Cross-linked Collagen Hydrogels Research Articles

Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix

Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1month of implantation. Statement of SignificanceWe reported a method for preparing autologous extracellular matrix scaffolds, murine collagen-Ph hydrogels, and demonstrated its suitability for use in supporting human progenitor cell-based formation of 3D vascular networks in vitro and in vivo. Results showed extensive human vascular networks can be generated within 7days, engineered vascular density inside collagen-Ph constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with existing vasculature to further support the survival of host muscle tissues. Moreover, optimized conditions of cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse.

Enhancing the stiffness of collagen hydrogels for delivery of encapsulated chondrocytes to articular lesions for cartilage regeneration.

This study investigated a dual crosslinking paradigm, consisting of (a) photocrosslinking with Rose Bengal (RB) and green light followed by (b) chemical crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) to enhance collagen gel stiffness. In group 1, 50 μL collagen constructs of 2% (w/v) type I collagen containing 10 μM RB were allowed to gel spontaneously at 37 °C. In group 2, the spontaneous gels were exposed to green light (532 nm). In group 3, the photochemically crosslinked gels were subsequently treated with a 1-h exposure to 33 mM EDC/6 mM NHS. Samples (n = 18) were subjected to 0.08% (w/v) collagenase digestion, and the storage modulus of samples was measured by rheometry. Viability of encapsulated chondrocytes was measured by live/dead assay. Chondrocytes were ≥ 95% viable in all constructs at 10 days in vitro. Resistance to collagenase digestion increased as; spontaneous gels (2 h) < photochemical gels (3-4 h) < dual crosslinked gels (>24 h). The storage modulus of dual-crosslinked constructs was increased 5-fold over both photocrosslinked and spontaneous gels. As the dual crosslinking paradigm did not reduce encapsulated chondrocyte viability, these crosslinked collagen hydrogels could be a useful tool for the practical delivery of encapsulated chondrocytes to articular defects.

Novel Physically Cross-Linked Collagen Hydrogel for Cardiovascular Tissue Regeneration

Novel Physically Cross-Linked Collagen Hydrogel for Cardiovascular Tissue Regeneration

Epoxy cross-linked collagen and collagen-laminin Peptide hydrogels as corneal substitutes.

A bi-functional epoxy-based cross-linker, 1,4-Butanediol diglycidyl ether (BDDGE), was investigated in the fabrication of collagen based corneal substitutes. Two synthetic strategies were explored in the preparation of the cross-linked collagen scaffolds. The lysine residues of Type 1 porcine collagen were directly cross-linked using l,4-Butanediol diglycidyl ether (BDDGE) under basic conditions at pH 11. Alternatively, under conventional methodology, using both BDDGE and 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as cross-linkers, hydrogels were fabricated under acidic conditions. In this latter strategy, Cu(BF4)2·XH2O was used to catalyze the formation of secondary amine bonds. To date, we have demonstrated that both methods of chemical cross-linking improved the elasticity and tensile strength of the collagen implants. Differential scanning calorimetry and biocompatibility studies indicate comparable, and in some cases, enhanced properties compared to that of the EDC/NHS controls. In vitro studies showed that human corneal epithelial cells and neuronal progenitor cell lines proliferated on these hydrogels. In addition, improvement of cell proliferation on the surfaces of the materials was observed when neurite promoting laminin epitope, IKVAV, and adhesion peptide, YIGSR, were incorporated. However, the elasticity decreased with peptide incorporation and will require further optimization. Nevertheless, we have shown that epoxy cross-linkers should be further explored in the fabrication of collagen-based hydrogels, as alternatives to or in conjunction with carbodiimide cross-linkers.

Preparation and characterization of macromolecule cross-linked collagen hydrogels for chondrocyte delivery

Collagen hydrogels are widely used in cartilage tissue engineering for their mimicked chondrogenic environment. Due to the rapid degradation nature and weak mechanical property, collagen hydrogels are often cross-linked in application. In this work, collagen hydrogels were soaked into oxidized alginate solution which used as macromolecular cross-linker to prepare the cross-linked hydrogels. Soaking method could retain the self-assemble property of collagen and also bring in a cross-linking network. The compressive modulus and degradation properties of collagen hydrogels were ameliorated after cross-linked, and chondrocytes encapsulated in the cross-linked hydrogels proliferated well and maintained the cell phenotype. This study implied that collagen hydrogels cross-linked by oxidized alginate may have a great potential for application in cartilage tissue engineering.

Crosslinked collagen hydrogels as corneal implants: Effects of sterically bulky vs. non-bulky carbodiimides as crosslinkers

We have previously shown that recombinant human collagen can be crosslinked with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) to fabricate transparent hydrogels possessing the shape and dimensions of the human cornea. These corneal implants have been tested in a Phase I human clinical study. Although these hydrogels successfully promoted corneal tissue and nerve regeneration, the gelling kinetics were difficult to control during the manufacture of the implants. An alternative carbodiimide capable of producing hydrogels of similar characteristics as EDC in terms of strength and biocompatibility, but with a longer gelation time would be a desirable alternative. Here, we compared the crosslinking kinetics and properties of hydrogels crosslinked with a sterically bulky carbodiimide, N-Cyclohexyl-N′-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), with that of EDC. CMC crosslinking was possible at ambient temperature whereas the EDC reaction was too rapid to control and had to be carried out at low temperatures. The highest tensile strength obtained using optimized formulations were equivalent, although CMC crosslinked hydrogels were found to be stiffer. The collagenase resistance of CMC crosslinked hydrogels was superior to that of EDC crosslinked hydrogels while biocompatibility was similar. We are also able to substitute porcine collagen with recombinant human collagen and show that the in vivo performance of both resulting hydrogels as full-thickness corneal implants is comparable in a mouse model of an orthotopic corneal graft. In conclusion, CMC is a viable alternative to EDC as a crosslinker for collagen-based biomaterials for use as corneal implants, and potentially for use in other tissue engineering applications.

Stimuli responsive deswelling of radiation synthesized collagen hydrogel in simulated physiological environment

Collagen hydrogels were prepared via radiation crosslinking. The simulated physiological environmental effects related to their biomedical applications on the volume phase transition of collagen hydrogel were studied, that is stimuli response to ions, temperature, and pH. The deswelling behavior of collagen hydrogel depends on the salt concentration, temperature, pH, and the hydrogel preparation procedure. Meanwhile, hydrogel structure related to the volume phase transition was investigated by FTIR, fluorescence spectrum, and HR-MAS NMR. Deswelling in salt solution caused little change on collagen conformation, and a denser network led to more significant tyrosine-derived fluorescence quenching. Hydrogen bonding between hydrated water and collagen polypeptide chain was dissociated and the activity of hydrophobic side chain increased, inducing a higher extent of contraction with the increasing of salt concentration. Moreover, salt solution treatments weakened the electrostatic interactions, side chains interactions, and hydrogen bonding of collagen hydrogel, which reduced the thermal stability of collagen hydrogel. Comparing with cell-free collagen hydrogel contraction, fibroblasts did not aggravate contraction of collagen hydrogel significantly. This study elucidated the deswelling mechanism of radiation crosslinked collagen hydrogel in simulated physiological environment and provides strategies for controlling the stimuli response of collagen hydrogel in biomedical application.

Pluronics as crosslinking agents for collagen: novel amphiphilic hydrogels

AbstractA series of Pluronic samples (L61, L121, F68, F108) were investigated as collagen crosslinking agents to determine their ability to improve the Young's modulus of a collagen hydrogel, while simultaneously serving as surfactants for single‐walled carbon nanotubes (SWNTs). The crosslinked collagen matrices were prepared by blending type I bovine collagen with either Pluronics or SWNTs dispersed in an aqueous Pluronic solution and crosslinked utilizing carbodiimide chemistry. The resulting material was a crosslinked collagen hydrogel with sufficient mechanical strength to be manipulated and transferred without damaging the matrix. Differential scanning calorimetry confirmed a change in the denaturation temperature for hydrogels prepared using Pluronic or Pluronic/SWNT solutions. Water uptake analysis confirmed the crosslinked matrices to be hydrogels. These collagen hydrogels produced with Pluronics as the crosslinking agents exhibited a Young's modulus 3 to 9 times greater than collagen hydrogels produced in the absence of any crosslinking agent, regardless of polymer molecular weight. However, non‐covalent incorporation of SWNTs was not found to affect the Young's modulus of the resulting collagen hydrogels at the incorporation levels achieved with the Pluronics surfactants. Copyright © 2010 Society of Chemical Industry

Reinforcement of collagen with covalently-functionalized single-walled carbon nanotube crosslinkers

Poly(ethyleneimine) (PEI) functionalized single-walled carbon nanotubes (SWNTs) and free PEI were investigated as collagen crosslinking agents to determine their ability to improve the Young's modulus of a collagen hydrogel. The crosslinked collagen matrices were prepared by blending Type I bovine collagen with either PEI-SWNT or free PEI and crosslinked utilizing carbodiimide chemistry. The resulting SWNT enriched material was a crosslinked collagen hydrogel with sufficient mechanical strength to be manipulated and transferred without damaging the matrix. Raman spectroscopy confirmed the presence of SWNTs within the PEI-SWNT/collagen hydrogels. In addition, it confirmed that crosslinking did not alter the SWNT delocalized network. Dynamic scanning calorimetry confirmed a change in the denaturation temperature for hydrogels prepared by PEI-SWNT or PEI crosslinkers. Water uptake analysis suggested very loosely crosslinked matrices were produced regardless of the crosslinking agent used. However, evaluating the Young's modulus, it was found that collagen hydrogels produced with PEI-SWNTs as the crosslinking agent had a modulus 4.5 to 9 times that of collagen hydrogels produced with PEI of the same amine concentration or in the absence of any crosslinking agent. In addition, PEI-SWNT crosslinked collagen hydrogels exhibited superior Young's modulus to control samples in which only the SWNTs or unlinked mixtures of SWNTs and PEI were used as crosslinkers. This comparative study confirms that PEI-SWNT crosslinked collagen gels exhibited Young's moduli significantly higher than collagen gels cross-linked with just nanotubes, PEI, or blends of nanotubes and PEI.