Crosslinking of collagen scaffolds promotes blood and lymphatic vascular stability

Abstract

The low stiffness of reconstituted collagen hydrogels has limited their use as scaffolds for engineering implantable tissues. Although chemical crosslinking has been used to stiffen collagen and protect it against enzymatic degradation in vivo, it remains unclear how crosslinking alters the vascularization of collagen hydrogels. In this study, we examine how the crosslinking agents genipin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) alter vascular stability and function in microfluidic type I collagen gels in vitro. Under moderate perfusion (~10 dyn/cm2 shear stress), tubes of blood endothelial cells exhibited indistinguishable stability and barrier function in untreated and crosslinked scaffolds. Surprisingly, under low perfusion (~5 dyn/cm2 shear stress) or nearly zero transmural pressure, microvessels in crosslinked scaffolds remained stable, while those in untreated gels rapidly delaminated and became poorly perfused. Similarly, tubes of lymphatic endothelial cells under intermittent flow were more stable in crosslinked gels than in untreated ones. These effects correlated well with the degree of mechanical stiffening, as predicted by analysis of fracture energies at the cell-scaffold interface. This work demonstrates that crosslinking of collagen scaffolds does not hinder normal endothelial cell physiology; instead, crosslinked scaffolds promote vascular stability. Thus, routine crosslinking of scaffolds may assist in vascularization of engineered tissues.

Physical and Chemical Signals That Promote Vascularization of Capillary-Scale Channels

“…6, 32, 36 We thus expected these physical properties to play a similar role in capillary-scale vascularization. The Darcy permeabilities of untreated and crosslinked collagen gels were 0.0097 ± 0.0025 μm 2 , and 0.0097 ± 0.0031 μm 2 , respectively.…”

“…Permeability was not significantly affected by crosslinking ( p = 0.87), matching previous results. 6 At these permeabilities, the pore size of the scaffolds is small enough to inhibit invasion of the gel by cells. 7 …”

“…Because a cell-permeant analog of cyclic AMP (dibutyryl cAMP) improves the stability of large (~120-μm-diameter) engineered microvessels 26, 38 , we hypothesized that elevated levels of intracellular cAMP would likewise improve the stability of newly formed endothelium in narrow (≤30-μm-diameter) channels. Moreover, substratum stiffness is a strong controller of endothelial cell function 4 ; in particular, stiff substrata promote vascular stability in ~120-μm-diameter channels 6 , and we expected the same to hold at the capillary scale. By optimizing these physical and chemical signals, we have developed a technique for rapid generation of perfusable “capillaries” with diameters as low as 20 μm in type I collagen gels.…”

“…6 Gel disks were untreated or crosslinked with 20 mM genipin for two hours at 25 ° C, and then washed extensively with PBS. Stainless steel or aluminum spheres (Precision Balls) were placed on top of gels submerged in PBS.…”

Extraction and Characterization of Collagen from Buffalo Skin for Biomedical Applications

“…Twentynine different types of collagen composed of at least 46 distinct polypeptide chains have been identified in vertebrates 3,4 . Collagens type I, II and III are the most abundant and well investigated for biomedical applications 5 . Type I collagen has been described as a natural scaffold and a potential candidate for tissue engineering and reconstructive medicine 6 .…”