Genipin Crosslinking Research Articles

Superior Cell Delivery Features of Genipin Crosslinked Polymeric Microcapsules: Preparation, In Vitro Characterization and Pro-Angiogenic Applications Using Human Adipose Stem Cells

The ability of mesenchymal stem cells to self-renew and differentiate into specialized cell lineages makes them promising tools for regenerative medicine. Local injection and use of scaffolds had been employed earlier to deliver these cells; yet, an optimal delivery system remains to be identified. Here, using genipin, which is a non-toxic natural cross linker for proteins, we prepared alginate-chitosan polymeric microcapsules (GCAC) to develop an efficient stem cell delivery system. We investigated the properties of this membrane along with the encapsulated adipose tissue-derived stem cells (ASCs) and compared that with the widely used alginate poly-lysine (APA) membranes. The GCAC membrane was able to support cell viability, augment cell growth, and showed better results under external rotational and osmotic pressures with about 30% of the ruptured capsules in comparison to 60% ruptured APA capsules. The membrane also provided immune-protection to the entrapped cells as demonstrated by the lymphocyte proliferation assay. The capsule also has potential for long-term storage. The encapsulated four million ASCs also showed steady secretion of approximately 4600pg vascular endothelial growth factor (VEGF) over 15-day time period comparable to that of free cells. Furthermore, the encapsulated ASCs showed around 3.8-fold increase in VEGF secretion after 72h hypoxic conditions in comparison to normoxic conditions. This increased VEGF expression resulted in improved angiogenic potential of the bioactive capsules as noted by enhanced endothelial cell growth. GCAC encapsulation also did not show any effect on their differentiation ability. Thus, because of these biocompatible and bioactive attributes, genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications.

Electrospun gelatin nanofibers: Optimization of genipin cross-linking to preserve fiber morphology after exposure to water

The development of suitable biomimetic three-dimensional scaffolds is a fundamental requirement of tissue engineering. This paper presents the first successful attempt to obtain electrospun gelatin nanofibers cross-linked with a low toxicity agent, genipin, and able to retain the original nanofiber morphology after water exposure. The optimized procedure involves an electrospinning solution containing 30wt.% gelatin in 60/40 acetic acid/water (v/v) and a small amount of genipin, followed by further cross-linking of the as-electrospun mats in 5% genipin solution for 7days, rinsing in phosphate-buffered saline and then air drying at 37°C. The results of scanning electron microscopy investigations indicated that the cross-linked nanofibers were defect free and very regular and they also maintained the original morphology after exposure to water. Genipin addition to the electrospinning solution dramatically reduced the extensibility of the as-electrospun mats, which displayed further remarkable improvements in elastic modulus and stress at break after successive cross-linking up to values of about 990 and 21MPa, respectively. The results of the preliminary in vitro tests carried out using vascular wall mesenchymal stem cells indicated good cell viability and adhesion to the gelatin scaffolds.

Genipin-crosslinked hydrophobical chitosan microspheres and their interactions with bovine serum albumin

In this work chitosan microspheres have been prepared by ion precipitation using sodium sulfate as precipitant, and crosslinked with natural crosslinker genipin. Then the microspheres were hydrophobically modified by covalent and ion interaction grafting of stearic acid and sodium stearate respectively. Results indicated that the there were still some amount of –NH 2 groups on the genipin-crosslinked chitosan microspheres. And the contact angles for hydrophobical graft microspheres were increased greatly from 27° to 104°, which indicates stearic chains have been grafted on the surface of the microspheres even only by a simple graft process based on ion interaction. The interactions of protein with the hydrophobically modified chitosan microspheres were investigated by surface absorption method using bovine serum albumin as a model protein. And results indicated that adsorption efficiency for bovine serum albumin of the fatty chain grafted chitosan microspheres was higher than that of the prestine one distinctly, due to the additional hydrophobic interaction. And the adsorbed protein could well-off be desorbed under a general condition.

The Effects of Exogenous Crosslinking on Hydration and Fluid Flow in the Intervertebral Disc Subjected to Compressive Creep Loading and Unloading

In vitro study of genipin crosslinking effect on disc water content changes under compressive loading and unloading. To investigate the influence of collagen crosslinking on hydration and fluid flow in different regions of intact discs, and to evaluate the nutritional implications. Age-related reductions of nutrient supply and waste product removal are critically important factors in disc pathogenesis. Diffusion and fluid flow are blocked by subchondral bone thickening, cartilaginous endplate calcification, loss of hydrophilic proteoglycans, and clogging of anular pores by degraded matrix molecules. Previous studies demonstrated increased hydraulic permeability and macromolecular transport through crosslinked collagenous matrices. Genipin has also demonstrated the capability to increase retention of proteoglycans. A total of 57 bovine lumbar motion segments were divided randomly into phosphate buffered saline and 0.33% genipin-soaked treatment groups. Water content changes were measured using a mass-loss technique in 3 intervertebral disc regions following successive stages of compressive loading and unloading (post-treatment, after 1 hour 750 N compression, and after a subsequent 24-hour period of nominal loading). Net flow of fluid into or out of a region was determined from the percentage change in mean water content from successive groups. Fluid flow to and from the nucleus doubled with genipin crosslinking. Relative to the buffer-only controls, overall net fluid flow increased 103% in the nucleus pulposus, 36% in the inner anulus, and was 31% less in the outer anulus of genipin treated discs. The effects of genipin crosslinking on matrix permeability and proteoglycan retention can alter hydration levels and fluid flow in the intervertebral disc. Resulting increases in fluid flow, including a doubling of flow to and from the nucleus, could lead to enhanced nutritional inflow and waste product outflow for the disc, and may have implications for emerging cell-based therapies.

Genipin-cross-linked kappa-carrageenan/carboxymethyl cellulose beads and effects on beta-carotene release

Beads of kappa-carrageenan/sodium carboxymethylcellulose were prepared based on different blend formulations using genipin, a natural and non-toxic cross-linking reagent. Different genipin concentrations (0.5, 1.0, 1.5 mM) were used to study the effects on swelling ratio of the beads in different pH values under simulated gastrointestinal tract condition (pH 1.2 and 7.4). Results have shown that the cross-linked beads possess lower swelling ability in all pH conditions and swelling ratio decreases with increasing genipin concentration (95.24% in pH 1.2; 100% in pH 7.4 at 0.5 mM genipin; 76.2% in pH 1.2; 85.71% in pH 7.4 at 1.5 mM genipin). It was also found the beads released beta-carotene slower and lesser after being cross-linked. Microstructure study shows that cross-linked beads exhibited smoother surface and more spherical shape compared to the native beads. This indicates that cross-linking of genipin has enhanced the beads network stability and their structure to be applied as suitable hydrogel.

Preparation of the new thermosensitive chitosan-based hydrogel containing SDF-1β, TGF-β1 and drug release behavior

Objective To investigate temperature sensitivity of the new thermosensitive chitosanbased hydrogel and drug release behavior. Methods 2% chitosan crosslinked with 0. 005% -0. 100% genipin within 10 min was added with 56% glycerphosphate to prepare the new thermosensitive chitosanbased hydrogel and investigate its temperature sensitivity. Results The optimal thermosensitive chitosanbased hydrogel could be prepared by using 2% chitosan 5 ml, 0. 03% genipin 0. 5 ml and 56% glycerphosphate 0. 55 ml. The hydrogel contained 400 μg/L stromal cell derived factor 1 β (SDF-1β) 0. 5 ml and 200 μg/L transforming growth factor-pi (TGF-pl) 0. 5 ml. The release curve was obtained to express its delivery property. Conclusion The fast gelable chitosan-based hydrogel can be obtained by genipin crosslinking. The hydrogel shows the thermosensitivity and sustained release profile of SDF-1 β and TGF-β1. Key words: Chitosan; Thermosensitive hydrogel; Genipin; Controlled release

Genipin‐cross‐linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications

In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.

Genipin-cross-linked silk fibroin microspheres prepared by the simple water-in-oil emulsion solvent diffusion method

This work describes for the first time the preparation of cross-linked and non-cross-linked silk fibroin (SF) microspheres using the simple water-in-oil emulsion solvent diffusion method. Aqueous SF solution and ethyl acetate were used as water and oil phases, respectively. Span80, oil-soluble emulsifier was found to induce the formation of SF microparticles that are completely spherical in shape and smooth in surface. SF microsphere sizes were found to depend upon various process parameters. The SF microsphere matrices showed predominantly random coil conformation. It was possible to fabricate genipin-cross-linked SF microspheres by cross-linking SF solution with genipin before microsphere formation. Both non-cross-linked and genipin-cross-linked SF microspheres contained porous structures. Percentage of dissolution in water decreased and density values of the SF microspheres increased when increasing the genipin ratio and cross-linking time. The genipin cross-linking induced SF conformational transition from random coil to β-sheet form but the size and shape of the SF microparticles did not change. It is suggested that these SF microspheres might be suitable microcarriers for hydrophilic drug delivery.

A New Green Thermoplastic Polymer with Improved Hydrophobic Character

AbstractSummary: A green thermoplastic polymer based on wheat flour was modified by the addition of a natural crosslinker genipin. Films of the polymer modified with different composition of genipin were prepared by extrusion. Free surface energy using contact angle method, moisture absorption test and hardness test were used to characterize the products. From moisture absorption and contact angle measurements, it was seen that for genipin amounts higher than 0.2% w/w, a hydrophobic character is achieved. Results obtained indicate enhancement in hydrophobic properties of the films.

The influence of exogenous cross-linking and compressive creep loading on intradiscal pressure

This study involves a biomechanical evaluation of a prospective injectable treatment for degenerative discs. The high osmolarity of the non-degenerated nucleus pulposus attracts water contributing to the hydrostatic behavior of the tissue. This intradiscal pressure is known to drop as fluid is exuded from the matrix due to compressive loading. The objective of this study was to compare the changes in intradiscal pressure in control and genipin cross-linked intervertebral discs. Thirty bovine lumbar motion segments were randomly divided into a phosphate-buffered saline control group and a 0.33% genipin group and soaked at room temperature for 2 days. A needle pressure sensor was held in the center of the disc while short-term and static creep compressive loads were applied. The control group demonstrated a 25% higher average intradiscal pressure compared to genipin-treated discs under 750 N compressive load (p=0.029). Depressurization during static compressive creep was 56% higher in the control than in the genipin group (p=0.014). These results suggest cross-linking induced changes in the poroelastic properties of the involved tissues affected the mechanics of compressive load support in the disc with lower levels of nucleus pressure, a corresponding decrease in the elastic expansion of the annulus, and an increased axial compressive loading of the inner and outer annulus tissues. It is possible that concurrent changes in hydraulic permeability and proteoglycan retention known to be associated with genipin cross-linking were also contributors to poroelastic changes. Reduction of peak pressures and moderation of pressure fluctuations could be beneficial relative to discogenic pain.

Preparation and characterization of genipin-cross-linked chitosan microparticles by water-in-oil emulsion solvent diffusion method

Chitosan (CS) microparticles with and without cross-linking were prepared by a water-in-oil emulsion solvent diffusion method without any surfactants. Aqueous CS solution and ethyl ace- tate were used as water and oil phases, respectively. Genipin was used as a cross-linker. Influ- ences of genipin ratios and cross-linking times on CS microparticle characteristics were investigated. Non-cross-linked and cross-linked CS microparticles were spherical in shape and rough in surface. Microparticle matrices showed porous structures. Surface roughness, mean par- ticle sizes and bulk density of CS microparticles increased and their dissolutions in acetic acid solution decreased when genipin ratio and cross- linking time increased.

Effect of adipic dihydrazide modification on the performance of collagen/hyaluronic acid scaffold

Collagen and hydrazide-functionalized hyaluronic acid derivatives were hybridized by gelating and genipin crosslinking to form composite hydrogel. The study contributed to the understanding of the effects of adipic dihydrazide modification on the physicochemical and biological properties of the collagen/hyaluronic acid scaffold. The investigation included morphology observation, mechanical measurement, swelling evaluation, and collagenase degradation. The results revealed that the stability of composites was increased through adipic dihydrazide modification and genipin crosslinking. The improved biocompatibility and retention of hyaluronic acid made the composite material more favorable to chondrocytes growing, suggesting the prepared scaffold might be high potential for chondrogenesis.

Regulating Fibrinolysis to Engineer Skeletal Muscle from the C2C12 Cell Line

Muscles engineered from transformed cells would be a powerful model for the study of muscle physiology by allowing long-term in vitro studies of muscle adaptation. However, previously described methods either take >5 weeks to produce a tissue or use collagen as a scaffold, which decreases the specific force of the muscle, making it hard to measure the function of the constructs. The aim of this study was to rapidly engineer muscle using the C2C12 cell line in fibrin, which has a stiffness similar to muscle tissue, allowing accurate functional testing. Both the protease inhibitor aprotinin and the natural cross-linker genipin increased the length of time that muscle could be cultured, with genipin increasing the time in culture to 10 weeks. The function of the tissues was significantly affected by the batch of serum (64-78%) or thrombin (41%), the differentiation medium (78%), and the seeding protocol (38%), but was unaffected by initial cell number. Strikingly, different C2C12 clones produced up to a 3.6-fold variation in force production. Under optimal conditions, the tissues form in 10.4+/-0.3 days and remain fully functional for 5 weeks over which time they continue to mature. The optimized model described here provides rapid, reliable, and functional tissues that will be useful in the study of skeletal muscle physiology.

Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films

Use of polyelectrolyte multi-layers as biomaterials for cell attachment has been limited due to their gel-like characteristics. Herein, we attempt to improve the cellular adhesion properties of multi-layer films, reduce their gel-like nature and rigidify them through chemical cross-linking with genipin; a natural and non-cytotoxic compound. Chitosan (CH), hyaluronan (HA) and alginate (Alg) were used to assemble [CH–HA]n CH and [CH–Alg]n CH films, and the effects of genipin cross-linking on the cell adhesion properties of these multi-layers were investigated. Atomic force microscopy (AFM) confirmed that cross-linking affected each of the films differently. Quartz crystal microbalance with dissipation (QCM-D) revealed that [CH–HA]10 CH films were very viscoelastic, with thicknesses in the range 350–450nm, while [CH–Alg]10 CH films only grew to thicknesses of ∼100nm. These differences were a result of the different growth regimes of these two polyelectrolyte systems. Cell adhesion studies using MC3T3 pre-osteoblasts and rat fibroblastic skin cells, carried out on both films demonstrated vast differences in cell adhesion. [CH–HA]n CH cross-linked films proved to be highly non-adhesive for pre-osteoblasts and fibroblastic skin cells. Conversely, cross-linking [CH–Alg]n CH films was shown to dramatically improve pre-osteoblast and rat fibroblastic skin cell adhesion, especially for high bi-layer numbers and using higher concentrations of cross-linker.

The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin–polysaccharide cardiac patches in vitro

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS–hyaluronic acid (SF/CS–HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR–FTIR spectra confirm the co-existence of CS or CS–HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS–HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded ( P < 0.001) that on culture wells after seven days of cultivation. Additionally, the relative growth rates of rMSCs on SF/CS and SF/CS–HA hybrid patches were significantly better ( P < 0.01) than that on SF patches that were also observed by using vimentin stain to the cells. For instance, the relative cell growth rates (%) in cell culture wells, SF, SF/CS and SF/CS–HA patches were 100%, 282.9 ± 6.5%, 337.0 ± 8.0% and 332.6 ± 6.6% ( n = 6, for all), respectively. For investigating the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real-time quantitative polymerase chain reaction (real-time PCR) analysis. The results of cardiomyogenic differentiation of induced rMSCs on SF/CS and SF/CS–HA hybrid patches significantly improved the expressions of cardiac genes of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P < 0.01 or better, n = 3) than those on SF patches and culture wells. Interestingly, the results of cardiac gene expressions of the cells on the SF/CS–HA hybrid patches were the most pronounced in promoting cardiomyogenic differentiations in this investigation. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS and SF/CS–HA hybrid patches were much pronounced compared with SF patches, indicating the improvements of cardiomyogenic differentiation on the hybrid patches. The results of this study demonstrate that the SF/CS and SF/CS–HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues.

Genipin Cross-Linked Fibrin Hydrogels for in vitro Human Articular Cartilage Tissue-Engineered Regeneration

Our objective was to examine the potential of a genipin cross-linked human fibrin hydrogel system as a scaffold for articular cartilage tissue engineering. Human articular chondrocytes were incorporated into modified human fibrin gels and evaluated for mechanical properties, cell viability, gene expression, extracellular matrix production and subcutaneous biodegradation. Genipin, a naturally occurring compound used in the treatment of inflammation, was used as a cross-linker. Genipin cross-linking did not significantly affect cell viability, but significantly increased the dynamic compression and shear moduli of the hydrogel. The ratio of the change in collagen II versus collagen I expression increased more than 8-fold over 5 weeks as detected with real-time RT-PCR. Accumulation of collagen II and aggrecan in hydrogel extracellular matrix was observed after 5 weeks in cell culture. Overall, our results indicate that genipin appeared to inhibit the inflammatory reaction observed 3 weeks after subcutaneous implantation of the fibrin into rats. Therefore, genipin cross-linked fibrin hydrogels can be used as cell-compatible tissue engineering scaffolds for articular cartilage regeneration, for utility in autologous treatments that eliminate the risk of tissue rejection and viral infection.

Genipin enhances the mechanical properties of tissue‐engineered cartilage and protects against inflammatory degradation when used as a medium supplement

Genipin is a naturally-derived biocompatible cross-linking agent commonly used to generate three dimensional tissue-engineered scaffolds or to fix biologically derived scaffolds prior to implantation. Here we propose a novel use for genipin as a long-term culture medium supplement to promote cross-linking of de novo cell products that are produced in engineered cartilage. We hypothesize that the application of genipin will stabilize the extracellular matrix components and increase the mechanical properties of developing engineered cartilage. Chondrocytes encapsulated in agarose hydrogel (a neutrally charged polysaccharide scaffold that is unaffected by genipin cross-linking) were cultured in a chemically-defined growth medium that was supplemented with varying concentrations of genipin (22 microM, 220 microM, 2200 microM) for various durations (continuous or intermittent). Tissues developed significantly higher mechanical properties (+28% dynamic modulus and +20% Young's modulus) by day 42 with genipin treatment compared to untreated controls. These increases were not immediate, but presented over culture time after genipin treatment. The genipin treated groups were also more resistant to cytokine-induced degradation with interleukin-1alpha; maintaining an E(Y) (+218%), G* (+390%) and glycosaminoglycan (GAG) content (+477%) over genipin-untreated constructs subjected to interleukin. We hypothesize two mechanisms through which the physical enhancement of tissue properties may be fostered: (1) by cross-link mediated reorganization and enhanced retention of cell-elaborated extracellular matrix components, and (2) through reduction of the loss of extracellular matrix components by increasing their resilience to catabolic degradation. These studies demonstrate a potential use of genipin as a medium supplement to develop enhanced engineered cartilage.

Development of a stable hydrogel network based on agar–kappa-carrageenan blend cross-linked with genipin

Blend of food hydrocolloids, viz. agar and kappa-carrageeenan (agar/kC), was treated with the natural cross-linker genipin in aqueous medium to impart functional stability. The genipin-fixed blend exhibited remarkable stability in the wide range of pH 1–12, as well as in Ringer's solution. Ratios of the hydrocolloids and genipin in the blend as well as in the cross-linked product were optimized (agar/kC 25:75 and agar/kC/genipin 25:75:0.8, w/w) on the basis of swelling properties. Compatibilities of unmodified blend (agar/kC) and the cross-linked blend product in aqueous solution were studied by measuring apparent and intrinsic viscosities and UV absorptions. Properties of agar/kC blend and the genipin cross-linked blend in solid state were evaluated by bulk and true density, pore volume, porosity, thermogravimetric analysis (TGA), swelling ability, X-ray diffraction, degradation rate in vitro, optical microscopy, scanning electron microscopy (SEM) and rheological measurements. The cross-linked blend exhibited higher viscosity, thermal stability and swelling ability as well as low weight loss ratio compared to those of the un-modified blend. The presence of genipin in the cross-linked polymer network was confirmed by MS/MS analysis. This genipin cross-linked blend presents an immense potential for food applications and in other pH-specific applications as well.

Characterization of Genipin Crosslinked Hydrogels Composed of Chitosan and Partially Hydrolyzed Poly(vinyl alcohol)

AbstractChitosan and 80 % hydrolyzed poly(vinyl alcohol) (PVA) were used to prepare semi-interpenetrating networks of varying ratios of the constituents. The hydrogels were crosslinked using genipin, a naturally occurring nontoxic crosslinking agent. The swelling behavior of these hydrogels was studied in deionized water at 25, 35 and 45 °C and in media of different pH at 25 °C. The swelling behavior of the gels was found to be dependent on temperature, pH of the medium and the amount of PVA present in the gel. States of water in the hydrogels swollen at 25 °C and pH 7 were determined using Differential Scanning Calorimetry (DSC). The amount of freezing water in the swollen hydrogels increased whereas the amount of nonfreezing bound water decreased with the increase in PVA concentration in the gels. The gelation behaviour of the pregel solutions were also studied.

Preparation and evaluation of ketorolac tromethamine gel containing genipin for periodontal diseases

Ketorolac tromethamine gel (KT gel) and ketorolac tromethamine gel containing genipin (KTG gel) were prepared and their therapeutic effects on periodontitis were evaluated. The skin permeation rate of ketorolac from the KT gel and KTG gel was 5.75+/-0.53 and 5.82 +/- 0.74 microg/cm2/ h, respectively. The skin permeation rate of genipin from the KTG gel was 10.13 +/- 1.47 microg/ cm2/h. The tensile strength of the KTG gel was larger than the KT gel. After 4 weeks, the periodontal pocket depth of the KTG gel group (3.22 +/- 0.20 mm) significantly decreased compared with the non-treated group (4.50 +/- 0.25 mm) and the KT group (3.84 +/- 00.26 mm). The KTG gel did not induce separation of the stratum corneum and subcutaneous tissue, and the collagen layers of the corium were closer, more fibrous, and showed longer connections than in the other groups. The KTG gel appears to be effective against gingivitis in the periodontal pocket through its increased anti-inflammatory activity and the crosslinking of genipin with the biological tissue.