Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.     

壳聚糖/介孔生物活性玻璃多孔膜的制备和表征

背景：壳聚糖和介孔生物玻璃都具有良好的生物相容性和止血性能,但壳聚糖止血作用有限,介孔生物玻璃粉体方式止血,给应用带来不便。 目的：制备壳聚糖/介孔生物玻璃复合多孔膜并检测材料的性能。 方法：采用冷冻干燥法制备壳聚糖/介孔生物玻璃复合多孔膜。 结果与结论：通过冷冻干燥法可以实现壳聚糖和介孔生物玻璃的均匀复合。制备的复合多孔膜的孔隙分布较均匀;多孔膜具有很好的吸水性,吸水率的大小与壳聚糖和介孔生物玻璃质量比相关;多孔膜的孔隙率高。     

Preparation and characterization of bioactive mesoporous calcium silicate-silk fibroin composite films

Composite films of bioactive mesoporous calcium silicate (MCS)/silk fibroin (SF) and conventional calcium silicate (CS)/SF were fabricated by the solvent casting method, and the structures and properties of the composite films were characterized. Results of field emission scanning electron microscope (FESEM) indicated that MCS or CS was uniformly dispersed in the SF films. The measurements of the water contact angles suggested that the incorporation of either MCS or CS into SF could improve the hydrophilicity of the composite films, and the former was more effective than the later. The bioactivity of the composite films was evaluated by soaking in a simulated body fluid (SBF), and the formation of a hydroxycarbonate apatite (HCA) layer was determined by XRD and FT-IR. The results showed that the MCS/SF composite films have significantly enhanced apatite-forming bioactivity compared with the CS/SF composite films owing to the highly specific surface area and pore volume of MCS. In vitro cell attachment and proliferation tests showed that the MCS/SF composite film was a good matrix for the growth of MG63 cells. Consequently, the MCS/SF composite film possessed excellent physicochemical and biological properties, indicating its potential application for bone tissue engineering by designing 3D scaffolds according to its corresponding composition.     

Facile and large-scale synthesis of curcumin/PVA hydrogel: effectively kill bacteria and accelerate cutaneous wound healing in the rat

The complicated synthesis procedure and limited preparation size of hydrogel inhibit its clinical application. Therefore, a facile preparation method for large-size hydrogel is required. In this study, a series of curcumin (Cur)/polyvinyl alcohol (PVA) hydrogel in a large size with different Cur concentrations is prepared by a facile physical-chemical crosslinking. The physicochemical properties, antibacterial performance and accelerating wound healing ability are evaluated with the aim of attaining a novel and effective wound dressing. The results show that the as-prepared hydrogel with the optimal Cur to PVA volume ratio of 1:5 (20% Cur/PVA) exhibits the best antibacterial abilities to E. coli (85.6%) and S. aureus (97%) than other hydrogels. When the volume ratio of Cur to PVA is 1:10 (10% Cur/PVA), the hydrogel can significantly accelerate the wound healing in rats, and successfully reconstruct intact and thickened epidermis during 14 day of healing of impaired wounds after histological examination. In one word, the present approach can shed new light on designing new type of hydrogels with promising applications in wound dressing.     

Fabrication and characterization of phlorotannins/poly (vinyl alcohol) hydrogel for wound healing application

Abstract

Phlorotannins (PH) derived from brown algae have been shown to have biological effects. However, the application of PH in biomedical materials has not been investigated. Here, we investigated the effects of PH on normal human dermal fibroblast (NHDF) proliferation and fabricated a composite hydrogel consisting PH and poly (vinyl alcohol) (PVA) (PVA/PH) by a freezing-thawing method for wound healing applications. Cell proliferation was significantly higher in the PH-treated (0.01 and 0.02%) cells than in non-treated cells. Based on the mechanical properties, the PVA/PH hydrogel had a significantly increased swelling ratio and ultimate strain compared to the PVA hydrogel, but the ultimate tensile strength and tensile modulus were decreased. Additionally, cell attachment and proliferation on the composites were evaluated using NHDFs. The results showed that after 1 and 5 days, cell attachment and proliferation were significantly increased on the PVA/PH hydrogel compared with that on the PVA hydrogel. The findings from this study suggest that the PVA/PH hydrogel may be a candidate biomedical material for wound healing applications.     

Fabrication and characterization of novel bacterial cellulose/alginate/gelatin biocomposite film

Hydrogels from bacterial, algal, and animal cells—bacterial cellulose (BC), alginate, and gelatin, respectively—were combined to fabricate a biocomposite film (BCAGG) via an eco-friendly casting technique. In addition, glycerol was added as a plasticizer to improve the elasticity and water absorption capacity of the film. In this study, BC pellicles were simply deconstructed into fibrils suspension and then reconstructed into films with a supplement of alginate, gelatin and glycerol. The physical appearance of fabricated films resembled native BC but possessed improved ductility, enhanced flexibility, higher water uptake ability and better biocompatibility. The film was found to resist tearing under suture pullout strength in a hydrated state. In vitro cytotoxicity tests showed that the film was cytocompatible. A cell study using a human keratinocyte culture demonstrated enhanced cell adhesion, spreading, and proliferation on the BCAGG film compared with BC/alginate film. The BCAGG film therefore has significant potential for use in biomedical applications, particularly in dermal treatment, skin tissue regeneration, and wound healing.     

Characteristics of calcium sulfate/gelatin composite biomaterials for bone repair

A novel hybrid biomaterial composed of calcium sulfate (CS) and gelatin (GEL) was prepared with the potential of being used as bone filler or scaffold owing to its osteoconduction. Such composite biomaterial, cross-linked or un-cross-linked, could provide a suitable absorbing rate and prevent the CS crystals migrating from the implant for tissue engineering. The structure of the composite was analyzed with infrared (IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). The results indicated that the crystal pattern of CS was affected by the addition of GEL. The GEL part affected the development of the CS dihydrate (CSD) crystal by slowing the conversion from CS hemihydrate (CSH) to CSD; thus, the composite actually contained CSD, CSH and GEL. The compressive strength of the CS/CLGEL composite was also investigated. The compressive strength was correlated to the weight proportions of CS in the CS/cross-linked GEL (CS/CLGEL) composite, and the highest compressive strength of 82 MPa was obtained for the composite containing 40 wt% CS. The in vitro absorption test and the SEM results showed that a porous scaffold was formed in situ with the absorption of CS in the CS/CLGEL composite in a certain time. Therefore, the CS/CLGEL composite material can be used as an in situ porous scaffold with a high initial mechanical strength, and the remaining porous GEL scaffold will enable further in-growth of cells. Human osteoblasts were cultured in contact with the CS/CLGEL composite and the primary results suggested that human osteoblasts could attach and spread on the surface of CS/CLGEL films. The preliminary animal model experiment was operated for assessing the potential of the CS/CLGEL composite as a biodegradable bone substitute. The primary results showed that the CS/CLGEL composite filler could promote new bone in-growth, which will stimulate further study.     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

Specific interactions between human fibroblasts and particular chondroitin sulfate molecules for wound healing

The chondroitin sulfates (CSs) constitute an important group of biomacromolecules in the extracellular matrix. However, limited information is available about their specific biological functions. This study aimed to define the interactions between cells and various types of CS. The effects of CSs on cellular activities and the cell cycle were evaluated using cell culture, RNA interference, real-time polymerase chain reaction, flow cytometry, wound healing and contraction models. The results showed that C-6-S promoted both cell proliferation and adhesion, while C-4-S promoted proliferation but inhibited adhesion. Moreover, knockdown of chondroitin inhibited cell proliferation and migration, as well as arresting cells in the G₂/M phase. Also, both C-4-S and C-6-S promoted wound closure in a two-dimensional wound model, whereas only C-6-S inhibited wound contraction in a three-dimensional wound model. This study illustrates that the interaction between cells and different CSs are specific and sulfate-group-dependent. These findings provide useful information for better applications of CSs for wound healing.     

Characterization and ornidazole release in vitro of a novel composite film prepared with chitosan/poly(vinyl alcohol)/alginate

To create a moist environment for rapid wound healing, a new C-P-A film with sustained antibacterial capacity had been developed by the casting/solvent evaporation method. This new type of C-P-A film consists of a chitosan top layer and sodium alginate sublayer separated by an ornidazole-incorporated poly(vinyl alcohol) layer, exhibited perfect binding characteristics among the three layers. Physical characterization of the C-P-A film showed that the triple-layerd film had excellent light transmittance, control of water vapor transmission rate, and fluid drainage ability promotion, compared with the single-layer film. From the in vitro release studies, about 90% of OD was released from the composite films within 60 min, and no significant difference was observed in cumulative release percentage with increases in the drug content. The composite film at low concentration of OD (1.0 mg/cm2) showed effective antimicrobial activity in the cultures of Staphylococcus aureus and Escherichia coli in agar plates. The results obtained in this work indicated that the new type of C-P-A composite film incorporated with ornidazole has the potential for wound dressing application.     

Effect of xenogenic immobilized bone matrix on the course of wound process

The effect of xenogenic demineralized bone matrix as a component of film coating on healing of a full-thickness skin wound was studied. Regenerative processes were more intensive in wounds dressed with a complex film containing demineralized bone matrix than in uncovered wounds and wounds covered with films without bone matrix. Film coating with antibiotics suppressed the development of wound microflora. In vitro experiments demonstrated different effects of demineralized bone matrix on some enzymes in rat skin and liver homogenates. Biological activity of xenogenic demineralized bone matrix as a component of wound coating is shown. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 448–450, April, 2006     

Proliferation of Mesenchymal Stem Cell on Chitosan Films Associated with Convex Micro-topography

Topographical micro-structures of material surfaces are known to influence cell behavior. In this study, alignment and proliferation of mesenchymal stem cells (MSCs) on chitosan (CS) films with different microtopography was investigated. A modified solvent–non-solvent method was used to produce micro-well polystyrene films which were used as templates for preparation of micro-hilled CS films. The alignment and proliferation of MSCs on CS films were characterized by scanning electron microscopy and the MTT assay, respectively. Polystyrene films with different micro-well diameters were produced under different conditions. We successfully produced CS films with micro-hills of 10 μm (CS10) and 5–30 μm (CS5-30) by a molding approach. MSCs cultured on CS10 covered the tightly packed micro-hills and flattened polygonally. In contrast, MSCs on CS5-30 mostly adhered between loosely packed micro-hills and demonstrated elongated extensions. The proliferation rate of MSCs on CS5-30 was highest on day 4 (66% on CS5-30 versus 46% on CS10 and 24% on smooth CS) and day 8 (106% on CS5-30 versus 38% on CS10 and 72% on smooth CS). It is reasonable to suggest that spatially and dimensionally dispersed micro-hills on CS films facilitate the alignment and proliferation of MSCs. This may be used to physically modify CS for tissue-engineering applications.     

Quercetin incorporated collagen matrices for dermal wound healing processes in rat

We have been developing antioxidants incorporated collagen matrix as a novel biomaterial for various biomedical applications. In this study we made use of quercetin incorporated collagenous matrix for dermal wound healing in rat. Quercetin incorporated collagen (QIC) treated groups were compared with control and collagen (CS) treated animals. QIC treated animal showed a better healing when compared to control and CS treated wound. The biochemical parameters like hydroxyproline, protein, uronic acid content in the healing wound, revealed that there is an increase in proliferation of cells in quercetin treated groups when compared to CS group and there is considerable increase in wound contraction when compared to CS treated group. In addition we adapted the antioxidant assay using 2,2'-azobisisobutryonitrile (AIBN) to assess in vitro antioxidant activity of QIC. The antioxidant studies indicate QIC quench the radicals more efficiently. These results suggested that quercetin incorporated collagen matrix could be a novel dressing material for dermal wound healing.     

Biosynthetic wound coatings as susbtrates for cell growth

Experimental studies of composite materials formed on the basis of fluorine-containing latex and bioactive polysaccharides showed that physicochemical properties of composite materials and their adhesion characteristics can be modulated by variations of polysaccharide-latex ratio and the nature of polysaccharides. The ratio of components ensuring the formation of biosynthetic films that meet the standards for modern wound coating and maintain adhesion and growth of substrate-dependent mammalian cells was determined. These materials can considerably increase the efficiency of treatment of extensive and deep skin wounds in cases when application of cell cultures is indicated. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 52–58, January, 2007     

Biocompatibility evaluation of dicalcium phosphate/calcium sulfate/poly (amino acid) composite for orthopedic tissue engineering in vitro and in vivo

In vitro cytocompatibility of ternary biocomposite of dicalcium phosphate (DCP) and calcium sulfate (CS) containing 40 wt% poly (amino acid) (PAA) was evaluated using L929 ?broblasts and MG-63 osteoblast-like cells. Thereafter, the biocompatibility of biocomposite in vivo was investigated using an implantation in muscle and bone model. In vitro L929 and MG-63 cell culture experiments showed that the composite and PAA polymer were noncytotoxic and allowed cells to adhere and proliferate. The scanning electron microscope (SEM) confirmed that two kinds of cells maintained their phenotype on all of samples surfaces. Moreover, the DCP/CS/PAA composite showed higher cellular viability than that of PAA; meanwhile, the cell proliferation and ALP activity were much higher when DCP/CS had added into PAA. After implanted in muscle of rabbits for 12 weeks, the histological evaluation indicated that the composite exhibited excellent biocompatibility and no inflammatory responses were found. When implanted into bone defects of femoral condyle of rabbits, the composite was combined directly with the host bone tissue without fibrous capsule tissue, which shown good biocompatibility and osteoconductivity. Thus, this novel composite may have potential application in the clinical setting.     

壳聚糖／魔芋葡甘聚糖共混膜细胞毒性和机械性能研究

目的利用天然高分子材料壳聚糖（CS）和魔芋葡甘聚糖（KGM）制备两者含量不同比例的生物膜,测定其细胞毒性和机械性能。方法流延法制备CS与KGM含量不同比例的混合膜。并将其与人脐带间充质干细胞联合培养,观察其细胞毒性：测定其断裂伸长率、拉伸强度及吸水率。结果CS/KGM含量比例分别为3：0、2：1、1：1和1：2制备的8种共混膜,具有良好的细胞毒性：膜平均厚度0．110mm,拉伸强度为35．84～48．17MPa,断裂伸长率为4．84％-6．25％,吸水率均随着CS含量增加而增加。结论CS／KGM含量比为3：0、2：1、1：1和1：2制备的共混膜,都具有良好的细胞毒性,初步具备了引导组织再生膜的基本性能。     

Bioactivity and mechanical properties of collagen composite membranes reinforced by chitosan and β-tricalcium phosphate

In this study, we analyzed the effects of varying concentrations of chitosan (CS) and β-tricalcium phosphate [β-TCP, Ca(3) (PO(4) )(2) ] on the mechanical and cell-adhesion properties of a collagen (CG) matrix for use in guided bone regeneration (GBR). Three different CS concentrations (0.5, 1, and 2%) and five different contents of β-TCP (0, 17, 29, 38, and 44%) were investigated. The composite membranes were analyzed by scanning electron microscopy and cell-adhesion, flexural-strength, and tear-strength assays. The results show that the cell-adhesion and mechanical properties of the composite membranes improved with increasing β-TCP and CS contents, yielding suitable levels of the adhesion of cells and adequate mechanical stability to ensure successful GBR. The CS adhered to the microsized β-TCP, which was distributed uniformly in the composite membranes. The β-TCP and CS have no negative effect on the cell morphology, viability, and proliferation and possess good biocompatibility. This study demonstrates that β-TCP/CS/CG composite membranes are good candidates for GBR membranes in future applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

创面复合液体敷料在皮肤创伤中的治疗研究

基于羧甲基壳聚糖的高生物相容性及聚乙烯醇缩丁醛的快速成膜,构建了一种创面复合液体敷料,并对其应用效果进行评价。首先应用羧甲基壳聚糖 (CMC)、聚乙烯醇缩丁醛 (PVB)和乙醇溶液,按照一定的比例,制备创面复合液体敷料。对其防水、透气、阻菌、细胞毒性进行性能研究及安全性评价。然后选择健康成年Sprague-Dawley(SD)大鼠40只,雌雄各半,构建大鼠创面模型,并将含有不同浓度的羧甲基壳聚糖(1.0、10.0、30.0 mg/mL)应用在其创面上,通过日常观察、HE染色等,研究创面复合液体敷料在皮肤创伤中的治疗效果。结果显示创面复合液体敷料上层膜液在1.8～2.3 mm 之间具有很好的防水透气性、阻菌性及生物兼容性。运用在动物模型上可以看到,第7 d含有10.0、30.0 mg/mL CMC组的大鼠创面愈合率分别为65.42%、67.38%,明显高于对照组且存在显著性差异(P< 0.01),14 d后含有10.0、30.0 mg/mL CMC组的大鼠创面愈合率已达到100%。HE 染色的第七天含有10.0、30.0 mg/mL CMC的创面复合液体敷料组中观察到有复层扁平的表皮和真皮的胶原纤维,第12 d组织开始出现内陷结构,含有厚实、粗糙胶原纤维的正常真皮与较薄的胶原纤维水平连接,表皮的复层鳞状上皮远远大于对照组中的三到四层。而且创面连接真皮结缔组织,它的表皮构成非常接近于正常皮肤组织。构建的创面复合液体敷料(10.0 mg/mL CMC)具备良好的防水、透气、阻菌性以及生物兼容性,随着羧甲基壳聚糖浓度的升高,治疗急性创面的效果越好,创面复合液体敷料能够对创面起到早期保护和促进愈合的效果。.     

脂肪间充质干细胞聚合物-细胞外基质复合物补片修复兔外耳道全层皮肤损伤的实验研究

目的 探讨脂肪间充质干细胞聚合物（adipose-derived stem cell aggregates,ADSC aggregates）-细胞外基质（extracellular matrix,ECM）复合物补片（ADSC aggregates-ECM）在兔外耳道全层皮肤损伤修复中的应用。 方法 将新西兰大白兔随机分为3组,空白组、对照组、实验组,每组10只。在大白兔的耳腹侧耳根处构建外耳道内壁全层皮肤缺损模型。对照组、实验组采用内植法将ECM支架、ADSC aggregates-ECM支架铺于明胶海绵与创面边缘之间,缝合伤口前确保支架完全覆盖创面。空白组不做处理。术后14 d,比较各组动物创面愈合率、创面组织细菌菌落阳性比例;免疫组化检测各组创面组织中细胞核增殖抗原（proliferating cell nuclear antigen,Ki67）的表达;免疫印迹实验检测各组创面组织中白细胞介素1β（IL-1β）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）的表达。 结果 与空白组相比,实验组、对照组动物的创面愈合率、Ki67阳性细胞比例明显升高,细菌菌落阳性比例、创面组织中IL-1β、TNF-α的表达明显下降,差异均有统计意义（P<0.05）;与对照组相比,实验组动物的创面愈合率明显升高、Ki67阳性细胞比例明显升高,细菌菌落阳性比例、创面组织中IL-1β、TNF-α的表达明显下降,差异均有统计意义（P<0.05）。 结论 ADSC aggregates-ECM补片能明显促进大白兔外耳道内壁全层皮肤缺损的修复。     

Effect of chitosan film containing basic fibroblast growth factor on wound healing in genetically diabetic mice

Basic fibroblast growth factor (bFGF) has been shown to stimulate wound healing. However, consistent delivery of bFGF has been problematic. We studied the stability of bFGF incorporated into a chitosan film as a delivery vehicle for providing sustained release of bFGF. The therapeutic effect of this system on wound healing in genetically diabetic mice was determined as a model for treating clinically impaired wound healing. A chitosan film was prepared by freeze-drying hydroxypropylchitosan (a water-soluble derivative of chitosan) acetate buffer solution. Growth factor was incorporated into films before drying by mixing bFGF solution with the hydroxypropylchitosan solution. bFGF activity remained stable for 21 days at 5 degrees C, and 86.2% of activity remained with storage at 25 degrees C. Full-thickness wounds were created on the backs of diabetic mice, and chitosan film or bFGF-chitosan film was applied to the wound. The wound was smaller in after 5 days in both groups, but the wound was smaller on day 20 only in the bFGF-chitosan group. Proliferation of fibroblasts and an increase in the number of capillaries were observed in both groups, but granulation tissue was more abundant in the bFGF-chitosan group. These results suggest that chitosan itself facilitates wound repair and that bFGF incorporated into chitosan film is a stabile delivery vehicle for accelerating wound healing.