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.

Self-expandable tubular collagen implants.

Collagen has been extensively used as a biomaterial, yet for tubular organ repair, synthetic polymers or metals (e.g., stents) are typically used. In this study, we report a novel type of tubular implant solely consisting of type I collagen, suitable to self-expand in case of minimal invasive implantation. Potential benefits of this collagen scaffold over conventional materials include improved endothelialization, biodegradation over time, and possibilities to add bioactive components to the scaffold, such as anticoagulants. Implants were prepared by compression of porous scaffolds consisting of fibrillar type I collagen (1.0-2.0% (w/v)). By applying carbodiimide cross-linking to the compressed scaffolds in their opened position, entropy-driven shape memory was induced. The scaffolds were subsequently crimped and dried around a guidewire. Upon exposure to water, crimped scaffolds deployed within 15-60 s (depending on the collagen concentration used), thereby returning to the original opened form. The scaffolds were cytocompatible as assessed by cell culture with human primary vascular endothelial and smooth muscle cells. Compression force required to compress the open scaffolds increased with collagen content from 16 to 32 mN for 1.0% to 2.0% (w/v) collagen scaffolds. In conclusion, we report the first self-expandable tubular implant consisting of solely type I collagen that may have potential as a biological vascular implant.

Ureteral Reconstruction in Goats Using Tissue-Engineered Templates and Subcutaneous Preimplantation.

Repair of long ureteral defects often requires long graft tissues and extensive surgery. This is associated with complications, including a lack of suitable tissue and graft site morbidity. Tissue engineering may provide an attractive alternative to the autologous graft tissues. In this study, ureteral repair using (preimplanted) tubular collagen-Vicryl templates was evaluated in a new goat model. Tubular templates were prepared from tubularized Vicryl meshes and 0.7% type-I collagen (length = 6 cm, inner diameter = 6 mm, wall thickness = 3 mm). In total, twelve goats were used and evaluated after 3 months. Eight goats were implanted with the collagen-Vicryl templates and in four goats the templates were first preimplanted in the subcutis and subsequently used as ureteral graft. Template implantation was successful in 92% of the goats(11/12). During follow-up, 82% of the animals (9/11) survived without signs of discomfort. Two animals were sacrificed prematurely due to kidney perforation by the stent and urine leakage. Two other animals presented with stenosis of the neoureter due to stent migration. After preimplantation, the templates were remodeled mostly to autologous tissue with similar mechanical characteristics as the native ureter. Goats grafted with preimplanted templates presented with predominantly healthy kidneys, whereas the goats grafted with the collagen-Vicryl templates presented with fibrotic and inflamed regions in the kidneys. The use of preimplanted tissue templates showed favorable results compared with direct functional implantation of the templates. Partial remodeling toward autologous tissue and similar mechanical characteristics likely improved the integration in the ureteral tissue. Preimplantation of tissue-engineered templates should therefore be considered when two-stage procedures using a nephrostomy catheter are indicated or when planning allows for additional time to treatment.

Tissue Engineering of the Urethra: A Systematic Review and Meta-analysis of Preclinical and Clinical Studies.

CONTEXT: Urethra repair by tissue engineering has been extensively studied in laboratory animals and patients, but is not routinely used in clinical practice. OBJECTIVE: To systematically investigate preclinical and clinical evidence of the efficacy of tissue engineering for urethra repair in order to stimulate translation of preclinical studies to the clinic. EVIDENCE ACQUISITION: A systematic search strategy was applied in PubMed and EMBASE. Studies were independently screened for relevance by two reviewers, resulting in 80 preclinical and 23 clinical studies of which 63 and 13 were selected for meta-analysis to assess side effects, functionality, and study completion. Analyses for preclinical and clinical studies were performed separately. Full circumferential and inlay procedures were assessed independently. Evaluated parameters included seeding of cells and type of biomaterial. EVIDENCE SYNTHESIS: Meta-analysis revealed that cell seeding significantly reduced the probability of encountering side effects in preclinical studies. Remarkably though, cells were only sparsely used in the clinic (4/23 studies) and showed no significant reduction of side effects. ln 21 out of 23 clinical studies, decellularized templates were used, while in preclinical studies other biomaterials showed promising outcomes as well. No direct comparison to current clinical practice could be made due to the limited number of randomized controlled studies. CONCLUSIONS: Due to a lack of controlled (pre)clinical studies, the efficacy of tissue engineering for urethra repair could not be determined. Meta-analysis outcome measures were similar to current treatment options described in literature. Surprisingly, it appeared that favorable preclinical results, that is inclusion of cells, were not translated to the clinic. Improved (pre)clinical study designs may enhance clinical translation. PATIENT SUMMARY: We reviewed all available literature on urethral tissue engineering to assess the efficacy in preclinical and clinical studies. We show that improvements to (pre)clinical study design is required to improve clinical translation of tissue engineering technologies.

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.

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.

Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.