Collagen Hydrogels Research Articles

Immune cells and inflammatory mediators cause endothelial dysfunction in a vascular microphysiological system.

Functional assessment of endothelium serves as an important indicator of vascular health and is compromised in vascular disorders including hypertension, atherosclerosis, and preeclampsia. Endothelial dysfunction in these cases is linked to dysregulation of the immune system involving both changes to immune cells and increased secretion of inflammatory cytokines. Herein, we utilize a well-established microfluidic device to generate a 3-dimensional vascular microphysiological system (MPS) consisting of a tubular blood vessel lined with human umbilical vein endothelial cells (HUVECs) to evaluate endothelial function measured via endothelial permeability and Ca2+ signaling. We evaluated the effect of a mixture of factors associated with inflammation and cardiovascular disease (TNFα, VEGF-A, IL-6 at 10 ng ml-1 each) on vascular MPS and inferred that inflammatory mediators contribute to endothelial dysfunction by disrupting the endothelial barrier over a 48 hour treatment and by diminishing coordinated Ca2+ activity over a 1 hour treatment. We also evaluated the effect of peripheral blood mononuclear cells (PBMCs) on endothelial permeability and Ca2+ signaling in the HUVEC MPS. HUVECs were co-cultured with PBMCs either directly wherein PBMCs passed through the lumen or indirectly with PBMCs embedded in the supporting collagen hydrogel. We revealed that phytohemagglutinin (PHA)-M activated PBMCs cause endothelial dysfunction in MPS both through increased permeability and decreased coordinated Ca2+ activity compared to non-activated PBMCs. Our MPS has potential applications in modeling cardiovascular disorders and screening for potential treatments using measures of endothelial function.

Amine-reactive crosslinking enhances type 0 collagen hydrogel properties for regenerative medicine.

Collagen is extensively utilised in regenerative medicine due to its highly desirable properties. However, collagen is typically derived from mammalian sources, which poses several limitations, including high cost, potential risk of immunogenicity and transmission of infectious diseases, and ethical and religious constraints. Jellyfish-sourced type 0 collagen represents a safer and more environmentally sustainable alternative collagen source. Thus, we investigated the potential of jellyfish collagen-based hydrogels, obtained from Rhizostoma pulmo (R. pulmo) jellyfish, to be utilised in regenerative medicine. A variety of R. pulmo collagen hydrogels (RpCol hydrogels) were formed by adding a range of chemical crosslinking agents and their physicochemical and biological properties were characterised to assess their suitability for regenerative medicine applications. The characteristic chemical composition of RpCol was confirmed by Fourier-transform infrared spectroscopy (FTIR), and the degradation kinetics, morphological, and rheological properties of RpCol hydrogels were shown to be adaptable through the addition of specific chemical crosslinking agents. The endotoxin levels of RpCol were below the Food and Drug Administration (FDA) limit for medical devices, thus allowing the potential use of RpCol in vivo. 8-arm polyethylene glycol succinimidyl carboxyl methyl ester (PEG-SCM)-crosslinked RpCol hydrogels preserved the viability and induced a significant increase in the metabolic activity of immortalised human mesenchymal stem/stromal cells (TERT-hMSCs), therefore demonstrating their potential to be utilised in a wide range of regenerative medicine applications.

Development of a Simple and Reproducible Cell-derived Orthotopic Xenograft Murine Model for Neuroblastoma.

Neuroblastoma is a common childhood cancer with poor survival for children with high-risk disease, and ongoing research to improve outcomes is needed. Patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM) are reliable models for oncologic research; however, they are resource-intensive, expensive, and require significant expertise to develop and maintain. We developed an orthotopic xenograft murine model of neuroblastoma that utilizes cryopreserved banks of human neuroblastoma cell lines, requires minimal equipment, and is easily reproducible. The neuroblastoma cell line NB1643 was obtained from the Children's Oncology Group (COG) Childhood Cancer Repository. Nod-SCID-gamma (NSG) mice underwent orthotopic injection of 2x106 NB1643 cells suspended in 10 μl of collagen hydrogel directly into the adrenal gland via an open retroperitoneal surgical approach. Mice were monitored by ultrasound and in vivo imaging system (IVIS) until the tumor reached the volume of the ipsilateral kidney. Tumor identity was confirmed by necropsy and histologic analysis. A total of 55 mice underwent surgery. Eight died due to anesthetic or surgical complications. 39/47 (78%) survivors grew primary adrenal tumors. Average anesthesia time was 30 min. Ultrasound and IVIS successfully characterized tumor growth in all mice. Average time to target tumor size was 5 weeks (range=3-9). Gross pathologic and histologic analysis confirmed adrenal tumors consistent with neuroblastoma in all mice with adrenal masses. A cell-derived orthotopic xenograft murine model can be successfully used to create an in vivo model of neuroblastoma. This model can be utilized in environments where PDX or GEMM models are not feasible.

A comparative analysis of stem cell differentiation on 2D and 3D substrates using Raman microspectroscopy.

Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.

A simple and rapid assay of lysosomal-targeting CDy6 for long-term real-time viability assessments in 2D and 3D in vitro culture models

CDy6, a BODIPY-derived compound, is used to label lysosomes and visualize proliferating cells. However, its effectiveness in long-term, real-time cell viability assays using 2D or 3D cell culture models is unclear. We evaluated the suitability of CDy6 by assessing cell health using human keratinocyte and fibroblast cell lines in both models. Cells were stained with CDy6 or other dyes and fluorescent images were obtained with confocal microscopy. CLV extracts derived from CDy6-stained HaCaT cells were also dissolved with DMSO and analyzed using a spectrometer. Furthermore, we added CDy6-stained collagen hydrogels to CCD-986sk cells, loaded them into a frame construction to establish a 3D dermal layer for long-term culture, and analyzed the status of the CLVs. The CLV method, also measured using a spectrometer, yielded results similar to MTT assay for validating viability. In contrast to calcein AM staining, the CLV method allows for both absorbance measurement and imaging under short-term and long-term culture conditions with less cytotoxicity. In conclusion, the CLV method provides a simple and sensitive tool for assessing the status of live cells in 2D and 3D in vitro cell culture models and can be used as an alternative to animal testing, such as with 3D artificial skin models.

One-step fabrication of an injectable antibacterial collagen hydrogel with in situ synthesized silver nanoparticles for accelerated diabetic wound healing

Developing antibacterial collagen-based wound dressings that target multidrug-resistant bacteria holds promise for healing diabetic wounds prone to infection, yet traditional approaches pose persistent challenges of cytotoxicity and uneconomical steps. To address these issues, we develop a biocompatible, eco-friendly, one-step cross-linking and reduction strategy, fabricating injectable antibacterial collagen hydrogels containing in situ-generated silver nanoparticles for diabetic wound care. By exposing the collagen-silver ion (Ag+) mixture to visible light, collagen-silver nanoparticle (Col I/AgNPs) hydrogels were one step created, eliminating the need for additional cross-linkers and toxic reducing agents. The synergistic interaction between collagen and silver ions was the first time investigated, revealing a pivotal redox reaction with a dual role that simultaneously initiated collagen cross-linking and reduced Ag+ to AgNPs. The resulting biocompatible Col I/AgNPs hydrogel was found to have multifunctional properties, exhibiting excellent injectability, enhanced biological activity, and outstanding broad-spectrum antimicrobial ability. Furthermore, animal experiments utilizing a diabetic full-thickness wound model demonstrated their excellent in vivo healing efficacies, capable of establishing efficient antibacterial barriers and serving as bioactive regenerative scaffolds. Our study proposes an innovative one-step approach to create injectable antibacterial collagen-silver nanoparticle hydrogels, highlighting their potential as advanced dressings for diabetic wound treatment and other biomedical applications.

Morphological features of bone regenerate when using bone plastic material based on collagen hydrogel in an experiment

Background: Bone-plastic materials of various origin are used when it is necessary to restore or increase the volume of bone tissue of the jaws. Collagen hydrogel of xenogenic origin is a relatively new form of materials used for bone grafting, and is of interest for studying its osteoplastic properties.
 Aim: Morphological evaluation of osteoplastic properties of a material based on collagen hydrogel in an experiment.
 Material and methods: to evaluate the osteoplastic properties of collagen hydrogel, an experimental study was conducted on laboratory animals. A model of a critical defect in the cranial vault of rats was chosen for the study. In the experimental group, the defect was filled with xenogenic bone matrix and collagen hydrogel. In the comparison group, the defect was filled with xenogenic bone matrix. morphological evaluation of bone regeneration of samples of the experimental group and the comparison group was performed at different study periods.
 Results and their discussion:
 After the first month of observation, the histological picture in the samples of the experimental group and the comparison group had no significant differences. After two months of the study, the formation of an osseomucoid and the appearance of an insignificant number of osteoblasts were observed in the experimental group, while in the comparison group the stromal component was represented only by collagen fibers. After three months of observation, the organization of the stromal component was determined in the comparison group, single osteoblasts were observed, whereas in the experimental group the stromal component was partially replaced by a bone plate, and the cellular composition was represented by osteoblasts, osteocytes and osteoclasts.
 Conclusion: The addition of collagen hydrogel to the bone plastic material provides not only acceleration of the process of osteoreparation, but also the formation of a regenerate having the correct histoarchitectonics. The issue of using collagen hydrogel as a component of bone-plastic materials is promising and requires further study.

Chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially modify the biophysical properties of collagen-based hydrogels

Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. This study focuses on the GAG molecules chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA). CS and DS are stereoisomers while HA is the only non-sulfated GAG. We characterized and decoupled the effects of these GAG molecules on the stiffness, transport, and matrix microarchitecture properties of type I collagen hydrogels using mechanical indentation testing, microfluidics, and confocal reflectance imaging, respectively. We complement these biophysical measurements with turbidity assays to profile collagen aggregate formation. Surprisingly, only HA enhanced the ECM indentation modulus, while all three GAGs had no effect on hydraulic permeability. Strikingly, we show that CS, DS, and HA differentially regulate the matrix microarchitecture of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs define key physical properties of the ECM, this work shows new ways in which stiffness measurements, microfluidics, microscopy, and turbidity kinetics can be used complementarily to reveal details of collagen self-assembly and structure. Statement of SignificanceCollagen and glycosaminoglycans (GAGs) are integral to the structure, function, and bioactivity of the extracellular matrix (ECM). Despite widespread interest in collagen-GAG composite hydrogels, there is a lack of quantitative understanding of how different GAGs alter the biophysical properties of the ECM across tissue, cellular, and subcellular length scales. Here we show using mechanical, microfluidic, microscopy, and analytical methods and measurements that the GAG molecules chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially regulate the mechanical, transport, and microstructural properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. As such, these results will inform improved design and utilization of collagen-based scaffolds of tailored composition, mechanical properties, molecular availability due to mass transport, and microarchitecture.

Development of an In Situ Photo-Crosslinking Antimicrobial Collagen Hydrogel for the Treatment of Infected Wounds.

Antimicrobial hydrogels have received considerable attention in the treatment of bacteria-infected wounds. Herein, we develop a neutral, soluble collagen via modification with maleic anhydride, serving as a hydrogel precursor. Maleic anhydride-modified collagen (ColME) could form a gel after exposure to UV light and be loaded with the antimicrobial agents, nisin and levofloxacin, to acquire antimicrobial ability. The ColME hydrogel containing nisin and levofloxacin had good cytocompatibility and effectively killed pathogenic bacterial strains, such as Escherichia coli and Staphylococcus aureus. The antimicrobial ColME hydrogels effectively supported the healing of a full-thickness skin wound infected with S. aureus in a mouse model. Our results demonstrate the potential of antimicrobial hydrogels as effective wound dressings via in situ photogelation for the healing of infected wounds.

Cornea‐Inspired Ultrasound‐Responsive Adhesive Hydrogel Patches for Keratitis Treatment

AbstractBiomedical patches offer significant potential for keratitis treatment. Building on advances in multifunctionality and biomimicry, an innovative, multifunctional hydrogel patch with high therapeutic efficacy, inspired by the native architecture and functions of the cornea, is introduced. By engineering a composite patch comprising recombinant human collagen (RHC) hydrogel, near‐field electrospinning (NFES) microfibers, and gold‐nanoparticle‐decorated tetragonal barium titanates (BTO@Au), structural mimicry, mechanical reinforcement, tissue‐specific adhesion, and bacterial inhibition are achieved. The RHC hydrogel recreates a three‐dimensional (3D) microenvironment that emulates the natural structure of the corneal tissue, demonstrating excellent tissue adhesion. Integrated within this hydrogel, the NFES microfibers, designed to emulate the orthogonal arrangement of native corneal stroma, not only reinforce the mechanical strength of the RHC hydrogel but also act as scaffolds to guide the aligned growth of human keratocytes. A unique aspect of this advanced patch is the incorporation of BTO@Au nanoparticles, which generate reactive oxygen species for effective bacterial eradication when subjected to ultrasound stimulation. Through in vivo studies on rat models with infected corneal wounds, this hydrogel patch exhibits superior therapeutic efficacy compared to the current treatment. It is posited that these cornea‐inspired ultrasound‐responsive adhesive hydrogel patches represent a significant scientific advancement with high potential for clinical applications.

Effect of Electrical Stimulation on PC12 Cells Cultured in Different Hydrogels: Basis for the Development of Biomaterials in Peripheral Nerve Tissue Engineering.

Extensive damage to peripheral nerves is a health problem with few therapeutic alternatives. In this context, the development of tissue engineering seeks to obtain materials that can help recreate environments conducive to cellular development and functional repair of peripheral nerves. Different hydrogels have been studied and presented as alternatives for future treatments to emulate the morphological characteristics of nerves. Along with this, other research proposes the need to incorporate electrical stimuli into treatments as agents that promote cell growth and differentiation; however, no precedent correlates the simultaneous effects of the types of hydrogel and electrical stimuli. This research evaluates the neural differentiation of PC12 cells, relating the effect of collagen, alginate, GelMA, and PEGDA hydrogels with electrical stimulation modulated in four different ways. Our results show significant correlations for different cultivation conditions. Electrical stimuli significantly increase neural differentiation for specific experimental conditions dependent on electrical frequency, not voltage. These backgrounds allow new material treatment schemes to be formulated through electrical stimulation in peripheral nerve tissue engineering.

Embedded macrophages induce intravascular coagulation in 3D blood vessel-on-chip

Macrophages are innate immune cells that prevent infections and help in wound healing and vascular inflammation. While these cells are natural helper cells, they also contribute to chronic diseases, e.g., by infiltrating the endothelial layer in early atherosclerosis and by promoting vascular inflammation. There is a crosstalk between inflammatory pathways and key players in thrombosis, such as platelets and endothelial cells – a phenomenon known as ‘thromboinflammation’. The role of the embedded macrophages in thromboinflammation in the context of vascular disease is incompletely understood. Blood vessels-on-chips, which are microfluidic vascular cell culture models, have been used extensively to study aspects of vascular disease, like permeability, immune cell adhesion and thrombosis. Blood perfusion assays in blood vessel-on-chip models benefit from multiple unique aspects of the models, such as control of microvessel structure and well-defined flow patterns, as well as the ability to perform live imaging. However, due to their simplified nature, blood vessels-on-chip models have not yet been used to capture the complex cellular crosstalk that is important in thromboinflammation. Using induced pluripotent stem cell-derived endothelial cells and polarized THP-1 monocytes, we have developed and systematically set up a 3D blood vessel-on-chip with embedded (lipid-laden) macrophages, which is created using sequential cell seeding in viscous finger patterned collagen hydrogels. We have set up a human whole blood perfusion assay for these 3D blood vessels-on-chip. An increased deposition of fibrin in the blood vessel-on-chip models containing lipid-laden macrophages was observed. We anticipate the future use of this advanced vascular in vitro model in drug development for early atherosclerosis or aspects of other vascular diseases.Graphical

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.

Effect of Phytic Acid Addition on the Structure of Collagen-Hyaluronic Acid Composite Gel.

Composite collagen gels with hyaluronic acid are developed tissue-engineered structures for filling and regeneration of defects in various organs and tissues. For the first time, phytic acid was used to increase the stability and improve the mechanical properties of collagen gels with hyaluronic acid. Phytic acid is a promising cross-linker for collagen hydrogels and is a plant-derived antioxidant found in rich sources of beans, grains, and oilseeds. Phytic acid has several benefits due to its antioxidant, anticancer, and antitumor properties. In this work, studies were carried out on the kinetics of the self-assembly of collagen molecules in the presence of phytic and hyaluronic acids. It was shown that both of these acids do not lead to collagen self-assembly. Scanning electron microscopy showed that in the presence of phytic and hyaluronic acids, the collagen fibrils had a native structure, and the FTIR method confirmed the chemical cross-links between the collagen fibrils. DSC and rheological studies demonstrated that adding the phytic acid improved the stability and modulus of elasticity of the collagen gel. The presence of hyaluronic acid in the collagen gel slightly reduced the effect of phytic acid. The presence of phytic acid in the collagen gel improved the stability of the scaffold, but, after 1 week of cultivation, slightly reduced the viability of mesenchymal stromal cells cultured in the gel. The collagen type I gel with hyaluronic and phytic acids can be used to replace tissue defects, especially after the removal of cancerous tumors.

Porous Polylactide Microparticles as Effective Fillers for Hydrogels.

High-strength composite hydrogels based on collagen or chitosan-genipin were obtained via mixing using highly porous polylactide (PLA) microparticles with diameters of 50-75 µm and porosity values of over 98%. The elastic modulus of hydrogels depended on the filler concentration. The modulus increased from 80 kPa to 400-600 kPa at a concentration of porous particles of 12-15 wt.% and up to 1.8 MPa at a filling of 20-25 wt.% for collagen hydrogels. The elastic modulus of the chitosan-genipin hydrogel increases from 75 kPa to 900 kPa at a fraction of particles of 20 wt.%. These elastic modulus values cover a range of strength properties from connective tissue to cartilage tissue. It is important to note that the increase in strength in this case is accompanied by a decrease in the density of the material, that is, an increase in porosity. PLA particles were loaded with C-phycocyanin and showed an advanced release profile up to 48 h. Thus, composite hydrogels mimic the structure, biomechanics and release of biomolecules in the tissues of a living organism.

Synthetic Collagen Hydrogels through Symmetric Self-Assembly of Small Peptides.

Animal-sourced hydrogels, such as collagen, are widely used as extracellular-matrix (ECM) mimics in tissue engineering but are plagued with problems of reproducibility, immunogenicity, and contamination. Synthetic, chemically defined hydrogels can avoid such issues. Despite the abundance of collagen in the ECM, synthetic collagen hydrogels are extremely rare due to design challenges brought on by the triple-helical structure of collagen. Sticky-ended symmetric self-assembly (SESSA) overcomes these challenges by maximizing interactions between the strands of the triple helix, allowing the assembly of collagen-mimetic peptides (CMPs) into robust synthetic collagen nanofibers. This optimization, however, also minimizes interfiber contacts. In this work, symmetric association states for the SESSA of short CMPs to probe their increased propensity for interfiber association are modelled. It is found that 33-residue CMPs not only self-assemble through sticky ends, but also form hydrogels. These self-assemblies behave with remarkable consistency across multiple scales and present a clear link between their triple-helical architecture and the properties of their hydrogels. The results show that SESSA is an effective and robust design methodology that enables the rational design of synthetic collagen hydrogels.

Investigating the Impact of Mechanical Properties and Cell-Collagen Interaction on NIH3T3 Function: A Comparative Study on Different Substrates and Culture Environments.

This study investigates the intricate dynamics of matrix stiffness, substrate composition, and cell-cell interactions and elucidates their cumulative effects on fibroblast behavior in different culture contexts. Three primary substrate types were examined: non-coated, collagen-coated, and collagen hydrogel, within both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid cultures. The research provides several key insights. First, 3D spheroid culture, which promotes robust cell-cell interactions, emerges as a critical factor in maintaining fibroblast functionality. Second, substrate stiffness significantly influences results, with the soft collagen hydrogel showing superior support for fibroblast function. Notably, fibroblasts cultured on collagen hydrogel in 2D exhibit comparable functionality to those in 3D, highlighting the importance of substrate mechanical properties. Third, surface composition, as exemplified by collagen coating, showed a limited effect compared to the other factors studied. These findings provide a basis for innovative applications in regenerative medicine, tissue engineering, and drug testing models, and offer valuable insights into harnessing the potential of fibroblasts and advancing biomedical sciences.

The Rapid Generation of Cell-Laden, FACS-Compatible Collagen Gels

A three-dimensional cell culture in hydrogel beads can support cell growth and differentiation into multi-cellular structures, and these gel beads could be used as building blocks for more complex three-dimensional assemblies. This requires hydrogel beads that are robust enough to sort via FACS yet can be degraded by cell-secreted enzymes. Collagen polymers form hydrogels that are excellent cell growth substrates; however, collagen-containing hydrogel beads typically include additional polymers that limit their degradation. Here, we introduce a simple microfluidic method to generate robust, sortable, cell-laden collagen hydrogel beads. We use on-device pH control to trigger collagen gelation without exposing cells to low pH, ensuring high cell viability. We fabricate microfluidic devices to generate droplets with a wide size range, as demonstrated by production of both small (~55 µm diameter) and large (~300 µm diameter) collagen gels. All hydrogels are sufficiently robust to allow for sorting using FACS. Moreover, high cell viability is maintained throughout the process.

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.