Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures.

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

Poly-l-arginine modifications alter the organization and secretion of collagen in SKH1-E mice.

Functionalized biomaterials interface with tissue upon implantation. There is a growing need to understand how materials properties influence this interaction so that efficient tissue engineering systems can be developed. In this study, we characterize collagen organization in response to functionalized glass beads implanted in SKH1-E mice. Poly-l-arginine (PLR) was modified with arginine derivatives to create a functionalized surface and was coated on glass beads. Tissue sections were removed 28 days post-implantation and were imaged using second harmonic generation (SHG) microscopy. These chemical modifications were able to alter the collagen distribution from highly aligned to disordered (17 ±â€¯6 to 78 ±â€¯1° full width at half-maximum (FWHM)) and the collagen III/I ratio (0.02 to 0.42). Principal component analysis (PCA) comparing the physical properties of the modifiers (e.g. hydrophobicity, molar volume, freely rotating bonds, polarizability) with the SHG analytically derived parameters (e.g. collagen III/I ratio, collagen orientation) was performed. Chemical properties of the PLR-like modifications including lipophilicity, along with the number of freely rotating bonds and the polarizability had significant effects on the collagen surrounding the implant, both in terms of collagen orientation as well as the production of collagen III. These findings demonstrate the possibility of tuning the foreign body response, in terms of collagen deposition and organization, to positively influence the acceptance of implanted biomaterials.

Degradation and Remodeling of Epitaxially Grown Collagen Fibrils.

INTRODUCTION­: The extracellular matrix (ECM) in the tumor microenvironment contains high densities of collagen that are highly aligned, resulting in directional migration called contact guidance that facilitates efficient migration out of the tumor. Cancer cells can remodel the ECM through traction force controlled by myosin contractility or proteolytic activity controlled by matrix metalloproteinase (MMP) activity, leading to either enhanced or diminished contact guidance. METHODS­: Recently, we have leveraged the ability of mica to epitaxially grow aligned collagen fibrils in order to assess contact guidance. In this article, we probe the mechanisms of remodeling of aligned collagen fibrils on mica by breast cancer cells. RESULTS­: We show that cells that contact guide with high fidelity (MDA-MB-231 cells) exert more force on the underlying collagen fibrils than do cells that contact guide with low fidelity (MTLn3 cells). These high traction cells (MDA-MB-231 cells) remodel collagen fibrils over hours, pulling so hard that the collagen fibrils detach from the surface, effectively delaminating the entire contact guidance cue. Myosin or MMP inhibition decreases this effect. Interestingly, blocking MMP appears to increase the alignment of cells on these substrates, potentially allowing the alignment through myosin contractility to be uninhibited. Finally, amplification or dampening of contact guidance with respect to a particular collagen fibril organization is seen under different conditions. CONCLUSIONS­: Both myosin II contractility and MMP activity allow MDA-MB-231 cells to remodel and eventually destroy epitaxially grown aligned collagen fibrils.

Second harmonic generation microscopy of collagen organization in tunable, environmentally responsive alginate hydrogels.

We fabricated photocrosslinked, environmentally responsive alginate hydrogels for tissue engineering applications. Methacrylated alginate (ALGMA) hydrogels were prepared across a variety and combination of ionic and covalent (chain growth, step growth, and mixed mode) crosslinking strategies to obtain a range of compressive moduli from 9.3 ± 0.2 kPa for the softest ionically crosslinked hydrogels to 22.6 ± 0.3 kPa for the dually crosslinked ionic mixed mode gels. The swelling behavior of the alginate hydrogels was significantly higher under basic pH conditions. Stiffer gels consistently swelled to a lesser degree compared to softer gels for all conditions. These hydrogels were stable - retaining >80% of their original mass for three weeks - when incubated in a basic solution of diluted sodium hydroxide, which mimicked accelerated degradation conditions. Encapsulated NIH/3T3 fibroblasts remained viable and proliferated significantly more in stiffer hydrogel substrates compared to softer gels. Additionally, the collagen secreted by encapsulated fibroblasts was quantifiably compared using second harmonic generation (SHG) imaging. Fibroblasts encapsulated in the softer hydrogels secreted significantly less collagen than the stiffer gels. The collagen in these softer gels was also more aligned than the stiffer gels. The ability to tune collagen organization using hydrogels has potential applications ranging from corneal wound healing where organized collagen is desired to epithelial wound scaffolds where a random organization is preferable.

Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues.

Contact guidance or bidirectional migration along aligned fibers modulates many physiological and pathological processes such as wound healing and cancer invasion. Aligned 2D collagen fibrils epitaxially grown on mica substrates replicate many features of contact guidance seen in aligned 3D collagen fiber networks. However, these 2D collagen self-assembled substrates are difficult to image through, do not have known or tunable mechanical properties and cells degrade and mechanically detach collagen fibrils from the surface, leading to an inability to assess contact guidance over long times. Here, we describe the transfer of aligned collagen fibrils from mica substrates to three different functionalized target substrates: glass, polydimethylsiloxane (PDMS) and polyacrylamide (PA). Aligned collagen fibrils can be efficiently transferred to all three substrates. This transfer resulted in substrates that were to varying degrees resistant to cell-mediated collagen fibril deformation that resulted in detachment of the collagen fibril field, allowing for contact guidance to be observed over longer time periods. On these transferred substrates, cell speed is lowest on softer contact guidance cues for both MDA-MB-231 and MTLn3 cells. Intermediate stiffness resulted in the fastest migration. MTLn3 cell directionality was low on soft contact guidance cues, whereas MDA-MB-231 cell directionality marginally increased. It appears that the stiffness of the contact guidance cue regulates contact guidance differently between cell types. The development of this collagen fibril transfer method allows for the attachment of aligned collagen fibrils on substrates, particularly flexible substrates, that do not normally promote aligned collagen fibril growth, increasing the utility of this collagen self-assembly system for the fundamental examination of mechanical regulation of contact guidance.

Collagen organization deposited by fibroblasts encapsulated in pH responsive methacrylated alginate hydrogels.

The pH of dermal wounds shifts from neutral during the inflammatory phase to slightly basic in the tissue remodeling phase. Stage specific wound treatment can be developed using environmentally responsive alginate hydrogels. The chemistry of these networks dictates swelling behavior. Here, we fabricated alginate hydrogels using chain growth, step growth, and combined mixed mode gelation methods to crosslink methacrylated alginate (ALGMA) and gain control over swelling responses. Methacrylation of the alginate network was confirmed through NMR spectroscopy. Strontium cations were introduced to fabricate stiffer, dually crosslinked hydrogels. Dual crosslinking significantly decreased the swelling response over the pH range of 3-9 for step growth and chain growth hydrogels, with no impact on mixed mode hydrogels. The extent of crosslinking altered the hydrogel degradation profiles under accelerated degradation conditions. Encapsulated NIH/3T3 fibroblasts in the different ALGMA hydrogels remained viable with greater cell proliferation in the stiffer gels. Collagen organization deposited by the NIH/3T3 fibroblasts was monitored using second harmonic generation (SHG) microscopy and was influenced by the crosslinking mechanism. Ionic chain growth and ionic mixed mode crosslinked ALGMA hydrogels caused relatively isotropic collagen organization, particularly 10 days post-cell encapsulation. Principal component analysis (PCA) was employed to uncover correlations between the observed properties. The ability of these environmentally responsive gels to induce isotropic collagen and respond to pH changes means they hold promise as phase specific wound dressings. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2934-2943, 2018.

The effect of polarized light on the organization of collagen secreted by fibroblasts.

Recent studies have demonstrated the beneficial effect of low-power lasers and polarized light on wound healing, inflammation, and the treatment of rheumatologic and neurologic disorders. The overall effect of laser irradiation treatment is still controversial due to the lack of studies on the biochemical mechanisms and the optimal parameters for the incident light that should be chosen for particular applications. Here, we study how NIH/3T3 fibroblasts respond to irradiation with linearly polarized light at different polarization angles. In particular, we examined vascular endothelial growth factor (VEGF) secretion, differentiation to myofibroblasts, and collagen organization in response to 800 nm polarized light at 0°, 45°, 90°, and 135° with a power density of 40 mW/cm2 for 6 min every day for 6 days. Additional experiments were conducted in which the polarization angle of the incident was changed every day to induce an isotropic distribution of collagen. The data presented here shows that polarized light can upregulate VEGF production, myofibroblast differentiation, and induce different collagen organization in response to different polarization angles of the incident beam. These results are encouraging and demonstrate possible methods for controlling cell response through the polarization angle of the laser light, which has potential for the treatment of wounds.

Elucidating the interactions and phytotoxicity of zinc oxide nanoparticles with agriculturally beneficial bacteria and selected crop plants.

There is a growing interest in the use of bioinoculants to assist mineral fertilizers in improving crop production and yield. Azotobacter and Pseudomonas are two agriculturally relevant strains of bacteria which have been established as efficient bioinoculants. An experiment involving addition of graded concentrations of zinc oxide (ZnO) nanoparticles was undertaken using log phase cultures of Azotobacter and Pseudomonas. Growth kinetics revealed a clear trend of gradual decrease with Pseudomonas; however, Azotobacter exhibited a twofold enhancement in growth with increase in the concentration of ZnO concentration. Scanning electron microscopy (SEM), supported by energy-dispersive X-ray (EDX) analyses, illustrated the significant effect of ZnO nanoparticles on Azotobacter by the enhancement in the abundance of globular biofilm-like structures and the intracellular presence of ZnO, with the increase in its concentration. It can be surmised that extracellular mucilage production in Azotobacter may be providing a barrier to the nanoparticles. Further experiments with Azotobacter by inoculation of wheat and tomato seeds with ZnO nanoparticles alone or bacteria grown on ZnO-infused growth medium revealed interesting results. Vigour index of wheat seeds reduced by 40-50% in the presence of different concentrations of ZnO nanoparticles alone, which was alleviated by 15-20%, when ZnO and Azotobacter were present together. However, a drastic 50-60% decrease in vigour indices of tomato seeds was recorded, irrespective of Azotobacter inoculation.

Methods for Implant Acceptance and Wound Healing: Material Selection and Implant Location Modulate Macrophage and Fibroblast Phenotypes.

This review focuses on materials and methods used to induce phenotypic changes in macrophages and fibroblasts. Herein, we give a brief overview on how changes in macrophages and fibroblasts phenotypes are critical biomarkers for identification of implant acceptance, wound healing effectiveness, and are also essential for evaluating the regenerative capabilities of some hybrid strategies that involve the combination of natural and synthetic materials. The different types of cells present during the host response have been extensively studied for evaluating the reaction to different materials and there are varied material approaches towards fabrication of biocompatible substrates. We discuss how natural and synthetic materials have been used to engineer desirable outcomes in lung, heart, liver, skin, and musculoskeletal implants, and how certain properties such as rigidity, surface shape, and porosity play key roles in the progression of the host response. Several fabrication strategies are discussed to control the phenotype of infiltrating macrophages and fibroblasts: decellularization of scaffolds, surface coatings, implant shape, and pore size apart from biochemical signaling pathways that can inhibit or accelerate unfavorable host responses. It is essential to factor all the different design principles and material fabrication criteria for evaluating the choice of implant materials or regenerative therapeutic strategies.