Objective: To explore the effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice. Methods: The experimental research method was adopted. The control hydrogel composed of polyvinyl alcohol and gelatin, and the water-soluble chitosan hydrogel composed of the aforementioned two materials and water-soluble chitosan were prepared by the cyclic freeze-thaw method. The fluidity of the two dressings in test tube before and after the first freeze-thawing was generally observed, and the difference in appearance of the final state of two dressings in 12-well plates were compared. According to random number table (the same grouping method below), the cell strains of L929 and HaCaT were both divided into water-soluble chitosan hydrogel group and control hydrogel group, respectively. After adding corresponding dressings and culturing for 24 h, the cell proliferation activity was measured using cell counting kit 8. Rabbit blood erythrocyte suspensions were divided into normal saline group, polyethylene glycol octyl phenyl ether (Triton X-100) group, water-soluble chitosan hydrogel group, and control hydrogel group, which were treated accordingly and incubated for 1 hour, and then the hemolysis degree of erythrocyte was detected by a microplate reader. Twenty-four female db/db mice aged 11-14 weeks were selected, and full-thickness skin defect wounds on their backs were inflicted and inoculated with the methicillin-resistant Staphylococcus aureus (MRSA), 72 h later, the mice were divided into blank control group, sulfadiazine silver hydrogel group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. On post injury day (PID) 0 (immediately), 7, 14, and 21, the healing of the wound was observed. On PID 14 and 21, the wound healing rate was calculated. On PID 14, MRSA concentration in wounds was determined. On PID 21, the wounds were histologically analyzed by hematoxylin and eosin staining; the expression of CD31 in the wounds was detected by immunofluorescence method, and its positive percentage was calculated. Raw264.7 cells were taken and divided into interleukin-4 (IL-4) group, blank control group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. At 48 h of culture, the percentages of CD206 positive cells were detected by flow cytometry. The number of samples was all 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, least significant difference test, and Dunnett T3 test. Results: Two dressings in test tube had certain fluidity before freeze-thawing and formed semi-solid gels after freeze-thawing for once. The final forms of two dressings in 12-well plates were basically stable and translucent sheets, with little difference in transparency. At 24 h of culture, the cell proliferation activities of L929 and HaCaT in water-soluble chitosan hydrogel group were significantly higher than those in control hydrogel group (with t values of 6.37 and 7.50, respectively, P<0.01). At 1 h of incubation, the hemolysis degree of erythrocyte in water-soluble chitosan hydrogel group was significantly lower than that in Triton X-100 group (P<0.01), but similar to that in normal saline group and control hydrogel group (P>0.05). On PID 0, the traumatic conditions of mice in the 4 groups were similar. On PID 7, more yellowish exudates were observed inside the wound in blank control group and control hydrogel group, while a small amount of exudates were observed in the wound in sulfadiazine silver hydrogel group and water-soluble chitosan hydrogel group. On PID 14, the wounds in blank control group and control hydrogel group were dry and crusted without obvious epithelial coverage; in sulfadiazine silver hydrogel group, the scabs fell off and purulent exudate was visible on the wound; in water-soluble chitosan hydrogel group, the base of wound was light red and obvious epithelial coverage could be observed on the wound. On PID 14, the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 21, the wound in water-soluble chitosan hydrogel group was completely closed, while the wounds in the other 3 groups were not completely healed; the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 14, the concentration of MRSA in the wound in water-soluble chitosan hydrogel group was significantly lower than that in blank control group (P<0.01), but similar to that in control hydrogel group and sulfadiazine silver hydrogel group (P>0.05). On PID 21, the new epidermis was severely damaged in blank control group; the epidermis on the wound in control hydrogel group also had a large area of defect; complete new epidermis had not yet being formed on the wound in sulfadiazine silver hydrogel group; the wound in water-soluble chitosan hydrogel group was not only completely covered by the new epidermis, the basal cells of the new epidermis were also regularly aligned. On PID 21, the percentage of CD31 positivity in the wound in water-soluble chitosan hydrogel group was (2.19±0.35)%, which was significantly higher than (0.18±0.05)% in blank control group, (0.23±0.06)% in control hydrogel group, and (0.62±0.25)% in sulfadiazine silver hydrogel group, all P<0.01. At 48 h of culture, the percentage of CD206 positive Raw264.7 cells in water-soluble chitosan hydrogel group was lower than that in IL-4 group (P>0.01) but significantly higher than that in blank control group and control hydrogel group (P<0.05 or P<0.01). Conclusions: The water-soluble chitosan hydrogel has good biosafety and can induce higher level of macrophage M2 polarization than control hydrogel without water-soluble chitosan, so it can enhance the repair effect of MRSA-infected full-thickness skin defect wounds in diabetic mice and promote rapid wound healing.
Mice ; Female ; Animals ; Rabbits ; Interleukin-4 ; Hydrogels/pharmacology* ; Wound Healing ; Chitosan/pharmacology* ; Diabetes Mellitus, Experimental ; Water ; Methicillin-Resistant Staphylococcus aureus ; Gelatin ; Polyvinyl Alcohol ; Hemolysis ; Saline Solution ; Eosine Yellowish-(YS) ; Hematoxylin ; Octoxynol ; Silver ; Phenyl Ethers ; Sulfadiazine

Pulp loss is accompanied by the functional impairment of defense, sensory, and nutrition supply. The approach based on endogenous stem cells is a potential strategy for pulp regeneration. However, endogenous stem cell sources, exogenous regenerative signals, and neovascularization are major difficulties for pulp regeneration based on endogenous stem cells. Therefore, the purpose of our research is to seek an effective cytokines delivery strategy and bioactive materials to reestablish an ideal regenerative microenvironment for pulp regeneration. In in vitro study, we investigated the effects of Wnt3a, transforming growth factor-beta 1, and bone morphogenetic protein 7 (BMP7) on human dental pulp stem cells (h-DPSCs) and human umbilical vein endothelial cells. 2D and 3D culture systems based on collagen gel, matrigel, and gelatin methacryloyl were fabricated to evaluate the morphology and viability of h-DPSCs. In in vivo study, an ectopic nude mouse model and an in situ beagle dog model were established to investigate the possibility of pulp regeneration by implanting collagen gel loading BMP7. We concluded that BMP7 promoted the migration and odontogenic differentiation of h-DPSCs and vessel formation. Collagen gel maintained the cell adhesion, cell spreading, and cell viability of h-DPSCs in 2D or 3D culture. The transplantation of collagen gel loading BMP7 induced vascularized pulp-like tissue regeneration in vivo. The injectable approach based on collagen gel loading BMP7 might exert promising therapeutic application in endogenous pulp regeneration.
Animals ; Bone Morphogenetic Protein 7/pharmacology* ; Cell Differentiation ; Cells, Cultured ; Collagen/pharmacology* ; Dental Pulp ; Dogs ; Endothelial Cells ; Gelatin ; Humans ; Methacrylates ; Mice ; Regeneration ; Stem Cells

Objective: To investigate the effects of methacrylic anhydride gelatin (GelMA) hydrogel loaded with silver and recombinant human basic fibroblast growth factor (rh-bFGF) on deep partial-thickness burn wounds in rabbits. Methods: The experimental research method was adopted. Low-concentration GelMA materials, medium-concentration GelMA materials and high-concentration GelMA materials containing different concentrations of methacrylic anhydride (MA) were prepared, after adding photoinitiator, low-concentration GelMA hydrogels, medium-concentration GelMA hydrogels, and high-concentration GelMA hydrogels were obtained, respectively. The nuclear magnetic resonance spectroscopy was performed to detect the hydrogen nuclear magnetic resonance spectra of the above-mentioned three concentrations of GelMA materials, and to calculate the degree of substitution according to the spectrum diagram. The three-dimensional microstructure and pore size of 3 types of above-mentioned GelMA hydrogels were detected by field emission scanning electron microscopy (FESEM), with 9 samples measured. According to the selected concentration of MA, ten kinds of solutions of GelMA with different concentration of silver (silver-containing GelMA) were synthesized, and the silver-containing GelMA solution of each concentration was divided into three parts, and then exposed to ultraviolet light lasting for 20, 25, and 35 s, respectively. After adding photoinitiator,the corresponding silver-containing GelMA hydrogels were obtained. The residual degradation rate of silver-containing GelMA hydrogel with different photocrosslinking times was detected by collagenase degradation method at degradation of 12, 24, 36, and 48 h; and the time required for complete degradation was detected, and the sample number was 5. The inhibition zone diameter of GelMA hydrogel under above screened photocrosslinking times containing 10 concentrations of silver against Staphylococcus aureus was measured to reflect its antibacterial ability, and the sample numbers were all 5. The silver-containing GelMA hydrogel with statistical significance compared with the antibacterial circle diameter of the silver-containing GelMA hydrogel containing the lowest concentration (no silver) was considered as having antibacterial activity. The three-dimensional microstructure and pore size of the silver-containing GelMA hydrogels with antibacterial activity and the lowest drug concentration selected were detected by FESEM, and the sample numbers were all 9. The freeze-dried alone GelMA hydrogel and the freeze-dried silver-containing GelMA hydrogel were soaked in phosphate buffer solution for 24 h, respectively, then the swelling rate of the two GelMA hydrogel were calculated and compared by weighing method, and the sample number was 5. GelMA hydrogel containing silver and rh-bFGF, namely compound hydrogel for short, was prepared according to the preliminary experiment and the above experimental results. The appearance of the composite hydrogel was observed in general, and its three-dimensional microstructure and pore size were detected by FESEM. The deep partial-thickness burn wound was made on the back of 30 rabbits (aged 4-6 months, female half and half). Meanwhile, with the rabbit head as the benchmark, the wounds on the left side of the spine were treated as composite hydrogel treatment group, and the wounds on the right side were treated as gauze control group, and which were treated accordingly. On post injury day (PID) 3, 7, 14, 21, and 28, the healing of wounds in the two groups was observed. On PID 7, 14, 21, and 28, the wound healing area was recorded and the healing rate was calculated, with a sample number of 30. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: The substitution degree among low-concentration GelMA materials, medium-concentration GelMA materials, and high-concentration GelMA materials was significantly different (F=1 628.00, P<0.01). The low-concentration GelMA hydrogel had a loose and irregular three-dimensional spatial network structure with a pore size of (60±17) μm; the medium-concentration GelMA hydrogel had a relatively uniform three-dimensional spatial network and pore size with a pore size of (45±13) μm; the high-concentration GelMA hydrogel had the dense and disordered three-dimensional spatial network with a pore size of (25±15) μm, the pore sizes of 3 types of GelMA hydrogels were significantly differences (F=12.20, P<0.01), and medium concentration of MA was selected for the concentration of subsequent materials. The degradability of silver-containing GelMA hydrogels with different concentrations of the same photocrosslinking time was basically same. The degradation residual rates of silver-containing GelMA hydrogels with 20, 25, and 35 s crosslinking time at 12 h were (74.2±1.7)%, (85.3±0.9)%, and (93.2±1.2)%, respectively; the residual rates of degradation at 24 h were (58.3±2.1)%, (65.2±1.8)%, and (81.4±2.6)%, respectively; the residual rates of degradation at 36 h were (22.4±1.9)%, (45.2±1.7)%, and (68.1±1.4)%, respectively; the residual rates of degradation at 48 h were (8.2±1.7)%, (32.4±1.3)%, and (54.3±2.2)%, respectively, and 20, 25, and 30 s photocrosslinking time required for complete degradation of silver-containing GelMA hydrogels were (50.2±2.4), (62.4±1.4), and (72.2±3.2) h, and the difference was statistically significant (F=182.40, P<0.01), 25 s were selected as the subsequent photocrosslinking time. The antibacterial diameters of 10 types of silver-containing GelMA hydrogels against Staphylococcus aureus from low to high concentrations were (2.6±0.4), (2.5±0.4), (3.2±0.4), (12.1±0.7), (14.8±0.7), (15.1±0.5), (16.2±0.6), (16.7±0.5), (16.7±0.4), and (16.7±0.6) mm, respectively, and which basically showed a concentration-dependent increasing trend, and the overall difference was statistically significant (F=428.70, P<0.01). Compared with the silver-containing GelMA hydrogel with the lowest concentration, the antibacterial circle diameters of other silver-containing GelMA hydrogels with antibacterial ability from low to high concentration were significantly increased (with t values of 26.35, 33.84, 43.65, 42.17, 49.24, 55.74, and 43.72, respectively, P<0.01). The silver-containing GelMA hydrogel with the antibacterial diameter of (12.1±0.7) mm had the lowest antibacterial activity against Staphylococcus aureus and the lowest drug loading concentration, and the concentration of silver was selected for the concentration of subsequent materials. The microscopic morphology of the silver-containing GelMA hydrogel containing silver element with a pore size of (45±13) μm had a regular and linear strip-like structure. After soaking for 24 h, the swelling ratio of silver-containing GelMA hydrogel was similar to that of alone GelMA hydrogel. The composite hydrogel was colorless, clear and transparent, and its three-dimensional microstructure was a regular and uniform grid, with a filament network structure inside, and the pore size of (40±21) μm. On PID 3, a large amount of necrotic tissue and exudate of rabbit wound in composite hydrogel group were observed, and scattered scabs, a small amount of necrotic tissue and exudate of rabbit wound in gauze control group were observed. On PID 7, the area of rabbit wound in composite hydrogel group was significantly reduced, and adhesion of rabbit wound and gauze in gauze control group was observed. On PID 14, In composite hydrogel group, the rabbit wound surface was ruddy, and the growth of granulation tissue was observed, and in gauze control group, the rabbit wound base was pale, and the blood supply was poor. On PID 21, the rabbit wounds in composite hydrogel group healed completely, and rabbit wound in gauze control group had healing trend. On PID 28, new hair could be seen on rabbit wound surface in composite hydrogel group; oval wound of rabbit in gauze control group still remained. On PID 7, 14, 21, and 28, the wound healing areas of rabbit in composite hydrogel group were significantly larger than those in gauze control group (with t values of 2.24, 4.43, 7.67, and 7.69, respectively, P<0.05 or P<0.01). Conclusions: The medium-concentration GelMA hydrogel has good physical and chemical properties in terms of swelling and degradability. The screened silver-containing GelMA hydrogels had the lowest antibacterial activity and the lowest drug loading concentration. Composite hydrogel can significantly shorten the healing time of deep partial-thickness burn wounds in rabbits.
Anhydrides ; Animals ; Anti-Bacterial Agents ; Burns/drug therapy* ; Female ; Fibroblast Growth Factor 2 ; Gelatin/pharmacology* ; Humans ; Hydrogels/pharmacology* ; Rabbits ; Recombinant Proteins ; Staphylococcal Infections ; Staphylococcus aureus

Objective: To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods: The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results: GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions: GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.
Anhydrides ; Animals ; Endothelial Cells ; Eosine Yellowish-(YS) ; Female ; Gelatin/pharmacology* ; Graphite ; Hematoxylin ; Humans ; Hydrogels/pharmacology* ; Methacrylates ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; Skin Abnormalities ; Vascular Endothelial Growth Factor A

BACKGROUNDThree-dimensional (3D) printing technology holds great promise for treating diseases or injuries that affect human bones with enhanced performance over traditional techniques. Different patterns of design can lead to various mechanical properties and biocompatibility to various degrees. However, there is still a long way to go before we can fully take advantage of 3D printing technologies.

METHODSThis study tailored 3D printed scaffolds with gelatin and platelets to maximize bone regeneration. The scaffolds were designed with special internal porous structures that can allow bone tissue and large molecules to infiltrate better into the scaffolds. They were then treated with gelatin and platelets via thermo-crosslinking and freeze-drying, respectively. Vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1 were measured at different time points after the scaffolds had been made. Cell proliferation and cytotoxicity were determined via cell counting kit-8 (CCK-8) assay.

RESULTSThere was a massive boost in the level of VEGF and TGF-β1 released by the scaffolds with gelatin and platelets compared to that of scaffolds with only gelatin. After 21 days of culture, the CCK-8 cell counts of the control group and treated group were significantly higher than that of the blank group (P < 0.05). The cytotoxicity test also indicated the safety of the scaffolds.

CONCLUSIONSOur experiments confirmed that the 3D printed scaffolds we had designed could provide a sustained-release effect for growth factors and improve the proliferation of preosteoblasts with little cytotoxicity in vitro. They may hold promise as bone graft substitute materials in the future.

3T3 Cells ; Animals ; Biocompatible Materials ; chemistry ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Gelatin ; chemistry ; Mice ; Printing, Three-Dimensional ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry ; Transforming Growth Factor beta1 ; chemistry ; pharmacology ; Vascular Endothelial Growth Factor A ; chemistry ; pharmacology

The purpose of this study is to investigate the effect of superparamagnetic chitosan FGF-2 gelatin microspheres (SPCFGM) on the proliferation and differentiation of mouse mesenchymal stem cells. The superparamagnetic iron oxide chitosan nanoparticles (SPIOCNs) were synthesized by means of chemical co-precipitation, combined with FGF-2. Then The SPCFGM and superparamagnetic chitosan gelatin microspheres (SPCGM) were prepared by means of crosslinking-emulsion. The properties of SPCFGM and SPIONs were measured by laser diffraction particle size analyser and transmisson electron microscopy. The SPCFGM were measured for drug loading capacity, encapsulation efficiency and release pharmaceutical properties in vitro. The C3H10 cells were grouped according to the different ingredients being added to the culture medium: SPCFGM group, SPCGM group and DMEM as control group. Cell apoptosis was analyzed by DAPI staining. The protein expression level of FGF-2 was determined by Western blot. The proliferation activity and cell cycle phase of C3H10 were examined by CCK8 and flow cytometry. The results demonstrated that both of the SPIOCNs and SPCFGM were exhibited structure of spherical crystallization with a diameter of (25 ± 9) nm and (140 ± 12) μm, respectively. There were no apoptosis cells in the three group cells. Both the protein expression level of FGF-2 and cell proliferation activity increased significantly in the SPCFGM group cells (P < 0.05). The SPCFGM is successfully constructed and it can controlled-release FGF-2, remained the biological activity of FGF-2, which can promote proliferation activity of C3H10 cells, and are non-toxic to the cell.
Animals ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Chitosan ; Fibroblast Growth Factor 2 ; pharmacology ; Gelatin ; Magnetite Nanoparticles ; Mesenchymal Stromal Cells ; drug effects ; Mice ; Microspheres ; Plasmids

In order to investigate the effects of HA whisker and carboxymethyl chitosan-gelatin(CMC-Gel) on the mechanical properties of porous calcium phosphate cement, a series of alpha-tricalcium phosphate (alpha-TCP), HA whisker and L-sodium glutamate porogen with different mass fractions were mixed, and setting liquid was added to them to prepare alpha-TCP/HA whisker composite porous bone cement. Then, the cement was immersed in a series of CMC-Gel solutions which had different weight ratios of CMC to Gel to prepare alpha-TCP/HA whisker/CMC-Gel composite porous bone cement. The compressive strengths and microstructure of cement were characterized by mechanical testing machine and SEM. The results showed that when the mass fraction of HA whisker is 4%, the compressive strength of alpha-TCP/HA whisker composite porous bone cement reaches 2.57MPa, which is 1.81 times that of alpha-TCP bone cement. When the weight ratio of CMC to Gel is 50:50, the compressive strength of alpha-TCP/HA whisker/CMC-Gel composite porous bone cement is 3. 34MPa, which is 2.35 times that of alpha-TCP bone cement, and the toughness of the composite cement is greatly improved as well.
Biocompatible Materials ; chemistry ; pharmacology ; Bone Cements ; chemical synthesis ; Calcium Phosphates ; chemistry ; Chitosan ; analogs & derivatives ; chemical synthesis ; chemistry ; Compressive Strength ; Gelatin ; chemistry ; Hydroxyapatites ; chemical synthesis ; chemistry ; Porosity

OBJECTIVEBy bioinformatics method, the effect in hematopoietic system of bioactive peptide HP-6, which was obtained from donkey serum albumin and is one of the major protein components from donkey-hide gelatin, was investigated.

METHODHuman bone marrow nucleated cells (hBMNCs) and murine bone marrow stromal cells (mBMSCs) were separated and cultured with different concentration of peptide HP-6 (0.000 15, 0.001 5, 0.015, 0.15, 1.5 micromol x L(-1)). The effect on promoting proliferation of cells related to hematopoiesis in bone morrow was detected and the ultrastructure of cells after treated by HP-6 was observed through transmission electron microscope. Hemorrhage anemia mouse model and anemia mouse model induced by cyclophosphamide were established, and randomly divided into peptide HP-6 groups which were administered respectively with different doses (1, 0.1, 0.01 mg x kg(-1)) by gavage, and control group which was administered with PBS by gavage. Peripheral blood components of all mice and bone morrow cells (BMC) number of mice induced by cyclophosphamide were evaluated.

RESULTPeptide HP-6 could concentration-related promote the proliferation of hBMNCs and mBMSCs, hBMNCs got the highest reproduction rate of 152.11% and mBMSCs also got 63.52% with the concentration of 0.15 micromol x L(-1), then the reproduction rate decreased while the concentration kept increasing. The transmission electron microscope showed that ultrastructure of cells was normal after treated by HP-6.1 mg x kg(-1) peptide HP-6 significantly increased peripheral platelet and protected mouse morrow injured by cyclophoshamide. 0.1 mg x kg(-1) peptide HP-6 significantly increased peripheral platelet with relative growth rate of 77.65%, increased peripheral white blood cells count and peripheral red blood cells count, also could protect mouse peripheral blood after treated by chemotherapeutics.

CONCLUSIONPeptide HP-6 could promote the proliferation of cells related to hematopoietic system, enhance mouse hemopoiesis function and the resistance to chemotherapeutic injury.

Anemia ; drug therapy ; Animals ; Cells, Cultured ; Dose-Response Relationship, Drug ; Equidae ; blood ; Female ; Gelatin ; pharmacology ; Hematopoiesis ; drug effects ; Hematopoietic System ; drug effects ; Humans ; Male ; Mice ; Peptides ; pharmacology ; Serum Albumin ; pharmacology

OBJECTIVETo observe the biochemical characters and antibiotic susceptibility of isolated Staphylococcus aureus (S. auerus) strains against some conventional and traditional antibiotics.

METHODSThirty post operative pathogenic isolated S. aureus strains were used in this study. Bacterial culture was done in Mueller-Hinton broth at 37 °C. Characters of these strains were determined by traditional biochemical tests such as hydrolysis test of gelatin, urea, galactose, starch and protein, and fermentation of lactose and sucrose. Antibiotic susceptibility were carried out by minimum inhibitory concentration test, minium bactericidal concentration test, disc agar diffusion test and brain heart infusion oxacillin screening agar.

RESULTSFrom this study, it was observed that 100% S. aureus isolates showed positive results in gelatin, urea and galactose hydrolysis test, 50% isolates were positive in starch hydrolysis test, 35% in protein hydrolysis test, 100% isolates in lactose fermenting test, but no isolate was positive in sucrose fermenting test. Antibiotic susceptibility testing suggested that 20% of isolates were resistant to kanamycin and 46.67% were resistant to oxacillin.

CONCLUSIONSThese findings show that all these isolates have gelatin, urea, galactose hydrolysis and lactose fermenting activity. 20% of these isolates were resistant to kanamycin and 46.67% were resistant to oxacillin.

Anti-Bacterial Agents ; pharmacology ; Disk Diffusion Antimicrobial Tests ; Galactose ; metabolism ; Gelatin ; metabolism ; Hydrolysis ; Microbial Sensitivity Tests ; Staphylococcus aureus ; drug effects ; isolation & purification ; metabolism ; Starch ; metabolism ; Urea ; metabolism

OBJECTIVETo prepare macroporous bone substitute composed of calcium phosphate cements and rhBMP-2 loaded gelatin microspheres, and to investigate ectopic osteoinduction of the composite.

METHODSAfter being prepared by improved emulsified cold-condensation method and crosslinked by 5% genipin solution,gelatin microspheres (GMs) were observed by scanning electron microscope (SEM) and loaded with rhBMP-2 by adsorption. Macroporous bone substitute was developed by mixing calcium phosphate cement (CPC) with 2.5% GMs, being as the experimental group,and CPC with rhBMP-2 was the control group. After the both composites had been soaked in the sodium chloride for 1 week or 3 weeks, compressive strength of the composites were tested, and the cross-sections were observed by SEM. Concentrations of rhBMP-2 in the solutions at different time by ELISA method and the cumulative drug release amount was calculated. The composites had been implanted in the muscle bags of the mouses for 3 weeks. Then the tissues around the materials were collected, stained by hematoxylin and eosin, and Ca and ALP in the tissues were also measured.

RESULTSGelatin microspheres were spherical with diameters of (62 +/- 18) microm. Macropores appeared in the experimental materials 1 week and 3 weeks after being soaked,and total porosity, macroporosity, cumulative release amount of rhBMP-2 in the experimental group were higher than that in the control. But compressive strength of the experimental group was lower than that of the control group 3 weeks after being soaked. Results of HE stain showed chondral formation in both groups, but there were more chondral tissues in the experiment group, and so were the concentrations of Ca and ALP.

CONCLUSIONMacroporous calcium phosphate cement can be prepared by using rhBMP-2 loaded gelatin microspheres, and it is an excellent bone substitute due to it's proterty of promoting rhBMP release and powerful ectopic osteoinduction.

Animals ; Bone Cements ; chemistry ; Bone Morphogenetic Protein 2 ; chemistry ; Bone Substitutes ; chemistry ; pharmacology ; Calcium Phosphates ; chemistry ; Gelatin ; chemistry ; Humans ; Male ; Mice ; Microspheres ; Osteogenesis ; drug effects ; Porosity ; Recombinant Proteins ; chemistry