Redifferentiation of Articular Chondrocytes by Hyperacute Serum and Platelet Rich Plasma in Collagen Type I Hydrogels.

Matrix-assisted autologous chondrocyte transplantation (MACT) for focal articular cartilage defects often fails to produce adequate cartilage-specific extracellular matrix in vitro and upon transplantation results in fibrocartilage due to dedifferentiation during cell expansion. This study aimed to redifferentiate the chondrocytes through supplementation of blood-products, such as hyperacute serum (HAS) and platelet-rich plasma (PRP) in vitro. Dedifferentiated monolayer chondrocytes embedded onto collagen type I hydrogels were redifferentiated through supplementation of 10% HAS or 10% PRP for 14 days in vitro under normoxia (20% O2) and hypoxia (4% O2). Cell proliferation was increased by supplementing HAS for 14 days (p < 0.05) or by interchanging from HAS to PRP during Days 7⁻14 (p < 0.05). Sulfated glycosaminoglycan (sGAG) content was deposited under both HAS, and PRP for 14 days and an interchange during Days 7⁻14 depleted the sGAG content to a certain extent. PRP enhanced the gene expression of anabolic markers COL2A1 and SOX9 (p < 0.05), whereas HAS enhanced COL1A1 production. An interchange led to reduction of COL1A1 and COL2A1 expression marked by increased MMP13 expression (p < 0.05). Chondrocytes secreted less IL-6 and more PDGF-BB under PRP for 14 days (p < 0.0.5). Hypoxia enhanced TGF-ß1 and BMP-2 release in both HAS and PRP. Our study demonstrates a new approach for chondrocyte redifferentiation.

Fibrillogenesis in collagen hydrogels accelerated by carboxylated microbeads.

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads.

3D Hydrogel Culture System Recapitulates Key Tuberculosis Phenotypes and Demonstrates Pyrazinamide Efficacy.

The mortality caused by tuberculosis (TB) infections is a global concern, and there is a need to improve understanding of the disease. Current in vitro infection models to study the disease have limitations such as short investigation durations and divergent transcriptional signatures. This study aims to overcome these limitations by developing a 3D collagen culture system that mimics the biomechanical and extracellular matrix (ECM) of lung microenvironment (collagen fibers, stiffness comparable to in vivo conditions) as the infection primarily manifests in the lungs. The system incorporates Mycobacterium tuberculosis (Mtb) infected human THP-1 or primary monocytes/macrophages. Dual RNA sequencing reveals higher mammalian gene expression similarity with patient samples than 2D macrophage infections. Similarly, bacterial gene expression more accurately recapitulates in vivo gene expression patterns compared to bacteria in 2D infection models. Key phenotypes observed in humans, such as foamy macrophages and mycobacterial cords, are reproduced in the model. This biomaterial system overcomes challenges associated with traditional platforms by modulating immune cells and closely mimicking in vivo infection conditions, including showing efficacy with clinically relevant concentrations of anti-TB drug pyrazinamide, not seen in any other in vitro infection model, making it reliable and readily adoptable for tuberculosis studies and drug screening.

A New Method for the Production of High-Concentration Collagen Bioinks with Semiautonomic Preparation.

It is believed that 3D bioprinting will greatly help the field of tissue engineering and regenerative medicine, as live patient cells are incorporated into the material, which directly creates a 3D structure. Thus, this method has potential in many types of human body tissues. Collagen provides an advantage, as it is the most common extracellular matrix present in all kinds of tissues and is, therefore, very natural for cells and the organism. Hydrogels with highly concentrated collagen make it possible to create 3D structures without additional additives to crosslink the polymer, which could negatively affect cell proliferation and viability. This study established a new method for preparing highly concentrated collagen bioinks, which does not negatively affect cell proliferation and viability. The method is based on two successive neutralizations of the prepared hydrogel using the bicarbonate buffering mechanisms of the 2× enhanced culture medium and pH adjustment by adding NaOH. Collagen hydrogel was used in concentrations of 20 and 30 mg/mL dissolved in acetic acid with a concentration of 0.05 and 0.1 wt.%. The bioink preparation process is automated, including colorimetric pH detection and adjustment. The new method was validated using bioprinting and subsequent cultivation of collagen hydrogels with incorporated stromal cells. After 96 h of cultivation, cell proliferation and viability were not statistically significantly reduced.

Collagen-ß-cyclodextrin hydrogels for advanced wound dressings: super-swelling, antibacterial action, inflammation modulation, and controlled drug release.

A key strategy in enhancing the efficacy of collagen-based hydrogels involves incorporating polysaccharides, which have shown great promise for wound healing. In this study, semi-interpenetrating polymeric network (semi-IPN) hydrogels comprised of collagen (Col) with the macrocyclic oligosaccharide ß-cyclodextrin (ß-CD) (20-80 wt.%) were synthesised. Fourier-transform infrared (FTIR) spectroscopy confirmed the successful fabrication of these Col/ß-CD hydrogels, evidenced by the presence of characteristic absorption bands, including the urea bond band at ∼1740 cm-1, related with collagen crosslinking. Higher ß-CD content was associated with increased crosslinking, higher swelling, and faster gelation. The ß-CD content directly influenced the morphology and semi-crystallinity. All Col/ß-CD hydrogels displayed superabsorbent properties, enhanced thermal stability, and exhibited slow degradation rates. Mechanical properties were significantly improved with contents higher than ß-CD 40 wt.%. These hydrogels inhibited the growth of Escherichia coli bacteria and facilitated the controlled release of agents, such as malachite green, methylene blue, and ketorolac. The chemical composition of the Col/ß-CD hydrogels did not induce cytotoxic effects on monocytes and fibroblast cells. Instead, they actively promoted cellular metabolic activity, encouraging cell growth and proliferation. Moreover, cell signalling modulation was observed, leading to changes in the expression of TNF-α and IL-10 cytokines. In summary, the results of this research indicate that these novel hydrogels possess multifunctional characteristics, including biocompatibility, super-swelling capacity, good thermal, hydrolytic, and enzymatic degradation resistance, antibacterial activity, inflammation modulation, and the ability to be used for controlled delivery of therapeutic agents, indicating high potential for application in advanced wound dressings.

Sclerostin Antibody-Loaded Dense Collagen Hydrogels Promote Critical-Size Bone Defect Repair.

The management of extensive bone loss remains a clinical challenge. Numerous studies are underway to develop a combination of biomaterials, biomolecules, and stem cells to address this challenge. In particular, the systemic administration of antibodies against sclerostin, a regulator of bone formation, was recently shown to enhance the bone repair efficiency of dense collagen hydrogels (DCHs) hosting murine dental pulp stem cells (mDPSCs). The aim of the present study was to assess whether these antibodies, encapsulated and released from DCHs, could promote craniofacial bone repair by the local inhibition of sclerostin. In vitro studies showed that antibody loading modified neither the hydrogel structure nor the viability of seeded mDPSCs. When implanted in a mouse calvaria critical-size bone defect, antibody-loaded DCHs showed repair capabilities similar to those of acellular unloaded DCHs combined with antibody injections. Importantly, the addition of mDPSCs provided no further benefit. Altogether, the local delivery of antisclerostin antibodies from acellular dense collagen scaffolds is highly effective for bone repair. The drastic reduction in the required amount of antibody compared to systemic injection should reduce the cost of the procedure, making the strategy proposed here a promising therapeutic approach for large bone defect repair.

Construction of Engineered Muscle Tissue Consisting of Myotube Bundles in a Collagen Gel Matrix.

Tissue engineering methods that aim to mimic the hierarchical structure of skeletal muscle tissue have been widely developed due to utilities in various fields of biology, including regenerative medicine, food technology, and soft robotics. Most methods have aimed to reproduce the microscopical morphology of skeletal muscles, such as the orientation of myotubes and the sarcomere structure, and there is still a need to develop a method to reproduce the macroscopical morphology. Therefore, in this study, we aim to establish a method to reproduce the macroscopic morphology of skeletal muscle by constructing an engineered muscle tissue (EMT) by culturing embryonic chicken myoblast-like cells that are unidirectionally aligned in collagen hydrogels with micro-channels (i.e., MCCG). Whole mount fluorescent imaging of the EMT showed that the myotubes were unidirectionally aligned and that they were bundled in the collagen gel matrix. The myotubes contracted in response to periodic electrostimulations with a frequency range of 0.5-2.0 Hz, but not at 5.0 Hz. Compression tests of the EMT showed that the EMT had anisotropic elasticity. In addition, by measuring the relaxation moduli of the EMTs, an anisotropy of relaxation strengths was observed. The observed anisotropies could be attributed to differences in maturation and connectivity of myotubes in the directions perpendicular and parallel to the long axis of the micro-channels of the MCCG.

Innovative Collagen Based Biopolymers Tested as Fertilizers for Poor Soils Amendment.

Improving soil quality is of growing interest and, among optimal solutions, the reuse and recycling of biopolymers of pelt waste from the tannery industry have been proposed, one of them being for collagen hydrolysate with micronutrients and polymers incorporated, to be used as fertilizers for poor soils rehabilitation. As functionalization agents, polyacrylamide, starch and dolomite were included into biopolymer matrixes in order to enhance their specific efficiency. These fertilizers were adequately characterized for their physical-chemical properties, including nutrient content, and tested on three poor soils, while a fourth sample of normal soil was chosen for comparative purposes. These soils were also characterized for their texture and physical-chemical properties in order to establish the fertility state of the soils as a function of nutrient content. In this respect, a series of agrochemical tests were developed at laboratory scale, simulating real agriculture environments in a vegetation room, where a significant plant growth in height was observed for all the agro-hydrogels with nutrients encapsulated, and multiplication of the nodosities number was observed in the case of the soybean culture. The most significant effect was obtained in the case of the fertilizer functionalized with starch. Finally, the application dose of the organic fertilizers for specific culture plants was estimated, such as field cultures (cereals, corn), field vegetables, vineyards or fruit-growing plantations. These agro-collagen fertilizers are particularly recommended for amendment of field cereals and vegetables. The novelty of this study mainly consists of the recovery and recycling of the pelt waste as efficient fertilizers after their adequate functionalization with synthetic or natural biopolymers.

The role of ADAM8 in the mechanophenotype of MDA-MB-231 breast cancer cells in 3D extracellular matrices.

The majority of investigations of cancer cells have been performed in an oversimplified 2D in vitro environment. In the last decade there is a trend toward more sophisticated 3D in vitro cell culture model systems that can bridge the existing gap between 2D in vitro and in vivo experiments in the field of biophysical and cell biological cancer cell research. Here, we hypothesize that the bidirectional interplay between breast cancer cells and their tumor microenvironment is critical for the outcome of the disease. Thereby, the tissue remodeling processes evoked by cancer cells are important for cancer cell-driven mechanical probing of their matrix environment and on cancer cell adhesion and motility. When remodeling processes have been explored, the emphasis was placed on matrix metalloproteinases and rather not on a disintegrin and metalloproteases (ADAMs). However, the role of ADAM8 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. Thus, in this study, we focus on the function of ADAM8 in matrix remodeling and migration of 3D extracellular matrix scaffolds. Therefore, human MDA-MB-231 breast carcinoma cells with ADAM8 knocked down, referred to as ADAM8-KD cells, as well as MDA-MB-231 scrambled control cells, referred to as ADAM8-Ctrl cells, have been used to examine their ability to interact with and migrate in dense extracellular 3D matrices. The fiber displacements, as the capacity of cells to deform the environmental 3D matrix scaffold, has been observed. ADAM8-KD cells displace collagen fibers more strongly than ADAM8-Ctrl cells. Moreover, ADAM8-KD cells migrated more numerous in 3D collagen matrices compared to ADAM8-Ctrl cells. The impairment of ADAM8 using the ADAM8 inhibitor BK-1361 led to significantly increased fiber displacements of ADAM8-Ctrl cells to the levels of ADAM8-KD cells. In contrast, the inhibitor had no effect on ADAM8-KD cells in terms of fiber displacements as well as on the quantitative characteristics of cell invasion of ADAM8-Ctrl cells, albeit the cells that were found in the matrix invaded considerably deeper. When matrix remodeling by cells is impaired through GM6001, a broad-band metalloproteinase inhibitor, the fiber displacements of both cell types increased. In fact, ADAM8 is known to degrade fibronectin in a direct and/or indirect manner. The supplementation of fibronectin before polymerization of the 3D collagen matrices caused an enhancement in fiber displacements as well as in cell invasion into fibronectin-collagen matrices of ADAM8-Ctrl cells, whereas the fiber displacements of ADAM8-KD cells did not change. However, fibrinogen and laminin supplementation induced an increase in fiber displacements of both cell types. Thus, the impact of fibronectin on selective increase in fiber displacement of ADAM8-Ctrl cells appears to be ADAM8-dependent. As a consequence, the presence of ADAM8 may provide an explanation for the longstanding controversial results of fibronectin enrichment on malignant progression of cancers such as breast cancer. Finally, ADAM8 is apparently essential for providing cell-driven fiber displacements of the extracellular matrix microenvironment, which fosters 3D motility in a fibronectin-rich environment. Contribution to the field. Currently, the role of ADAM8 has been explored in 2D or at maximum 2.5D in vitro cell culture motility assays. However, the mechanical characteristics of these two cell types have not been examined. In this study, the function of ADAM8 in breast cancer is refined by providing in vitro cell investigations in 3D collagen fiber matrices of various conditions. ADAM8 has been shown to be involved in the reduced generation of fiber displacements and in influencing breast cancer cell migration. However, especially in the presence of fibronectin in 3Dcollagen fiber matrices, the fiber displacements of ADAM8-Ctrl cells are increased.

Spreading of P301S Aggregated Tau Investigated in Organotypic Mouse Brain Slice Cultures.

Tau pathology extends throughout the brain in a prion-like fashion through connected brain regions. However, the details of the underlying mechanisms are incompletely understood. The present study aims to examine the spreading of P301S aggregated tau, a mutation that is implicated in tauopathies, using organotypic slice cultures. Coronal hippocampal organotypic brain slices (170 µm) were prepared from postnatal (day 8-10) C57BL6 wild-type mice. Collagen hydrogels loaded with P301S aggregated tau were applied to slices and the spread of tau was assessed by immunohistochemistry after 8 weeks in culture. Collagen hydrogels prove to be an effective protein delivery system subject to natural degradation in 14 days and they release tau proteins up to 8 weeks. Slices with un- and hyperphosphorylated P301S aggregated tau demonstrate significant spreading to the ventral parts of the hippocampal slices compared to empty collagen hydrogels after 8 weeks. Moreover, the spread of P301S aggregated tau occurs in a time-dependent manner, which was interrupted when the neuroanatomical pathways are lesioned. We illustrate that the spreading of tau can be investigated in organotypic slice cultures using collagen hydrogels to achieve a localized application and slow release of tau proteins. P301S aggregated tau significantly spreads to the ventral areas of the slices, suggesting that the disease-relevant aggregated tau form possesses spreading potential. Thus, the results offer a novel experimental approach to investigate tau pathology.

3D Porous Collagen Matrices-A Reservoir for In Vitro Simultaneous Release of Tannic Acid and Chlorhexidine.

The treatment of wounds occurring accidentally or as a result of chronic diseases most frequently requires the use of appropriate dressings, mainly to ensure tissue regeneration/healing, at the same time as treating or preventing potential bacterial infections or superinfections. Collagen type I-based scaffolds in tandem with adequate antimicrobials can successfully fulfill these requirements. In this work, starting from the corresponding hydrogels, we prepared a series of freeze-dried atelocollagen type I-based matrices loaded with tannic acid (TA) and chlorhexidine digluconate (CHDG) as active agents with a broad spectrum of antimicrobial activity and also as crosslinkers for the collagen network. The primary aim of this study was to design an original and reliable algorithm to in vitro monitor and kinetically analyze the simultaneous release of TA and CHDG from the porous matrices into an aqueous solution of phosphate-buffered saline (PBS, pH 7.4, 37 °C) containing micellar carriers of a cationic surfactant (hexadecyltrimethylammonium bromide, HTAB) as a release environment that roughly mimics human extracellular fluids in living tissues. Around this central idea, a comprehensive investigation of the lyophilized matrices (morpho-structural characterization through FT-IR spectroscopy, scanning electron microscopy, swelling behavior, resistance against the collagenolytic action of collagenase type I) was carried out. The kinetic treatment of the release data displayed a preponderance of non-Fickian-Case II diffusion behavior, which led to a general anomalous transport mechanism for both TA and CHDG, irrespective of their concentrations. This is equivalent to saying that the release regime is not governed only by the gradient concentration of the releasing components inside and outside the matrix (like in ideal Fickian diffusion), but also, to a large extent, by the relaxation phenomena of the collagen network (determined, in turn, by its crosslinking degree induced by TA and CHDG) and the dynamic capacity of the HTAB micelles to solubilize the two antimicrobials. By controlling the degree of physical crosslinking of collagen with a proper content of TA and CHDG loaded in the matrix, a tunable, sustainable release profile can be obtained.

Dual drug delivery collagen vehicles for modulation of skin fibrosisin vitro.

Single molecule drug delivery systems have failed to yield functional therapeutic outcomes, triggering investigations into multi-molecular drug delivery vehicles. In the context of skin fibrosis, although multi-drug systems have been assessed, no system has assessed molecular combinations that directly and specifically reduce cell proliferation, collagen synthesis and transforming growth factorß1 (TGFß1) expression. Herein, a core-shell collagen type I hydrogel system was developed for the dual delivery of a TGFßtrap, a soluble recombinant protein that inhibits TGFßsignalling, and Trichostatin A (TSA), a small molecule inhibitor of histone deacetylases. The antifibrotic potential of the dual delivery system was assessed in anin vitroskin fibrosis model induced by macromolecular crowding (MMC) and TGFß1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography analyses revealed that ∼50% of the TGFßtrap and ∼30% of the TSA were released from the core and shell compartments, respectively, of the hydrogel system after 10 d (longest time point assessed) in culture. As a direct consequence of this slow release, the core (TGFßtrap)/shell (TSA) hydrogel system induced significantly (p< 0.05) lower than the control group (MMC and TGFß1) collagen type I deposition (assessed via SDS-PAGE and immunocytochemistry),αsmooth muscle actin (αSMA) expression (assessed via immunocytochemistry) and cellular proliferation (assessed via DNA quantification) and viability (assessed via calcein AM and ethidium homodimer-I staining) after 10 d in culture. On the other hand, direct TSA-TGFßsupplementation induced the lowest (p< 0.05) collagen type I deposition,αSMA expression and cellular proliferation and viability after 10 d in culture. Our results illustrate the potential of core-shell collagen hydrogel systems for sustained delivery of antifibrotic molecules.

pH Modification of High-Concentrated Collagen Bioinks as a Factor Affecting Cell Viability, Mechanical Properties, and Printability.

The 3D bioprinting of cell-incorporated gels is a promising direction in tissue engineering applications. Collagen-based hydrogels, due to their similarity to extracellular matrix tissue, can be a good candidate for bioink and 3D bioprinting applications. However, low hydrogel concentrations of hydrogel (<10 mg/mL) provide insufficient structural support and, in highly concentrated gels, cell proliferation is reduced. In this study, we showed that it is possible to print highly concentrated collagen hydrogels with incorporated cells, where the viability of the cells in the gel remains very good. This can be achieved simply by optimizing the properties of the bioink, particularly the gel composition and pH modification, as well as by optimizing the printing parameters. The bioink composed of porcine collagen hydrogel with a collagen concentration of 20 mg/mL was tested, while the final bioink collagen concentration was 10 mg/mL. This bioink was modified with 0, 5, 9, 13, 17 and 20 µL/mL of 1M NaOH solution, which affected the resulting pH and gelling time. Cylindrical samples based on the given bioink, with the incorporation of porcine adipose-derived stromal cells, were printed with a custom 3D bioprinter. These constructs were cultivated in static conditions for 6 h, and 3 and 5 days. Cell viability and morphology were evaluated. Mechanical properties were evaluated by means of a compression test. Our results showed that optimal composition and the addition of 13 µL NaOH per mL of bioink adjusted the pH of the bioink enough to allow cells to grow and divide. This modification also contributed to a higher elastic modulus, making it possible to print structures up to several millimeters with sufficient mechanical resistance. We optimized the bioprinter parameters for printing low-viscosity bioinks. With this experiment, we showed that a high concentration of collagen gels may not be a limiting factor for cell proliferation.

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.

Anti-inflammatory cytokine-eluting collagen hydrogel reduces the host immune response to dopaminergic cell transplants in a rat model of Parkinson's disease.

In cell replacement approaches for Parkinson's disease, the intracerebral implantation of dopamine neuron-rich grafts generates a neuroinflammatory response to the grafted cells that contributes to its varied outcome. Thus, the aim of the present study was to fabricate an anti-inflammatory cytokine-eluting collagen hydrogel capable of delivering interleukin (IL)-10 to the brain for reduction of the neuroinflammatory response to intracerebral cellular grafts. In vitro assessment revealed that cross-linker concentration affected the microstructure and gelation kinetics of the hydrogels and their IL-10 elution kinetics, but not their cytocompatibility or the functionality of the eluted IL-10. In vivo evaluation revealed that the hydrogels were capable of delivering and retaining IL-10 in the rat striatum, and reducing the neuroinflammatory (microglial) response to hydrogel-encapsulated grafts. In conclusion, IL-10-eluting collagen hydrogels may have beneficial anti-inflammatory effects in the context of cellular brain repair therapies for Parkinson's disease and should be investigated further.

Collagen-Based Thiol-Norbornene Photoclick Bio-Ink with Excellent Bioactivity and Printability.

As the essential foundation of bioprinting technology, cell-laden bio-ink is confronted with the inevitable contradiction between printability and bioactivity. For example, type I collagen has been widely applied for its excellent biocompatibility; however, its relatively low self-assembly speed restricts the performance in high-precision bioprinting of cell-laden structures. In this study, we synthesize norbornene-functionalized neutral soluble collagen (NorCol) by the reaction of acid-soluble collagen (Col) and carbic anhydride in the aqueous phase. NorCol retains collagen triple-helical conformation and can be quickly orthogonally cross-linked to build a cell-laden hydrogel via a cell-friendly thiol-ene photoclick reaction. Moreover, the additional carboxyl groups produced in the reaction of carbic anhydride and collagen obviously improve the solubility of NorCol in neutral buffer and miscibility of NorCol with other polymers such as alginate and gelatin. It enables hybrid bio-ink to respond to multiple stimuli, resulting in continuous cross-linked NorCol networks in hybrid hydrogels. For the first time, the collagen with a triple helix structure and gelatin can be mixed and printed, keeping the integrity of the printed construct after gelatin's dissolution. The molecular interaction among giant collagen molecules allows NorCol hydrogel formation at a low concentration, which leads to excellent cell spreading, migration, and proliferation. These properties give NorCol flexible formability and excellent biocompatibility in temperature-, ion-, and photo-based bioprinting. We speculate that NorCol is a promising bio-ink for emerging demands in tissue engineering, regenerative medicine, and personalized therapeutics.

Growth environment influences B16.F10 mouse melanoma cell response to gene electrotransfer.

We developed and characterized a 3D collagen hydrogel model for B16.F10 melanoma tumors. Cells in this 3D environment exhibited lower proliferation than cells in the conventional 2D culture environment. Interestingly, the basal expression levels of several genes varied when compared to conventionally grown cells. In each growth environment, a significant number of melanoma cells were transfected by plasmid electroporation (electrotransfer), although expression could only be ascertained on the surface of the 3D constructs. Cellular responses to plasmid entry as demonstrated by pro-inflammatory cytokine and chemokine upregulation varied based on the growth environment, as did the mRNA levels of several putative DNA-specific pattern recognition receptors (DNA sensors). Unexpectedly, when plasmid DNA was delivered while cells where attached in the 2D or 3D environments, the mRNAs of the DNA sensor p204 and the inflammatory mediator TNFα were regulated in cells receiving pulses only. However, we were unable to confirm coordinate upregulation of TNFα and p204 proteins. This study confirms that cell responses differ significantly based on their environment, and demonstrates the difficulty of extending experimental observations between cell environments.

Predicting Bone Formation in Mesenchymal Stromal Cell-Seeded Hydrogels Using Experiment-Based Mathematical Modeling.

In vitro bone formation by mesenchymal stromal cells encapsulated in type-1 collagen hydrogels is demonstrated after a 28-day in vitro culture period. Analysis of the hydrogels is carried out by X-ray microcomputed tomography, histology, and immunohistochemistry, which collectively demonstrates that bone formation in the hydrogels was quantifiably proportional to the initial collagen concentration, and subsequently the population density of seeded cells. This was established by varying the initial collagen concentration at a constant cell seeding density (3 × 105 cells/0.3 mL hydrogel), and separately varying cell seeding density at a constant collagen concentration (1 mg/mL). Using these data, a mathematical model is presented for the total hydrogel volume and mineralization volume based on the observed linear contraction dynamics of cell-seeded collagen gels. The model parameters are fitted by comparing the predictions of the mathematical model for the hydrogel and mineralized volumes on day 28 with the experimental data. The model is then used to predict the hydrogel and mineralization volumes for a range of hydrogel collagen concentrations and cell seeding densities, providing comprehensive input/output descriptors for generating mineralized hydrogels for bone tissue engineering. It is proposed that this quantitative approach will be a useful tool for generating in vitro manufactured bone tissue, defining input parameters that yield predictable output measures of tissue maturation. Impact statement This article describes a simple yet powerful quantitative description of in vitro tissue-engineered bone by combining experimental data with mathematical modeling. The overall aim of the article is to examine what is currently known about cell-mediated collagen contraction, and demonstrate that this phenomenon can be exploited to tailor bone formation by choosing a specific set of input parameters in the form of cell seeding density and collagen hydrogel concentration. Our study utilizes a clinically relevant cell source (human mesenchymal stem cells) with a biomaterial that has received regulatory approval for use in humans (collagen type 1), and hence could be useful for clinical applications, as well as furthering our understanding of cell/extracellular matrix interactions in determining in vitro bone tissue formation.

X-Ray Diffraction Imaging of Corneal Ultrastructure.

X-ray scattering enables the structure of collagen-rich tissues, such as the cornea, to be examined at both the molecular and fibrillar level. The high-intensity X-rays available at synchrotron radiation sources, coupled with minimal sample preparation requirements, facilitates the rapid generation of high-quality X-ray scattering data from corneal tissue at a close-to-physiological state of hydration. Analysis of resulting X-ray scatter patterns allows one to quantify numerous structural parameters relating to the average diameter, lateral arrangement and alignment of collagen fibrils within the cornea, as well as the axial and lateral arrangements of collagen molecules within the fibrils. Here we describe the typical experimental setup and considerations involved in the collection of X-ray scattering data from corneal tissue.

Proteomic profiling of striatal tissue of a rat model of Parkinson's disease after implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells.

Parkinson's disease (PD) is the most common neurodegenerative disorder of movement worldwide. To date, only symptomatic treatments are available. Implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells (hUC-MSCs) is being developed as a novel therapeutic approach to potentially modify PD progression. However, implanted collagen scaffolds may induce a host tissue response. To gain insight into such response, hUC-MSCs were encapsulated into collagen hydrogels and implanted into the striatum of hemi-Parkinsonian male Sprague-Dawley rats. One or 14 days after implantation, the area of interest was dissected using a cryostat. Total protein extracts were subjected to tryptic digestion and subsequent LC-MS/MS analyses for protein expression profiling. Univariate and multivariate analyses were performed to identify differentially expressed protein profiles with subsequent gene ontology and pathway analysis for biological interpretation of the data; 2,219 proteins were identified by MaxQuant at 1% false discovery rate. A high correlation of label-free quantification (LFQ) protein values between biological replicates (r = .95) was observed. No significant differences were observed between brains treated with encapsulated hUC-MSCs compared to appropriate controls. Proteomic data were highly robust and reproducible, indicating the suitability of this approach to map differential protein expression caused by the implants. The lack of differences between conditions suggests that the effects of implantation may be minimal. Alternatively, effects may only have been focal and/or could have been masked by nonrelevant high-abundant proteins. For follow-up assessment of local changes, a more accurate dissection technique, such as laser micro dissection, and analysis method are recommended.