Potential of gelatin hydrogel nonwoven fabrics (Genocel) as a skin substitute in a diabetic mouse skin defect model.

Background: Gelatin hydrogel nonwoven fabrics (Genocel) are three-dimensional gelatin scaffolds that provide cells with space for proliferation, migration, and differentiation. They are expected to be an effective wound healing modality to treat intractable wounds, such as diabetic foot ulcers, because they enhance early neovascularization when used as a skin substitute. In this study, we explored the healing process of Genocel applied to skin defects in diabetic mice and compared it with that of a conventional skin substitute, Pelnac. Methods: Genocel and Pelnac sheets were used to treat skin defects on the backs of diabetic mice. On days 7 and 14, the remaining wound area was evaluated and specimens were harvested for HE, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue formation, capillary formation, and macrophage infiltration. Results: Wounds treated with Genocel showed a wound healing process comparable to that of wounds treated with Pelnac. No significant differences were observed in the remaining wound area, neoepithelial length, granulation formation, number of pan-macrophages, or M2 ratio on days 7 and 14. The only significant difference was the number of induced M2 macrophages, which was higher in Pelnac group than in the Genocel group on day 7 (p < 0.05). Conclusions: Genocel showed similar healing effects in diabetic wounds as Pelnac and is considered an effective wound management modality for diabetic ulcers.

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Modified gelatin hydrogel nonwoven fabrics (Genocel) as a skin substitute in murine skin defects.

Introduction: From previous research, an emerging material composed of gelatin hydrogel nonwoven fabric (Genocel) has shown potential as a skin substitute, by improving neovascularization promotion in the early phase of wound healing. However, Genocel was inferior in terms of granulation formation compared to Pelnac. To solve this problem, we modified the manufacturing process of Genocel to reduce its water content, extend the degradation time (Genocel-L), and evaluate its healing process as a skin substitute. Methods: Genocel with a low water content (Genocel-L) was prepared and the difference in water content compared to that of the conventional Genocel was confirmed. Degradation tests were performed using collagenase and compared among Genocel-L, Genocel, and Pelnac sheets. In the in vivo study, sheets of Genocel-L or Pelnac were applied to skin defects created on the backs of C57BL/6JJcl mice. On days 7, 14, and 21, the remaining wound area was evaluated and specimens were harvested for Hematoxylin and Eosin, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue, capillary formation, and macrophage infiltration. Results: Genocel-L had a lower water content than the conventional Genocel and a slower degradation than Genocel and Pelnac. In the in vivo experiment, no significant differences were observed between Genocel-L and Pelnac in relation to the wound area, neoepithelium length, granulation formation, and the number of newly formed capillaries. The area of newly formed capillaries in the Pelnac group was significantly larger than that in the Genocel-L group on day 21 (p < 0.05). Regarding macrophage infiltration, significantly more M2 macrophages were induced in the Pelnac group on days 14 and 21, and the M2 ratio was larger in the Pelnac group (p < 0.05) during the entire process. Conclusions: Genocel-L has a lower water content and slower degradation rate than the conventional Genocel. Genocel-L had equivalent efficacy as a skin substitute to Pelnac, and can therefore be considered feasible for use as a skin substitute. However, a manufacturing method that can further modify Genocel-L is required to recover its early angiogenic potential.

Development of gelatin hydrogel nonwoven fabrics (Genocel®) as a novel skin substitute in murine skin defects.

Introduction: Genocel is an emerging material, used in cell culture, with high mechanical strength and good cytocompatibility. Based on these characteristics, Genocel is considered a promising skin substitute for wound healing. In this study, we explored the possibility of using Genocel as a skin substitute for murine skin defects and compared it with a conventional skin substitute. Methods: Sheets of Genocel and Pelnac were applied to skin defects created on the backs of mice. On days 7, 14, and 21, the remaining wound area was evaluated and specimens were harvested for HE, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue, capillary formation, and macrophage infiltration. Results: No significant differences in the wound area or neoepithelium length were observed between groups. The number of newly formed capillaries in the Genocel group was significantly higher than that in the Pelnac group on day 7 (p < 0.05). In contrast, granulation tissue formation in the Pelnac group was greater than that in the Genocel group on day 14 (p < 0.05). Regarding macrophage infiltration, the pan-macrophage number, M2 macrophage number, and M2 ratio in the Pelnac group were higher than those in the Genocel group on day 14 (p < 0.05). In other aspects, the two materials displayed comparable behavior. Conclusions: Genocel can be used as a skin substitute equivalent to the conventional one. In addition, Genocel accelerated capillary formation, which is more appropriate than conventional treatments for chronic skin ulcers, such as diabetic ulcers.

Gelatin hydrogel nonwoven fabrics of a cell culture scaffold to formulate 3-dimensional cell constructs.

The objective of this study is to evaluate the possibility of gelatin hydrogel nonwoven fabrics (GHNF) of a cell culture scaffold to formulate 3-dimensional (3D) cell construct. The thickness of cell construct is about 1 mm and the cells inside are live and bio-active, irrespective of their internal distribution. The GHNF were prepared by the solution blow method of gelatin, following by dehydrothermal crosslinking. The GHNF showed a mechanical strength strong enough not to allow the shape to deform even in a wet state. The wet GHNF also showed resistance against repeated compression. After human mesenchymal stromal cells (hMSC) were seeded and cultured, the inner distribution in GHNF, the apoptosis, hypoxia inducible factor (HIF)-1α, Ki67, collagen or sulfated glycosaminoglycan (sGAG) secretion of cells were evaluated. The hMSC proliferated inside the GHNF with time while a homogeneous distribution in the number of cells proliferated from the surface to the 1000 µm depth of GHNF was observed. The number of apoptosis and HIF-1α positive cells was significantly low compared with that of polypropylene nonwoven fabrics with the similar fiber diameters and intra-structure. The GHNF were degraded during cell culture, and completely replaced by collagen and sGAG secreted. It is concluded that the GHNF is a promising cell culture scaffold for 3D cell constructs.

The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication.

Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases alpha and epsilon onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase alpha. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.

A Gelatin Hydrogel Nonwoven Fabric Facilitates Metabolic Activity of Multilayered Cell Sheets.

IMPACT STATEMENT: This study introduces the utility of gelatin hydrogel nonwoven fabrics (GHNFs) for cell sheet engineering. The GHNF had the mechanical property strong enough to hold by forceps even in the swollen condition. The cell sheet harvest and transfer processes were performed simpler and faster than those without using the GHNF. The GHNF facilitates the metabolic activity of three-layered cell sheets, and the cell migration from cell sheets into the GHNF was observed. The GHNF is a promising material used to support cell sheets during the process of assemble formulation and contributes to the improved biological functions of tissue-like cell constructs.

Involvement of Ecdysone Signaling in the Expression of the doublesex Gene during Embryonic Development in the Silkworm, Bombyx mori.

Steroid hormones, represented by estrogen and testosterone, act as sex hormones that play an essential role in the sexual differentiation of vertebrates. However, it remains unclear whether ecdysteroids, typical steroid hormones in insects, function as sex hormones. In this study, we investigated whether ecdysteroids or ecdysone signals are involved in the sexual differentiation of the silkworm (Bombyx mori) embryo. Quantitative analysis using LC-MS/MS demonstrated that there was no significant difference in the 20-hydroxyecdysone (20E) titer between sexes during embryonic development. Consistent with this result, expression levels of 2 genes encoding ecdysteroid-phosphate phosphatase (EPPase) and ecdysone 20-hydroxylase (E20OHase), which are essential for the biosynthesis of ecdysone and 20E in eggs, did not show a significant difference between male and female embryos. Expression levels of ecdysone receptor (EcR) and E75, which is one of a small set of genes induced directly by 20E, were also similar between the 2 sexes. However, knockdown of EPPase and one isoform of EcR (EcR-A) resulted in decreased expression of Bombyx doublesex (Bmdsx), a master regulatory gene for sexual differentiation of the silkworm in both male and female embryos. In vitro analysis with cultured testes revealed that expression levels of Bmdsx were increased in a dose-dependent manner of the ecdysone analog, ponasterone A. These results suggest that ecdysone signaling may play a role in indirectly regulating the expression of some genes involved in sexual differentiation through inducing expression of Bmdsx in the silkworm.