Chitosan-alginate as scaffolding material for cartilage tissue engineering

Tissue compatibility of chitosan-alginate scaffolds was studied in vitro in terms of cell morphology, proliferation, and functionality using HTB-94 cells. The scaffold has an interconnected 3D porous structure, and was fabricated by thermally induced phase separation followed by freeze drying. Cell proliferation on the chitosan-alginate scaffold was found to be faster than on a pure chitosan scaffold. After cell culture for 2 weeks in vitro, the cells on the chitosan scaffold gradually assumed a fibroblast-like morphology while the cells on the chitosan-alginate scaffold retained their spherical morphology throughout the period of study. SDS-PAGE electrophoresis and Western blot assays for proteins extracted from cells grown on scaffolds indicated that production of cartilage-specific collagen type II, a marker for chondrocytic phenotype, increased from week 2 to week 3 on the chitosan-alginate scaffold but decreased on the chitosan scaffold. This study suggested that chitosan-alginate scaffolds promote cell proliferation, enhance phenotype expression of HTB-94 chondrocytes, and may potentially serve as an improved alternative to chitosan scaffolds for cartilage tissue engineering.     

壳聚糖与海藻酸钙双相陶瓷骨支架的机械性能及细胞相容性

BACKGROUND: Hydroxyapatite/β-tricalcium phosphate biphasic ceramic bone has good cell compatibility, but its mechanical properties are poor. OBJECTIVE: To construct chitosan/ or calcium alginate/biphasic ceramic bone scaffolds and to detect their mechanical properties and cytocompatibility. METHODS: Different concentrations of chitosan (2%, 4%, 7%, 10%) or calcium alginate (3%, 4%, 5%, 7%) were mixed with biphasic ceramic bone to prepare chitosan/biphasic ceramic bone scaffold and calcium alginate/biphasic ceramic bone scaffold. Their morphology and structure, coagulation time, anti-dissolution properties, shear force, compressive strength and cell compatibility were detected. RESULTS AND CONCLUSION: (1) Coagulation time: with the concentration increase, the initial and final setting time of these two kinds of composite scaffolds were prolonged to some extent. (2) Scanning electron microscopy: these two kinds of composite scaffolds showed porous microstructures with different pore sizes. (3) Anti-dissolution properties: the calcium alginate/biphasic ceramic bone scaffold (3%, 4%, 5%, 7%) and chitosan/biphasic ceramic bone scaffold (7%, 10%) had good anti-dissolution properties in the liquid. (4) Mechanical strength: with the concentration increase, the shear force and compressive strength of the calcium alginate/biphasic ceramic bone scaffold were reduced. (5) Cell compatibility: the cytotoxicity of chitosan/ or calcium alginate/biphasic ceramic bone scaffolds was graded as 0-1 or 2-3, respectively. These results show that the chitosan/biphasic ceramic bone scaffold has better mechanical properties and cell compatibility than the calcium alginate/biphasic ceramic bone scaffold.      

Potential use of collagen-chitosan-hyaluronan tri-copolymer scaffold for cartilage tissue engineering

A three-dimensional biodegradable porous scaffold plays a vital role in a tissue engineering approach. Collagen, chitosan and hyaluronan (HA) are natural extracellular matrix (ECM) or similarity, and may provide appropriate environment for the generation of cartilage-like tissue. In this study, we prepared a collagen/chitosan/HA tri-copolymer porous scaffold by freezing and lyophilization to evaluate physico-chemical properties of the tri-copolymer scaffold and its capacity to sustain chondrocytes proliferation and differentiation in vitro. The results show that the mechanical strength, the resistance to enzymatic degradation, and the waterblinding capacity were improved when chitosan and hyaluronan were incorporated into a collagen scaffold. After 21 days of culture, the porous scaffold had been surfaced with cartilaginous tissue. DNA and glycosaminoglycan (GAG) contents were significantly higher during culture periods in collagen/ chitosan/hyaluronan matrix compared to collagen alone matrix, and most seeded cells preserved the chondrocytic phenotype during culture within the scaffold. The collagen/chitosan/hyaluronan tri-copolymer scaffold has potential applications in a cartilage tissue engineering scaffold field.     

Hydrogel nerve conduits produced from alginate/chitosan complexes

Nerve conduits (NCs) represent a promising alternative to conventional treatments for peripheral nerve repair. Materials for NC production should be biodegradable, possess adequate mechanical properties, and allow for exchange of nutrients. To this aim, we developed biodegradable NC made of a hydrogel that consisted of the oppositely charged polysaccharides alginate and chitosan. Swelling and permeation studies, as well as rheological measurements, served to characterize the NC. The alginate/chitosan NC showed high water uptake (84% w/w) and permitted permeation of fluorescent-labeled dextrans in a molecular weight dependent manner. The NC fulfilled the mechanical specifications without further crosslinking. The soft NC can be expected to preclude nerve compression (storage modulus of about 40 kPa), but possess sufficient mechanical strength. In combination with remarkable tear resistance, the NC affords easy surgical handling.     

Alginate/lactose-modified chitosan hydrogels: a bioactive biomaterial for chondrocyte encapsulation

A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.     

Evaluation of sodium alginate for bone marrow cell tissue engineering

Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.     

Influence of processing parameters on pore structure of 3D porous chitosan-alginate polyelectrolyte complex scaffolds

Fabrication of porous polymeric scaffolds with controlled structure can be challenging. In this study, we investigated the influence of key experimental parameters on the structures and mechanical properties of resultant porous chitosan-alginate (CA) polyelectrolyte complex (PEC) scaffolds, and on proliferation of MG-63 osteoblast-like cells, targeted at bone tissue engineering. We demonstrated that the porous structure is largely affected by the solution viscosity, which can be regulated by the acetic acid and alginate concentrations. We found that the CA PEC solutions with viscosity below 300 Pa.s yielded scaffolds of uniform pore structure and that more neutral pH promoted more complete complexation of chitosan and alginate, yielding stiffer scaffolds. CA PEC scaffolds produced from solutions with viscosities below 300 Pa.s also showed enhanced cell proliferation compared with other samples. By controlling the key experimental parameters identified in this study, CA PEC scaffolds of different structures can be made to suit various tissue engineering applications.     

Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads

The enzyme bovine carbonic anhydrase (BCA) has been immobilized in the chitosan-alginate system for the first time, to catalyze the conversion of CO2 to HCO3-. Chitosan-coated alginate beads are a biodegradable and environmentally benign matrix, chosen for application of the enzyme in a novel biomimetic CO2 sequestration system. The feasibility of the system and immobilization of the enzyme were demonstrated in our earlier studies. Optimization of the matrix to improve the retention time of the enzyme in an encapsulated form is the subject of the present study. The improvement in the molecular weight cut-off of the beads was accomplished by adjusting the cross-linking conditions, coating composition, and molecular weight of the system. The quantity of enzyme released from the system was measured by a Bio-Rad protein assay. Poly-L-lysine was also used as a coating reagent for comparison purposes. The presence of a coating on the alginate beads was verified by Kjeldahl analyses. The difference in the microstructures of alginate and chitosan/alginate beads was demonstrated by SEM studies. Mineralization of the chitosan/alginate matrix in the presence of CaCO3 was also studied by FT-IR, to assess the possibility of using the beads continuously in a bioreactor.     

Study of bilineage differentiation of human-bone-marrow-derived mesenchymal stem cells in oxidized sodium alginate/N-succinyl chitosan hydrogels and synergistic effects of RGD modification and low-intensity pulsed ultrasound

The level of formation of new bone and vascularization in bone tissue engineering scaffold implants is considered as a critical factor for clinical application. In this study, an approach using an RGD-grafted oxidized sodium alginate/N-succinyl chitosan (RGD–OSA/NSC) hydrogel as a scaffold and low-intensity pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation of new bone and vascularization. An in vitro study of endothelial and osteogenic differentiations of human-bone-marrow-derived mesenchymal stem cells (hMSCs) was conducted to evaluate it. The results showed that RGD–OSA/NSC composite hydrogels presented good biological properties in attachment, proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones; similar results were obtained for alkaline phosphatase activity/staining and mineralized nodule formation assay of osteogenic induction and immunohistochemical test of endothelial induction. The positive synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was observed. These findings suggest that the hybrid use of RGD modification and LIPUS might provide one approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering scaffold implants.     

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.     

Design and fabrication of a biodegradable, covalently crosslinked shape-memory alginate scaffold for cell and growth factor delivery

The successful use of transplanted cells and/or growth factors for tissue repair is limited by a significant cell loss and/or rapid growth factor diffusion soon after implantation. Highly porous alginate scaffolds formed with covalent crosslinking have been used to improve cell survival and growth factor release kinetics, but require open-wound surgical procedures for insertion and have not previously been designed to readily degrade in vivo. In this study, a biodegradable, partially crosslinked alginate scaffold with shape-memory properties was fabricated for minimally invasive surgical applications. A mixture of high and low molecular weight partially oxidized alginate modified with RGD peptides was covalently crosslinked using carbodiimide chemistry. The scaffold was compressible 11-fold and returned to its original shape when rehydrated. Scaffold degradation properties in vitro indicated ～85% mass loss by 28 days. The greater than 90% porous scaffolds released the recombinant growth factor insulin-like growth factor-1 over several days in vitro and allowed skeletal muscle cell survival, proliferation, and migration from the scaffold over a 28-day period. The compressible scaffold thus has the potential to be delivered by a minimally invasive technique, and when rehydrated in vivo with cells and/or growth factors, could serve as a temporary delivery vehicle for tissue repair.     

Organic/inorganic hybrid network structure nanocomposite scaffolds based on grafted chitosan for tissue engineering

We describe the first study of structure-processing-property relationship in organic/inorganic hybrid network structure nanocomposite scaffolds based on grafted chitosan for bone tissue engineering. Chitosan was first grafted with propylene oxide to form hydroxypropylated chitosan, which was subsequently linked with ethylene glycol functionalized nanohydroxyapatite to form an organic/inorganic network structure. The resulting scaffold was characterized by a highly porous structure and significantly superior physico-chemical, mechanical and biological properties compared to pure chitosan. The scaffolds exhibited high modulus, controlled swelling behavior and reduced water uptake, but the water retention ability was similar to pure chitosan scaffold. MTT assay studies confirmed the non-cytotoxic nature of the scaffolds and enabled degradation products to be analyzed. The nanocomposite scaffolds were biocompatible and supported adhesion, spreading, proliferation and viability of osteoblasts cells. Furthermore, the cells were able to infiltrate and colonize into the pores of the scaffolds and establish cell-cell interactions. The study suggests that hydroxypropylation of chitosan and forming a network structure with a nano-inorganic constituent is a promising approach for enhancing physico-chemical, functional and biological properties for utilization in bone tissue engineering applications.     

Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment

Three-dimensional (3D) scaffolds hold great potential for stem cell-based therapies. Indeed, recent results have shown that biomimetic scaffolds may enhance cell survival and promote an increase in the concentration of therapeutic cells at the injury site. The aim of this work was to engineer an original polymeric scaffold based on the respective beneficial effects of alginate and chitosan. Formulations were made from various alginate/chitosan ratios to form opposite-charge polyelectrolyte complexes (PECs). After freeze-drying, the resultant matrices presented a highly interconnected porous microstructure and mechanical properties suitable for cell culture. In vitro evaluation demonstrated their compatibility with mesenchymal stell cell (MSC) proliferation and their ability to maintain paracrine activity. Finally, the in vivo performance of seeded 3D PEC scaffolds with a polymeric ratio of 40/60 was evaluated after an acute myocardial infarction provoked in a rat model. Evaluation of cardiac function showed a significant increase in the ejection fraction, improved neovascularization, attenuated fibrosis as well as less left ventricular dilatation as compared to an animal control group. These results provide evidence that 3D PEC scaffolds prepared from alginate and chitosan offer an efficient environment for 3D culturing of MSCs and represent an innovative solution for tissue engineering.     

Fabrication, characterization, and biocompatibility of single-walled carbon nanotube-reinforced alginate composite scaffolds manufactured using freeform fabrication technique

Composite polymeric scaffolds from alginate and single-walled carbon nanotube (SWCNT) were produced using a freeform fabrication technique. The scaffolds were characterized for their structural, mechanical, and biological properties by scanning electron microscopy, Raman spectroscopy, tensile testing, and cell-scaffold interaction study. Three-dimensional hybrid alginate/SWCNT tissue scaffolds were fabricated in a multinozzle biopolymer deposition system, which makes possible to disperse and align SWCNTs in the alginate matrix. The structure of the resultant scaffolds was significantly altered due to SWCNT reinforcement, which was confirmed by Raman spectroscopy. Microtensile testing presented a reinforcement effect of SWCNT to the mechanical strength of the alginate struts. Ogden constitutive modeling was utilized to predict the stress-strain relationship of the alginate scaffold, which compared well with the experimental data. Cellular study by rat heart endothelial cell showed that the SWCNT incorporated in the alginate structure improved cell adhesion and proliferation. Our study suggests that hybrid alginate/SWCNT scaffolds are a promising biomaterial for tissue engineering applications.     

Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads

The enzyme bovine carbonic anhydrase (BCA) has been immobilized in the chitosan-alginate system for the first time, to catalyze the conversion of CO₂ to HCO₃-. Chitosan-coated alginate beads are a biodegradable and environmentally benign matrix, chosen for application of the enzyme in a novel biomimetic CO₂ sequestration system. The feasibility of the system and immobilization of the enzyme were demonstrated in our earlier studies [1–3]. Optimization of the matrix to improve the retention time of the enzyme in an encapsulated form is the subject of the present study. The improvement in the molecular weight cut-off of the beads was accomplished by adjusting the crosslinking conditions, coating composition, and molecular weight of the system. The quantity of enzyme released from the system was measured by a Bio-Rad protein assay. Poly-L-lysine was also used as a coating reagent for comparison purposes. The presence of a coating on the alginate beads was verified by Kjeldahl analyses. The difference in the microstructures of alginate and chitosan/alginate beads was demonstrated by SEM studies. Mineralization of the chitosan/alginate matrix in the presence of CaCO₃ was also studied by FT-IR, to assess the possibility of using the beads continuously in a bioreactor.     

京尼平交联3D打印胶原/壳聚糖支架的表征北大核心

背景:胶原/壳聚糖支架需交联才能达到相应力学性能,有研究表示调节交联剂浓度可以在一定范围内调控支架的理化性能。目的:探究京尼平浓度对胶原/壳聚糖支架理化性能的影响,制备理化性能可调节的组织工程支架。方法:将胶原和壳聚糖粉末分别溶于弱酸后混合均匀,作为打印墨水,利用生物3D打印机低温打印胶原支架与胶原/壳聚糖支架,经冻干、中和处理后分别以1,3,5 mmol/L的京尼平进行交联。检测各组支架的表观结构稳定性、抗拉能力、溶胀性能、降解性能与生物相容性。结果与结论:①将支架在PBS中浸泡3 d后,对比未交联的冻干支架,交联后胶原支架表面维持规则的孔结构,但是支架出现明显变形;交联后胶原/壳聚糖支架表面结构规则,仅1 mmol/L京尼平交联的胶原/壳聚糖支架存在轻微变形。②随着京尼平浓度的增加,各组支架的力学性能增加,并且对应交联浓度下的胶原/壳聚糖支架力学性能好于胶原支架。③随着京尼平浓度的增加,胶原支架的溶胀率下降,胶原/壳聚糖支架的溶胀率无明显变化。④浸泡于胶原酶溶液中后,不同浓度京尼平交联的胶原支架在1 h内被完全降解,胶原/壳聚糖支架的降解速率随京尼平浓度的增加而降低,均呈现先快速后平缓的趋势。⑤将骨髓间充质干细胞接种于各组交联支架3 d后,1,3 mmol/L京尼平交联的胶原/壳聚糖支架(或胶原支架)上的细胞数量明显多于5 mmol/L京尼平交联的胶原/壳聚糖支架(P<0.05)。⑥结果表明,京尼平可在一定范围调节胶原/壳聚糖支架理化性能,其中3 mmol/L京尼平交联的胶原/壳聚糖支架具有较好的力学性能、抗酶解能力与生物相容性。     

Developing an alginate/chitosan hybrid fiber scaffold for annulus fibrosus cells

A fibrous scaffold made of alginate or alginate/chitosan was fabricated for annulus fibrosus (AF) cell culture using a wet-spinning and lyophilization technique. The scaffolds were evaluated using several in vitro tests. Scanning electron microscopy showed the scaffold fibers generally aligned in one direction with individual fiber diameters varying between 40-100 microm. The alginate/chitosan hybrid scaffold exhibited a slower degradation rate, while both scaffold types did not display any cyto-toxicity to 3T3 fibroblasts and could maintain canine AF cell growth. The AF cells retained their spherical shape within the fibrous scaffold at the beginning of the culture period and formed into cell clusters at later times. Specific extracellular matrix molecules, including collagen I, collagen II, and aggrecan, could be detected in the AF cell clusters. These results demonstrate the feasibility of using this hybrid alginate/chitosan scaffold for AF cell culture, and the potential application for intervertebral disc tissue engineering.     

Chitosan-alginate films prepared with chitosans of different molecular weights.

Chitosan-alginate polyelectrolyte complex (CS-AL PEC) is water insoluble and more effective in limiting the release of encapsulated materials compared to chitosan or alginate. Coherent CS-AL PEC films have been prepared in our laboratory by casting and drying suspensions of chitosan-alginate coacervates. The objective of this study was to evaluate the properties of the CS-AL PEC films prepared with chitosans of different molecular weights. Films prepared with low-molecular-weight chitosan (Mv 1.30 x 10(5)) were twice as thin and transparent, as well as 55% less permeable to water vapor, compared to films prepared with high-molecular-weight chitosan (Mv 10.0 x 10(5)). It may be inferred that the low-molecular-weight chitosan reacted more completely with the sodium alginate (M(v) 1.04 x 10(5)) than chitosan of higher molecular weight. A threshold molecular weight may be required, because chitosans of Mv 10.0 x 10(5) and 5.33 x 10(5) yielded films with similar physical properties. The PEC films exhibited different surface properties from the parent films, and contained a higher degree of chain alignment with the possible formation of new crystal types. The PEC films exhibited good in vitro biocompatibility with mouse and human fibroblasts, suggesting that they can be further explored for biomedical applications.     

Preparation and characterization of a chitosan/galactosylated hyaluronic acid/heparin scaffold for hepatic tissue engineering

Cell culture microenvironment and hepatocyte-specific three-dimensional tissue-engineering scaffold play important roles for bioartificial liver devices. In the present study, highly porous sponge scaffolds composed of chitosan (CS) and galactosylated hyaluronic acid (GHA, galactose moieties were covalently coupled with hyaluronic acid through ethylenediamine), were prepared by freezing-drying technique. Because the growth factors specifically bind to heparin with a high affinity and biological stability of the growth factors are modulated by heparin. Heparin was added into CS/GHA scaffold under mild conditions. The effects of heparin on the morphology, structure, porosity, mechanical properties of the CS/GHA/heparin scaffold were studied. CS/GHA scaffold containing heparin maintains the porous structure and good mechanical properties. Furthermore, addition of heparin with the growth factors into the scaffold resulted in a significantly improved the microenvironment of cell growth and prolonged liver functions of the hepatocytes such as albumin secretion, urea synthesis and ammonia elimination. These results indicate that this CS/GHA/heparin scaffold is a potential candidate for liver tissue engineering.