角蛋白/海藻酸钠/聚丙烯酰胺水凝胶皮肤敷料的制备及创口修复研究

为了获得更为理想的皮肤创口修复敷料,在海藻酸钠(SA)和聚丙烯酰胺(PAM)水凝胶的基础上复合人发角蛋白(KTN),制得KTN/SA/PAM水凝胶皮肤敷料.用电子万能测试机、扫描电子显微镜等对其进行表征,结果显示,KTN/SA/PAM水凝胶皮肤敷料拉伸强度为42.41 kPa,弹性模量11.19 kPa,接近人体皮肤组...     

Effects of nonwoven mats of Di-O-butyrylchitin and related polymers on the process of wound healing

The aim of the study was to observe the effects of dibutyrylchitin (DBC) on the repair processes and to explain the mechanisms of its action in comparison with other dressing materials made of butyrylchitin (BC), regenerated chitin (RC), and chitosan. The results showed that DBC implanted subcutaneously to the rats increased weight of the granulation tissue. Increased cell number isolated from the wound and cultured on the DBC films was also revealed. The DBC was proved to reduce also the necrotic cells number in the culture. DBC elevates the glycosaminoglycans (GAG) level in the granulation tissue. The total collagen content in the wound was not influenced by all applied dressing materials. However, a low level of the poorly polymerized soluble collagen in the wounds treated with DBC and BC indicated better polymerization of the remaining part of that protein. Both DBC and chitosan increased the weight of granulation tissue. However, chitosan contrary to DBC lowered GAG content and increased water capacity in the wound. The study documents the beneficial influence of DBC on the repair, which could be explained by the modification of the extracellular matrix and cells number. The best effects were observed after application of DBC with [eta] DBC-1 = 1.75 dL/g.     

Influence of genetic drift on patterns of genetic variation: The footprint of aquaculture practices in Sparus aurata (Teleostei: Sparidae)

Aquaculture finfish production based on floating cage technology has raised increasing concerns regarding the genetic integrity of natural populations. Accidental mass escapes can induce the loss of genetic diversity in wild populations by increasing genetic drift and inbreeding. Farm escapes probably represent an important issue in the gilthead sea bream (Sparus aurata), which accounted for 76.4% of total escapees recorded in Europe during a 3‐year survey. Here, we investigated patterns of genetic variation in farmed and wild populations of gilthead sea bream from the Western Mediterranean, a region of long gilthead sea bream farming. We focused on the role that genetic drift may play in shaping these patterns. Results based on microsatellite markers matched those observed in previous studies. Farmed populations showed lower levels of genetic diversity than wild populations and were genetically divergent from their wild counterparts. Overall, farmed populations showed the smallest effective population size and increased levels of relatedness compared to wild populations. The small broodstock size coupled with breeding practices that may favour the variance in individual reproductive success probably boosted genetic drift. This factor appeared to be a major driver of the genetic patterns observed in the gilthead sea bream populations analysed in the present study. These results further stress the importance of recommendations aimed at maintaining broodstock sizes as large as possible and equal sex‐ratios among breeders, as well as avoiding unequal contributions among parents.     

Novel Biodegradable Porous Scaffold Applied to Skin Regeneration

Skin wound healing is an important lifesaving issue for massive lesions. A novel porous scaffold with collagen, hyaluronic acid and gelatin was developed for skin wound repair. The swelling ratio of this developed scaffold was assayed by water absorption capacity and showed a value of over 20 g water/g dried scaffold. The scaffold was then degraded in time- and dose-dependent manners by three enzymes: lysozyme, hyaluronidase and collagenase I. The average pore diameter of the scaffold was 132.5±8.4 µm measured from SEM images. With human skin cells growing for 7 days, the SEM images showed surface fractures on the scaffold due to enzymatic digestion, indicating the biodegradable properties of this scaffold. To simulate skin distribution, the human epidermal keratinocytes, melanocytes and dermal fibroblasts were seeded on the porous scaffold and the cross-section immunofluorescent staining demonstrated normal human skin layer distributions. The collagen amount was also quantified after skin cells seeding and presented an amount 50% higher than those seeded on culture wells. The in vivo histological results showed that the scaffold ameliorated wound healing, including decreasing neutrophil infiltrates and thickening newly generated skin compared to the group without treatments.     

Isolation and characterization of polymorphic microsatellite markers in Pagellus bogaraveo,and cross‐species amplification in Sparus aurata and Dicentrarchus labrax

Several new fish species are currently being included in breeding programmes. However, as specific molecular markers have not yet been developed, this represents a commercial handicap with respect to traditional aquaculture species such as gilthead sea bream or Atlantic salmon. In the present study, 12 new microsatellite loci were developed for blackspot sea bream (Pagellus bogaraveo) that show high levels of polymorphism, especially useful in parentage assignment and individual identification. In addition, cross‐amplification was obtained for two important species for Spanish aquaculture, gilthead sea bream and sea bass.     

In vitro reconstruction of full thickness human skin on a composite collagen material

Rapid progress of in vitro techniques in the lastyears enabled the creation of organotypic skin cultures offering newpossibilities in wound treatment. Rebuilding of graft is one of the keyelementsof successful outcome of the procedure.In search for the best scaffold for organotypic skin culture, the novelcomposite xenogenic collagen based material with unique properties has beencreated and used to reconstitute full thickness human skin invitro. Based on our long established technology used for theproduction of collagen dressings for the treatment of burns, this novel,composite material offers excellent growth support of highly biodegradablespongy layer, combined with mechanical strength of collagen membrane. Themodulation of collagen properties was accomplished by consecutive treatmentwithhigh temperature and gamma irradiation. The use of the substrate enabled toobtain organotypic culture that resembles full thickness skin with fibroblastslayer and well-developed multilayer epithelium. Our new material offers easyhandling of obtained graft during surgery along with accelerated cell growth andcontrolled biodegradation of the culture support.     

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.     

Preparation of porcine small intestinal submucosa sponge and their application as a wound dressing in full-thickness skin defect of rat

Small intestinal submucosa (SIS) sponge was prepared by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The prepared SIS sponges exhibited elastic and soft property on touch and were ease to handle. The SIS sponges have the pore diameter of 100-200 microm and an interconnective porous structure. The SIS sponges exhibited high water absorption ability over 8000%. The water uptake of SIS sponges decreased as SIS concentration used to manufacture SIS sponge increased. In wound healing test, SIS sponge attained uniform adherence to the wound surface. The SIS sponges absorbed higher extent of exudation for wound than that covered with Tegaderm as control. Wound area contracted above 80% at the 21st postoperative day. The SIS sponge treated wound was almost completely covered with a thin layer of epidermis at 4 weeks. In addition, the dermal collagen in the wound regenerated at only SIS sponges treated wounds. The progress of granulous tissue formation was faster in SIS sponges as wound dressing than in Tegaderm. In conclusion, we found that the SIS sponges might be a potential material as a wound dressing.     

Identification of the depth of burn injury by collagen stainability

Heat-denatured collagen in burned skin stains red instead of blue in Masson's trichrome stain. This change in stainability corresponds to the loss of birefringence in slides examined in polarized light. The depth of the abnormal staining of the skin slices was proportional to the time and temperature of the heat exposure. It is concluded that the change in collagen stainability from blue to red relates to the loss of crystallinity or parallel alignment of the collagen fibers. It is further proposed that change in the stainability of collagen in the burns could be used to delineate the depth of the thermal skin injury or the effectiveness of the surgical excision or debridement of the wound by dressing materials.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Transplantation of bone-marrow-derived mesenchymal and epidermal stem cells contribute to wound healing with different regenerative features

A new concept for wound therapy is the initiation of the regeneration of epidermal and dermal layers with appendages for skin function recovery. Bone-marrow-derived mesenchymal and epidermal stem cells (BMSCs and SSCs) are hypothesized to be able to home toward or to be transplanted to wound sites for skin repair and regeneration, but this awaits confirmation by further experimental and clinical evidence. In this study, the influence of the transplantation of BMSCs and SSCs with porous gelatin-β-tricalcium phosphate sponge as scaffolds on wound re-epithelization, collagen synthesis, skin tensile strength recovery, and skin appendage regeneration has been investigated. The transplantation of BMSCs or SSCs significantly accelerates wound re-epithelization, stimulates dermal collagen synthesis, and exhibits the trend to enhance the tensile strength recovery of skin. Furthermore, regenerative features of BMSCs and SSCs have been identified in activating blood vessel and hair follicle formation, respectively. These results not only provide experimental evidence for the application of BMSCs and SSCs as promising therapeutics for clinical wound treatment, but also display their characteristics in activating distinct skin appendage regeneration, which might have novel applications in skin tissue engineering.     

Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.     

一种可止血的富血小板血浆壳聚糖/丝素蛋白多孔伤口敷料的制备及性能表征

为了进一步提高伤口敷料的止血性能,文中在生物相容性良好的壳聚糖溶液中引入含有多种生长因子的人源性富血小板血浆(Humanplatelet-richplasma,hPRP),并加入不同体积比例(1∶1、1∶3、3∶1、1∶0)的丝素蛋白溶液以提高材料的多孔性与止血性,通过冷冻干燥法制备不同配比的hPRP-壳聚糖/丝素蛋白敷料,并将纯壳聚糖敷料作为对照组,研究hPRP和丝素蛋白对敷料的止血性能的影响以及丝素蛋白对PRP中生长因子控制释放的影响。结果表明,在壳聚糖敷料中引入hPRP对敷料的止血性有所提高,但对敷料的多孔结构及吸水率无明显改善,若在hPRP-壳聚糖溶液中按照体积比为1∶1的比例加入丝素蛋白溶液,会得到具有较为均匀的多孔结构的敷料,敷料的孔隙率与吸水率分别可达到86.83%±3.84%与1 474%±114%,且该比例的敷料在快速止血性能上表现优异。此外,加入丝素蛋白与壳聚糖比例为1∶1的PRP敷料能有效减少PRP中生长因子在初始阶段的爆裂释放。因此,含hPRP的壳聚糖/丝素蛋白复合敷料有望成为一种能快速止血且能促进伤口愈合的新型伤口敷料。     

Biodegradable collagen from Scomberomorus lineolatus skin for wound healing dressings and its application on antibiofilm properties

The major resolution of the study was to develop a dynamic form of natural biopolymer material to improve the wound healing by inhibition of biofilm formation on the surface. The extraction of collagen was effectively prepared from Scomberomorus lineolatus fish skin. Lyophilized collagen sheet was liquefied in 0.5M acetic acid to form acidic solubilized collagen (ASC) for further analysis. Physicochemical characterization of ASC was performed by various techniques using a standard protocol. The yield of ASC form S.lineolatus is higher (21.5%) than the previous reported studies. The effect of collagen solubility is gradually decreases with increasing concentration of NaCl and collagen is mostly soluble in acidic pH conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of ASC contains α chain composition of α₁ and α₂ subunits and was characterized as type I collagen. Ultraviolet absorption was regulated as the appropriate wavelength to optimize the collagen. Fourier-transform infrared spectroscopy and X-ray diffraction confirmed that the isolated collagen is a triple-helical structure. The biofilm formation of Pseudomonas aeruginosa was significantly reduced by collagen incorporated with isolated 3,5,7-trihydroxyflavone (collagen-TF) sheet up to 70%. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay executed on fibroblast cell lines (L929) shows that the collagen-TF sheet was 100% compatible to enrich the cell adhesion and proliferation. The current study was the first report to extract, purify, and characterize ASC from S. lineolatus fish skin and characterize as type I collagen. Based on the result, we design the natural biodegradable collagen loaded with TF compound (collagen-TF) for antibiofilm properties. Compared with different sources of polymer, fish skin collagen is more effective and can be used as a biopolymer sheet for wound healing, food, drug delivery, tissue engineering, and pharmaceutical application.     

Functional extracellular matrix hydrogel modified with MSC‐derived small extracellular vesicles for chronic wound healing

ObjectivesDiabetic wound healing remains a global challenge in the clinic and in research. However, the current medical dressings are difficult to meet the demands. The primary goal of this study was to fabricate a functional hydrogel wound dressing that can provide an appropriate microenvironment and supplementation with growth factors to promote skin regeneration and functional restoration in diabetic wounds.Materials and MethodsSmall extracellular vesicles (sEVs) were bound to the porcine small intestinal submucosa‐based hydrogel material through peptides (SC‐Ps‐sEVs) to increase the content and achieve a sustained release. NIH3T3 cell was used to evaluate the biocompatibility and the promoting proliferation, migration and adhesion abilities of the SC‐Ps‐sEVs. EA.hy926 cell was used to evaluate the stimulating angiogenesis of SC‐Ps‐sEVs. The diabetic wound model was used to investigate the function/role of SC‐Ps‐sEVs hydrogel in promoting wound healing.ResultsA functional hydrogel wound dressing with good mechanical properties, excellent biocompatibility and superior stimulating angiogenesis capacity was designed and facilely fabricated, which could effectively enable full‐thickness skin wounds healing in diabetic rat model.ConclusionsThis work led to the development of SIS, which shows an unprecedented combination of mechanical, biological and wound healing properties. This functional hydrogel wound dressing may find broad utility in the field of regenerative medicine and may be similarly useful in the treatment of wounds in epithelial tissues, such as the intestine, lung and liver.

Schematic illustration showing synthesis of the SC‐Ps scaffold dressing and nanoscale sEVs loaded SC‐Ps scaffold dressing and the potential application of the dressings in diabetic wound healing and skin reconstruction.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis

Bacterial cellulose has been used in the food industry for applications such as low-calorie desserts, salads, and fabricated foods. It has also been used in the paper manufacturing industry to enhance paper strength, the electronics industry in acoustic diaphragms for audio speakers, the pharmaceutical industry as filtration membranes, and in the medical field as wound dressing and artificial skin material. In this study, different types of plastic composite support (PCS) were implemented separately within a fermentation medium in order to enhance bacterial cellulose (BC) production by Acetobacter xylinum. The optimal composition of nutritious compounds in PCS was chosen based on the amount of BC produced. The selected PCS was implemented within a bioreactor to examine the effects on BC production in a batch fermentation. The produced BC was analyzed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), and dynamic mechanical analysis (DMA). Among thirteen types of PCS, the type SFYR+ was selected as solid support for BC production by A. xylinum in a batch biofilm reactor due to its high nitrogen content, moderate nitrogen leaching rate, and sufficient biomass attached on PCS. The PCS biofilm reactor yielded BC production (7.05 g/L) that was 2.5-fold greater than the control (2.82 g/L). The XRD results indicated that the PCS-grown BC exhibited higher crystallinity (93%) and similar crystal size (5.2 nm) to the control. FESEM results showed the attachment of A. xylinum on PCS, producing an interweaving BC product. TGA results demonstrated that PCS-grown BC had about 95% water retention ability, which was lower than BC produced within suspended-cell reactor. PCS-grown BC also exhibited higher T _max compared to the control. Finally, DMA results showed that BC from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young's modulus when compared to the control BC. The results clearly demonstrated that implementation of PCS within agitated fermentation enhanced BC production and improved its mechanical properties and thermal stability.     

Cell therapy for skin wound using fibroblast encapsulated poly(ethylene glycol)-poly(L-alanine) thermogel

As a new application of a thermogel, a poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA) gel encapsulating fibroblasts was investigated for wound healing. The fibroblasts were encapsulated by the temperature sensitive sol-to-gel transition of the polymer aqueous solution. Under the in vitro three-dimensional (3D) cell culture condition, the PEG-L-PA thermogel was comparable with Matrigel for cell proliferation and was significantly better than Matrigel for collagen types I and III formation. After confirming the excellent 3D microenvironment of the PEG-L-PA thermogel for fibroblasts, in vivo wound healing was investigated by injecting the cell-suspended polymer aqueous solution on incisions of rat skin, where the cell-encapsulated gel was formed in situ. Compared with the phosphate buffered saline treated system and the cell-free PEG-L-PA thermogel, the cell-encapsulated PEG-L-PA thermogel not only accelerated the wound closure but also improved epithelialization and the formation of skin appendages such as keratinocyte layer (epidermis), hair follicles, and sebaceous glands. The results demonstrate the potential of thermogels for cell therapy as an injectable tissue-engineering scaffold.