A salt-based method to adapt stiffness and biodegradability of porous collagen scaffolds.

Type I collagen scaffolds for tissue reconstruction often have impaired mechanical characteristics such as limited stiffness and lack of strength. In this study, a new technique is presented to fine-tune stiffness and biodegradability of collagen scaffolds by treatment with concentrated salt solutions. Collagen scaffolds were prepared by a casting, freezing and lyophilization process. Scaffolds were treated with 90% saturated salt solutions, the salts taken from the Hofmeister series, followed by chemical crosslinking. Treatment with salts consisting of a divalent cation in combination with a monovalent anion, e.g. CaCl2, resulted in fast shrinkage of the scaffolds up to approximately 10% of the original surface area. Effective salts were mostly at the chaotropic end of the Hofmeister series. Shrunken scaffolds were more than 10 times stiffer than non-shrunken control scaffolds, and displayed reduced pore sizes and swollen, less organized collagen fibrils. The effect could be pinpointed to the level of individual collagen molecules and indicates the shrinking effect to be driven by disruption of stabilizing hydrogen bonds within the triple helix. No calcium deposits remained in CaCl2 treated scaffolds. Subcutaneous implantation in rats showed similar biocompatibility compared to H2O and NaCl treated scaffolds, but reduced cellular influx and increased structural integrity without signs of major degradation after 3 months. In conclusion, high concentrations of chaotropic salts can be used to adjust the mechanical characteristics of collagen scaffolds without affecting biocompatibility. This technique may be used in regenerative medicine to stiffen collagen scaffolds to better comply with the surrounding tissues, but may also be applied for e.g. slow release drug delivery systems.

Tubular collagen scaffolds with radial elasticity for hollow organ regeneration.

Tubular collagen scaffolds have been used for the repair of damaged hollow organs in regenerative medicine, but they generally lack the ability to reversibly expand in radial direction, a physiological characteristic seen in many native tubular organs. In this study, tubular collagen scaffolds were prepared that display a shape recovery effect and therefore exhibit radial elasticity. Scaffolds were constructed by compression of fibrillar collagen around a star-shaped mandrel, mimicking folds in a lumen, a typical characteristic of empty tubular hollow organs, such as ureter or urethra. Shape recovery effect was introduced by in situ fixation using a star-shaped mandrel, 3D-printed clamps and cytocompatible carbodiimide crosslinking. Prepared scaffolds expanded upon increase of luminal pressure and closed to the star-shaped conformation after removal of pressure. In this study, we applied this method to construct a scaffold mimicking the dynamics of human urethra. Radial expansion and closure of the scaffold could be iteratively performed for at least 1000 cycles, burst pressure being 132±22mmHg. Scaffolds were seeded with human epithelial cells and cultured in a bioreactor under dynamic conditions mimicking urination (pulse flow of 21s every 2h). Cells adhered and formed a closed luminal layer that resisted flow conditions. In conclusion, a new type of a tubular collagen scaffold has been constructed with radial elastic-like characteristics based on the shape of the scaffold, and enabling the scaffold to reversibly expand upon increase in luminal pressure. These scaffolds may be useful for regenerative medicine of tubular organs. STATEMENT OF SIGNIFICANCE: In this paper, a new type I collagen-based tubular scaffold is presented that possesses intrinsic radial elasticity. This characteristic is key to the functioning of a number of tubular organs including blood vessels and organs of the gastrointestinal and urogenital tract. The scaffold was given a star-shaped lumen by physical compression and chemical crosslinking, mimicking the folding pattern observed in many tubular organs. In rest, the lumen is closed but it opens upon increase of luminal pressure, e.g. when fluids pass. Human epithelial cells seeded on the luminal side adhered well and were compatible with voiding dynamics in a bioreactor. Collagen scaffolds with radial elasticity may be useful in the regeneration of dynamic tubular organs.

Novel tubular constructs for urinary diversion: a biocompatibility study in pigs.

The use of bowel tissue for urinary diversion can be associated with severe complications, and regenerative medicine may circumvent this by providing an engineered conduit. In this study, a novel tubular construct was identified for this purpose. Three constructs (diameter 15 mm) were prepared from type I collagen and either (a) a semi-biodegradable Vypro II polymer (COL-Vypro), (b) a rapidly biodegradable Vicryl polymer (COL-Vicryl) or (c) an additional collagenous layer (COL-DUAL). After freezing, lyophilization and crosslinking, all constructs showed a porous structure with a two-fold higher strength for the polymer-containing constructs. These constructs were connected to full bladder defects of 11 female pigs and evaluated after 1 (n = 4) or 3 months (n = 5). With respect to surgical handling, the polymer-containing constructs were superior. All pigs voided normally without leakage and the survival rate was 82%. For the implanted COL-Vypro constructs (8/9), stone formation was observed. COL-DUAL and COL-Vicryl showed better biocompatibility and only small remnants were found 1 month post-implantation. Histological and immunohistochemical analysis showed the best regeneration for COL-Vicryl with respect to urothelium; muscle pedicles and elastin formation were best developed in the COL-Vicryl constructs. In this study, COL-Vicryl constructs were superior in both biocompatibility and bladder tissue regeneration and have high potential for artificial urinary diversions. Copyright © 2016 John Wiley & Sons, Ltd.

Design of an elasticized collagen scaffold: A method to induce elasticity in a rigid protein.

UNLABELLED: Type I collagen is widely applied as a biomaterial for tissue regeneration. In the extracellular matrix, collagen provides strength but not elasticity under large deformations, a characteristic crucial for dynamic organs and generally imparted by elastic fibers. In this study, a methodology is described to induce elastic-like characteristics in a scaffold consisting of solely type I collagen. Tubular scaffolds are prepared from collagen fibrils by a casting, molding, freezing and lyophilization process. The lyophilized constructs are compressed, corrugated and subsequently chemically crosslinked with carbodiimide in the corrugated position. This procedure induces elastic-like properties in the scaffolds that could be repeatedly stretched five times their original length for at least 1000 cycles. The induced elasticity is entropy driven and can be explained by the introduction of hydrophobic patches that are disrupted upon stretching thus increasing the hydrophobic-hydrophilic interface. The scaffolds are cytocompatible as demonstrated by fibroblast cell culture. In conclusion, a new straightforward technique is described to endow unique elastic characteristics to scaffolds prepared from type I collagen alone. Scaffolds may be useful for engineering of dynamic tissues such as blood vessels, ligaments, and lung. STATEMENT OF SIGNIFICANCE: In this research report, a methodology is presented to introduce elasticity to biomaterials consisting of only type I collagen fibrils. The method comprises physical compression and corrugation in combination with chemical crosslinking. By introducing elasticity to collagen biomaterials, their application in regenerative medicine may be expanded to dynamic organs such as blood vessels, ligaments and lung. The combination of strength and elasticity in one single natural biomaterial may also "simplify" the design of new scaffolds.

Scaffolds for whole organ tissue engineering: Construction and in vitro evaluation of a seamless, spherical and hollow collagen bladder construct with appendices.

UNLABELLED: The field of regenerative medicine has developed promising techniques to improve current neobladder strategies used for radical cystectomies or congenital anomalies. Scaffolds made from molecularly defined biomaterials are instrumental in the regeneration of tissues, but are generally confined to small flat patches and do not comprise the whole organ. We have developed a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold, mimicking the shape of the whole bladder, and with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized, with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. Human and porcine bladder urothelial and smooth muscle cells were able to attach to the scaffold and maintained their phenotype in vitro. The closed luminal side and the porous outside of the scaffold facilitated the formation of an urothelial lining and infiltration of smooth muscle cells, respectively. The cells aligned according to the provided scaffold template. The technology used is highly adjustable (shape, size, materials) and may be used as a starting point for research to an off-the-shelf medical device suitable for neobladders. STATEMENT OF SIGNIFICANCE: In this study, we describe the development of a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold mimicking the shape of the whole bladder with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. The closed luminal surface and the porous exterior of the scaffold facilitated the formation of a urothelial lining and infiltration of smooth muscle cells, respectively. The applied technology is highly adjustable (shape, size, materials) and can be the starting point for research to an off-the-shelf medical device suitable for neobladders.

Directing collagen fibers using counter-rotating cone extrusion.

The bio-inspired engineering of tissue equivalents should take into account anisotropic morphology and the mechanical properties of the extracellular matrix. This especially applies to collagen fibrils, which have various, but highly defined, orientations throughout tissues and organs. There are several methods available to control the alignment of soluble collagen monomers, but the options to direct native insoluble collagen fibers are limited. Here we apply a controlled counter-rotating cone extrusion technology to engineer tubular collagen constructs with defined anisotropy. Driven by diverging inner and outer cone rotation speeds, collagen fibrils from bovine skin were extruded and precipitated onto mandrels as tubes with oriented fibers and bundles, as examined by second harmonic generation microscopy and quantitative image analysis. A clear correlation was found whereby the direction and extent of collagen fiber alignment during extrusion were a function of the shear forces caused by a combination of the cone rotation and flow direction. A gradual change in the fiber direction, spanning +50 to -40°, was observed throughout the sections of the sample, with an average decrease ranging from 2.3 to 2.6° every 10µm. By varying the cone speeds, the collagen constructs showed differences in elasticity and toughness, spanning 900-2000kPa and 19-35mJ, respectively. Rotational extrusion presents an enabling technology to create and control the (an)isotropic architecture of collagen constructs for application in tissue engineering and regenerative medicine.

Collagen-Vicryl scaffolds for reconstruction of the diaphragm in a large animal model.

Current methods for closure of congenital diaphragmatic hernia using patches are unsatisfactory, and novel collagen-based scaffolds have been developed, and successfully applied in a rat model. However, for translation to the human situation constructs must be evaluated in larger animal models. We developed collagen scaffolds enforced with Vicryl, loaded either with or without the muscle stimulatory growth factor insulin-like growth factor 1 (IGF1). We describe our steps to a surgical method to implant these scaffolds into a diaphragmatic defect in 1.5­3 week old lambs, and evaluate the scaffolds 6 months after implantation. Omentum was attached to the scaffold. At sacrifice, eventration of the implantation site was observed in all animals with a thin layer of tissue separating the abdomen from the thorax. Histologically, no scaffold remnants could be observed. Fatty tissue surrounded by fibrous tissue was seen, resembling encapsulated omentum, with collagen-rich tissue present between this tissue and the original diaphragmatic muscle. Outcomes were not different for scaffolds with or without IGF1. In conclusion, the scaffolds integrated well into the surrounding tissue, but slower degrading materials are needed to prevent eventrations.

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.

Regenerative medicine for the respiratory system: distant future or tomorrow's treatment?

Regenerative medicine (RM) is a new field of biomedical science that focuses on the regeneration of tissues and organs and the restoration of organ function. Although regeneration of organ systems such as bone, cartilage, and heart has attracted intense scientific research over recent decades, RM research regarding the respiratory system, including the trachea, the lung proper, and the diaphragm, has lagged behind. However, the last 5 years have witnessed novel approaches and initial clinical applications of tissue-engineered constructs to restore organ structure and function. In this regard, this article briefly addresses the basics of RM and introduces the key elements necessary for tissue regeneration, including (stem) cells, biomaterials, and extracellular matrices. In addition, the current status of the (clinical) application of RM to the respiratory system is discussed, and bottlenecks and recent approaches are identified. For the trachea, several initial clinical studies have been reported and have used various combinations of cells and scaffolds. Although promising, the methods used in these studies require optimization and standardization. For the lung proper, only (stem) cell-based approaches have been probed clinically, but it is becoming apparent that combinations of cells and scaffolds are required to successfully restore the lung's architecture and function. In the case of the diaphragm, clinical applications have focused on the use of decellularized scaffolds, but novel scaffolds, with or without cells, are clearly needed for true regeneration of diaphragmatic tissue. We conclude that respiratory treatment with RM will not be realized tomorrow, but its future looks promising.