elastin and genipin

Physically crosslinked hydrogels with thixotropic properties attract considerable attention in the biomedical research field because their self-healing nature is useful in cell encapsulation, as injectable gels, and as bioinks for three-dimensional (3D) bioprinting. Here, we report the formation of thixotropic hydrogels containing nanofibers of double-hydrophobic elastin-like polypeptides (ELPs). The hydrogels are obtained with the double-hydrophobic ELPs at 0.5 wt%, the concentration of which is an order of magnitude lower than those for previously reported ELP hydrogels. Although the kinetics of hydrogel formation is slower for the double-hydrophobic ELP with a cell-binding sequence, the storage moduli G' of mature hydrogels are similar regardless of the presence of a cell-binding sequence. Reversible gel-sol transitions are demonstrated in step-strain rheological measurements. The degree of recovery of the storage modulus G' after the removal of high shear stress is improved by chemical crosslinking of nanofibers when intermolecular crosslinking is successful. This work would provide deeper insight into the structure-property relationships of the self-assembling polypeptides and a better design strategy for hydrogels with desired viscoelastic properties.

Topics: Amino Acid Sequence; Cross-Linking Reagents; Elastic Modulus; Elastin; Hydrogels; Hydrophobic and Hydrophilic Interactions; Iridoids; Nanofibers; Peptides; Rheology

Deposition of calcium phosphate minerals on the elastin-rich medial layers of arteries can cause severe cardiovascular complications. There are no available treatments for medial calcification, and the mechanism of mineral formation on elastin layers is still unknown. We recently developed an in vitro model of medial calcification using cross-linked elastin-like polypeptide (ELP) membranes immersed in simulated body fluid (SBF). While mineral phase evolution matched that observed in a mouse model of medial calcification, the long incubation required was a practical limitation of this model. Using higher SBF ion concentrations could be a solution to speed up mineral deposition, but its effect on the mineralization process is still not well understood. Here we analyze mineral formation and phase transformation on ELP membranes immersed in high concentration SBF. We show that while mineral deposition is significantly accelerated in these conditions, the chemistry and morphology of the minerals deposited on the ELP membranes and the overall mineralization process are strongly affected. Overall, this work suggests that while the use of low concentration SBF in this in vitro model is more appropriate to study medial calcification associated with the loss of calcification inhibitors, higher SBF ion concentration may be more relevant to study medial calcification in patients with life-threatening diseases such as chronic kidney disease.

Topics: Apatites; Biomimetic Materials; Calcium; Crystallization; Elastin; Escherichia coli; Iridoids; Membranes, Artificial; Peptides; Sodium

Genipin is a natural plant-derived compound that covalently cross-links biopolymers into lattice networks with good biocompatibility, controllable swelling, and mechanical properties. This protocol describes the genipin cross-linking of elastic proteins, including tropoelastin and elastin-based polypeptides, through steps of elastin phase-separation upon addition of salt and heat, centrifugation to rapidly concentrate the dense protein phase, and incubation. This method is applicable for the fabrication of elastic materials suitable for use as scaffolds for biomedical applications.

Topics: Cross-Linking Reagents; Elastin; Iridoids; Molecular Structure; Peptides

Coronary angioplasty is the most widely used technique for removing atherosclerotic plaques in blood vessels. The regeneration of the damaged intima layer after this treatment is still one of the major challenges in the field of cardiovascular tissue engineering. Different polymers have been used in scaffold manufacturing in order to improve tissue regeneration. Elastin-mimetic polymers are a new class of molecules that have been synthesized and used to obtain small diameter fibers with specific morphological characteristics. Elastin-like polymers produced by recombinant techniques and called elastin-like recombinamers (ELRs) are particularly promising due to their high degree of functionalization. Generally speaking, ELRs can show more complex molecular designs and a tighter control of their sequence than other chemically synthetized polymers Rodriguez Cabello et al (2009 Polymer 50 5159-69, 2011 Nanomedicine 6 111-22). For the fabrication of small diameter fibers, different ELRs were dissolved in 2,2,2-fluoroethanol (TFE). Dynamic light scattering was used to identify the transition temperature and get a deep characterization of the transition behavior of the recombinamers. In this work, we describe the use of electrospinning technique for the manufacturing of an elastic fibrous scaffold; the obtained fibers were characterized and their cytocompatibility was tested in vitro. A thorough study of the influence of voltage, flow rate and distance was carried out in order to determine the appropriate parameters to obtain fibrous mats without beads and defects. Moreover, using a rotating mandrel, we fabricated a tubular scaffold in which ELRs containing different cell adhesion sequences (mainly REDV and RGD) were collected. The stability of the scaffold was improved by using genipin as a crosslinking agent. Genipin-ELRs crosslinked scaffolds  show a good stability and fiber morphology. Human umbilical vein endothelial cells  were used to assess the in vitro bioactivity of the cell adhesion domains within the backbone of the ELRs.

Topics: Amino Acid Sequence; Atherosclerosis; Biocompatible Materials; Cell Adhesion; Cell Survival; Cross-Linking Reagents; Dynamic Light Scattering; Elastin; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Microscopy, Electron, Scanning; Nanofibers; Polymers; Tissue Engineering; Tissue Scaffolds

Recombinant elastin-like protein polymers are increasingly being investigated as component materials of a variety of implantable medical devices. This is chiefly a result of their favorable biological properties and the ability to tailor their physical and mechanical properties. In this report, we explore the potential of modulating the water content, mechanical properties, and drug release profiles of protein films through the selection of different crosslinking schemes and processing strategies. We find that the selection of crosslinking scheme and processing strategy has a significant influence on all aspects of protein polymer films. Significantly, utilization of a confined, fixed volume, as well as vapor-phase crosslinking strategies, decreased protein polymer equilibrium water content. Specifically, as compared to uncrosslinked protein gels, water content was reduced for genipin (15.5%), glutaraldehyde (GTA, 24.5%), GTA vapor crosslinking (31.6%), disulfide (SS, 18.2%) and SS vapor crosslinking (25.5%) (P<0.05). Distinct crosslinking strategies modulated protein polymer stiffness, strain at failure and ultimate tensile strength (UTS). In all cases, vapor-phase crosslinking produced the stiffest films with the highest UTS. Moreover, both confined, fixed volume and vapor-phase approaches influenced drug delivery rates, resulting in decreased initial drug burst and release rates as compared to solution phase crosslinking. Tailored crosslinking strategies provide an important option for modulating the physical, mechanical and drug delivery properties of protein polymers.

Topics: Cross-Linking Reagents; Disulfides; Drug Delivery Systems; Elastin; Fibronectins; Glutaral; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Mechanical Phenomena; Sirolimus; Water

Decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the biochemical and structural properties of the extracellular matrix are still to be found. For this aim, bioreactors could be an alternative means to dynamically treat the biological samples, automatically controlling all the variables involved in the process and speeding up the entire procedure in order to deal with a suitable scaffold within a limited time period. This paper presents the characterization of rat tracheae decellularized in dynamic conditions, implementing a detergent-enzymatic method, previously considered. Only 6 cycles were enough to generate a tracheal matrix that was histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers was well preserved, such as the epithelial cilia, the luminal basement membrane and the main matrix components. The elastin content decreased, even if not significantly, after the decellularization protocol. Mechanical properties of the treated tissues were slightly affected by the procedure, and were partially recovered after crosslinking with genipin, a naturally-derived agent. The use of bioreactors could enhance the decellularization procedure of tissues/organs, but a careful selection of the processing parameters is needed in order to prevent large modifications compared to the native condition.

Topics: Animals; Biocompatible Materials; Biomechanical Phenomena; Cross-Linking Reagents; Elastin; Extracellular Matrix; Humans; Iridoids; Male; Materials Testing; Mesenchymal Stem Cells; Rats, Inbred BN; Trachea

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture - in particular of collagenous and elastic fiber structure -and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.

Topics: Adult; Biomechanical Phenomena; Cross-Linking Reagents; Elastin; Extracellular Matrix; Humans; Iridoids; Male; Materials Testing; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Middle Aged; Neovascularization, Physiologic; Time Factors; Tissue Donors; Tissue Engineering; Tissue Preservation; Tissue Scaffolds; Trachea

Polypeptides based on the alternating hydrophobic and cross-linking domain structure of human elastin are capable of undergoing self-assembly to produce polymeric matrices with unique biological and mechanical properties. Here, we test the initial feasibility of using a genipin cross-linked elastin-based material as an acellular plug in the treatment of an osteochondral defect in the rabbit knee. Full-thickness defects in the weight-bearing surface of the medial femoral condyle in 18 New Zealand White rabbits were surgically produced and press fitted with cylindrical pads composed of genipin cross-linked elastin-like polypeptides, with identical wounds in the opposite knee left untreated as controls. The biocompatibility of the material, overall wound healing and regeneration of subchondral tissue was assessed at 2, 4 and 6weeks by histological evaluation, synovial fluid analysis and microcomputerized tomography scanning. Histological analysis revealed the regeneration of subchondral bone at the periphery of the material, with evidence of hyaline-like overgrowth across the apical surface in 11/16 cases. Pads developed tight contacts with host tissue and appeared completely biocompatible, with no evidence of localized immune response or increased inflammation compared to controls. The material was stable to 6weeks, with an aggregate elastic modulus calculated at approximately 470kPa when tested under confined compression. Further studies are required to assess material degradation over time and long-term replacement with repair tissue.

Topics: Animals; Biocompatible Materials; Cross-Linking Reagents; Drug Implants; Elastic Modulus; Elastin; Hardness; Iridoid Glycosides; Iridoids; Materials Testing; Osteochondritis; Peptides; Rabbits; Surface Properties; Treatment Outcome

Elastin is an elastomeric, self-assembling extracellular matrix protein with potential for use in biomaterials applications. Here, we compare the microstructural and tensile properties of the elastin-based recombinant polypeptide (EP) EP20-244 crosslinked with either genipin (GP) or pyrroloquinoline quinone (PQQ). Recombinant EP-based sheets were produced via coacervation and subsequent crosslinking. The micron-scale topography of the GP-crosslinked sheets examined with atomic force microscopy revealed the presence of extensive mottling compared with that of the PQQ-crosslinked sheets, which were comparatively smoother. Confocal microscopy showed that the subsurface porosity in the GP-crosslinked sheets was much more open. GP-crosslinked EP-based sheets exhibited significantly greater tensile strength (P < or = 0.05). Mechanistically, GP appears to yield a higher crosslink density than PQQ, likely due to its capacity to form short-range and long-range crosslinks. In conclusion, GP is able to strongly modulate the microstructural and mechanical properties of elastin-based polypeptide biomaterials forming membranes with mechanical properties similar to native insoluble elastin.

Topics: Cross-Linking Reagents; Elastin; Iridoid Glycosides; Iridoids; Lysine; Microscopy, Atomic Force; Microscopy, Confocal; Peptides; PQQ Cofactor; Solvents; Tensile Strength; Water