Implantable biodegradable sponges: effect of interpolymer complex formation of chitosan with gelatin on the release behavior of tramadol hydrochloride

The effect of interpolymer complex formation between positively charged chitosan and negatively charged gelatin (Type B) on the release behavior of tramadol hydrochloride from biodegradable chitosan-gelatin sponges was studied. Mixed sponges were prepared by freeze-drying the cross-linked homogenous stable foams produced from chitosan and gelatin solutions where gelatin acts as a foam builder. Generation of stable foams was optimized where concentration, pH of gelatin solution, temperature, speed and duration of whipping process, and, chitosan-gelatin ratio drastically affect the properties and the stability of the produced foams. The prepared sponges were evaluated for their morphology, drug content, and microstructure using scanning electron microscopy, mechanical properties, uptake capacity, drug release profile, and their pharmacodynamic activity in terms of the analgesic effect after implantation in Wistar rats.

It was revealed that whipping 7% (w/w) gelatin solution, of pH 5.5, for 15 min at 25°C with a stirring speed of 1000 rpm was the optimum conditions for stable gelatin foam generation. Moreover, homogenous, uniform chitosan-gelatin foam with small air bubbles were produced by mixing 2.5% w/w chitosan solution with 7% w/w gelatinsolution in 1:5 ratio. Indeed, polyionic complexation between chitosan and gelatin overcame the drawbacks of chitosan sponge mechanical properties where, pliable, soft, and compressible sponge with high fluid uptake capacity was produced at 25°Cand 65% relative humidity without any added plasticizer. Drugreleasestudies showed a successful retardation of the incorporated drug where the t_50% values of the dissolution profiles were 0.55, 3.03, and 4.73 hr for cross-linked gelatin, un-cross-linked chitosan-gelatin, and cross-linked chitosan-gelatin sponges, respectively. All the release experiments followed Higuchi's diffusion mechanism over 12 hr. The achieved drug prolongation was a result of a combined effect of both cross-linking and polyelectrolyte complexation between chitosan and gelatin. The analgesic activity of the implanted tramadol hydrochloride mixed chitosan-gelatin sponge showed reasonable analgesic effect that was maintained for more than 8 hr. Therefore, the use of chitosan and gelatin together appears to allow the formulator to manipulate both the drug release profiles and the mechanical properties of the sponge that could be effectively implanted.     

壳聚塘-明胶共混膜的制备及机械性能测试

以共混方法制备了壳聚糖-明胶共混薄膜,并对共混薄膜的机械性能进行了测试。研究表明:壳聚糖-明胶共混膜的混合比例可以改变共混膜的力学强度,共混膜随明胶含量增加其弹性模量和拉伸断裂应力逐渐减小,壳聚糖溶液与明胶共混的体积比为4∶6时,拉伸强度达到最小值,而断裂伸长率在壳聚糖与明胶体积比为6∶4的时候达到了最大值。     

Preparation and bioactivity evaluation of hydroxyapatite-titania/chitosan-gelatin polymeric biocomposites

Biocomposites consisting of hydroxyapatite (HA) and natural polymers such as collagen, chitosan, chitin,and gelatin have been extensively investigated. However, studies on the combination of HA and titania with chitosan and gelatin have not been conducted yet. Novel biodegradable hydroxyapatite-titania/chitosan-gelatin polymeric composites were fabricated. In this work, our results are concerning with the preparation and characterization of HA powder and HA filler containing titania powder (10 and 30%) with a chitosan and gelatin copolymer matrix. The present research focuses on characterizing the structure of this novel class of biocomposites. Thermogravimetric analysis (TGA), X-ray diffraction (XRD), and Fourier Transformed Infrared Spectroscopy (FT-IR), Scanning electron microscopy (SEM-EDAX) were employed to assess the produced composites. The mechanical properties in terms of compressive strength and hardness test were also investigated. The in vitro study in simulated body fluid (SBF) was performed to assess the bioactivity of composites. The results proved that apatite resembling natural bone are formed faster and greater in the case the composite of HA containing 10% titania into chitosan-gelatin polymeric matrix when they are soaked in a simulated body fluid (SBF) than the composite containing 30% titania. The biocomposites containing HA with 10% titania are expected to be attractive for bioapplications as bone substitutes and scaffolds for tissue engineering in future.     

聚乳酸复合纳米纤维创面敷料的制备及性能

采用静电纺丝技术制备了聚乳酸(PLLA)纳米纤维毡、壳聚糖/PLLA纳米纤维毡和明胶/PLLA纳米纤维毡。利用扫描电镜(SEM)、图像分析软件等手段研究了纳米纤维微观形貌,并研究各种创面敷料的吸水性、保水性和水蒸汽通透性等性能。结果表明,壳聚糖/PLLA、明胶/PLLA复合纳米纤维毡的吸水性和保水性有显著提高,水蒸汽通透性略有下降,是理想的创面敷料材料。     

Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly (P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.     

Preparation and properties of a chitosan-based carrier of corneal endothelial cells

A novel chitosan-based membrane that was made of hydroxypropyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane, such as transparency, equilibrium water content, permeability, mechanical properties, protein absorption ability, hydrophilicity and surface morphology, were determined. To study the effects of the membrane on cell attachment and growth, rabbit corneal endothelial cells were cultured on this artificial membrane. The biodegradability and biocompatibility of the blend membrane were in vivo evaluated by its implantation into the muscle of the rats. Glucose permeation results demonstrated that the blend membrane had higher glucose permeability than natural human cornea. Scanning electron microscopy (SEM) analysis of the membranes demonstrated that no fibrils were observed. As a result, the optical transparency of the membrane was as good as the natural human cornea. The average value of tensile strength of the membrane was 13.71 MPa for dry membrane and 1.48 MPa for wet membrane. The value of elongation at break of the wet was 45.64%. The cultured rabbit corneal endothelial cells formed a monolayer on the blend membrane which demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow. In addition, the membranes in vivo showed a good bioabsorption property. The mild symptoms of inflammation at sites of treatment could be resolved as the implant was absorbed by the host. The results of this study demonstrated that the hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.     

The use of gas plasma treatment to improve the cell-substrate properties of a skin substitute made of poly(ether)/poly(ester) copolymers

The objective of this study was to improve the cell-substrate interactions between skin cells and a biodegradable elastomeric matrix, part of a cell-seeded skin substitute for the treatment of large-scale deep dermal skin defects (i.e. burn wounds). Polyactive, a synthetic biodegradable elastomeric copolymer, was used as the constituent of the bilayered matrix. It consists of a dense toplayer seeded with epidermal keratinocytes and a macroporus underlayer, which may be seeded with dermal fibroblasts. Although former studies demonstrated the suitability of the copolymer as a substrate for these skin-derived cell types, we aimed to improve the bilayered matrix' seeding efficiency. Using radio frequency glow discharge (RFGD) pretreatment significantly improved the adherence and growth of SVK14 epithelial cells seeded on the dense copolymeric toplayers and on non-tissue grade plastics, approximating tissue culture polystyrene values. With scanning electron microscopy (SEM), early epithelial cell-substrate interactions were investigated. Seeding efficiency and growth of dermal fibroblasts into the porous underlayers was improved as was visualized with the SEM and confocal scanning laser microscopy. It is concluded that RFGD pretreatment is a cost-effective measurement for improving cell-substrate properties of the investigated copolymers.     

Fabrication and characters of a corneal endothelial cells scaffold based on chitosan

A novel chitosan-based membrane that made of hydroxyethyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane including transparency, equilibrium water content, ion and glucose permeability were determined. The results showed that the optical transparency of the membrane was as good as the natural human cornea. The water content of this scaffold was 81.32% which was remarkably close to the native cornea. The membrane had a good ion permeability and its glucose permeability was even higher than natural human cornea. The cultured rabbit corneal endothelial cells formed a monolayer on the membrane. The results demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow on it. In addition, the membranes in vivo could be degraded steadily with less inflammation and showed a good histocompatibility. These results demonstrated that the hydroxyethyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.     

PAA influence on chitosan membrane calcification

This work discusses the influence of polyacrylic acid (PAA) on chitosan in vitro calcification. Calcium phosphate deposition on porous and dense (non-porous) chitosan membranes treated or not with PAA has been studied using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), X-ray diffraction, potentiometric titration and attenuated total reflectance infrared spectroscopy (ATR-IR). The experiments were carried out by soaking chitosan membranes in simulated body fluids (SBF), 1.0 and 1.5 times the human serum salt concentration, at 36.5 °C during 7 days at pH 7.4 or 7.8. SEM micrographs showed that the use of these growth solutions resulted in substrates with significant coverage with calcium phosphate. PAA led to a higher coverage even at lower pH (7.4), but the morphological and chemical nature of deposits was remarkably different if compared to deposits on pure chitosan membranes. Although PAA has shown to promote a more intense deposition, probably by offering ion-binding sites adequate for nucleation, it does not necessarily determine a well-defined crystal growth orientation.     

Nanofibers prepared by needleless electrospinning technology as scaffolds for wound healing

Electrospun gelatin and poly-ε-caprolactone (PCL) nanofibers were prepared using needleless technology and their biocompatibility and therapeutic efficacy have been characterized in vitro in cell cultures and in an experimental model of a skin wound. Human dermal fibroblasts, keratinocytes and mesenchymal stem cells seeded on the nanofibers revealed that both nanofibers promoted cell adhesion and proliferation. The effect of nanofibers on wound healing was examined using a full thickness wound model in rats and compared with a standard control treatment with gauze. Significantly faster wound closure was found with gelatin after 5 and 10 days of treatment, but no enhancement with PCL nanofibers was observed. Histological analysis revealed enhanced epithelialisation, increased depth of granulation tissue and increased density of myofibroblasts in the wound area with gelatin nanofibers. The results show that gelatin nanofibers produced by needleless technology accelerate wound healing and may be suitable as a scaffold for cell transfer and skin regeneration.     

Preparation of reduced graphene oxide/gelatin composite films with reinforced mechanical strength

Graphene oxide (GO) was reduced by chitosan/gelatin solution and added to gelatin (Gel) to fabricate reduced graphene oxide/gelatin (RGO/Gel) films by a solvent-casting method using genipin as cross-linking agent. The structure and properties of the films were characterized by scanning electron microscopy (SEM), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA) and UV–vis spectroscopy. The addition of RGO increased the tensile strength of the RGO/Gel films in both dry and wet states, but decreased their elongation at break. The incorperation of RGO also decreased the swelling ability of the films in water. Cell cultures were carried out in order to test the cytotoxicity of the films. The cells grew and reproduced well on the RGO/Gel films, indicating that the addition of RGO has no negative effect on the compatibility of the gelatin. Therefore, the reduced graphene oxide/gelatin composite is a promising biomaterial with excellent mechanical properties and good cell compatibility.     

Fabrication and development of artificial osteochondral constructs based on cancellous bone/hydrogel hybrid scaffold

Using tissue engineering techniques, an artificial osteochondral construct was successfully fabricated to treat large osteochondral defects. In this study, porcine cancellous bones and chitosan/gelatin hydrogel scaffolds were used as substitutes to mimic bone and cartilage, respectively. The porosity and distribution of pore size in porcine bone was measured and the degradation ratio and swelling ratio for chitosan/gelatin hydrogel scaffolds was also determined in vitro. Surface morphology was analyzed with the scanning electron microscope (SEM). The physicochemical properties and the composition were tested by using an infrared instrument. A double layer composite scaffold was constructed via seeding adipose-derived stem cells (ADSCs) induced to chondrocytes and osteoblasts, followed by inoculation in cancellous bones and hydrogel scaffolds. Cell proliferation was assessed through Dead/Live staining and cellular activity was analyzed with IpWin5 software. Cell growth, adhesion and formation of extracellular matrix in composite scaffolds blank cancellous bones or hydrogel scaffolds were also analyzed. SEM analysis revealed a super porous internal structure of cancellous bone scaffolds and pore size was measured at an average of 410 ± 59 μm while porosity was recorded at 70.6 ± 1.7 %. In the hydrogel scaffold, the average pore size was measured at 117 ± 21 μm and the porosity and swelling rate were recorded at 83.4 ± 0.8 % and 362.0 ± 2.4 %, respectively. Furthermore, the remaining hydrogel weighed 80.76 ± 1.6 % of the original dry weight after hydration in PBS for 6 weeks. In summary, the cancellous bone and hydrogel composite scaffold is a promising biomaterial which shows an essential physical performance and strength with excellent osteochondral tissue interaction in situ. ADSCs are a suitable cell source for osteochondral composite reconstruction. Moreover, the bi-layered scaffold significantly enhanced cell proliferation compared to the cells seeded on either single scaffold. Therefore, a bi-layered composite scaffold is an appropriate candidate for fabrication of osteochondral tissue.     

Silk‐based microcarriers: current developments and future perspectives

Cell‐seeded microcarriers (MCs) are currently one of the most promising topics in biotechnology. These systems are supportive structures for cell growth and expansion that allow efficient nutrient and gas transfer between the media and the attached cells. Silk proteins have been increasingly used for this purpose in the past few years due to their biocompatibility, biodegradability and non‐toxicity. To date, several silk fibroin spherical MCs in combination with alginate, gelatin and calcium phosphates have been reported with very interesting outcomes. In addition, other silk‐based three‐dimensional structures such as microparticles with chitosan and collagen, as well as organoids, have been increasingly studied. In this study, the physicochemical and biological properties of these biomaterials, as well as the recent methodologies for their processing and for cell culture, are discussed. The potential biomedical applications are also addressed. In addition, an analysis of the future perspectives is presented, where the potential of innovative silk‐based MCs processing technologies is highlighted.Inspec keywords: biodegradable materials, proteins, calcium compounds, gelatin, biomedical materials, cellular biophysics, molecular biophysicsOther keywords: supportive structures, cell growth, gas transfer, attached cells, silk proteins, biodegradability, nontoxicity, silk fibroin spherical MCs, gelatin, calcium phosphates, silk‐based three‐dimensional structures, chitosan, collagen, physicochemical properties, biological properties, cell culture, silk‐based microcarriers, cell‐seeded microcarriers, biotechnology, efficient nutrient transfer, biocompatibility, alginate, biomedical applications     

Preparation and characterization of pH sensitive sugar mediated (polyethylene glycol/chitosan) membrane

Novel biodegradable membrane based on chitosan matrix was prepared and characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and swelling test. Native sugar, which is commonly used in human life, was utilized to prepare the crosslinked hydrophilic chitosan-polyethylene glycol (PEG) polyblend. According to TGA and FTIR results, the chemical reaction occurred in imine bonds (C=N) between sugar and amino groups in chitosan. Chitosan blending with high swelling capacity of PEG increased the water affinity and reacted with sugar decreased the water affinity. The equilibrium water content (EWC) value of the sugar mediated membrane is in the sequence of sucrose>D-fructose>glucose and they all present much lower water uptake ability than polyblend. The chitosan was not degraded by lysozyme, but all of the sugar-mediated membranes were susceptible to lysozyme. Scanning electron microscope (SEM) morphology shows that the degradation rate not only was controlled by the chemical complexation between sugar and polyblends, but the surface morphology of membranes also has great influence. Sucrose-mediated membrane supports the attachment and growth of NIH 3T3 fibroblasts. The pH-sensitive and well degradable property of the sucrose-mediated membrane can be applied for biomedical application.     

壳聚糖/聚乳酸/聚羟基丁酸酯人工硬脑膜的制备与性能研究

以壳聚糖、聚乳酸、聚(R)-3-羟基丁酸甲酯等为主要成膜物质,用流延法制得新型薄膜材料。研究表明,当壳聚糖∶明胶∶聚乳酸为1∶1∶1时,得到膜的抗拉强度比其他的可生物降解膜都大,达到27.346MPa;当壳聚糖∶明胶∶聚(R)-3-羟基丁酸甲酯为1∶1∶1时,其抗拉强度达到24.363Mpa,防漏性能达到80h。经模拟体内降解研究,在人工脑脊液的环境中浸置44d后,降解率分别为20%左右,降解速度较慢,可以保持其结构6～7周,有利于引导组织再生材料的基本要求。因此该复合膜有望成为医用可生物降解人工硬脑膜。     

壳聚糖改性纤维素共混复合物的制备及性能

利于壳聚糖共混改性纤维素,介绍了共混改性复合物的制备方法,并利用红外光谱(FT-IR)、扫描电子显微(SEM)、x射线衍射(XRD)、热失重分析(TGA)、平衡水分含量及力学性能的测试对共混复合物的特征进行研究.结果表明,在共混复合物中纤维素和壳聚糖具有良好的相容性,没有相分离发生;共混壳聚糖后,随着壳聚糖含量的增加,...     

壳聚糖-硫酸软骨素共混膜与兔角膜基质细胞相容性的研究

为了探讨硫酸软骨素对壳聚糖膜与兔角膜基质细胞相容性的影响,在制备了不同壳聚糖-硫酸软骨素共混膜的基础上,以壳聚糖-硫酸软骨素共混膜作为载体培养兔角膜基质细胞,研究兔角膜基质细胞在共混膜上贴附、生长和代谢的情况,并观察了细胞的形态结构.结果发现,硫酸软骨素的混入可以有效地提高兔角膜基质细胞在膜上的贴附和生长速度,促进蛋白质的代谢,降低共混膜对细胞的损伤,有利于细胞在膜上长成密集单层,预示了以一定比例混入硫酸软骨素可以显著提高壳聚糖膜与兔角膜基质细胞的相容性,壳聚糖-硫酸软骨素共混膜具有作为兔角膜基质细胞培养和移植载体的潜能.     

Chitosan–gelatin biopolymers as carrier substrata for limbal epithelial stem cells

The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH–G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH–G 20:80 membranes than other proportions. Consequently, CH–G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH–G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH–G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.     

Formation of bone-like apatite layer on chitosan fiber mesh scaffolds by a biomimetic spraying process

Bone-like apatite coating of polymeric substrates by means of biomimetic process is a possible way to enhance the bone bonding ability of the materials. The created apatite layer is believed to have an ability to provide a favorable environment for osteoblasts or osteoprogenitor cells. The purpose of this study is to obtain bone-like apatite layer onto chitosan fiber mesh tissue engineering scaffolds, by means of using a simple biomimetic coating process and to determine the influence of this coating on osteoblastic cell responses. Chitosan fiber mesh scaffolds produced by a previously described wet spinning methodology were initially wet with a Bioglass((R))-water suspension by means of a spraying methodology and then immersed in a simulated body fluid (SBF) mimicking physiological conditions for one week. The formation of apatite layer was observed morphologically by scanning electron microscopy (SEM). As a result of the use of the novel spraying methodology, a fine coating could also be observed penetrating into the pores, that is clearly within the bulk of the scaffolds. Fourier Transform Infrared spectroscopy (FTIR-ATR), Electron Dispersive Spectroscopy (EDS) and X-ray diffraction (XRD) analysis also confirmed the presence of apatite-like layer. A human osteoblast-like cell line (SaOs-2) was used for the direct cell contact assays. After 2 weeks of culture, samples were observed under the SEM. When compared to the control samples (unmodified chitosan fiber mesh scaffolds) the cell population was found to be higher in the Ca-P biomimetic coated scaffolds, which indicates that the levels of cell proliferation on this kind of scaffolds could be enhanced. Furthermore, it was also observed that the cells seeded in the Ca-P coated scaffolds have a more spread and flat morphology, which reveals an improvement on the cell adhesion patterns, phenomena that are always important in processes such as osteoconduction.     

体外壳聚糖-明胶-生长因子缓释支架对骨髓间充质干细胞增殖的影响

制备具有缓释功能的壳聚糖-明胶-碱性成纤维细胞生长因子(bFGF)-肝细胞生长因子(HGF)三维大孔支架,探讨两种生长因子在体外对骨髓间充质干细胞(BMSCs)增殖的协同作用。方法:采用冷冻干燥法,将不同比例的壳聚糖、明胶、bFGF和HGF依次混合,使其成为具有一定孔径的三维缓释支架。取SD大鼠乳鼠股骨和胫骨骨髓,分离、培养BMSCs。取生长状态良好的第三代BMSCs,将其接种于96孔板后,加入支架混合培养,5d后进行MTT细胞增殖测定。结果:在与含1μg/mL的bFGF支架混合培养,以及与同时含1μg/mL的bFGF、1μg/mL的HGF支架混合培养后,BMSCs增殖明显(P<0.05),但这二组间无统计学差异(P>0.05)。分别与含0.1μg/mL的bFGF支架、0.01μg/mL的bFGF支架、1μg/mL的HGF支架混合培养后,细胞增殖无统计学意义(P>0.05)。结论:壳聚糖-明胶可作为生长因子缓释支架材料;bFGF具有促进BMSCs增殖的作用,促进作用的大小与加入bFGF的量有关;HGF对BMSCs不具有增殖作用;在实验浓度范围内bFGF和HGF体外促进BMSCs增殖上不具有协同性。