Viscoelasticity of diverse biological samples quantified by Acoustic Force Microrheology (AFMR).

In the context of soft matter and cellular mechanics, microrheology - the use of micron-sized particles to probe the frequency-dependent viscoelastic response of materials - is widely used to shed light onto the mechanics and dynamics of molecular structures. Here we present the implementation of active microrheology in an Acoustic Force Spectroscopy setup (AFMR), which combines multiplexing with the possibility of probing a wide range of forces ( ~ pN to ~nN) and frequencies (0.01-100 Hz). To demonstrate the potential of this approach, we perform active microrheology on biological samples of increasing complexity and stiffness: collagen gels, red blood cells (RBCs), and human fibroblasts, spanning a viscoelastic modulus range of five orders of magnitude. We show that AFMR can successfully quantify viscoelastic properties by probing many beads with high single-particle precision and reproducibility. Finally, we demonstrate that AFMR to map local sample heterogeneities as well as detect cellular responses to drugs.

Resveratrol-Ampicillin Dual-Drug Loaded Polyvinylpyrrolidone/Polyvinyl Alcohol Biomimic Electrospun Nanofiber Enriched with Collagen for Efficient Burn Wound Repair.

Background: The healing of burn wounds is a complicated physiological process that involves several stages, including haemostasis, inflammation, proliferation, and remodelling to rebuild the skin and subcutaneous tissue integrity. Recent advancements in nanomaterials, especially nanofibers, have opened a new way for efficient healing of wounds due to burning or other injuries. Methods: This study aims to develop and characterize collagen-decorated, bilayered electrospun nanofibrous mats composed of PVP and PVA loaded with Resveratrol (RSV) and Ampicillin (AMP) to accelerate burn wound healing and tissue repair. Results: Nanofibers with smooth surfaces and web-like structures with diameters ranging from 200 to 400 nm were successfully produced by electrospinning. These fibres exhibited excellent in vitro properties, including the ability to absorb wound exudates and undergo biodegradation over a two-week period. Additionally, these nanofibers demonstrated sustained and controlled release of encapsulated Resveratrol (RSV) and Ampicillin (AMP) through in vitro release studies. The zone of inhibition (ZOI) of PVP-PVA-RSV-AMP nanofibers against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was found 31±0.09 mm and 12±0.03, respectively, which was significantly higher as compared to positive control. Similarly, the biofilm study confirmed the significant reduction in the formation of biofilms in nanofiber-treated group against both S. aureus and E. coli. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis proved the encapsulation of RSV and AMP successfully into nanofibers and their compatibility. Haemolysis assay (%) showed no significant haemolysis (less than 5%) in nanofiber-treated groups, confirmed their cytocompatibility with red blood cells (RBCs). Cell viability assay and cell adhesion on HaCaT cells showed increased cell proliferation, indicating its biocompatibility as well as non-toxic properties. Results of the in-vivo experiments on a burn wound model demonstrated potential burn wound healing in rats confirmed by H&E-stained images and also improved the collagen synthesis in nanofibers-treated groups evidenced by Masson-trichrome staining. The ELISA assay clearly indicated the efficient downregulation of TNF-alpha and IL-6 inflammatory biomarkers after treatment with nanofibers on day 10. Conclusion: The RSV and AMP-loaded nanofiber mats, developed in this study, expedite burn wound healing through their multifaceted approach.

Development of bilayer tissue-engineered scaffolds: combination of 3D printing and electrospinning methodologies.

Although different fabrication methods and biomaterials are used in scaffold development, hydrogels and electrospun materials that provide the closest environment to the extracellular matrix have recently attracted considerable interest in tissue engineering applications. However, some of the limitations encountered in the application of these methods alone in scaffold fabrication have increased the tendency to use these methods together. In this study, a bilayer scaffold was developed using 3D-printed gelatin methacryloyl (GelMA) hydrogel containing ciprofloxacin (CIP) and electrospun polycaprolactone (PCL)-collagen (COL) patches. The bilayer scaffolds were characterized in terms of chemical, morphological, mechanical, swelling, and degradation properties; drug release, antibacterial properties, and cytocompatibility of the scaffolds were also studied. In conclusion, bilayer GelMA-CIP/PCL-COL scaffolds, which exhibit sufficient porosity, mechanical strength, and antibacterial properties and also support cell growth, are promising potential substitutes in tissue engineering applications.

Enhancing chondrogenic differentiation of mesenchymal stem cells through synergistic effects of cellulose nanocrystals and plastic compression in collagen-based hydrogel for cartilage formation.

Collagen-based (COL) hydrogels could be a promising treatment option for injuries to the articular cartilage (AC) becuase of their similarity to AC native extra extracellular matrix. However, the high hydration of COL hydrogels poses challenges for AC's mechanical properties. To address this, we developed a hydrogel platform that incorporating cellulose nanocrystals (CNCs) within COL and followed by plastic compression (PC) procedure to expel the excessive fluid out. This approach significantly improved the mechanical properties of the hydrogels and enhanced the chondrogenic differentiation of mesenchymal stem cells (MSCs). Radially confined PC resulted in higher collagen fibrillar densities together with reducing fibril-fibril distances. Compressed hydrogels containing CNCs exhibited the highest compressive modulus and toughness. MSCs encapsulated in these hydrogels were initially affected by PC, but their viability improved after 7 days. Furthermore, the morphology of the cells and their secretion of glycosaminoglycans (GAGs) were positively influenced by the compressed COL-CNC hydrogel. Our findings shed light on the combined effects of PC and CNCs in improving the physical and mechanical properties of COL and their role in promoting chondrogenesis.

A highly biocompatible and bioactive transdermal nano collagen for enhanced healing of UV-damaged skin.

Skin damage caused by excessive UV radiation has gradually become one of the most prevalent skin diseases. Collagen has gradually found applications in the treatment of UV-damaged skin; however, their high molecular weight greatly limits their capacity to permeate the skin barrier and repair the damaged skin. Nano collagen has garnered growing attentions in the mimicking of collagen; while the investigation of its skin permeability and wound-healing capability remains vacancies. Herein, we have for the first time created a highly biocompatible and bioactive transdermal nano collagen demonstrating remarkable transdermal capacity and repair efficacy for UV-damaged skin. The transdermal nano collagen exhibited a stable triple-helix structure, effectively promoting the adhesion and proliferation of fibroblasts. Notably, the transdermal nano collagen displayed exceptional penetration capabilities, permeating fibroblast and healthy skin. Combo evaluations revealed that the transdermal nano collagen contributed to recovering the intensity and TEWL values of UV-damaged skin to normal level. Histological analysis further indicated that transdermal nano collagen significantly accelerated the repair of damaged skin by promoting the collagen regeneration and fibroblasts activation. This highly biocompatible and bioactive transdermal nano collagen provides a novel substituted strategy for the transdermal absorption of collagen, indicating great potential applications in cosmetics and dermatology.

The Legionella collagen-like protein employs a distinct binding mechanism for the recognition of host glycosaminoglycans.

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires' disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual trimer arrangement with a positively charged external surface and negatively charged solvent exposed internal cavity. Through molecular dynamics simulations, we show how the glycosaminoglycan chondroitin-4-sulphate associates with the Lcl-CTD surface via distinct binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate-binding mechanism.

Optimizing decellularization protocols for human thyroid tissues: a step towards tissue engineering and transplantation.

Hypothyroidism is caused by insufficient stimulation or disruption of the thyroid. However, the drawbacks of thyroid transplantation have led to the search for new treatments. Decellularization allows tissue transplants to maintain their biomimetic structures while preserving cell adhesion, proliferation, and differentiation. This study aimed to decellularize human thyroid tissues using a structure-preserving optimization strategy and present preliminary data on recellularization. Nine methods were used for physical and chemical decellularization. Quantitative and immunohistochemical analyses were performed to investigate the DNA and extracellular matrix components of the tissues. Biomechanical properties were determined by compression test, and cell viability was examined after seeding MDA-T32 papillary thyroid cancer (PTC) cells onto the decellularized tissues. Decellularized tissues exhibited a notable decrease (<50 ng mg-1DNA, except for Groups 2 and 7) compared to the native thyroid tissue. Nonetheless, collagen and glycosaminoglycans were shown to be conserved in all decellularized tissues. Laminin and fibronectin were preserved at comparatively higher levels, and Young's modulus was elevated when decellularization included SDS. It was observed that the strain value in Group 1 (1.63 ± 0.14 MPa) was significantly greater than that in the decellularized tissues between Groups 2-9, ranging from 0.13 ± 0.03-0.72 ± 0.29 MPa. Finally, viability assessment demonstrated that PTC cells within the recellularized tissue groups successfully attached to the 3D scaffolds and sustained metabolic activity throughout the incubation period. We successfully established a decellularization optimization for human thyroid tissues, which has potential applications in tissue engineering and transplantation research. Our next goal is to conduct recellularization using the methods utilized in Group 1 and transplant the primary thyroid follicular cell-seeded tissues into anin vivoanimal model, particularly due to their remarkable 3D structural preservation and cell adhesion-promoting properties.

Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip.

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

Construction of Dome-Shaped 3D Corneal Epithelial Tissue Models Based on Eyeball-Shaped Gel Microspheres.

By overcoming interspecies differences and mimicking the in vivo microenvironment, three-dimensional (3D) in vitro corneal models have become a significant novel tool in contemporary ophthalmic disease research. However, existing 3D corneal models struggle to replicate the actual human corneal environment, especially the dome-shaped physiological structure with adjustable curvature. Addressing these challenges, this study introduces a straightforward method for fabricating collagen/chitosan-alginate eyeball-shaped gel microspheres with a Janus structure via a two-phase aqueous system, used subsequently to construct in vitro 3D corneal epithelial tissue models. By adjusting the diameter ratio of collagen/chitosan to alginate droplets, we can create eyeball-shaped gel microspheres with varying curvatures. Human corneal epithelial cells were seeded on the surfaces of these microspheres, leading to the formation of in vitro 3D corneal epithelial tissues characterized by dome-like multilayers and tight junctions. Additionally, the model demonstrated responsiveness to UVB exposure through the secretion of reactive oxygen species (ROS) and proinflammatory factors. Therefore, we believe that in vitro 3D corneal epithelial tissue models with dome-shaped structures hold significant potential for advancing ophthalmic research.

Bioactive Peptide Profiling in Collagen Hydrolysates: Comparative Analysis Using Targeted and Untargeted Liquid Chromatography-Tandem Mass Spectrometry Quantification.

The investigation of collagen hydrolysates (CHs) is essential due to their widespread use in health, cosmetic, and therapeutic industries, attributing to the presence of bioactive dipeptides (DPs) and tripeptides (TPs). This study developed a novel targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with propyl chloroformate (PCF) derivatization to measure three bioactive peptides-Hydroxyprolyl-glycine (Hyp-Gly), Glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp), and Prolyl-hydroxyproline (Pro-Hyp)-in CHs, with strong correlation coefficients (0.992, 1.000, and 0.995, respectively) and low limits of detection (LODs) of 1.40, 0.14, and 1.16 µM, respectively. Untargeted data-dependent acquisition (DDA) analyses measured peptide size distribution, while amino acid analysis assessed nutritional content. The analysis of ten commercial CHs revealed similar amino acid profiles but varied peptide lengths, indicating diverse hydrolysis conditions. Products with higher proportions of smaller peptides showed elevated levels of the targeted bioactive peptides, suggesting that a smaller peptide size may increase bioactivity. These findings can inform the optimization of CH supplements, providing consumers with detailed peptide content for more informed choices. Data are available via ProteomeXchange with the identifier PXD051699.

Development of an Optical System for Strain Drop Measurement of Osteosarcoma Cells on Substrates with Different Stiffness.

Adherent cells perceive mechanical feedback from the underlying matrix and convert it into biochemical signals through a process known as mechanotransduction. The response to changes in the microenvironment relies on the cell's mechanical properties, including elasticity, which was recently identified as a biomarker for various diseases. Here, we propose the design, development, and characterization of a new system for the measurement of adherent cells' strain drop, a parameter correlated with cells' elasticity. To consider the interplay between adherent cells and the host extracellular matrix, cell stretching was combined with adhesion on substrates with different stiffnesses. The technique is based on the linear stretching of silicone chambers, high-speed image acquisition, and feedback for image centering. The system was characterized in terms of the strain homogeneity, impact of collagen coating, centering capability, and sensitivity. Subsequently, it was employed to measure the strain drop of two osteosarcoma cell lines, low-aggressive osteoblast-like SaOS-2 and high-aggressive 143B, cultured on two different substrates to recall the stiffness of the bone and lung extracellular matrices. Results demonstrated good substrate homogeneity, a negligible effect of the collagen coating, and an accurate image centering. Finally, the experimental results showed an average strain drop that was lower in the 143B cells in comparison with the SaOS-2 cells in all the tested conditions.

So Let's Design a Peptide . . . A Physician's Approach.

This paper outlines a process undertaken by a physician to design a peptide aimed at impacting the extracellular matrix. From a position of very little expertise, a new peptide was designed with amino acid constituents based on the structural proteins collagen and elastin. Sequencing was also considered, given the periodic repetition observed in these proteins, and a peptide with reasonable molecular weight and physical characteristics was designed using available software. The sequence of events concerning intellectual property, functionality investigation, and eventual use of the peptide in new formulations is detailed. This may be of interest to physicians who consider this exercise out of the scope of the usual practice. J Drugs Dermatol. 2024;23(5):347-352.    doi:10.36849/JDD.7921.

Innovative Vancomycin-Loaded Hydrogel-Based Systems - New Opportunities for the Antibiotic Therapy.

Purpose: Surgical site infections pose a significant challenge for medical services. Systemic antibiotics may be insufficient in preventing bacterial biofilm development. With the local administration of antibiotics, it is easier to minimize possible complications, achieve drugs' higher concentration at the injured site, as well as provide their more sustained release. Therefore, the main objective of the proposed herein studies was the fabrication and characterization of innovative hydrogel-based composites for local vancomycin (VAN) therapy. Methods: Presented systems are composed of ionically gelled chitosan particles loaded with vancomycin, embedded into biomimetic collagen/chitosan/hyaluronic acid-based hydrogels crosslinked with genipin and freeze-dried to serve in a flake/disc-like form. VAN-loaded carriers were characterized for their size, stability, and encapsulation efficiency (EE) using dynamic light scattering technique, zeta potential measurements, and UV-Vis spectroscopy, respectively. The synthesized composites were tested in terms of their physicochemical and biological features. Results: Spherical structures with sizes of about 200 nm and encapsulation efficiencies reaching values of approximately 60% were obtained. It was found that the resulting particles exhibit stability over time. The antibacterial activity of the developed materials against Staphylococcus aureus was established. Moreover, in vitro cell culture study revealed that the surfaces of all prepared systems are biocompatible as they supported the proliferation and adhesion of the model MG-63 cells. In addition, we have demonstrated significantly prolonged VAN release while minimizing the initial burst effect for the composites compared to bare nanoparticles and verified their desired physicochemical features during swellability, and degradation experiments. Conclusion: It is expected that the developed herein system will enable direct delivery of the antibiotic at an exposed to infections surgical site, providing drugs sustained release and thus will reduce the risk of systemic toxicity. This strategy would both inhibit biofilm formation and accelerate the healing process.

Sustained Secretion of CCL21 via an Implantable Cell Reservoir Hydrogel Enhances the Systemic Antitumor Effect of Radiotherapy.

The combination of radiotherapy (RT) and immunotherapy shows promise in improving the clinical treatment of solid tumors; however, it faces challenges of low response rates and systemic toxicity. Herein, an implantable alginate/collagen hydrogel encapsulating C-C motif ligand 21 (CCL21)-expressing dendritic cells (CCL21-DCs@gel) was developed to potentiate the systemic antitumor effects of RT. The hydrogel functioned as a suitable reservoir for in vivo culture and proliferation of CCL21-DCs, thereby enabling sustained CCL21 release. The local CCL21 gradient induced by CCL21-DCs@gel significantly enhanced the efficacy of RT in suppressing primary tumor growth and inhibiting distant metastasis across several mouse models. Furthermore, the combination of RT with CCL21-DCs@gel provided complete prophylactic protection to mice. Mechanistic investigations revealed that CCL21-DCs@gel potentiated RT by promoting tumor lymphangiogenesis and attracting immune cell infiltration into the tumor. Collectively, these results suggest that CCL21-DCs@gel is a promising adjunct to RT for effectively eradicating tumors and preventing tumor recurrence.

Human-Derived collagen hydrogel as an antibiotic vehicle for topical treatment of bacterial biofilms.

The complexity of chronic wounds creates difficulty in effective treatments, leading to prolonged care and significant morbidity. Additionally, these wounds are incredibly prone to bacterial biofilm development, further complicating treatment. The current standard treatment of colonized superficial wounds, debridement with intermittent systemic antibiotics, can lead to systemic side-effects and often fails to directly target the bacterial biofilm. Furthermore, standard of care dressings do not directly provide adequate antimicrobial properties. This study aims to assess the capacity of human-derived collagen hydrogel to provide sustained antibiotic release to disrupt bacterial biofilms and decrease bacterial load while maintaining host cell viability and scaffold integrity. Human collagen harvested from flexor tendons underwent processing to yield a gellable liquid, and subsequently was combined with varying concentrations of gentamicin (50-500 mg/L) or clindamycin (10-100 mg/L). The elution kinetics of antibiotics from the hydrogel were analyzed using liquid chromatography-mass spectrometry. The gel was used to topically treat Methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens in established Kirby-Bauer and Crystal Violet models to assess the efficacy of bacterial inhibition. 2D mammalian cell monolayers were topically treated, and cell death was quantified to assess cytotoxicity. Bacteria-enhanced in vitro scratch assays were treated with antibiotic-embedded hydrogel and imaged over time to assess cell death and mobility. Collagen hydrogel embedded with antibiotics (cHG+abx) demonstrated sustained antibiotic release for up to 48 hours with successful inhibition of both MRSA and C. perfringens biofilms, while remaining bioactive up to 72 hours. Administration of cHG+abx with antibiotic concentrations up to 100X minimum inhibitory concentration was found to be non-toxic and facilitated mammalian cell migration in an in vitro scratch model. Collagen hydrogel is a promising pharmaceutical delivery vehicle that allows for safe, precise bacterial targeting for effective bacterial inhibition in a pro-regenerative scaffold.

Investigation of collagen reconstruction mechanism in skin wound through dual-beam laser welding: Insights from multi-spectroscopy, molecular dynamics simulation, and finite element multiphysics simulation.

Since the mechanism underlying real-time acquisition of mechanical strength during laser-induced skin wound fusion remains unclear, and collagen is the primary constituent of skin tissue, this study investigates the structural and mechanical alterations in collagen at temperatures ranging from 40 °C to 60 °C using various spectroscopic techniques and molecular dynamics calculations. The COMSOL Multiphysics coupling is employed to simulate the three-dimensional temperature field, stress-strain relationship, and light intensity distribution in the laser thermal affected zone of skin wounds during dual-beam laser welding process. Raman spectroscopy, synchronous fluorescence spectroscopy and circular dichroism measurement results confirm that laser energy activates biological activity in residues, leading to a transformation in the originally fractured structure of collagen protein for enhanced mechanical strength. Molecular dynamics simulations reveal that stable hydrogen bonds form at amino acid residues within the central region of collagen protein when the overall temperature peak around the wound reaches 60 °C, thereby providing stability to previously fractured skin incisions and imparting instantaneous strength. However, under a 55 °C system, Type I collagen ensures macrostructural stability while activating biological properties at amino acid bases to promote wound healing function; this finding aligns with experimental analysis results. The COMSOL simulation outcomes also correspond well with macroscopic morphology after laser welding samples, confirming that by maintaining temperatures between 55 °C-60 °C during laser welding of skin incisions not only can certain instantaneous mechanical strength be achieved but irreversible thermal damage can also be effectively controlled. It is anticipated that these findings will provide valuable insights into understanding the healing mechanism for laser-welded skin wounds.

Study of the collagen tissue nanostructure by analyzing the echo decay obtained using the MRI technique.

The multicomponent relaxation observed in nuclear magnetic resonance experiments in biological tissues makes it difficult to establish a correlation between specific relaxation times and tissue structural parameters. The analysis of a nanostructure (the characteristic size of 10-1000 nm) is usually based on formulas for relaxation times which depend on structural parameters at the atomic or molecular levels in the size range of 0.1-5 nm. We have recently developed an analysis method in which relaxation times' anisotropy in a sample is explicitly related to its structure of nanocavities containing a liquid or gas. However, the method is based on the analysis of experimental data on the anisotropy of relaxation times obtained by using the standard NMR technique and rotating the sample relative to a magnetic field and requires a series of experiments. In the present study, to address this challenge, we develop a new method of analysis of a multi-exponential magnetic resonance signal that does not require determining relaxation times and eliminates the sample rotation and the necessity of a series of experiments. Using the magnetic resonance imaging (MRI) technique, the total signal from the whole sample was obtained as a sum of the signals (echo decays) from all voxels. In contrast to previous research, the volumes of nanocavities and their angular distribution can be obtained by analyzing a single total signal for the entire cartilage. In addition, within the framework of this approach, it is possible to identify the reason for the multicomponent nature of relaxation. The proposed method for analyzing a single multi-exponential signal (transverse relaxation) was implemented on cartilage. Using the signal, three anatomical zones of cartilage were studied, revealing significant structural differences between them. The proposed method not only avoids the need for sample rotation but also enables repeated application of layer-by-layer magnetic resonance imaging with micron resolution. The study results allow us to suggest that water molecules contributing to the echo decay are more likely located in nanocavities formed by the fibrillar structure rather than inside the fibrils.

Harnessing oriented arrangement of collagen fibers by 3D printing for enhancing mechanical and osteogenic properties of mineralized collagen scaffolds.

As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.

Development of a Static Avascular and Dynamic Vascular Human Skin Equivalent Employing Collagen/Keratin Hydrogels.

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Multi-acrylamides improve bond stability through collagen reinforcement under physiological conditions.

OBJECTIVES: Acrylamides were shown to significantly improve bonding stability in adhesive restorations, but the reinforcement mechanism has not been fully elucidated. We tested the hypothesis that hydrogen bonding reinforcement of the collagen network (with secondary or tertiary acrylamides), as well as degree of crosslinking of the polymer network (with di- or tri-functional acrylamides), can be two of the factors at play. METHODS: Two-step total etch adhesives comprising UDMA (60 wt%) and 40 wt% of: TAAEA, TMAAEA (secondary, tertiary tri-acrylamides), BAAP, DEBAAP (secondary, tertiary di-acrylamides) or HEMA (mono-methacrylate - control) were formulated. Simulated composite restorations (n = 5) were tested after cyclic mechanical and biological (S. mutans biofilm) challenges. Gap formation before and after aging was assessed with SEM imaging. Micro-tensile bond strength (µTBS, n = 6) was assessed after seven-day incubation in water or S. mutans-containing culture medium. Collagen reinforcement was assessed with hydroxyproline assay (n = 10) and rheology (n = 3). Data were analyzed with one-way/two-way ANOVA/Tukey's test (alpha=5%). RESULTS: Gap formation increased and bond strength decreased for all monomers after biofilm incubation (p < 0.001). Except for DEBAAP, secondary and tertiary di/tri-acrylamides showed lower occlusal gap width values, but no significant differences overall gap length compared to HEMA. µTBS increased for tri-acrylamides compared with HEMA. Samples treated with multi-acrylamides had lower concentration of hydroxyproline (by-product of collagen degradation) (p < 0.001), except for DEBAAP, which showed values close to HEMA (p > 0.05). Dentin shear modulus increased for all acrylamides after 72 h, especially TMAAEA. SIGNIFICANCE: In general, multi-acrylamides promote collagen reinforcement, leading to reduced gap formation, and stabilize the bond strength under physiological conditions.