Chitosan–gelatin scaffolds for tissue engineering: Physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP–buffalo embryonic stem cells

The favorable cellular response of newly developed cell line, buffalo embryonic stem (ES) cells to three-dimensional biodegradable chitosan–gelatin composite scaffolds with regard to stem-cell-based tissue engineering is described. Chitosan–gelatin composites were characterized by a highly porous structure with interconnected pores, and the mechanical properties were significantly enhanced. Furthermore, X-ray diffraction study indicated increased amorphous content in the scaffold on the addition of gelatin to chitosan. To develop a transfectant of green fluorescence protein (GFP)–buffalo ES cell, transfection of GFP plasmid to the cell was carried out via the electroporation procedure. In comparison with pure chitosan, cell spreading and proliferation were greater in highly visualized GFP-expressing cell–chitosan–gelatin scaffold constructs. The relative comparison of biological response involving cell proliferation and viability on the scaffolds suggests that blending of gelatin in chitosan improved cellular efficiency. Studies involving scanning electron and fluorescence microscopy, histological observations and flow cytometer analysis of the constructs implied that the polygonal cells attached to and penetrated the pores, and proliferated well, while maintaining their pluripotency during the culture period for 28 days. Chitosan–gelatin scaffolds were cytocompatible with respect to buffalo ES cells. The study underscores for the first time that chitosan–gelatin scaffolds are promising candidates for ES-cell-based tissue engineering.     

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.     

Cultured cell-derived extracellular matrix scaffolds for tissue engineering

Cell-derived extracellular matrix (ECM) scaffolds have received considerable interest for tissue engineering applications. In this study, ECM scaffolds derived from mesenchymal stem cell (MSC), chondrocyte, and fibroblast were prepared by culturing cells in a selectively removable poly(lactic-co-glycolic acid) (PLGA) template. These three types of ECM scaffolds were used for in vitro cultures of MSC and fibroblasts to examine their potential as scaffolds for cartilage and skin tissue engineering. The MSC were cultured in MSC- and chondrocyte-derived ECM scaffolds. The ECM scaffolds supported cell adhesion, promoted both cell proliferation and the production of ECM and demonstrated a stronger stimulatory effect on the chondrogenesis of MSC compared with a conventional pellet culture method. Histological and immunohistochemical staining indicated that cartilage-like tissues were regenerated after the MSC were cultured in ECM scaffolds. Fibroblasts were cultured in the fibroblast-derived ECM scaffolds. Fibroblasts proliferated and produced ECM to fill the pores and spaces in the scaffold. After 2 weeks of culture, a uniform multilayered tissue was generated with homogenously distributed fibroblasts. Cell-derived ECM scaffolds have been demonstrated to facilitate tissue regeneration and will be a useful tool for tissue engineering.     

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.     

Fabrication of polycaprolactone scaffolds using a sacrificial compression-molding process

A method of compression-molding fine-powder blends of polycaprolactone (PCL) and poly(ethylene oxide) (PEO) and subsequently dissolving the PEO phase was investigated to prepare porous PCL scaffolds. Different mixing ratios of the two polymers from 20 to 70% PCL were used to study the effect of the mixing ratio on the morphology formation of the scaffold. The mixing ratio was found to play an important role in affecting the porosity of the scaffold and the size of pores. Murine embryonic stem cell derived osteogenic cells were utilized to test the suitability of these scaffolds in tissue engineering applications. The seeded cells were able to colonize and grow in these scaffolds. Based on the overall consideration of morphology, mechanical performance, and ability for cell attachment and proliferation, the scaffolds with approximately 30-40% PCL appear to be an appropriate choice for tissue engineering. These findings suggest that sacrificial compression-molding of PCL-PEO powder blends can be used in the generation of biocompatible scaffolds with controllable porosity and pore size and may be used for in vitro tissue engineering applications.     

Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis

Combining bovine collagen with chitosan followed by freeze-drying has been shown to produce porous scaffolds suitable for skin and connective tissue engineering applications. In this study collagen extracted from porcine and avian skin was compared with bovine collagen for the production of tissue engineered scaffolds. A similar purity of the collagen extracts was shown by electrophoresis, confirming the reliability of the extraction process. Collagen was solubilized, cross-linked by adding chitosan to the solution and freeze-dried to generate a porous structure suitable for tissue engineering applications. Scaffold porosity and pore morphology were shown to be source dependant, with bovine collagen and avian collagen resulting into the smallest and largest pores, respectively. Scaffolds were seeded with dermal fibroblasts and cultured for 35 days to evaluate the suitability of the different collagen–chitosan scaffolds for long-term tissue engineered dermal substitute maturation in vitro. Cell proliferation and scaffold biocompatibility were found to be similar for all the collagen–chitosan scaffolds, demonstrating their capability to support long-term cell adhesion and growth. The scaffolds contents was assessed by immunohistochemistry and showed increased deposition of extracellular matrix by the cells as a function of time. These results correlate with measurements of the mechanical properties of the scaffolds, since both the ultimate tensile strength and tensile modulus of the cell seeded scaffolds had increased by the end of the culture period. This experiment demonstrates that porcine and avian collagen could be used as an alternative to bovine collagen in the production of collagen–chitosan scaffolding materials.     

In vitro cell infiltration and in vivo cell infiltration and vascularization in a fibrous, highly porous poly(D,L-lactide) scaffold fabricated by cryogenic electrospinning technique

One of the obstacles limiting the application of electrospun scaffolds for tissue engineering is the nanoscale pores that inhibit cell infiltration. In this article, we describe a technique that uses ice crystals as templates to fabricate cryogenic electrospun scaffolds (CES) with large three-dimensional and interconnected pores using poly(D,L-lactide) (PLA). Manipulating the humidity of the electrospinning environment the pore sizes are controlled. We are able to achieve pore sizes ranging from 900 +/- 100 microm(2) to 5000 +/- 2000 microm(2) depending on the relative humidity used. Our results show that cells infiltrated the CES up to 50 microm in thickness in vitro under static culture conditions whereas cells did not infiltrate the conventional electrospun scaffolds. In vivo studies demonstrated improved cell infiltration and vascularization in the CES compared with conventionally prepared electrospun scaffolds. In gaining control of the pore characteristics, we can then design CES that are optimized for specific tissue engineering applications.     

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.     

Structure-process-property relationship of the polar graphene oxide-mediated cellular response and stimulated growth of osteoblasts on hybrid chitosan network structure nanocomposite scaffolds

We here describe the structure-process-property relationship of graphene oxide-mediated proliferation and growth of osteoblasts in conjunction with the physico-chemical, mechanical, and structural properties. Chitosan-graphene network structure scaffolds were synthesized by covalent linkage of the carboxyl groups of graphene oxide with the amine groups of chitosan. The negatively charged graphene oxide in chitosan scaffolds was an important physico-chemical factor influencing cell-scaffold interactions. Furthermore, it was advantageous in enhancing the biocompatibility of the scaffolds and the degradation products of the scaffolds. The high water retention ability, hydrophilic nature, and high degree of interconnectivity of the porous structure of chitosan-graphene oxide scaffolds facilitated cell attachment and proliferation and improved the stability against enzymatic degradation. The cells infiltrated and colonized the pores of the scaffolds and established cell-cell interactions. The interconnectivity of the porous structure of the scaffolds helps the flow of medium throughout the scaffold for even cell adhesion. Moreover, the seeded cells were able to infiltrate inside the pores of chitosan-graphene oxide scaffolds, suggesting that the incorporation of polar graphene oxide in scaffolds is promising for bone tissue engineering.     

Three-dimensional honeycomb-patterned chitosan/poly(L-lactic acid) scaffolds with improved mechanical and cell compatibility

Micro-size patterned surfaces trigger specific biological responses such as the promotion of cell growth, cell migration, cell differentiation, and ECM production. The aim of this work was to elaborate three-dimensional scaffolds with honeycomb patterned surfaces and large open pores, and to study the influence of surface patterning on cell behavior. In this study, we used water droplets as porogen material to prepare a novel type of chitosan sponge with large open pores on its surface. The sponges obtained were then immersed into 6 wt % Poly(L-lactic acid) chloroform solution to obtain honeycomb patterned composite porous scaffolds. The morphology and mechanical properties were characterized with SEM and compression testing. The fibroblast behaviors in scaffolds were analyzed with SEM, VG, PAS, live-dead staining, and flow cytometer. Results showed that these composite scaffolds possessed better mechanical properties and hierarchical porous structure than pure chitosan sponges. Cell culture revealed that the honeycomb patterned surface had positive influences on fibroblast behaviors, wherein the cell adhesion, proliferation, ECM secretion and viability were improved dramatically. Such a hierarchical composite scaffold would be a suitable candidate for tissue engineering purposes.     

透明质酸修饰的壳聚糖复合支架用于神经组织工程可行性研究

目的探索壳聚糖复合支架经透明质酸（HA）及多聚赖氨酸（PLL）修饰后,神经元在体外复合支架黏附情况,以及将不同复合支架植入鼠脑,观察支架对大鼠脑组织炎症反应、胶质纤维表达的差异。方法孔隙为（70.32±15.33）μm壳聚糖复合支架分别用质量浓度0.05mg/mLHA和质量浓度2mg/mLPLL进行表面涂镀法修饰,继而在上述两组及未修饰复合支架上进行小鼠神经细胞培养,1d后对支架细胞固定行扫描电子显微镜形态学观察,记录两组各30例支架高倍视野下细胞黏附数量;另将复合支架植入大鼠脑皮层损伤区,术后不同时间点分别取脑组织支架行苏木精-伊红染色、免疫组织化学观察炎性细胞及胶质纤维的表达情况。数据用SAS9.1.3统计软件处理,P〈0.05为差异有统计学意义。结果 HA修饰的壳聚糖复合支架上细胞黏附较多[扫描电子显微镜,×1700,（24.9±4.5）/高倍镜],而经PLL修饰的壳聚糖复合支架上细胞黏附相对较少[扫描电子显微镜,×1700,（19.2±3.2）/高倍镜],未修饰支架上细胞碎片较多,并且细胞聚集,细胞总数少;在体内用HA修饰的壳聚糖复合支架组与其他组相比,支架周围炎症反应轻、支架周围胶质纤维酸性蛋白（GFAP）阳性细胞数少。所获得数据分析后差异均有显著统计学意义（P〈0.01）。结论 HA修饰的壳聚糖复合支架能够提高体外神经元黏附,并且在体内观察炎性细胞,胶质纤维表达均优越于PLL修饰的壳聚糖复合支架、单纯壳聚糖复合支架。HA修饰的壳聚糖复合支架在神经组织工程应用中更具有研发前景。     

Electrospun collagen–chitosan nanofiber: A biomimetic extracellular matrix for endothelial cell and smooth muscle cell

Electrospinning of collagen and chitosan blend solutions in a 1,1,1,3,3,3-hexafluoroisopropanol/trifluoroacetic acid (v/v, 90/10) mixture was investigated for the fabrication of a biocompatible and biomimetic nanostructure scaffold in tissue engineering. The morphology of the electrospun collagen–chitosan nanofibers was observed by scanning electron microscopy (SEM) and stabilized by glutaraldehyde (GTA) vapor via crosslinking. Fourier transform infrared spectra analysis showed that the collagen–chitosan nanofibers do not change significantly, except for enhanced stability after crosslinking by GTA vapor. X-ray diffraction analysis implied that both collagen and chitosan molecular chains could not be crystallized in the course of electrospinning and crosslinking, and gave an amorphous structure in the nanofibers. The thermal behavior and mechanical properties of electrospun collagen–chitosan fibers were also studied by differential scanning calorimetry and tensile testing, respectively. To assay the biocompatibility of electrospun fibers, cellular behavior on the nanofibrous scaffolds was also investigated by SEM and methylthiazol tetrazolium testing. The results show that both endothelial cells and smooth muscle cells proliferate well on or within the nanofiber. The results indicate that a collagen–chitosan nanofiber matrix may be a better candidate for tissue engineering in biomedical applications such as scaffolds.     

Enhancing cell penetration and proliferation in chitosan hydrogels for tissue engineering applications

The aim of this study was to develop a process to create highly porous three-dimensional (3D) chitosan hydrogels suitable for tissue engineering applications. Chitosan was crosslinked by glutaraldehyde (0.5 vol %) under high pressure CO(2) at 60 bar and 4 °C for a period of 90 min. A gradient-depressurisation strategy was developed, which was efficient in increasing pore size and the overall porosity of resultant hydrogels. The average pore diameter increased two fold (59 μm) compared with the sample that was depressurised after complete crosslinking and hydrogel formation (32 μm). It was feasible to achieve a pore diameter of 140 μm and the porosity of hydrogels to 87% by addition of Acacia gum (AG) as a surfactant to the media. The enhancement in porosity resulted in an increased swelling ratio and decreased mechanical strength. On hydrogels with large pores (>90 μm) and high porosities (>85%), fibroblasts were able to penetrate up to 400 μm into the hydrogels with reasonable viabilities (~80%) upon static seeding. MTS assays showed that fibroblasts proliferated over 14 days. Furthermore, aligned microchannels were produced within porous hydrogels to further promote cell proliferation. The developed process can be easily used to generate homogenous pores of controlled sizes in 3D chitosan hydrogels and may be of use for a broad range of tissue engineering applications.     

A healing method of tympanic membrane perforations using three-dimensional porous chitosan scaffolds

Both surgical tympanoplasty and paper patch grafts are frequently procedured to heal tympanic membrane (TM) perforation or chronic otitis media, despite their many disadvantages. In this study, we report a new healing method of TM perforation by using three-dimensional (3D) porous chitosan scaffolds (3D chitosan scaffolds) as an alternative method to surgical treatment or paper patch graft. Various 3D chitosan scaffolds were prepared; and the structural characteristics, mechanical property, in vitro biocompatibility, and healing effects of the 3D chitosan scaffolds as an artificial TM in in vivo animal studies were investigated. A 3D chitosan scaffold of 5 wt.% chitosan concentration showed good proliferation of TM cells in an in vitro study, as well as suitable structural characteristics and mechanical property, as compared with either 1% or 3% chitosan. In in vivo animal studies, 3D chitosan scaffold were able to migrate through the pores and surfaces of TM cells, thus leading to more effective TM regeneration than paper patch technique. Histological observations demonstrated that the regenerated TM with the 3D chitosan scaffold consisted of three (epidermal, connective tissue, and mucosal) layers and were thicker than normal TMs. The 3D chitosan scaffold technique may be an optimal healing method used in lieu of surgical tympanoplasty in certain cases to heal perforated TMs.     

Chitosan–poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: In vitro degradation and in vivo bone regeneration studies

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation.     

Porogen-based solid freeform fabrication of polycaprolactone-calcium phosphate scaffolds for tissue engineering

Drop on demand printing (DDP) is a solid freeform fabrication (SFF) technique capable of generating microscale physical features required for tissue engineering scaffolds. Here, we report results toward the development of a reproducible manufacturing process for tissue engineering scaffolds based on injectable porogens fabricated by DDP. Thermoplastic porogens were designed using Pro/Engineer and fabricated with a commercially available DDP machine. Scaffolds composed of either pure polycaprolactone (PCL) or homogeneous composites of PCL and calcium phosphate (CaP, 10% or 20% w/w) were subsequently fabricated by injection molding of molten polymer-ceramic composites, followed by porogen dissolution with ethanol. Scaffold pore sizes, as small as 200 microm, were attainable using the indirect (porogen-based) method. Scaffold structure and porosity were analyzed by scanning electron microscopy (SEM) and microcomputed tomography, respectively. We characterized the compressive strength of 90:10 and 80:20 PCL-CaP composite materials (19.5+/-1.4 and 24.8+/-1.3 Mpa, respectively) according to ASTM standards, as well as pure PCL scaffolds (2.77+/-0.26 MPa) fabricated using our process. Human embryonic palatal mesenchymal (HEPM) cells attached and proliferated on all scaffolds, as evidenced by fluorescent nuclear staining with Hoechst 33258 and the Alamar Blue assay, with increased proliferation observed on 80:20 PCL-CaP scaffolds. SEM revealed multilayer assembly of HEPM cells on 80:20 PCL-CaP composite, but not pure PCL, scaffolds. In summary, we have developed an SFF-based injection molding process for the fabrication of PCL and PCL-CaP scaffolds that display in vitro cytocompatibility and suitable mechanical properties for hard tissue repair.     

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.     

Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants

Three-dimensional macroporous calcium phosphate bioceramics embedded with porous chitosan sponges were synthesized to produce composite scaffolds with high mechanical strength and a large surface/volume ratio for load-bearing bone repairing and substitutes. The macroporous calcium phosphate bioceramics with pore diameters of 300 microm to 600 microm were developed using a porogen burnout technique, and the chitosan sponges were formed inside the pores of the bioceramics by first introducing chiosan solution into the pores followed by a freeze-drying process. Our scanning electron microscopy results showed that the pore size of chitosan sponges formed inside the macroporous structure of bioceramics was approximately 100 microm, a structure favorable for bone tissue in-growth. The compressive modulus and yield stress of the composite scaffolds were both greatly improved in comparison with that of HA/beta-TCP scaffolds. The simulated body fluid (SBF) and cell culture experiments were conducted to assess the bioactivity and biocompatibility of the scaffolds. In the SBF tests, a layer of randomly oriented needle-like apatite crystals formed on the scaffold surface after sample immersion in SBF, which suggested that the composite material has good bioactivity. The cell culture experiments showed that MG63 osteoblast cells attached to the composite scaffolds, proliferated on the scaffold surface, and migrated onto the pore walls, indicating good cell biocompatibility of the scaffold. The cell differentiation on the composite scaffolds was evaluated by alkaline phosphatase (ALP) assay. Compared with the control in tissue culture dishes, the cells had almost the same ALP activity on the composite scaffolds during the first 11 days of culture.