In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.     

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) improved osteogenic differentiation of the human induced pluripotent stem cells while considered as an artificial extracellular matrix

Cocell polymers can be the best implants for replacing bone defects in patients. The pluripotent stem cells produced from the patient and the nanofibrous polymeric scaffold that can be completely degraded in the body and its produced monomers could be also usable are the best options for this implant. In this study, electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers were fabricated and characterized and then osteogenic differentiation of the human-induced pluripotent stem cells (iPSCs) was investigated while cultured on PHBV scaffold. MTT results showed that cultured iPSCs on PHBV proliferation were increased compared to those cultured on tissue culture polystyrene (TCPS) as the control. Alkaline phosphatase (ALP) activity and calcium content were also significantly increased in iPSCs cultured on PHBV compared to the cultured on TCPS under osteogenic medium. Gene expression evaluation demonstrated that Runx2, collagen type I, ALP, osteonectin, and osteocalcin were upregulated in iPSCs cultured on PHBV scaffold in comparison with those cultured on TCPS for 2 weeks. Western blot analysis have shown that osteocalcin and osteopontin expression as two major osteogenic markers were increased in iPSCs cultured on PHBV scaffold. According to the results, nanofiber-based PHBV has a promising potential to increase osteogenic differentiation of the stem cells and iPSCs-PHBV as a cell-co-polymer construct demonstrated that has a great efficiency for use as a bone tissue engineered bioimplant.     

Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitosan scaffolds for skin regeneration

The purpose of this study was to evaluate hybrid poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/chitosan nanofibrous mats as scaffolds for skin engineering. In vitro studies were carried out to test the potential of the scaffolds for fibroblasts adhesion, viability, and proliferation (L929 cell line). The in vivo performance was also studied in a full-thickness wound healing model. PHBV/chitosan 4:1 (w/w) exhibited a higher in vitro biocompatibility and a better ability for cell adhesion and growth, compared to PHBV/chitosan 2:3 (w/w). The in vivo assay also revealed the better performance of this scaffold, improving the wound healing process in rats.     

Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase

Background

Wound healing is a highly dynamic process that requires signaling from the extracellular matrix to the fibroblasts for migration and proliferation, and closure of the wound. This rate of wound closure is impaired in diabetes, which may be due to the increased levels of the precursor for advanced glycation end products, 3-deoxyglucosone (3DG). Previous studies suggest a differential role for p38 mitogen-activated kinase (MAPK) during wound healing; whereby, p38 MAPK acts as a growth kinase during normal wound healing, but acts as a stress kinase during diabetic wound repair. Therefore, we investigated the signaling cross-talk by which p38 MAPK mediates wound healing in fibroblasts cultured on native collagen and 3DG-collagen.

Methodology/Principal Findings

Using human dermal fibroblasts cultured on 3DG-collagen as a model of diabetic wounds, we demonstrated that p38 MAPK can promote either cell growth or cell death, and this was dependent on the activation of AKT and ERK1/2. Wound closure on native collagen was dependent on p38 MAPK phosphorylation of AKT and ERK1/2. Furthermore, proliferation and collagen production in fibroblasts cultured on native collagen was dependent on p38 MAPK regulation of AKT and ERK1/2. In contrast, 3DG-collagen decreased fibroblast migration, proliferation, and collagen expression through ERK1/2 and AKT downregulation via p38 MAPK.

Conclusions/Significance

Taken together, the present study shows that p38 MAPK is a key signaling molecule that plays a significantly opposite role during times of cellular growth and cellular stress, which may account for the differing rates of wound closure seen in diabetic populations.     

The effect of redox signaling on extracellular matrix changes in diabetic wounds leading to amputation

Introduction& Objectives: Redox signaling is a critical regulator in the process of wound healing. This signaling pathway can be effective in the development or healing of diabetic ulcers through the ECM.In this study, the structure of extracellular matrix investigated in relation to redox signaling in the tissue of patients with diabetic ulcers that lead to organ amputation.Materials and methodsThe case-control design on diabetic patients ulcers as case group and non-diabetic limb ischemia as control were used.Hematoxylin-eosin, trichrome, and elastin staining methods were used for pathological evaluations of ECM. MDA, total thiol, and SOD levels were measured using ELISA kits to assess the oxidative stress level. Also, NO level was measured by using ELISA kits in both groups. Expression levels of genes MMP2, MMP9, and HIF were detected using real-time PCR with SYBR-green assay.ResultsThe pathological results showed an increase in the thickness of collagen and elastin fibers. Lipids atrophy was visible in the tissue isolated from the diabetic wound group. The amount of MAD to evaluate the level of lipid oxidation in patients with diabetic Ulcer was significantly higher than the control group(p < 0.01). Thiol level was significantly lower in the diabetic ulcer group than in the control group(p < 0.0001). The expression of metalloproteinases 2 and 9 genes in the tissues isolated from diabetic ulcers was lower than the control group(p < 0.0001). While the expression of the HIF gene in this group was higher than the control group(p < 0.0001).ConclutionIn the diabetic wound, the HIF secretion due to hypoxic conditions is beneficial for matrix deposition and prevents protease activity, but if the hypoxia persists, it can lead to ECM deposition subsequently increases the tissue pressure, increases of the collagen I-to-collagen III ratio in collagen accumulation that due to more hypoxia , lipidsAtrophy and eventually amputation.     

Growth modulation of fibroblasts by chitosan-polyvinyl pyrrolidone hydrogel: Implications for wound management?

Wounds in adults and fetuses differ in their healing ability with respect to scar formation. In adults, wounds lacking the epidermis exhibit excess collagen production and scar formation. Fibroblasts synthesize and deposit a collagen rich extracellular matrix. The early migration and proliferation of fibroblasts in the wound area is implicated in wound scarring. We have synthesized a hydrogel from chitosan-polyvinyl pyrrolidone (PVP) and examined its effect on fibroblast growth modulation in vitro. The hydrogel was found to be hydrophilic as seen from its octane contact angle (141.2+/-0.37 degrees). The hydrogel was non-toxic and biocompatible with fibroblasts and epithelial cells as confirmed by the 3(4,5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) as-say. It showed dual properties by supporting growth of epithelial cells (SiHa) and selectively inhibiting fibro-blast (NIH3T3) growth. Growth inhibition of fibroblasts resulted from their inability to attach on to the hydrogel. These findings are supported by image analysis, which revealed a significant difference (P<0.05) between the number of fibroblasts attached to the hydrogel in tissue culture as compared to tissue culture treated polystyrene (TCPS) controls. However, no significant difference was observed (P>0.05) in the number of epithelial (SiHa) cells attached on to the hydrogel as compared to the TCPS control. Although in vivo experiments are awaited, these findings point to the possible use of chitosan-PVP hydrogels in wound-management.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm⁻¹ can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.     

Acceleration of diabetic wound healing with chitosan-crosslinked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired STZ diabetic rats

In order to develop a better wound-dressing to enhance diabetic wound healing, we have examined the biochemical and biophysical features of chitosan-crosslinked collagen sponge (CCCS) and pre-clinically evaluated the CCCS containing recombinant human acidic fibroblast growth factor (CCCS/FGF) in accelerating diabetic wound healing as compared to collagen sponge alone and FGF alone. Collagen crosslinked with chitosan showed several advantages required for wound dressing, including the uniform and porous ultrastructure, less water-imbibition, small interval porosity, high resistance to collagenase digestion and slow release of FGF from CCCS/FGF. Therapeutic effect of the new wound-dressing containing FGF (i.e.: CCCS/FGF) on diabetic wound healing was examined in type 1 diabetic rat model in which hyperglycemia was induced by single dose of streptozotocin (STZ) and persisted for two months. The CCCS/FGF provided the most efficiently therapeutic effect among various treatments, showing the shortest healing time (14 days in the CCCS/FGF-treated group as compared to 18~21 days in other treatment groups), the quickest tissue collagen generation, the earliest and highest TGF-beta1 expression and dermal cell proliferation (PCNA expression). All these results suggest that CCCS/FGF is an ideal wound-dressing to improve the recovery of healing-impaired wound such as diabetic skin wound, which provides a great potential use in clinics for diabetic patients in the future.     

Effect of sodium diphenylhydantoin on skin wound healing in rats

This study evaluated the effect of phenytoin (sodium diphenylhydantoin) on skin wound healing in a rat model. The study was divided into two parts. In part I, 20 mul of phenytoin (10 mg/ml) was subcutaneously injected into the 3-cm dorsal full-thickness incisional wounds of 14 rats on postoperative days 0, 3, and 6. Twelve rats that received saline injections were used as the controls. The skin samples were harvested and tested for tensile strength and histology. An additional 12 rats with the same incisional wounds were tested for chemokine gene expressions. In part II, 20 mul of phenytoin (10 mg/ml) was applied topically once a day on a 4 x 4 cm area of the open dorsal wounds of 10 rats. Saline was applied to the wounds of the 10 control group rats. The wounds were measured weekly. The results showed that the average tensile strength of the phenytoin-treated wound was 0.49 +/- 0.08 MPa compared with the control group at 0.02 +/- 0.01 MPa (p < 0.05). The density ratio of chemokine monocyte chemotactic protein (MCP-1) to beta-actin in the phenytoin-treated group was also significantly higher than in the control group (p < 0.05). Histologic analysis of the phenytoin group showed a large amount of fibroblast proliferation, collagen synthesis, and neovascularization. Phenytoin-treated wounds were also smaller at 1 to 6 weeks postoperatively than the control group wounds. The authors conclude that the administration of phenytoin can promote wound healing and significantly increase MCP-1 expression. Phenytoin-treated wounds showed significant increase in collagen deposition and neovascularization, which resulted in an increased wound tensile strength and accelerated healing of both open and closed wounds.     

Fibroblast growth factor 2-functionalized collagen matrices for skeletal muscle tissue engineering

Fibroblast growth factor 2 (FGF2) protein plays important roles in wound healing and tissue regeneration. Collagen is clinically used for wound care applications. We investigated the potential value of FGF2-functionalized collagen matrices for skeletal muscle tissue engineering. When C2C12 cells were treated with FGF2, cell adhesion increased after 3 and 5 days compared to the control (P < 0.05). Wound healing activity of FGF2 was slightly higher than the control through cell migration. Cell proliferation activity of FGF2-functionalized collagen matrices on C2C12 cells also increased. Taken together, FGF2 stimulated C2C12 myoblast growth by promoting cell adhesion, proliferation and wound healing activity after injury. The potential effect of FGF2-functionalized collagen matrices was also observed. Thus FGF2 stimulates skeletal muscle development and regeneration, thereby leading to potential utility for skeletal muscle tissue engineering.     

LncRNA H19 Drives Proliferation of Cardiac Fibroblasts and Collagen Production via Suppression of the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β Axis

The aim of this study was to investigating whether lncRNA H19 promotes myocardial fibrosis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis. Patients with atrial fibrillation (AF) and healthy volunteers were included in the study, and their biochemical parameters were collected. In addition, pcDNA3.1-H19, si-H19, and miR-29a/b-3p mimic/inhibitor were transfected into cardiac fibroblasts (CFs), and proliferation of CFs was detected by MTT assay. Expression of H19 and miR-29a/b-3p were detected using real-time quantitative polymerase chain reaction, and expression of α-smooth muscle actin (α-SMA), collagen I, collagen II, matrix metalloproteinase-2 (MMP-2), and elastin were measured by western blot analysis. The dual luciferase reporter gene assay was carried out to detect the sponging relationship between H19 and miR-29a/b-3p in CFs. Compared with healthy volunteers, the level of plasma H19 was significantly elevated in patients with AF, while miR-29a-3p and miR-29b-3p were markedly depressed (P < 0.05). Serum expression of lncRNA H19 was negatively correlated with the expression of miR-29a-3p and miR-29b-3p among patients with AF (r_s = –0.337, r_s = –0.236). Moreover, up-regulation of H19 expression and down-regulation of miR-29a/b-3p expression facilitated proliferation and synthesis of extracellular matrix (ECM)-related proteins. SB431542 and si-VEGFA are able to reverse the promotion of miR-29a/b-3p on proliferation of CFs and ECM-related protein synthesis. The findings of the present study suggest that H19 promoted CF proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis, and provide support for a potential new direction for the treatment of AF.     

Multiphoton microscopy observations of 3D elastin and collagen fiber microstructure changes during pressurization in aortic media

Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal–circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.     

3-D collagen-dependent cell surface expression of MT1-MMP and MMP-2 activation regardless of integrin β1 function and matrix stiffness

Matrix metalloproteinases (MMPs) play roles in spatially dynamic processes, including morphogenesis, wound healing, and tumor invasion. Three-dimensional (3-D) type I collagen stimulates cellular activation of MMP-2, however, the mechanisms underlying this are controversial. The present study investigated mechanisms for 3-D collagen-induced MMP-2 activation in highly invasive human malignant mesothelioma cells. MMP-2 was effectively activated by cells cultured in 3-D collagen but not in 2-D collagen, whereas MMP-2 activation was not regulated by the flexibility of collagen. The 3-D collagen did not largely increase the gene expression of MMP-2 and MT1-MMP. However, MT1-MMP exposed to the cell surface was much increased by 3-D collagen, and loss of MT1-MMP abolished MMP-2 activation in response to 3-D collagen. MT1-MMP and integrin β1 translocated to pericellular regions interacting with collagen-coated microbeads, however their localization was different. Importantly, inhibition of integrin β1 function and expression did not affect 3-D collagen-induced cell surface localization of MT1-MMP and MMP-2 activation. Our results strongly suggest that 3-D collagen scaffolding may provide opportunity for direct and multivalent interaction with MT1-MMP, by which MMP-2 activation occur in abundant cell surface MT1-MMP-dependent manner, rather than a manner regulated by matrix stiffness and integrin β1 function.     

Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase

The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing.     

The elastin-laminin receptor functions as a mechanotransducer in vascular smooth muscle

Laminin and elastin, two major constituents of the extracellular matrix, bind to cells via the elastin-laminin receptor (ELR), a receptor distinct from integrins. Despite the ubiquitous nature of elastin and laminin in the matrix, the consequences of activation of the ELR are unknown. Because integrins are capable of mechanosensitive transduction, we hypothesized that the ELR would exert a similar function. Accordingly, we examined the effects of cyclical stretch on canine coronary smooth muscle gene expression and proliferation that are mediated by the ELR. Northern blot analyses showed a 31% decrease in serum-induced expression of c-fos when cells were stretched for 30 min on elastin, but no change in expression was observed on collagen. Serum-induced proliferation of stretched cells was markedly attenuated on elastin when compared with collagen. Both the molecular (decreased c-fos expression) and biological (decreased proliferation) responses on elastin were restored after blockade of the ELR with the elastin fragment hexapeptide (valine-glycine-valine-alanine-proline-glycine, VGVAPG). The inhibition was specific for this peptide, as another hydrophobic hexapeptide (valine-serine-leucine-serine-proline-glycine, VSLSPG) did not inhibit the responses. These results demonstrate that cyclic stretch inhibits c-fos expression and proliferation of coronary vascular smooth muscle cells grown on elastin matrixes, a mechanosensitive response that is transduced by the ELR.     

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.     

Clock gene Per2 modulates epidermal tissue repair in vivo

Wound healing can be influenced by genes that control the circadian cycle, including Per2 and BMAL1, which coordinate the functions of several organs, including the skin. The aim of the study was to evaluate the role of PER2 during experimental skin wound healing. Two groups (control and Per2-KO), consisting of 14 male mice each, were anesthetized by inhalation, and two 6 mm wounds were created on their dorsal skin using a punch biopsy. A silicone ring was sutured around the wound perimeter to restrict contraction. The wound healing process was clinically measured daily (closure index) until complete wound repair. On Day 6, histomorphometric analysis was performed using the length and thickness of the epithelial migration tongue, in addition to counting vessels underlying the lesion by immunofluorescence assay and maturation of collagen fibers through picrosirius staining. Bromodeoxyuridine (BrdU) incorporation and quantification were performed using the subcutaneous injection technique 2 h before euthanasia and through immunohistochemical analysis of the proliferative index. In addition, the qualitative analysis of myofibroblasts and periostin distribution in connective tissue was performed by immunofluorescence. Statistically significant differences were observed in the healing time between the experimental groups (means: 15.5 days for control mice and 13.5 days for Per2-KO; p = 0.001). The accelerated healing observed in the Per2-KO group (p < 0.05) was accompanied by statistical differences in wound diameter and length of the migrating epithelial tongue (p = 0.01) compared to the control group. Regarding BrdU immunoreactivity, higher expression was observed in the intact epithelium of Per2-KO animals (p = 0.01), and this difference compared to control was also present, to a lesser extent, at the wound site (p = 0.03). Immunofluorescence in the connective tissue underlying the wound showed a higher angiogenic potential in the Per2-KO group in the intact tissue area and the wound region (p < 0.01), where increased expression of myofibroblasts was also observed. Qualitative analysis revealed the distribution of periostin protein and collagen fibers in the connective tissue underlying the wound, with greater organization and maturation during the analyzed period. Our research showed that the absence of the Per2 gene positively impacts the healing time of the skin in vivo. This acceleration depends on the increase of epithelial proliferative and angiogenic capacity of cells carrying the Per2 deletion.     

Transforming growth factor-beta isoforms differently stimulate proalpha2 (I) collagen gene expression during wound healing process in transgenic mice

The role of many growth factors and cytokines in the process of wound healing has been intensively investigated in recent two decades. Among them, transforming growth factor-betas (TGF-betas) are well known to have a potent stimulatory effect on collagen synthesis as shown in various in vivo experimental systems. In the present study, we examined the effects of various growth factors on the promoter activity of the proalpha2 (I) collagen gene (COL1A2) during the wound healing process. For this purpose, we utilized transgenic mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase or a bacterial beta-galactosidase gene. These mice exhibited normal phenotypic expression and the wound healing process was not impaired. Full thickness wounds were made by punch biopsy. We examined the effects of TGF-beta1, -beta2, -beta3, basic fibroblast growth factor, platelet-derived growth factor, and connective tissue growth factor by applying them locally to the open wound every 2 days. Among the growth factors examined, all of the three isoforms of TGF- exhibited a more potent stimulatory effect on COL1A2 promoter activity than did other factors. In addition, while TGF-beta1 and -beta2 significantly increased the number of fibroblasts which were positive for X-Gal staining, TGF-beta3 treatment did not change the number of beta-galactosidase expressing cells. Accumulation of collagen fibers was observed to the same extent in the mice treated with TGF-beta1 and those with TGF-beta3. These findings suggest that TGF-beta1 and -beta3 have similar but not identical regulatory mechanisms of COL1A2 expression, and that their pathophysiological roles in wound healing might be different from each other.     

In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds

The in vitro responses of Schwann cells (RT4-D6P2T, a schwannoma cell line derived from a chemically induced rat peripheral neurotumor) on various types of electrospun fibrous scaffolds of some commercially available biocompatible and biodegradable polymers, i.e., poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polycaprolactone (PCL), poly(l-lactic acid) (PLLA), and chitosan (CS), were reported in comparison with those of the cells on corresponding solution-cast film scaffolds as well as on a tissue-culture polystyrene plate (TCPS), used as the positive control. At 24 h after cell seeding, the viability of the attached cells on the various substrates could be ranked as follows: PCL film > TCPS > PCL fibrous > PLLA fibrous > PHBV film > CS fibrous approximately CS film approximately PLLA film > PHB film > PHBV fibrous > PHB fibrous. At day 3 of cell culture, the viability of the proliferated cells on the various substrates could be ranked as follows: TCPS > PHBV film > PLLA film > PCL film > PLLA fibrous > PHB film approximately PCL fibrous > CS fibrous > CS film > PHB fibrous > PHBV fibrous. At approximately 8 h after cell seeding, the cells on the flat surfaces of all of the film scaffolds and that of the PCL nanofibrous scaffold appeared in their characteristic spindle shape, while those on the surfaces of the PHB, PHBV, and PLLA macrofibrous scaffolds also appeared in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds.