the bTEBV in the home-made extruder.
(A) The extruder devise with its main parts, the plunger (p), syringe (s), molder (m) and the prepared bTEBV in the hardening tube (ht) is shown. (B, D, G) The ready fabricated bTEBV is pulled over a tube outlet of the pressure device (pd). In (G) the cell culture medium that has been pumped into the vessels is colored in purple. Close up of the (C) central part of the extruder, with the syringe (s) and the molder (m), and (E, F, H) the finally fabricated bTEBVs with an inner diameter of 0.8 mm are depicted.
Fig 5.
Changing the aperture disk within the extruder housing from the one with a 0.8 mm central stab resulting in a smaller material extrusion to aperture discs with a 4 mm central stab as well as equally enlarged material outlets allows the fabrication of bTEBV with an inner diameter of 4 mm (right vessel) instead of the 0.8 mm sized bTEBV (inner diameter). Left vessel: commercially available vessel grafts, totally prepared from non-biological polymers.
Fig 6.
Pressure testing of the bTEBV.
Here the small sized vessels, 1.8 mm (inner diameter 0.8 mm) were examined. (A) Device for studying the stability of the vessels. The two ends of the respective bTEBV were connected via hoses to the connecting tubes (c) that are in full close with the pump (p). The manometer (ma) was connected with the water flow by a Y connection (y). Colored (purple) medium was alternatively pumped into the vessels; it was temperature-controlled at 37°C, using a thermostat (t). (B to E) During the pumping cycle the outer diameter of the vessels varies between 1.80 mm and 2.05 mm.
Fig 7.
Comparison of the burst pressures of vessels.
Physiological human saphenous veins show an average burst pressure of 1680±307 mbar, while the internal mammary arteries measure values between 2031±872 and 4225±1368 mbar. In comparison the bTEBV, fabricated here, display a lower resistance with 850±155 mbar for the larger vessels (of 6 mm) and with 145±24 mbar for the smaller ones (of 1.8 mm). The values for the human human saphenous veins as well as the human internal mammary arteries are taken from the literature [14,73–77].
Fig 8.
Determination of the elastic modulus of the biomaterial, used for fabrication of the bTEBV.
Hardening of the material was performed using 2.5% CaCl2 in 70% ethanol. The measurements were performed at 10°C (open bars), 30°C (closed) or 50°C (cross-hatched), using the ferrule-top nanoindenter. The determinations were performed in saline; the moduli are given in kPa. Significant values with respect to the values measured at 15 s and 30°C are marked; * P < 0.01.
Fig 9.
Influence of the different additives to the universal, basic scaffold on cell growth/viability.
The scaffold is prepared of N,O-CMC, silica, polyP and alginate/gelatin in a fixed sequence. The adhesion-promoting oligo/polymers, the polycationic poly-l-lysine and poly-d-lysine as well as His/Gly-tagged RGD were incorporated into the scaffold as described under “Material and methods”. The biomaterial was printed to 0.95 to 1.0 cm discs and placed into 48 well plates. After an incubation period of 7 d the growth/viability of the cells was determined using the XTT assay and the absorbance was measured at 490 nm. Both the “control-scaffold” (cont-sca) and the bioactive scaffolds “poly(l-Lys)-scaffold” (poly(l-Lys)-sca), “poly(d-Lys)-scaffold” (poly(d-Lys)-sca) and “RGD-scaffold” (RGD-sca) were examined. After an incubation period of 7 d the amount of insoluble tetrazolium salt formed was determined. The standard errors of the means (SEM) are indicated (n = 10 experiments); * P < 0.05.
Fig 10.
Density of EA.HY926 cells grown on different scaffolds for 2 weeks.
Left panel: staining with DRAQ5; right panel: staining for the cytoskeleton structures, actin; the fluorescent immunostained cells are shown. Density of the cells (A and B) onto “control-scaffold”, (C and D) onto “poly(d-Lys)-scaffold”, (E and F) onto “poly(l-Lys)-scaffold”, (G and H) onto “RGD-scaffold” and finally (I and J) onto a scaffold containing both poly(l-Lys) and His/Gly-tagged RGD.
Fig 11.
Expression of CD31 in EA.HY926 cells after cultivation on different scaffolds.
The EA.HY926 cells were incubated for 9 d in culture flasks und routine conditions (control), onto “control-scaffold” (cont-scaf), “poly(d-Lys)-scaffold” (poly(d-Lys)-scaf), “poly(l-Lys)-scaffold” (poly(l-Lys)-scaf) and “RGD-scaffold” (RGD-scaf). Then the cells were collected, the RNA isolated and subjected to RT-qPCR. The expression level determined for CD31 was correlated with the level for GAPDG. The means ± SD are shown (5 experiments/time point).
Fig 12.
Composition of the polyanionic, printable scaffold material.
The basic scaffold of the bioprinted discs or biofabricated vessels (bTEBV) are fabricated by N,O-CMC, alginate, polyP and silica. Due to ionic linkages the cation Ca2+ crosslinks those polymers and forms a more solid implant material. The durable hydrogel formed is proposed to be metabolically dissolved, via glycosidases and alkaline phosphatases (as well as carbonic anhydrases) to the respective monomers. The exchange of Ca2+ by Na+ is assumed to be mediated by both inorganic and organic chelators.