Gelatin Substrate Research Articles

Hydrodynamics of a dual-orifice needle-free jet injector

In the recent literature, it has been shown that an enhanced, multi-faceted, balanced immune response can be generated using a combined intradermal (ID) and intramuscular (IM) delivery of vaccines, especially for novel DNA vaccines. However, needle-based injection for a combined ID/IM delivery may require separate injections, which may prove to be time consuming, and impractical for mass immunizations. In contrast, a novel multi-orifice jet injector could be used to deliver drugs at multiple penetration depths simultaneously, which may provide advantages such as ease of operation, elimination of sharps, and short injection timeframe (O (10 m)).Here, we consider a dual-orifice geometry with both a wide orifice (200 μm–400 μm) for IM drug delivery and a narrow orifice (100 μm) for ID drug delivery. Using numerical simulations, we found that, at a fixed upstream pressure, jet velocities through wide and narrow orifices do not vary significantly for low-viscosity fluids (≲4%). However, it was previously hypothesized that the jet power, Pj=π8ρdj2vj3, is a more appropriate parameter to characterize tissue penetration depth. Thus, the jet power could vary by a factor of ∼10, yielding different depths for the two orifices. Using non-dimensional analysis via Euler (Eu) and Reynolds (Re) numbers, we characterize the role of orifice geometry and driving pressure, to generate geometry-specific correlations in the Eu–Re parameter space to estimate the jet velocity and pressure losses. We also elucidate the orifice size ratios that are best suited for fractional doses of ∼100 μL to intradermal tissue from a total injection of 1 ml.Preliminary experiments to visualize the penetration of wide and narrow jet streams into gelatin and pork substrates showed that the wide jet stream leads to substantially greater penetration depth than the narrow jet stream. In summary, we provide the initial feasibility of simultaneous delivery of a drug to multiple penetration depths from the same injection cartridge using the needle-free jet injection technique.

Flexible, self-healing, and degradable polymeric dielectrics cross-linked through metal–ligand for resistive memory device

The ability to self-heal is a crucial feature in nature, where living organisms can repair themselves when subjected to minor injuries. With an increasing emphasis on environmental sustainability, the concept of biomimetic self-healing polymeric materials has emerged as a prominent trend, promising to significantly extend the lifespan and reliability of products. Studies have shown that one-third of proteins in living organisms require metal cofactors to function properly. It is known that protein-metal interactions can enhance the performance of certain biomaterials, and different choices of metals and ligands can create diverse material properties, influencing characteristics such as hardness, toughness, adhesion, and self-healing abilities. Gelatin is a natural polymer derived from the hydrolysis of collagen, and its unique amino acid structure has led to a wide range of applications. In this research, by introducing aluminum ions that form metal coordination complexes with the carboxyl groups in gelatin, an elastic network with self-healing properties was constructed. This gelatin-based material was utilized as an insulating layer in resistive switching devices. Furthermore, by employing a gelatin substrate of the same composition, the device demonstrated strong interfacial adhesion. The device based on the self-healing gelatin film exhibited excellent electrical performance and mechanical properties. Even after self-healing, it maintained a high ON/OFF ratio of up to 105 and a concentrated distribution of switching parameters. Supported by compelling physical and electrical evidence, this study showcases significant development opportunities for biomimetic materials in green electronic devices.

Utilization of Pickering emulsion stabilized by chitin nanofibers for improving water and oxygen resistance of gelatin films

The hydrophilicity of gelatin films obviously limits their application. In this work, novel protonated chitin nanofiber was prepared with green methods, surficial deacetylation combined with acidic and mechanical treatment. Composite films with excellent water and oxygen barrier properties were successfully prepared by blending O/W Pickering emulsions stabilized by chitin nanofibers with gelatin substrates. The films were characterized by Fourier-transform infrared, X-ray diffraction, mechanical properties, water and oxygen permeability, moisture content, water solubility, water contact angle and optical properties. The results demonstrated that the amino groups of chitin nanofibers bound to the carboxyl groups of gelatin via electrostatic interactions, which contributing to the excellent mechanical and barrier properties of composite films. The elongation at break of composite film (the concentration of chitin nanofiber was 0.2 wt%) was 2.66 times of gelatin film. And the water vapor and oxygen permeability of composite films lowered to 65.9 % and 52.9 %, respectively. The introduction of O/W Pickering emulsion significantly enhanced the hydrophobicity of gelatin-based film and the chitin nanofibers played a positive role in stabilizing the gelatin chains to a certain extent. This study expands the application fields of gelatin-based films and provides valuable technical route for the preparation of barrier films.

Gelatin methacrylate based liquid dressing with antibacterial and hemostasis properties

For incompressible and irregularly shaped wounds, developing a novel dressing based on photo-crosslinked hydrogel that has both hemostasis and antibacterial functions is highly desirable. In this study, photoreactive substrate gelatin methacrylate (GelMA) and Poly (ethylene glycol) diacrylate (PEGDA) were combined with water-soluble quaternary ammonium antibacterial monomers (DMC). Interestingly, they can quickly change from liquid to hydrogel under visible light at 405 nm within 1 min, which is suitable for small incompressible wounds. Regarding the biosafety of the dressing, cell viability and blood viability test indicated that the GelMA/PEGDA/DMC3 dressing has no cytotoxicity toward L929 cells and red blood cells. The antibacterial performance test proved that the addition of DMC significantly improved the antibacterial performance of the dressing. Moreover, the GelMA/PEGDA/DMC3 dressing showed rapid hemostasis ability in tail clipping hemostasis test and liver hemostasis test. Taken together, this new type of convenient hydrogel dressing exhibits great potential as a quick hemostasis and antibacterial wound dressing candidate for small bleeding wounds.

A Model for Membrane Degradation Using a Gelatin Invadopodia Assay.

One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.

Design of biodegradable gelatin resistive memory with remarkable performance

For electronic systems, the substrate can be treated as a cornerstone of the overall device structure. With the growth of green electronics, the development of biodegradable substrates is of great significance. Gelatin substrates demonstrate transparency, low temperature, low cost, and fully biodegradable. In particular, gelatin of porcine source (GP) possesses excellent elasticity and high amount of hydroxyproline, which significantly improves the structural stability and memory performance of the device. The device with Ag/gelatin/Ag structure on GP substrate has illustrated non-volatile memory characteristics to be reckoned with an ON/OFF ratio of over 105, uniformly distributed switching parameters (<20 %), and steady retention time (>104 s). It is striking that the resistive switching of the GP-based device maintains stability under various curvature bending stresses (ON/OFF ratio of approximately 105) and displays repeatable resistive states undergoes 1000 cycles of bending tests, not only provides excellent structural reliability and robust mechanical flexibility but also effectively suppresses leakage current in devices, which is beneficial for flexible memory applications. Furthermore, the device possesses outstanding thermal stability, and numerous resistive switching behaviors even at 70 °C are observed. The conductive mechanism may be attributed to the ionic migration in the Ag ions chelating in amine groups of gelatin on the basis of the compelling electrical evidence for Ag/gelatin/Ag/GP devices. The biomass substrate disintegrates entirely in PBS solution without toxic residues, providing a novel design guideline for biomedical applications inspired by implantable, interventional medical, and wearable electronics.

Thermally Degradable Split-Ring Resonator Made on a Gelatin Substrate with Enhanced Dielectric and Conductive Properties

Ingestible electronics, taken orally for monitoring organs, bacteria, and cells, are innovative wireless diagnostic and therapeutic technologies. Whereas residual in-body devices may require surgical removal, food-based sensors are seen as a way of ensuring safety. This study reports a split-ring resonator (SRR) whose resonance can be observed wirelessly. It is fabricated on a thermoresponsive gelatin substrate with a collaged edible metal leaf. Gelatin is a highly water-absorbing material, which may affect the resonator performance. According to high-frequency structural simulations, this resonator requires a low dielectric loss, <0.05, and high conductivity, >106 S/m. We found that these targets were achieved by completely drying the gelatin film and transferring multiple stacked metal leaves onto the film. Thin metal sheets were glued using an oleogel made of beeswax and olive oil, and the use of a hydrophobic material also aided in the transfer. Thus, we gained insights into the criteria for fabricating transient resonators with thermodegradability. SRRs made with edible materials are a technology that works in harmony with people and the environment and could lead to wireless devices or micromachines for the healthcare, agriculture, and livestock industries.

Matrix metalloproteinase activity during methamphetamine cued relapse.

Relapse to drug seeking involves transient synaptic remodelling that occurs in response to drug-associated cues. This remodelling includes activation of matrix metalloproteinases (MMPs) to initiate catalytic signalling in the extracellular matrix in the nucleus accumbens core (NAcore). We hypothesized that MMP activity would be increased in the NAcore during cue-induced methamphetamine (meth) seeking in a rat model of meth use and relapse. Male and female rats had indwelling jugular catheters and bilateral intracranial cannula targeting the NAcore surgically implanted. Following recovery, rats underwent meth or saline self-administration (6h/day for 15 days) in which active lever responding was paired with a light + tone stimulus complex, followed by home cage abstinence. Testing occurred after 7 or 30 days of abstinence. On test day, rats were microinjected with a fluorescein isothiocyanate (FITC)-quenched gelatin substrate that fluoresces following cleavage by MMP-2,9, allowing for the quantification of gelatinase activity during cued-relapse testing. MMP-2,9 activity was significantly increased in the NAcore by meth cues presentation after 7 and 30 days of abstinence, indicating that remodelling by MMPs occurs during presentation of meth associated cues. Surprisingly, although cue-induced seeking increased between Days 7 and 30, MMP-2,9 activity did not increase. These findings indicate that although MMP activation is elicited during meth cue-induced seeking, MMP activation did not parallel the meth seeking that occurs during extended drug abstinence.

EFFECTS OF BACTERIAL LIPOPOLYSACCHARIDE AND SHIGA TOXIN ON INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STEM CELLS.

Background : Mesenchymal stem cells (MSCs) can be activated by different bacterial toxins. Lipopolysaccharides and Shiga Toxin (Stx) are the main toxins necessary for hemolytic uremic syndrome development. The main etiological event in this disease is endothelial damage that causes glomerular destruction. Considering the repairing properties of MSC, we aimed to study the response of MSC derived from induced pluripotent stem cells (iPSC-MSC) to LPS and/or Stx and its effect on the restoration of injured endothelial cells. Methods : iPSC-MSC were treated with LPS and or/Stx for 24 h and secretion of cytokines, adhesion, and migration were measured in response to these toxins. In addition, conditioned media from treated iPSC-MSC were collected and used for proteomics analysis and evaluation of endothelial cell healing and tubulogenesis using human microvascular endothelial cells 1 as a source of endothelial cells. Results : The results obtained showed that LPS induced a proinflammatory profile on iPSC-MSC, whereas Stx effects were less evident, even though cells expressed the Gb 3 receptor. Moreover, LPS induced on iPSC-MSC an increment in migration and adhesion to a gelatin substrate. Addition of conditioned media of iPSC-MSC treated with LPS + Stx, decreased the capacity of human microvascular endothelial cells 1 to close a wound, and did not favor tubulogenesis. Proteomic analysis of iPSC-MSC treated with LPS and/or Stx revealed specific protein secretion patterns that support the functional results described. Conclusions : iPSC-MSC activated by LPS acquired a proinflammatory profile that induces migration and adhesion to extracellular matrix proteins but the addition of Stx did not activate any repair program to ameliorate endothelial damage, indicating that the use of iPSC-MSC to regenerate endothelial injury caused by LPS and/or Stx in hemolytic uremic syndrome could not be the best option to consider to regenerate a tissue injury.

Luminescent mechanism analyzing and metal-ion sensing of Eu(III)-organic ligands complex doped gelatin-based films and hydrogels

A series of gelatin-based composite films (gf) were prepared by doping Eu(TTA)3(H2O)2, Eu(TTA)3bipy, or Eu(TTA)3phen respectively into type-E or -B gelatin film substrate. All of B-gf and the E-gf doped Eu(TTA)3phen were realized with red luminescence under 365 nm excitation, while only slight blue luminescence originated from gelatin substrate can be observed in E-gf doped Eu(TTA)3(H2O)2 or Eu(TTA)3bipy. A UV-absorption competition between substrate and dopant was confirmed to decide the luminescent properties of gf. Weaker UV absorption of B-gelatin ensures that Eu(TTA)3, regardless of ancillary ligand, can obtain sufficient excitation energy for luminescence emission. While only Eu(TTA)3phen possesses a strong enough ability to absorb UV against E-gelatin. Based on the optimal matching principle of substrate and dopant, luminescent gelatin-based hydrogel (gh) doped Eu(TTA)3phen was prepared. A ‘dual effect’ mechanism was proposed for explaining metal-ion sensing applications. Among various metal ions, furthermore, both B and E types of Eu(TTA)3phen@gh exhibited similar significant luminescence quenching for Fe3+ with a detection limit of about 1.15 μmol/L.

The effect of liquid-liquid extraction on metabolite detection and analysis using NMR spectroscopy in human synovial fluid

The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible.

Investigation of Some Bioactivities and Odor Components of Jasminum officinale Linn. (Oleaceae): A Valuable Tool for Cosmetic Product Design

In this study, researches were carried out on the protease enzyme activity of Jasminum officinale Linn. flower which grows naturally in Muğla and its surroundings. In addition, fragrance components in the content of jasmine flower were determined. It was aimed to be used in perfume making based on the harmony of white jasmine flower with other flowers and the concept of note. Protease enzyme was purified from J. officinale flower using TPP (Three Phase Partitioning) method. Optimal pH and optimal temperature for the enzyme, Km and Vmax values for casein, azokazaein, gelatin, hemoglobin, and azoalbumin substrates were determined. SDS-PAGE was used to check the purity of the protease enzyme purified from the J. officinale. The molecular weight of the enzyme was calculated as 21.386 kDa using gel filtration chromatography. The phenolic content was also determined. It has been determined that the content of jasmine flower can be used in perfume design which is the most prestigious product of the cosmetic industry.

Gelatinase Microbial Morphology from Leather Defect

Microbes attach in pickle leather, wet blue leather, and upper shoeleather have been identified to have positive profile to produce extracellular protease enzymes with pH values of acidic to basic on 15% gelatin and 2% commercial skimmed milk as substrates. Defective tanned leather was added to a microbial growth solution enriched with 2% gelatin substrate and incubated for 7 days at room temperature on an open rotary shaker at 120 rpm and then stored at room temperature while waiting for gelatinase testing. There were 5 morphological colonies of microbial gelatinase from defective tanned skin, all of which were white.

Development and performance evaluation of a two-stage cascade impactor equipped with gelatin filter substrates for the collection of multi-sized particulate matter

This study presents the development and evaluation of a high flow rate gelatin cascade impactor (GCI) to collect different PM particle sizes on water-soluble gelatin substrates. The GCI operates at a flow rate of 100 lpm, and consists of two impaction stages, followed by a filter holder to separate particles in the following diameter ranges: >2.5 μm, 0.2–2.5 μm, and <0.2 μm. Laboratory characterization of the GCI performance was conducted using monodisperse polystyrene latex (PSL) particles as well as polydisperse ammonium sulfate, sodium chloride, and ammonium nitrate aerosols to obtain the particle collection efficiency curves for both impaction stages. In addition to the laboratory characterization, we performed concurrent field experiments to collect PM2.5 employing both GCI equipped with gelatin filter and personal cascade impactor sampler (PCIS) equipped with PTFE filter for further toxicological analysis using macrophage-based reactive oxygen species (ROS) and dithiothreitol consumption (DTT) assays. Our results showed that the experimentally determined cut-point diameters for the first and second impaction stages were 2.4 μm and 0.21 μm, respectively, which agreed with the theoretical predictions. Although the GCI has been developed primarily to collect particles on gelatin filters, the use of a different type of substrate (i.e., quartz) led to similar particle separation characteristics. The findings of the field tests demonstrated the advantage of using the GCI in toxicological studies due to its ability to collect considerable PM-toxic constituents, as corroborated by the DTT and ROS values for the GCI-collected particles which were 26.44 nmol/min/mg PM and 8813.2 μg Zymosan Units/mg PM, respectively. These redox activity values were more than twice those of particles collected concurrently on PTFE filter using the PCIS. This high-flow-rate impactor can collect considerable amounts of size-fractionated PM on water-soluble filters (i.e., gelatin), which can completely dissolve in water allowing for the extraction of soluble and insoluble PM species for further toxicological analysis.

Dual mode fluorometric and colorimetric detection of matrix metalloproteinase MMP-9 as a cancer biomarker based on AuNPs@gelatin/AuNCs nanocomposite

Matrix metalloproteinase (MMPs) are proteolytic enzymes which considered as important enzymes and their higher expression is associated with some malignant progression. In the present study, a novel and sensitive method was developed for detection of MMP-9 based on its gelatinase activity. Green emitting gold nanoclusters (AuNCs) has been synthesized by gelatin substrate and then AuNPs@gelatin/AuNCs nanocomposite structure were prepared through coating of gold nanoparticles (AuNPs) with gelatin/AuNCs. Characterization and morphology of synthesized nanocomposite showed successful formation of AuNPs@gelatin/AuNCs. Constructed nanocomposite exhibited internal FRET occurrence and fluorescence quenching of AuNCs due to proximity of AuNCs/AuNPs structures. Hydrolysis effect of MMPs enzyme on AuNPs@gelatin/AuNCs in saline buffer induced changes in surface plasmon resonance (SPR) of gold nanoparticles and also enhanced the emitted fluorescence by AuNCs due to inhibition of internal FRET process. The proposed platform served as the efficient approach for semi-quantitative detection of MMP-9 enzyme by naked eye and precise activity detection with LOD of 2 ng/mL calorimetrically and 0.25 ng/mL fluorometrically. The approach was also showed convenient recovery for detection of MMP-9 enzyme in human serum matrix. Our work provides an efficient, convenient and practical tool for simple identification and precise detection of MMP-9 enzyme.

Numerical and Experimental Analysis of Shear Stress Influence on Cellular Viability in Serpentine Vascular Channels.

3D bioprinting has emerged as a tool for developing in vitro tissue models for studying disease progression and drug development. The objective of the current study was to evaluate the influence of flow driven shear stress on the viability of cultured cells inside the luminal wall of a serpentine network. Fluid–structure interaction was modeled using COMSOL Multiphysics for representing the elasticity of the serpentine wall. Experimental analysis of the serpentine model was performed on the basis of a desirable inlet flow boundary condition for which the most homogeneously distributed wall shear stress had been obtained from numerical study. A blend of Gelatin-methacryloyl (GelMA) and PEGDA200 PhotoInk was used as a bioink for printing the serpentine network, while facilitating cell growth within the pores of the gelatin substrate. Human umbilical vein endothelial cells were seeded into the channels of the network to simulate the blood vessels. A Live-Dead assay was performed over a period of 14 days to observe the cellular viability in the printed vascular channels. It was observed that cell viability increases when the seeded cells were exposed to the evenly distributed shear stresses at an input flow rate of 4.62 mm/min of the culture media, similar to that predicted in the numerical model with the same inlet boundary condition. It leads to recruitment of a large number of focal adhesion point nodes on cellular membrane, emphasizing the influence of such phenomena on promoting cellular morphologies.

Interfacial Reinitiation of Free Radicals Enables the Regeneration of Broken Polymeric Hydrogel Actuators

Interfacial Reinitiation of Free Radicals Enables the Regeneration of Broken Polymeric Hydrogel Actuators

Development of scaffold-free vascularized pancreatic beta-islets in vitro models by the anchoring of cell lines to a bioligand-functionalized gelatine substrate

Bioengineered pancreatic β-islets have been widely advocated for the research and treatment of diabetes by offering both suitable cell culture models for the study of the pathology and the testing of new drugs and a therapy in those patients no longer responding to insulin administration and as an alternative to the shortage of donors for organ and islet transplantation. Unlike most of the studies published so far where pancreatic islets of pancreatic β-cells are encapsulated in hydrogels, this study demonstrate the formation of bioengineered pancreatic islets through cell anchoring to a gelatine-based biomaterial, PhenoDrive-Y, able to mimic the basement membrane of tissues. Through simple culture conditions, PhenoDrive-Y led human pancreatic β-cell lines and human umbilical endothelial cell lines to form organized structures closely resembling the natural vascularized pancreatic islets. When compared to gelatine, the cultures in presence of PhenoDrive-Y show higher degree of organization in tissue-like structures, a more pronounced endothelial sprouting and higher expression of typical cell markers. Noticeably, when challenged by hyperglycaemic conditions, the cells embedded in the PhenoDrive-Y assembled spheroids responded with higher levels of insulin production. In conclusion, the present work demonstrates the potential of PhenoDrive-Y as substrate for the development of bioengineered vascularized pancreatic islets and to be particularly suitable as a model for in vitro studies and testing of new therapeutics.Graphical abstract

A Biodegradable Gelatin Substrate and its Application for Crack Suppression of Flexible Gelatin Resistive Memory Device

AbstractIn this work, gelatin is used not only as a substrate for the memory device but also as the resistive layer. This lightweight gelatin substrate (GS) will retain all of the desirable properties of plastic substrates, with the added advantages of being renewable, biodegradable, and inexpensive. GS is totally degraded after 18 days, showing excellent biodegradability in dry soil that can reduce the environmental impact effectively. Moreover, due to the substrate and resistive layer used of the same materials, it can be observed by the scanning electron microscope analysis that the films on GS both have smooth interfaces regardless of the gelatin layer or Ag electrodes after bending 1000 cycles. A strong interfacial adhesion leads to an efficient stress transfer underload, resulting in better structural stability and bending performance of the In/gelatin/Ag/GS device than those of the In/gelatin/Ag/PET device. The In/gelatin/Ag/GS device shows a stable retention time of more than 103 s and uniform current distribution, as well as the device with bending stress is capable to execute the ON/OFF ratio of over 104. This result exhibits the fabricating processes without a vacuum system and has broaden the application study of gelatin.

Zymography and Reverse Zymography for Testing Proteases and Their Inhibitors.

Zymography is a powerful technique for the assay of different hydrolases that act upon any biological macromolecule. In particular, zymography is used to assay the activities of serine proteases, e.g., matrix metalloproteinases (MMPs), and reverse zymography is used for tissue inhibitors of metalloproteinases (TIMPs) in multifarious experimental samples. Zymography is a method of electrophoretic separation of proteases under non-reducing conditions in a polyacrylamide gel containing substrate. The resolved proteins are renatured by exchange of the anionic detergent with a nonionic one, and the gel is incubated in a specific buffer for the specific proteases. After staining the gel by Coomassie blue staining solution, the proteolytic activities are visualized as clear colorless bands against a dark background. In contrast, reverse zymography is a parallel technique to detect protease inhibitors. In addition to substrate gelatin, proteases (i.e., MMPs) are also incorporated in proper ratio into the polyacrylamide gel. After electrophoresis, during the developing step, the MMPs specifically digest the substrate in regions where TIMPs are absent. Thus, inhibitors/TIMP is represented as dark zones of inhibition against a transparent background after staining. In this chapter, common troubleshoots during sample preparation, running zymography, and data interpretation are discussed. Notes are specified to enhance the sensitivity of the methods. In conclusion, zymography could be crucial for enzyme assay at the nanogram level and for the improvement of new investigative techniques for diseases such as endometriosis, rheumatoid arthritis, osteoarthritis, tumor invasion, and inflammation.