Cross-linked Collagen Hydrogels Research Articles

Effects of Phenolics on the Physicochemical and Structural Properties of Collagen Hydrogel.

In the current era, the treatment of collagen hydrogels with natural phenolics for the improvement in physicochemical properties has been the subject of considerable attention. The present research aimed to fabricate collagen hydrogels cross-linked with gallic acid (GA) and ellagic acid (EA) at different concentrations depending on the collagen dry weight. The structural, enzymatic, thermal, morphological, and physical properties of the native collagen hydrogels were compared with those of the GA/EA cross-linked hydrogels. XRD and FTIR spectroscopic analyses confirmed the structural stability and reliability of the collagen after treatment with either GA or EA. The cross-linking also significantly contributed to the improvement in the storage modulus, of 435 Pa for 100% GA cross-linked hydrogels. The thermal stability was improved, as the highest residual weight of 43.8% was obtained for the hydrogels cross-linked with 50% GA in comparison with all the other hydrogels. The hydrogels immersed in 30%, 50%, and 100% concentrations of GA also showed improved swelling behavior and porosity, and the highest resistance to type 1 collagenase (76.56%), was obtained for 50% GA cross-linked collagen hydrogels. Moreover, GA 100% and EA 100% obtained the highest denaturation temperatures (Td) of 74.96 °C and 75.78 °C, respectively. In addition, SEM analysis was also carried out to check the surface morphology of the pristine collagen hydrogels and the cross-linked collagen hydrogels. The result showed that the hydrogels cross-linked with GA/EA were denser and more compact. However, the improved physicochemical properties were probably due to the formation of hydrogen bonds between the phenolic hydroxyl groups of GA and EA and the nitrogen atoms of the collagen backbone. The presence of inter- and intramolecular cross-links between collagen and GA or EA components and an increased density of intermolecular bonds suggest potential hydrogen bonding or hydrophobic interactions. Overall, the present study paves the way for further investigations in the field by providing valuable insights into the GA/EA interaction with collagen molecules.

Biocompatible hydrogels comprised of collagen, chitosan and polyurethane with potential applications for wound healing and controlled drug release

AbstractChitosan and collagen are two valuable biopolymers for the synthesis and design of biomaterials. In this work, we evaluate the effect of the concentration of chitosan on the physicochemical and biological properties of polyurethane‐crosslinked collagen hydrogels. Chitosan was added with a proportion ranging from 0 to 40 wt%. Depending on the aminoglycoside content, semi‐interpenetrating polymeric networks (semi‐IPNs) or completely IPNs were generated; this is attributed to the occlusion of the chitosan granules in the reticulated fibrillar matrix. The presence of chitosan in these hydrogels provides antibacterial capacity against pathogenic microorganisms such as Escherichia coli, which is corroborated by inhibition halo experiments. Hydrogels with 40 wt% of chitosan only showed improvement in the gelation time and mechanical performance, indicating a dependent relation of these properties with respect to the aminopolysaccharide content. Hydrogels with 10 wt% of chitosan showed the best biomedical performance. They also have the highest reticulation, swelling capability and chemical stability against degradation events such as hydrolysis and proteolysis. Further, they have the best biocompatibility for human monocytes, and they stimulate the secretion of monocyte chemotactic protein‐1 and tumor necrosis factor‐alpha. Also, these hydrogels showed a controlled release of ketorolac at pH 7 and 37 °C. The results of these in vitro biological tests demonstrate the potential application of novel biopolymer‐based hydrogels in chronic wound healing due to their antibacterial capacity and modulation of the biological response of specialized cells in dermal repair treatments, as well as for the design of smart materials for the controlled release of drugs. © 2023 Society of Industrial Chemistry.

Engineering large and geometrically controlled vascularized nerve tissue in collagen hydrogels to restore large-sized volumetric muscle loss

Developing scalable vascularized and innervated tissue is a critical challenge for the successful clinical application of tissue-engineered constructs. Collagen hydrogels are extensively utilized in cell-mediated vascular network formation because of their naturally excellent biological properties. However, the substantial increase in hydrogel contraction induced by populated cells limits their long-term use. Previous studies attempted to mitigate this issue by concentrating collagen pre-polymer solutions or synthesizing covalently crosslinked collagen hydrogels. However, these methods only partially reduce hydrogel contraction while hindering blood vessel formation within the hydrogels. To address this challenge, we introduced additional support in the form of a supportive spacer to counteract the contraction forces of populated cells and prevent hydrogel contraction. This approach was found to promote cell spreading, resist hydrogel contraction, control hydrogel/tissue geometry, and even facilitate the engineering of functional blood vessels and host nerve growth in just one week. Subsequently, implanting these engineered tissues into muscle defect sites resulted in timely anastomosis with the host vasculature, leading to enhanced myogenesis, increased muscle innervation, and the restoration of injured muscle functionality. Overall, this innovative strategy expands the applicability of collagen hydrogels in fabricating large vascularized nerve tissue constructs for repairing volumetric muscle loss (∼63 %) and restoring muscle function.

Bacterial phototoxicity of lumichrome photocrosslinked collagen hydrogels

In response to the World Health Organization's (WHO) call to develop alternative antibacterial methods, we investigate endogenous compounds and biopolymers. Lumichrome (LC) is an endogenous, lipophilic degradation product of riboflavin (vitamin B2) that can act as a photosensitizer for antimicrobial applications. LC is also a photocrosslinker of collagen. Crosslinked collagen hydrogels were prepared with two types of cyclodextrins (CDs), namely (2-hydroxypropyl)-β-CD (HPβCD) and -γ-CD (HPγCD), to improve the aqueous solubility of LC. The release of LC from the collagen gels and the phototoxic effect of UVA-irradiated LC against planktonic Staphylococcus (S.) aureus were investigated. Irradiation of LC hydrogels complexed with either CD resulted in similar levels of bacterial death despite the HPγCD gels containing half the LC concentration of the HPβCD gels. This effect may be explained by the higher complexation efficiency and stability of the LC-HPβCD complex, slowing down the LC release. Following treatment, small colony variants of S. aureus were observed. The study is a proof of concept of the release and phototoxic effects of an endogenous photocrosslinker embedded in a biopolymer as a sustainable antibacterial alternative to conventional therapy of, e.g., infected wounds.

Tailoring structural properties, mechanical behavior and cellular performance of collagen hydrogel through incorporation of cellulose nanofibrils and cellulose nanocrystals: A comparative study

Physically cross-linked collagen hydrogel is a desirable scaffold for tissue engineering applications especially where 3D cell encapsulation is considered. To overcome its shortcomings such as weak mechanical properties and also provide additional benefits, nanofiller reinforcement could be applied. This study was conducted to compare physical properties and cellular performance of physically cross-linked collagen hydrogel reinforced with cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs). Addition of both nanofillers drastically changed the hydrogel properties depending on the type and concentration. As a general trend, both CNCs and CNFs resulted in reduction in gelation time, enhancement in compressive strength, increase in swelling ratio, decrease in weight loss and improvement of injectability. The highest impact for CNCs was achieved when 5 % was applied, while the maximum impact for CNFs was observed for 3 % content (related to the collagen solid weight). By comparing two types of nanocellulose, CNCs showed higher impact on all properties. In-vitro cell compatibility with fibroblasts showed that CNFs did not adversely affected the viability and morphology of the cells while the CNCs improved cell viability and developed more elongated cell morphology.

Suspended Collagen Hydrogels to Replicate Human Colonic Epithelial Cell Interactions with Immune Cells.

The human colon plays a critical role in fluid and salt absorption and harbors the largest immune compartment. There is a widespread need for in vitro models of human colon physiology with its innate immune system. A method is described to produce a cassette with a network of struts supporting a suspended, non-chemically cross-linked collagen hydrogel scaffold compatible with the co-culture of primary gastrointestinal epithelium and migratory inflammatory cells. The epithelial monolayer cultured on the suspended collagen possesses a population of polarized and differentiated cells similar to that present in vivo. This epithelial layer displays proper barrier function with a transepithelial electrical resistance (TEER) ≥ 1,500 Ω cm2 and an apparent permeability ≤10-5 cm2 s-1 . Immune cells plated on the basal face of the scaffold transmigrated over a period of 24h to the epithelial layer in response to epithelial production of IL-8 induced by luminal stimulation of Clostridium difficile Toxin A. These studies demonstrate that this in vitro platform possesses a functional primary colonic epithelial layer with an immune cell compartment capable of recruitment in response to pro-inflammatory cues coming from the epithelium.

Tuning of 2D cultured human fibroblast behavior using lumichrome photocrosslinked collagen hydrogels

Collagen is extensively used in fabrication of hydrogels for biomedical applications but needs improvement after its isolation from tissues due to slow gelation and poor mechanical properties. Crosslinking could tailor such properties. Collagen has previously been crosslinked by chemical or photochemical methods. Chemical crosslinkers are often toxic, and the crosslinking reaction is difficult to control. Photochemical crosslinkers are usually biocompatible compounds that are activated upon irradiation. Riboflavin (vitamin B2), a photochemical crosslinker of collagen, photodegrades to lumichrome upon irradiation. Cyclodextrins have previously been used to increase the aqueous solubility of lumichrome and regulate collagen self-assembly. In this study, lumichrome dissolved by cyclodextrin complexation was used as a photochemical crosslinker of collagen. Lumichrome photocrosslinking reduced the gelation time to 10 s, compared to 90 min for physical crosslinking. The formed hydrogels exhibited increased elasticity, water absorption properties and water holding capacity compared to physically crosslinked collagen hydrogels and riboflavin photocrosslinked collagen hydrogels. Fibroblasts achieved a myofibroblastic phenotype when cultivated in 2D on lumichrome photocrosslinked gels as observed from histology. These biocompatible photocrosslinked hydrogels could have potential applications in biomedical applications, such as wound healing.

Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Crosslinking effect of dialdehyde cholesterol modified starch nanoparticles on collagen hydrogel

Crosslinking is frequently used to improve the inherent poor physicochemical properties of collagen. However, local flocculation and irregular crosslinking of collagen would be unavoidably occurred once contacting with crosslinking agents due to widespread complex interactions. Herein, dialdehyde starch-based nanoparticles were developed to crosslink collagen as a new strategy. Starch was conjugated with cholesterol chloroformate before periodate oxidation to obtain dialdehyde cholesterol modified starch (DACS). DACS self-assembled into nanoparticles (DACSNPs) and crosslinked with collagen to fabricate collagen hydrogels (DACSNPs-Col). DACSNPs-Col hydrogels exhibited faster gelation rate, better uniform porous structure, higher mechanical properties and better degradation stability than dialdehyde starch crosslinked hydrogels. Significantly, DACSNPs-Col hydrogels show homogeneous structure, improved mechanical properties, low cytotoxicity, well blood compatibility, high cell adhesion and proliferation. Overall, the oxidized polysaccharide nanoparticles crosslinked collagen hydrogels have homogeneous and compact microstructure and improved physicochemical properties, which show potential application prospect in the field of tissue engineering scaffold.

Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

X-ray tomography analysis reveals the influence of graphene on porous morphology of collagen cryogels

X-ray micro-tomography based analysis of porous hydrogel has gained a wide attention recently. It provides an advantage in three-dimensional analysis of pore morphometric and interconnection within the hydrogel network. We have fabricated highly elastic graphene crosslinked collagen hydrogel using cryogelation technique. The influence of graphene as a nano-crosslinker on the overall porosity and inter-connections between the pores in collagen cryogels was determined using X-ray micro-tomography. We have evaluated the effect of different concentration of amino-functionalized graphene nano-crosslinker on collagen cryogels porosity, pore volume, interconnectivity density, fractal dimensions and pore wall thickness. This study, reveals that the use of graphene as a nano-crosslinker have improved micro-architecture as compare to collagen cryogels in the absence of graphene for tissue engineering applications.

Role of epigallocatechin gallate in collagen hydrogels modification based on physicochemical characterization and molecular docking

Collagen Type I derived from fish is mainly limited by its poor physicochemical properties for further applications. In this study, we developed epigallocatechin gallate (EGCG) cross-linked collagen hydrogels (EC hydrogels) to realize physicochemical improvements, basing on the interaction mechanism between collagen and EGCG. The integrity of collagen framework with slight secondary structure change in the presence of EGCG was confirmed. The stronger stability of collagen fibrils was proved by slower swelling ratio, declined enzymatic degradation, improved thermal analysis and mechanical test due to EGCG modification. To illustrate the potential mechanism between collagen and EGCG, molecular docking was used to identify both covalent (CN bond, between lysine of collagen and C2-ring B of EGCG) and non-covalent bonds (hydrogen bond and hydrophobic interaction) within in EC hydrogel. Taken together, this work would offer some insights into the further study about the interaction between EGCG and collagen.

Phototunable interpenetrating polymer network hydrogels to stimulate the vasculogenesis of stem cell-derived endothelial progenitors

Vascularization of engineered scaffolds remains a critical obstacle hindering the translation of tissue engineering from the bench to the clinic. We previously demonstrated the robust micro-vascularization of collagen hydrogels with induced pluripotent stem cell (iPSC)-derived endothelial progenitors; however, physically cross-linked collagen hydrogels compact rapidly and exhibit limited strength. We have synthesized an interpenetrating polymer network (IPN) hydrogel comprised of collagen and norbornene-modified hyaluronic acid (NorHA) to address these challenges. This dual-network hydrogel combines the natural cues presented by collagen's binding sites and extracellular matrix (ECM)-mimicking fibrous architecture with the in situ modularity and chemical cross-linking of NorHA. We modulated the IPN hydrogel's stiffness and degradability by varying the concentration and sequence, respectively, of the NorHA peptide cross-linker. Rheological characterization of the photo-mediated gelation process revealed that the IPN hydrogel's stiffness increased with cross-linker concentration and was decoupled from the bulk NorHA content. Conversely, the swelling of the IPN hydrogel decreased linearly with increasing cross-linker concentration. Collagen microarchitecture remained relatively unchanged across cross-linking conditions, although the addition of NorHA delayed collagen fibrillogenesis. Upon iPSC-derived endothelial progenitor encapsulation, robust, lumenized microvascular networks developed in IPN hydrogels over two weeks. Subsequent computational analysis showed that an initial rise in stiffness increased the number of branch points and vessels, but vascular growth was suppressed in high stiffness IPN hydrogels. These results suggest that an IPN hydrogel consisting of collagen and NorHA is highly tunable, compaction resistant, and capable of supporting vasculogenesis.

Tunable Fast Relaxation in Imine-Based Nanofibrillar Hydrogels Stimulates Cell Response through TRPV4 Activation.

As a key mechanical signal of natural extracellular matrix (ECM), stress relaxation plays an essential role in cell fate decision. However, the biomimetic matrix with fast stress relaxation and its cellular response mechanism have received little attention. Meanwhile, the nanofibrillar architecture which is conductive to mechanical transduction has invariably been ignored in the previous viscoelastic matrix design. Herein, by introducing a dynamic covalent imine bond into a physically cross-linked collagen hydrogel, we prepared bionic fast-relaxing nanofibrillar hydrogels with relaxation time less than 10 s. Through a single control of imine bond content, we realized fine-tuning of the relaxation rate while maintaining a constant initial modulus and fiber density. Using MC3T3-E1 cells as a model, we then proved that the nanofibrillar matrix with fast relaxation mechanics can effectively promote cell spreading and differentiation. In particular, TRPV4 as a molecular sensor of matrix viscoelasticity was demonstrated to regulate cell fate on the nanofibrillar hydrogels by mediating calcium influx. It is expected that the material design principle combining both nanofibrillar structure and tunable fast-relaxation can provide a more broadly adaptable materials platform for simulating natural ECM mechanical cues, and the investigation of the TRPV4 ion channel mediated cellular response will facilitate discovery of more fundamental mechanisms in tissue growth and development.

Activation of dendritic cells by crosslinked collagen hydrogels (artificial corneas) varies with their composition

Activated T cells are known to promote fibrosis, a major complication limiting the range of polymeric hydrogels as artificial corneal implants. As T cells are activated by dendritic cells (DC), minimally activating hydrogels would be optimal. In this study, we evaluated the ability of a series of engineered (manufactured/fabricated) and natural collagen matrices to either activate DC or conversely induce DC apoptosis in vitro. Bone marrow DC were cultured on a series of singly and doubly crosslinked hydrogels (made from recombinant human collagen III [RHCIII] or collagen mimetic peptide [CMP]) or on natural collagen-containing matrices, MatrigelTM and de-cellularised mouse corneal stroma. DC surface expression of major histocompatibility complex Class II and CD86 as well as apoptosis markers were examined. Natural matrices induced low levels of DC activation and maintained a "tolerogenic" phenotype. The same applied to singly crosslinked CMP-PEG gels. RHCIII gels singly crosslinked using either N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with the coinitiator N-hydroxy succinimide (EDC-NHS) or N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide metho-p-toulenesulfonate with NHS (CMC-NHS) induced varying levels of DC activation. In contrast, however, RHCIII hydrogels incorporating an additional polymeric network of 2-methacryloyloxyethyl phosphorylcholine did not activate DC but instead induced DC apoptosis, a phenomenon observed in natural matrices. This correlated with increased DC expression of leukocyte-associated immunoglobulin-like receptor-1. Despite low immunogenic potential, viable tolerogenic DC migrated into and through both natural and manufactured RHCIII gels. These data show that the immunogenic potential of RHCIII gels varies with the nature and composition of the gel. Preclinical evaluation of hydrogel immunogenic/fibrogenic potential is recommended.

Non-toxic chemically crosslinked collagen hydrogels for cell delivery

Non-toxic chemically crosslinked collagen hydrogels for cell delivery

Comparison of covalently and physically cross-linked collagen hydrogels on mediating vascular network formation for engineering adipose tissue

Timely tissue vascularization and integration of engineered tissues into a patient plays an important role in the successful translation of engineered tissues into clinically relevant therapies. To decrease the time needed to vascularize an engineered adipose tissue, suitable local microenvironments provided by hydrogels to support cell-based functional vascular network formation have been investigated. Using the same biomolecule in solution, two types of hydrogels can be obtained: a “physical hydrogel” which is thermal-induced self-assemble fibril initiation and growth, due to amino and carboxyl telopeptides on collagen chains, and a “chemical hydrogel” which results from the covalently cross-linking of the side chains induced by one step enzyme mediation in aqueous solution. In this paper, we compare the capability of engineering vascular network and large-sized vascularized adipose tissue in vivo in different types of collagen hydrogels, physical and chemical crosslinking. The relationships between vascular network formation and hydrogel properties for the two types of hydrogels are discussed. Finally, we successfully engineered a vascularized adipose tissue construct (∼877.6 adipocytes/mm2; 94% area of a construct) in the absence of exogenous cytokines in chemical covalently crosslinking cell-laden hydrogel. These results show manipulating the polymerized methods of a hydrogel could not only modulate vascular network formation, but also regenerate adipose tissue in vivo.

Dental pulp stem cell-derived chondrogenic cells demonstrate differential cell motility in type I and type II collagen hydrogels

Background ContextAdvances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation. PurposeThe purpose of this study was to determine the motility of DPSC and DPSC-derived chondrogenic cells in type I and type II collagen hydrogels. Study Design/SettingThe time lapse imaging that recorded cell migration was analyzed to quantify the cell migration velocity and distance. MethodsThe cell viability of DPSCs in native or poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG)-crosslinked type I and type II collagen hydrogels was determined using LIVE/DEAD cell viability assay and AlamarBlue assay. DPSCs were differentiated into chondrogenic cells. The migration of DPSCs and DPSC-derived chondrogenic cells in these hydrogels was recorded using a time lapse imaging system. This study was funded by the Regional Institute on Aging and Wichita Medical Research and Education Foundation, and the authors declare no competing interest. ResultDPSCs showed high cell viability in non-crosslinked and crosslinked collagen hydrogels. DPSCs migrated in collagen hydrogels, and the cell migration speed was not significantly different in either type I collagen or type II collagen hydrogels. The migration speed of DPSC-derived chondrogenic cells was higher in type I collagen hydrogel than in type II collagen hydrogel. Crosslinking of type I collagen with 4S-StarPEG significantly reduced the cell migration speed of DPSC-derived chondrogenic cells. ConclusionsAfter implantation of collagen hydrogels encapsulating DPSCs or DPSC-derived chondrogenic cells, the cells can potentially migrate from the hydrogels and migrate into the NP tissue. This study also explored the differential cell motility of DPSCs and DPSC-derived chondrogenic cells in these collagen hydrogels.

Cross-linked Collagen Hydrogel Matrix Resisting Contraction To Facilitate Full-Thickness Skin Equivalents.

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.

Isolation and characterization of pepsin solubilized type II collagen from porcine cartilage and cartilagious fish for intervertebral disc (IVD) regeneration

Event Abstract Back to Event Isolation and characterization of pepsin solubilized type II collagen from porcine cartilage and cartilagious fish for intervertebral disc (IVD) regeneration Zhuning Wu1, 2*, Anne Maria Mullen3, Abhay Pandit2 and Dimitrios Zeugolis1, 2 1 National University of Ireland Galway (NUI Galway), Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Ireland 2 National University of Ireland Galway (NUI Galway), Centre for Research in Medical Devices (CÚRAM), Ireland 3 Food Research Centre, Teagasc, Ireland Introduction: Collagen is the main component of ECM with 29 types identified. Type II collagen presents supportive function in cartilaginous tissues. Since type II collagen is the major component of nucleus pulposus in intervertebral disc, the potential of injectable type II collagen hydrogel as a cell carrier for intervertebral disc regeneration is being investigated in this project. At this stage, we isolated and characterised type II collagen from porcine cartilage nad cartilaginous fish. Type II collagen was cross-linked with 4arm and 8arm StarPEG. We hypothesise that pure type II collagen can be obtained from cartilaginous tissue and 8arm StarPEG crosslinking will increase the mechanical, biological and biomedical properties of collagen hydrogel. Materials and Methods: Tyep II collagen was extracted from porcine trachea, ear, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4ºC. SDS-PAGE was used to reveal the number and size of collagen chains. Thermal stability was tested by differential scanning calorimetry (DSC). Enzymatic degradation was assessed by collagenase assay. TNBSA assay will be carried out to test the free amine group in cross-linked collagen hydrogel. Human chondrocyte cell phenotype maintenance will be assessed with proteina and gene assays. Results: Conclusions: Pure type II collagen can be extracted from porcine cartilage and cartilaginous fish. Further biological assays will be carried out to characterise collagen enzymatic degradation and thermal stability. The authors would like to thank Teagasc, Irish Agriculture and Food Development Authority, Funding agency for providing financial support for this project.