Seamless vascularized large-diameter tubular collagen scaffolds reinforced with polymer knittings for esophageal regenerative medicine.

A clinical demand exists for alternatives to repair the esophagus in case of congenital defects, cancer, or trauma. A seamless biocompatible off-the-shelf large-diameter tubular scaffold, which is accessible for vascularization, could set the stage for regenerative medicine of the esophagus. The use of seamless scaffolds eliminates the error-prone tubularization step, which is necessary when emanating from flat scaffolds. In this study, we developed and characterized three different types of seamless tubular scaffolds, and evaluated in vivo tissue compatibility, including vascularization by omental wrapping. Scaffolds (luminal Ø âˆ¼ 1.5 cm) were constructed using freezing, lyophilizing, and cross-linking techniques and included (1) single-layered porous collagen scaffold, (2) dual-layered (porous+dense) collagen scaffold, and (3) hybrid scaffold (collagen+incorporated polycaprolacton knitting). The latter had an ultimate tensile strength comparable to a porcine esophagus. To induce rapid vascularization, scaffolds were implanted in the omentum of sheep using a wrapping technique. After 6 weeks of biocompatibility, vascularization, calcification, and hypoxia were evaluated using immunohistochemistry. Scaffolds were biocompatible, and cellular influx and ingrowth of blood vessels were observed throughout the whole scaffold. No calcification was observed, and slight hypoxic conditions were detected only in the direct vicinity of the polymer knitting. It is concluded that seamless large-diameter tubular collagen-based scaffolds can be constructed and vascularized in vivo. Such scaffolds provide novel tools for esophageal reconstruction.

Characterization of polyurethane scaffold surface functionalization with diamines and heparin.

Polyurethane scaffolds (PUs) have a good biocompatibility but lack cell recognition sites. In this study, we functionalized the surface of a PU, P(D/L)LA and PCL (50:50) containing urethane segments, with heparin. The first step in this functionalization, aminolysis, lead to free amine groups on the surface of the PU. Free amine content was determined to be 6.4 nmol/mL/mg scaffold, a significant increase of 230%. Subsequently, heparin was crosslinked. Immunohistochemistry demonstrated the presence of heparin homogeneous throughout the 3D porous scaffold. Young's modulus decreased significantly till 50% of the native stiffness after aminolysis and did not change after heparin crosslinking. Contact angle on PU films significantly decreased from 82.7° to 64.3° after heparin crosslinking, indicating a more hydrophilic surface. This functionalization beholds great potential for tissue engineering purposes. When used in a load-bearing environment, caution is necessary due to reduction in mechanical stiffness.

Evaluation of methods for the construction of collagenous scaffolds with a radial pore structure for tissue engineering.

Type I collagen is used widely as a biomaterial. The structure of collagenous biomaterials, including pore sizes and general architecture, can be varied by a number of techniques. In this study, we developed a method to construct flat fibrillar type I collagen scaffolds, 6 cm in diameter and with a radially orientated pore structure, by the use of directional freezing. Different methodologies were tested, the optimal one being freezing of a collagen suspension inside-out, using a centrally positioned liquid nitrogen-cooled tube. Pore sizes could be varied by the use of different tube materials. Use of aluminium tubes resulted in radial scaffolds with a pore size of 20-30 µm, whereas use of stainless steel produced radial scaffolds with 70-100 µm pore sizes. Brass- and copper-based tubes produced scaffolds with less homogeneous radial pores, pore sizes being 90-100 and 50-80 µm, respectively. Fibreglass tubes gave even less uniformity (pore size 100-150 µm). Scaffolds were free of cracks, except in case of aluminium. Scaffolds with a radial inner structure may be especially suitable for tissue engineering of organs with a radial scaffold structure, such as the diaphragm.

Organ-specific tubular and collagen-based composite scaffolds.

The body contains a number of organs characterized by a tubular shape. In this study, we explored several methodologies for the construction of collagenous tubular scaffolds and films with defined (ultra)structure, length, diameter, orientation, and molecular composition. Standardization of molding, casting, freezing, and lyophilizing techniques using inexpensive materials and methods resulted in controllable fabrication of a wide variety of tubular and tissue-specific tubular scaffolds and films. Analysis included immunohistochemical and (ultra)structural examination. Handling and suturability were found adequate for tissue engineering applications.

Urethral reconstruction of critical defects in rabbits using molecularly defined tubular type I collagen biomatrices: key issues in growth factor addition.

Tubular type I collagen biomatrices with and without growth factors (GFs) were constructed and evaluated in a rabbit model for critical urethral defects. Porous tubular biomatrices with an inner diameter of 3 mm were prepared using highly purified collagen fibrils and were crosslinked with or without heparin. Heparinized biomatrices were supplemented with the heparin-binding GFs vascular endothelial GF, fibroblast GF-2, and heparin-binding epidermal GF. Biomatrices with and without GFs were used to replace a critical 1 cm urethral segment in rabbits (n = 32). All animals showed normal urination without urinary retention. General histology and immunohistology of graft areas (2, 4, 12, and 24 weeks after implantation) indicated that all biomatrices were replaced by urethra-like structures with normal appearing cytokeratin-positive urothelium surrounded by vascularized tissue. The GF-containing biomatrices showed an increase in extracellular matrix deposition, neovascularization, urothelium, glands, granulocytes, and fibroblasts, compared with biomatrices without GF. GFs substantially improved molecular features of healing but failed to be superior in functional outcome. Retrograde urethrography indicated a normal urethral caliber in case of biomatrices without GF, but a relative narrowing of the urethra at 2 weeks postsurgery and diverticula after 4 weeks in case of biomatrices with GF. In conclusion, tubular acellular type I collagen biomatrices were successful in repairing urethral lesions in artificial urethral defects, and inclusion of GF has a profound effect on regenerative processes.