Evaluation of a novel collagen-gelatin scaffold for achieving the sustained release of basic fibroblast growth factor in a diabetic mouse model

Efficacy of the dual controlled release of HGF and bFGF impregnated with a collagen/gelatin scaffold

We have developed collagen/gelatin sponges (CGS) with a gelatin concentration of 10 wt% to sustain the release of basic fibroblast growth factor (bFGF). The objective of this study is to elucidate the efficacy of CGS impregnated with different concentrations of bFGF, using mouse skin defects. CGSs impregnated with normal saline solution (NSS) or bFGF solution (1, 7, 14, or 50 μg/cm) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, and total area of newly formed capillaries in CGS were evaluated. The group of CGS with 7-μg/cm bFGF was significantly superior to the NSS group in all evaluated items. CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation 2 or 3 times earlier than existing artificial dermis. The combination of CGS and bFGF could solve the problem of the existing artificial dermis and be very promising for the treatment of skin defects.

The objective of this study was to compare the effectiveness of the collagen-gelatin sponge (CGS) with that of the collagen sponge (CS) in dermis-like tissue regeneration. CGS, which achieves the sustained release of basic fibroblast growth factor (bFGF), is a promising material in wound healing. In the present study, we evaluated and compared CGSs and conventional CSs. We prepared 8 mm full-thickness skin defects on the backs of rats. Either CGSs or CSs were impregnated with normal saline solution (NSS) or 7 μg/cm2 of bFGF solution and implanted into the defects. At 1 and 2 weeks after implantation, tissue specimens were obtained from the rats of each group (n = 3, total n = 24). The wound area, neoepithelial length, dermis-like tissue area, and the number and area of capillaries were evaluated at 1 and 2 weeks after implantation. There were no significant differences in the CGS without bFGF and CS groups. Significant improvements were observed in the neoepithelial length, the dermis-like tissue area, and the number of newly formed capillaries in the group of rats that received CGSs impregnated with bFGF. The effects on epithelialization, granulation, and vascularization of wound healing demonstrated that, as a scaffold, CGSs are equal or superior to conventional CSs.

Skin MG was prepared from the skin of C57BL/6JJcl mice using Rigeneracons and administered onto Pelnac Gplus and Integra sheets. The amount of MG suspension impregnated in Pelnac Gplus and Integra was evaluated. Pelnac Gplus and Integra sheets combined with MG were applied to murine defects, and wound area, neoepithelium length, granulation tissue formation, and newly formed capillaries were compared with the control groups on days 7 and 14. The weight percentage of the MG absorbed by Pelnac Gplus and Integra was 88.8% ± 3.5% and 28.2% ± 7.0%, respectively (P < 0.05). In the in vivo experiment, the area of newly formed granulation tissue and both the number and area of newly formed capillaries in the PelnacG + MG group were significantly larger than those in the control group at 14 days after implantation (P < 0.05). Skin MG was successfully impregnated into Pelnac Gplus by simple administration but not into Integra. Administration of skin MG into the Pelnac Gplus promoted granulation formation and angiogenesis. Pelnac Gplus was more suitable than Integra in the combination therapy.

Collagen-Gelatin Scaffold Impregnated with bFGF Accelerates Palatal Wound Healing of Palatal Mucosa in Dogs

We have developed a collagen/gelatin sponge (CGS) that can provide a sustained release of basic fibroblast growth factor (bFGF). In our previous study, it was shown that CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation two or three times earlier than an existing collagen sponge. In this study, adipogenesis was evaluated using CGSs disseminated with adipose tissue-derived stem cells (ASCs). Human ASCs were primarily isolated from human adipose tissue that was obtained during breast cancer surgery with informed consent at Kyoto University Hospital. ASCs were isolated from collagenase digests of adipose tissue. ASCs were labelled with PKH26. CGSs (8 mm diameter × 3 mm thickness) were impregnated with bFGF (0.1, 1, 7, 14 µg/cm(2) ) or normal saline solution. Then the labelled cells were disseminated (passage 3) on CGSs at a seeding density of 1 × 10(5) cells/cm(2) and implanted into the back subcutis of nude mice. Six weeks after implantation, adipogenesis at the administered site was evaluated. Immunohistological staining with von Willebrand factor (vWf) was performed to evaluate newly formed capillaries. Newly formed adipose tissue was observed macroscopically and histologically in all groups. The weight and area of regenerated adipose tissue were largest in the 1 µg/cm(2) bFGF group. Under a fluorescent microscope, newly formed adipose tissue in the bFGF-administered group was PKH-positive. These findings show that ASCs differentiated and formed adipose tissue. In this study, we showed that our CGSs impregnated with bFGF could be used as scaffolds with ASCs for adipogenesis.

Vocal fold scar remains a therapeutic challenge. Basic fibroblast growth factor (bFGF) was reported to have regenerative effects for vocal fold scar, although it has the disadvantage of rapid absorption in vivo. A collagen-gelatin sponge (CGS) can compensate for the disadvantage by providing a sustained release system. The current study evaluated the efficacy of CGS combined with bFGF on vocal fold scar, using rat fibroblasts for an in vitro model and a canine in vivo model. We prepared fibroblasts from scarred vocal folds (sVFs) in rats and showed that bFGF accelerated cell proliferation and suppressed expression levels of cleaved caspase 3 and α-smooth muscle actin. Has 1, Has 3, Fgf2, Hgf and Vegfa mRNA levels were significantly upregulated, while Col1a1 and Col3a1 were dose-dependently downregulated, with a maximum effect at 100 ng/ml bFGF. In an in vivo assay, 6 weeks after lamina propria stripping, beagles were divided into three groups: CGS alone (CGS group); CGS with bFGF (7 µg/cm2 ; CGS + bFGF group); or a sham-treated group. Vibratory examination revealed that the glottal gap was significantly reduced in the bFGF group and the two implanted groups, whereas the CGS + bFGF group showed higher mucosal wave amplitude. Histological examination revealed significantly restored hyaluronic acid and elastin redistribution in the CGS + bFGF group and reductions in dense collagen deposition. These results provide evidence that CGS and bFGF combination therapy may have therapeutic potential and could be a promising tool for treating vocal fold scar. Copyright © 2015 John Wiley & Sons, Ltd.

Objective Reactive gliosis which can promote the recovery of retina function at early stage of trauma,and basic fibroblast growth factor（bFGF） can activate Muller cells to accelerate healing of blunt trauma. Present study was to investigate the effect bFGF on Vimentin expression in Mtiller cells in experimental rabbit retinal blunt trauma. Methods An animal model of retinal blunt trauma was established in 24 rabbits by contusion of 3 J with a free falling iron bar. The model rabbits were divided into bFGF group,normal saline control group,trauma group and 2 normal rabbits were as control group. 10 μL of bFGF （2 μg） was intravitreonsly injected in bFGF group and the equal volume of normal saline was used in the normal saline control group after 3 days of retinal blunt trauma at a 2-day interval. The experimental rabbits were sacrificed and eyeballs were enucleated in 3 hours,1 day,3 days,7 days and 14 days to evaluate the Vimcntin expression in Muller cells by immunochemistry. Results The expression of Vimentin in MUller cells was increased markedly whatever in the intensity or quantity and showed a time- depended manner in both bFGF group and normal saline group. The statistically significance differences were seen in Vimentin expression between bFGF group and normal saline group in 1 day,3 days,7 days and 14 days after retinal blunt trauma （P 〈 0.05,P 〈 0.01 ）. Conclusion bFGF can enhance the expression of Vimentin in Muller ceils and activate MUller cells to accelerate repairing of retinal blunt trauma. Key words: bFGF; Muller cells; Vimentin

Fibroblast Growth Factor-Impregnated Collagen-Gelatin Sponge Improves Keratinocyte Sheet Survival.

<b>Objective:</b> To explore the effects of lappaconitine (LA) on pain and inflammatory response of severely burned rats and the mechanism. <b>Methods:</b> Forty SD rats were divided into healthy+ normal saline group, sham injury+ normal saline group, pure burn group, burn+ LA group, and healthy+ LA group according to the random number table (the same dividing method below), with 8 rats in each group. Rats in pure burn and burn+ LA groups were inflicted with about 32% total body surface area deep partial-thickness scald (hereinafter referred to as burn) on the back and right hind. Rats in sham injury+ normal saline group were sham injured. Rats in burn+ LA group were intraperitoneally injected with 1 g/L LA solution in the dosage of 4 mL/kg at 2.0 h before injury and post injury hour (PIH) 0 (immediately), 24.0, 48.0, and 72.0. Rats in healthy+ LA group were intraperitoneally injected with LA solution in the same dose at the same time points as above, and rats in healthy+ normal saline and sham injury+ normal saline groups were intraperitoneally injected with normal saline in the dose of 4 mL/kg at the same time points as above. At 1.5 h before injury and PIH 12.5, 24.5, 36.5, 48.5, and 72.5, the paw withdrawal mechanical threshold (PWMT) of injured rats was detected, and their pain behaviors were observed. The same observation and detection were conducted in rats without injury in the two groups at the same time points as above. Another 32 SD rats were divided into normal saline group, trinitrophenyl (TNP)-ATP group, minocyline group, pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) group, with 8 rats in each group, and all the rats were inflicted with the same burn injury as above. At PIH 48.0, rats in normal saline group were intrathecally injected with 10 μL normal saline; rats in TNP-ATP group were intrathecally injected with 10 μL TNP-ATP in the concentration of 30 nmol/μL; rats in minocyline group were intrathecally injected with 10 μL minocyline in the concentration of 5 g/L; rats in PPADS group were intrathecally injected with 10 μL PPADS in the concentration of 10 nmol/μL. The PWMT of rats was detected at 0.5 h before injection and 0.5 h after. At PIH 72.5, the tissue in the dorsal horn of spinal cord of rats in sham injury+ normal saline, pure burn, and burn+ LA groups was harvested to observe the co-expression of P2X(4) receptor and OX42 receptor with immunofluorescent staining and to observe the expression of P2X(4) receptor and count the positive cells with immunohistochemical staining. The venous blood was harvested for determination of serum content of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) with enzyme-linked immunosorbent assay. The same observation and determination were conducted in rats without injury in the two groups at the same time point as above. Data were processed with one-way analysis of variance, analysis of variance for repeated measurement, SNK test, paired <i>t</i> test, and Bonferroni correction. <b>Results:</b> (1) There were no abnormal activity in rats of healthy+ normal saline, sham injury+ normal saline, healthy+ LA groups at all time points. Until PIH 72.5, rats in pure burn group were in poor mental state; red and swollen manifestation and blister were observed in burn wounds on the back and right hind; imbalance in gait, lick, bite, and scratch were observed occasionally. Fewer behaviors such as lick, bite, and limp were observed in rats in burn+ LA group than in pure burn group, and the red and swollen manifestation in wounds of rats in burn+ LA group dissipated faster than that in pure burn group. (2) At 1.5 h before injury, there were no significant differences in the PWMT values of rats in healthy+ normal saline, sham injury+ normal saline, pure burn, burn+ LA, and healthy+ LA groups (<i>F</i>=0.106, <i>P</i>>0.05). PWMT values of rats in pure burn group were significantly lower than those in the other 4 groups at all post injury time points (with <i>P</i> values below 0.05). PWMT values of rats in burn+ LA group were significantly lower than those in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups at all post injury time points (with <i>P</i> values below 0.05). (3) At 0.5 h before injection, PWMT values of rats in normal saline, TNP-ATP, PPADS, and minocyline groups were close, respectively 15.3±0.8, 15.1±1.0, 15.3±0.9, and 15.6±1.1 (<i>F</i>=0.343, <i>P</i>>0.05). At 0.5 h after injection, PWMT values of rats in normal saline group and PPADS group were respectively 15.2±1.2 and 14.8±1.0, which were significantly lower than 20.8±1.4 and 26.3±1.0 in TNP-ATP group and minocyline group respectively (with <i>P</i> values below 0.05). PWMT values of rats in normal saline and PPADS groups were similar before and after injection (with <i>t</i> values respectively 0.073 and -0.772, <i>P</i> values above 0.05), while those of rats in TNP-ATP and minocyline groups were higher after injection than before injection (with <i>t</i> values respectively -10.180 and -20.813, <i>P</i> values below 0.01). (4) At PIH 72.5, co-expression of P2X(4) receptor and OX42 receptor was observed in a few microglias of rats in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups, while co-expression of P2X(4) receptor and OX42 receptor was observed in a large number of microglias of rats in pure burn and burn+ LA groups. At PIH 72.5, more P2X(4) receptor positive cells were observed in rats in pure burn group than in the other 4 groups (with <i>P</i> values below 0.05), and more P2X(4) receptor positive cells were observed in rats in burn+ LA group than in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups (with <i>P</i> values below 0.05). (5) At PIH 72.5, the serum content of TNF-α and IL-1β of rats in pure burn group was significantly higher than that in the other 4 groups (with <i>P</i> values below 0.001). The serum content of TNF-α and IL-1β of rats in burn+ LA group was significantly lower than that in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups (with <i>P</i> values below 0.001). <b>Conclusions:</b> LA has significant analgesic effects on severely burned rats, and it can ameliorate the excessive inflammational situation. The mechanism may be related to its inhibition of expression of P2X(4) receptor in microglias in the dorsal horn of spinal cord and reduction in the release of inflammatory factors TNF-α and IL-1β.

Objective: To explore the performance of hyaluronic acid biogel and its effect on healing of infected burn wounds in mice. Methods: This study was an experimental study. The hyaluronic acid biogel prepared in phosphate buffered saline (PBS) was soaked in PBS. Then the liquid absorption rate of hyaluronic acid biogel at 1, 2, 3, 4, and 5 min of soaking was calculated. After mixing hyaluronic acid biogel in powder form with PBS to form a gel, the mechanical modulus of the hyaluronic acid biogel was tested using a rotational rheometer. The hyaluronic acid biogel solution at final mass concentrations of 5, 10, and 20 g/L along with PBS were added to Staphylococcus aureus and Escherichia coli bacteria culture solution, respectively, then after incubation for 12 h, dilution plating was performed on Lysogeny Broth agar medium. The colony growth on the medium was observed by a colony counter after culture for 24 h. Twenty-four male BALB/c mice aged 8-10 weeks underwent dorsal burn wound creation followed by necrotic tissue excision. A infected burn wound model was established by locally instilling Staphylococcus aureus bacteria culture solution. With the successful modeling being confirmed at 24 h after incubation, the mice were divided into 4 groups (n=6) according to the random number table method. The wounds in normal saline group received normal saline, while the wounds in Jingwanhong ointment group, hydrocolloid dressing group, and hyaluronic acid biogel group received Jingwanhong ointment, hydrocolloid dressing, and hyaluronic acid biogel, respectively, for 14 d. At treatment day 7, 10, and 14, the remaining wound area was measured and the percentage of residual wound area was calculated. At treatment day 7, the number of Staphylococcus aureus colonies on wounds were counted by a colony counter. All the experiments used a sample size of 3. Results: The hyaluronic acid biogel achieved the highest liquid absorption rate of 387.9% within 2 min of soaking in PBS, and stabilized at over 300.0% within 5 min of soaking. Frequency scanning of the mechanical modulus revealed that the hyaluronic acid biogel's elastic modulus remained stable above 250 Pa and increased with rising angular frequency. Amplitude scanning of the mechanical modulus indicated that at an angular frequency of 10 rad/s, the linear viscoelastic range of the hyaluronic acid biogel was close to 1 000%. After culture for 24 h, the medium added with PBS was almost completely covered with Staphylococcus aureus and Escherichia coli. The medium added with hyaluronic acid biogel solution at final mass concentrations of 5, 10, and 20 g/L showed no significant growth of Staphylococcus aureus or Escherichia coli. At treatment day 7, 10, and 14, the percentage of residual wound area of mice in hyaluronic acid biogel group was significantly lower than that in normal saline, Jingwanhong ointment, and hydrocolloid dressing groups (with P values all <0.05). At treatment day 14, the percentage of residual wound area of mice in hydrocolloid dressing group was significantly lower than that in normal saline and Jingwanhong ointment groups (with P values both <0.05). At treatment day 7, the number of Staphylococcus aureus colonies on wounds of mice in hyaluronic acid biogel group ((4.3±0.6) colonies) was significantly lower than that in normal saline group ((2 400.7±225.4) colonies), Jingwanhong ointment group ((899.0±57.0) colonies), and hydrocolloid dressing group ((11.7±5.7) colonies), with P values all <0.05. Conclusions: Hyaluronic acid biogel exhibits an excellent capacity to absorb liquid and form hydrogel, and promotes healing of infected burn wounds in mice by eliminating Staphylococcus aureus.

Efficacy of the Controlled Release of Concentrated Platelet Lysate from a Collagen/Gelatin Scaffold for Dermis-Like Tissue Regeneration

Efficacy of gelatin gel sheets sustaining epidermal growth factor for murine skin defects

We developed a novel skin regeneration therapy combining nevus tissue inactivated by high hydrostatic pressure (HHP) in the reconstruction of the dermis with a cultured epidermal autograft (CEA). The issue with this treatment is the unstable survival of CEA on the inactivated dermis. In this study, we applied collagen/gelatin sponge (CGS), which can sustain the release of basic fibroblast growth factor (bFGF), to the inactivated skin in order to accelerate angiogenesis. Murine skin grafts from C57BL6J/Jcl mice (8 mm in diameter) were prepared, inactivated by HHP and cryopreserved. One month later, the grafts were transplanted subcutaneously onto the back of other mice and covered by CGS impregnated with saline or bFGF. Grafts were harvested after one, two and eight weeks, at which point the engraftment was evaluated through the histology and angiogenesis-related gene expressions were determined by real-time polymerase chain reaction. Histological sections showed that the dermal cellular density and newly formed capillaries in the bFGF group were significantly higher than in the control group. The relative expression of FGF-2, PDGF-A and VEGF-A genes in the bFGF group was significantly higher than in the control group at Week 1. This study suggested that the angiogenesis into grafts was accelerated, which might improve the engraftment of inactivated dermis in combination with the sustained release of bFGF by CGSs.

Novel Collagen/Gelatin Scaffold with Sustained Release of Basic Fibroblast Growth Factor: Clinical Trial for Chronic Skin Ulcers