A Micro-Scale Surface-Structured PCL Scaffold Fabricated by a 3D Plotter and a Chemical Blowing Agent

To study cell responses, polymeric scaffolds with a controllable pore size and porosity have been fabricated using rapid-prototyping methods. However, the scaffolds fabricated by rapid prototyping have very smooth surfaces, which tend to discourage initial cell attachment. Initial cell attachment, migration, differentiation and proliferation are strongly dependent on the chemical and physical characteristics of the scaffold surface. In this study, we propose a three-dimensional (3D) plotting method supplemented with a chemical blowing agent to produce a surface-modified 3D scaffold in which the surface is inscribed with nano- and micro-sized pores. The chemically-blown 3D polymeric scaffold exhibited positive qualities, including the compressive modulus, hydrophilicity and initial cell adhesion. Cell cultures on the scaffolds demonstrated that chondrocytes interacted better with the surface-modified scaffold than with a normal 3D scaffold.     

Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning

Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS therefore have the potential to be used as scaffold material for a wide variety of biological tissues and organs, as an alternative to electrospinning.     

Nano-fibrous poly(L-lactic acid) scaffolds with interconnected spherical macropores

Biodegradable polymers have been used extensively as scaffolding materials to regenerate new tissues. These scaffolds should possess certain physical characteristics including a three-dimensional structure, high porosity with an interconnected pore structure, and a suitable surface structure for cell attachment, proliferation, and differentiation. To mimic the fibrous architecture of type I collagen, nano-fibrous matrices have been created in our laboratory using a phase-separation technique of poly(L-lactic acid) (PLLA) solutions. In addition, biodegradable scaffolds with controlled interconnected spherical pore networks have been fabricated in our laboratory. In this work, these two techniques were combined to yield scaffolds with highly interconnected spherical macroporous structures and nano-fibrous architectures. Paraffin spheres were first fabricated with a dispersion method, and were thermally bonded to form an interconnected mold. PLLA solutions were cast over the paraffin sphere assembly and were thermally phase-separated to form nano-fibrous matrices. After leaching out the paraffin, synthetic nano-fibrous extracellular matrices with interconnected spherical pores were yielded. By utilizing this fabrication process, we are able to control the architecture of the scaffolds at several different levels, including the macroscopic shape of the scaffold, the spherical pore size, interfiber distance, and the fiber diameter at the nano-size scale. The inter-pore connectivity could be controlled by varying the heat treatment time of the paraffin spheres, and mechanical properties could be controlled by varying the porosity of the scaffolds. With an interconnected macroporous structure that promotes cell seeding throughout the interstices of the scaffold, and a synthetic collagen-like matrix, these novel matrices may be an excellent scaffold for tissue engineering.     

Macroporous and nanofibrous hyaluronic acid/collagen hybrid scaffold fabricated by concurrent electrospinning and deposition/leaching of salt particles

A three-dimensional (3-D) macroporous and nanofibrous hyaluronic acid (HA) scaffold was fabricated by an electrospinning process combined with a salt leaching technique. HA and collagen were dissolved in a sodium hydroxide (NaOH)/N,N-dimethyl formamide (DMF) solvent mixture at a concentration of 10 wt.% and successfully electrospun into a nanofiber web with a soft, fluffy structure by the combined effects of numerous minijet evolutions and their subsequent vertical growth. To our knowledge, the formation of an extensive fluffy nanofiber morphology is the first time as a single route has been used to spontaneously generate a 3-D nanofibrous structure. By the simultaneous deposition of salt particulates as a porogen during electrospinning and subsequent chemical cross-linking and salt leaching, a water-swellable HA-based scaffold retaining a macroporous and nanofibrous geometry could be produced. Bovine chondrocytes were cultured on the HA/collagen scaffold to assessing the scaffold's cytocompatibility. The results revealed that cellular adhesion and proliferation were enhanced in proportion to the content of collagen, and the seeded chondrocytes maintained the roundness characteristic of a chondroblastic morphology.     

Synergic effects of nanofiber alignment and electroactivity on myoblast differentiation

The interactions between cells and materials play critical roles in the success of new scaffolds for tissue engineering, since chemical and physical properties of biomaterials regulate cell adhesion, proliferation, migration, and differentiation. We have developed nanofibrous substrates that possess both topographical cues and electroactivity. The nanofiber scaffolds were fabricated through the electrospinning of polycaprolactone (PCL, a biodegradable polymer) and polyaniline (PANi, a conducting polymer) blends. We investigated the ways in which those properties influenced myoblast behaviors. Neither nanofiber alignment nor PANi concentration influenced cell growth and proliferation, but cell morphology changed significantly from multipolar to bipolar with the anisotropy of nanofibers. According to our analyses of myosin heavy chain expression, multinucleate myotube formation, and the expression of differentiation-specific genes (myogenin, troponin T, MHC), the differentiation of myoblasts on PCL/PANi nanofibers was strongly dependent on both nanofiber alignment and PANi concentration. Our results suggest that topographical cues and the electroactivity of nanofibers synergistically stimulate muscle cell differentiation to make PCL/PANi nanofibers a suitable scaffold material for skeletal tissue engineering.     

Fabricating Microparticles/Nanofibers Composite and Nanofiber Scaffold with Controllable Pore Size by Rotating Multichannel Electrospinning

Polymeric nanofibers fabricated via electrospinning are regarded as promising scaffolds for biomimicking a native extracellular matrix. However, electrospun scaffolds have poor porosity, resulting in cells being unable to infiltrate into the scaffolds but grow only on its surface. In this study, we modified regular electrospinning into rotating multichannel electrospinning (RM-ELSP) to produce microparticles and nanofibers simultaneously. Gelatin nanofibers (0.1–1 μm) and polycaprolactone (PCL) microparticles (0.5–10 μm) were formed and well-mixed. Adjusting the concentration of PCL and/or gelatin, we can fabricate various microparticles/nanofibers composites with different sizes of PCL particles and different diameters of gelatin nanofibers depending on their concentrations (2–10%) during electrospinning. Using PCL particles as a pore generator, we obtained gelatin nanofiber scaffolds with controllable pore size and porosity. Cells adhere and grow into the scaffold easily during in vitro cell culture.     

新型纳米纤维支架的细胞生物相容性

背景：高孔隙率聚己内酯纳米纤维支架具有适合血管平滑肌细胞黏附、增殖的多级孔径结构,具有良好的细胞生物相容性。 目的：探讨高孔隙率聚己内酯静电纺丝纳米纤维支架的细胞相容性。 方法：根据支架的制作工艺不同分为传统支架组、新型纳米纤维支架组两组,另设单纯细胞组为对照组。采用组织块贴壁法体外原代培养兔主动脉平滑肌细胞并进行传代,用3~6代细胞作为实验用种子细胞。应用WST-1法测定平滑肌细胞黏附率、增殖力,光镜及扫描电镜观察细胞形态,评估支架的细胞生物相容性。 结果与结论：高孔隙率聚己内酯纳米纤维支架对细胞形态无明显影响,新型支架上的种子细胞黏附、增殖及代谢活性情况较传统支架好。提示,高孔隙率聚己内酯静电纺丝纳米纤维支架具有较高的细胞相容性。     

The influence of electrospun aligned poly(epsilon-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes

Current treatment options for restoring large skeletal muscle tissue defects due to trauma or tumor ablation are limited by the host muscle tissue availability and donor site morbidity of muscle flap implantation. Creation of implantable functional muscle tissue that could restore muscle defects may bea possible solution. To engineer functional muscle tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(epsilon-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal muscle cells may provide implantable functional muscle tissues for patients with large muscle defects.     

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.     

Poly(L-lactic acid)/hydroxyapatite hybrid nanofibrous scaffolds prepared by electrospinning

Poly(L-lactic acid)/hydroxyapatite hybrid nanofibrous scaffolds were prepared via electrospinning. The structure and morphology of the scaffolds were investigated using scanning electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The experimental results showed that the average diameter of hybrid nanofiber was similar to that of pure poly(L-lactic acid) fiber, but a new surface bonding (COO^?) was formed in hybrid nanofiber which made the surface of the fiber coarse. The weight loss and water uptake of pure poly(L-lactic acid) scaffolds increased continuously and the viscosity-average molecular weight decreased in the phosphate buffer solution as time passed, while those of hybrid scaffolds were very much slowed down because the dissolving of hydroxyapatite particles acted as a physical barrier and blocked off the entry of water. The biocompatibility of the scaffold has been investigated by human osteosarcoma MG-63 cell culture on the scaffold. The preliminary results showed that cells were well adhered and proliferated better on the hybrid scaffolds than pure scaffolds.     

The Effect of Hybridization of Hydrogels and Poly(L-lactide-co-ε-caprolactone) Scaffolds on Cartilage Tissue Engineering

For repairing cartilage defects by cartilage tissue engineering, it is important that engineered cartilage that is fabricated with scaffolds and cells can maintain the biological and physiological functions of cartilage, and also can induce three-dimensional spatial organization of chondrocytes. In this sense, hydrogels such as fibrin gels (FG) and hyaluronan (HA) are widely used for application in cartilage treatment. However, the use of hydrogels alone as a scaffold has a physical weakness; the mechanical properties of hydrogels are too weak to endure complex loading in the body. In this study, for mimicking a native cartilage microenvironment, we made cell–hybrid scaffold constructs with poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds and hydrogels to guide three-dimensional spatial organization of cells and extracellular matrix. A highly elastic scaffold was fabricated from PLCL with 85% porosity and 300–500 μm pore size using a gel-pressing method. The mixture of rabbit chondrocytes and hydrogels was seeded on PLCL scaffolds, and was subcutaneously implanted into nude mice for up to eight weeks. The cell seeding efficiency of the hybrid scaffolds with FG or HA was higher than that of the PLCL scaffolds. From in vivo studies, the accumulation of cartilaginous extracellular matrices of constructs, which was increased by hybridization of hydrogels and PLCL scaffolds, showed that the cell–hybrid scaffold constructs formed mature and well-developed cartilaginous tissue. In conclusion, the hybridization of hydrogels and PLCL scaffold for three-dimensional spatial organization of cells would provide a biomimetic environment where cartilage tissue growth is enhanced and facilitated. It can enhance the production of cartilaginous extracellular matrices and, consequently, improve the quality of the cartilaginous tissue formed.     

Pores Created by Laser Surface Modification of Poly(vinylalcohol)-Collagen with Glycosaminoglycan Scaffold for Cell Culture in Tissue Engineering

A PVA-GAG-COL composite scaffold is fabricated by polyvinyl alcohol (PVA),glycosaminoglycan (GAG) and collagen (COL).Laser surface modification technology is used to make holes on the surface of the sc...     

Poly(L-lactic acid)/hydroxyapatite hybrid nanofibrous scaffolds prepared by electrospinning

Poly(L-lactic acid)/hydroxyapatite hybrid nanofibrous scaffolds were prepared via electrospinning. The structure and morphology of the scaffolds were investigated using scanning electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The experimental results showed that the average diameter of hybrid nanofiber was similar to that of pure poly(L-lactic acid) fiber, but a new surface bonding (COO-) was formed in hybrid nanofiber which made the surface of the fiber coarse. The weight loss and water uptake of pure poly(L-lactic acid) scaffolds increased continuously and the viscosity-average molecular weight decreased in the phosphate buffer solution as time passed, while those of hybrid scaffolds were very much slowed down because the dissolving of hydroxyapatite particles acted as a physical barrier and blocked off the entry of water. The biocompatibility of the scaffold has been investigated by human osteosarcoma MG-63 cell culture on the scaffold. The preliminary results showed that cells were well adhered and proliferated better on the hybrid scaffolds than pure scaffolds.     

Novel biodegradable electrospun membrane: scaffold for tissue engineering

Nonwoven fibrous matrixes have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The objective of this study was fabrication of nanofibrous matrix from novel biodegradable poly(p-dioxanone-co-L-lactide)-block-poly(ethylene glycol) (PPDO/PLLA-b-PEG) copolymer, and to examine cell proliferation, morphology of cell-matrix interaction with the electrospun nanofibrous matrix. The electrospun structure composed of PPDO/PLLA-b-PEG fibers with an average diameters of 380 nm, median pore size 8 microm, porosity more than 80% and mechanical strength 1.4 MPa, is favorable for cell-matrix interaction and supports the active biocompatibility of the structure. NIH 3T3 fibroblast cell seeded on the structure tend to maintain phenotypic shape and guided growth according to nanofiber orientation. Good capability of the nanofibrous structure for supporting the cell attachment and proliferation are observed. This novel biodegradable scaffold will be applicable for tissue engineering based upon its unique architecture, which acts to support and guide cell growth.     

Repair of mandibular defects using MSCs-seeded biodegradable polyester porous scaffolds

PLLA, PLA-PEG and PLGA porous scaffolds with pore size ranging from 100 to 250 microm and porosity over 85% were fabricated by a solution-casting/salt-leaching method. The porous structure and porosity of the scaffold were mainly dependent on volume fraction and size of the porogens of NaCl particles. The effects of the polymeric materials on the cell culture behavior and bone formation in vitro in their scaffolds were studied. In vitro cell culture in the scaffolds of the three polymers demonstrated that mesenchymal stem cells (MSCs) had a good adhesion and spread. The composite matrixes cultured for several days possessed preliminary functions of tissue-engineering bone, with signs of the calcium knur formation and the expression of osteocalcin and collagen I in mRNA, especially that of PLA-PEG and PLGA. These cell-loaded porous scaffolds showed effective repair of mandibular defect of rabbits in vivo. Contrastive experiments demonstrated that the MSCs/PLGA scaffold owned better ability facilitating for the MSCs proliferation, differentiation and defect repair. These composite scaffolds can be a potential effective tool for treating mandibular and other bone defects.     

Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration

Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.     

Electrospun P(LLA-CL) nanofiber: a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation

Poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] with L-lactide to epsilon-caprolactone ratio of 75 to 25 has been electrospun into nanofibers. The relationship between electrospinning parameters and fiber diameter has been investigated. The fiber diameter decreased with decreasing polymer concentration and with increasing electrospinning voltage. The X-ray diffractometer and differential scanning colorimeter results suggested that the electrospun nanofibers developed highly oriented structure in CL-unit sequences during the electrospinning process. The biocompatibility of the nanofiber scaffold has been investigated by culturing cells on the nanofiber scaffold. Both smooth muscle cell and endothelial cell adhered and proliferated well on the P(LLA-CL) nanofiber scaffolds.     

Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers

In this study, we successfully developed three-dimensional scaffolds fabricated from the chitosan-based hyaluronic acid hybrid polymer fibers, which can control the porous structure. To determine the adequate pore size for enhancing the chondrogenesis of cultured cells, we compared the behaviors of rabbit chondrocytes in scaffolds comprising different pore sizes (100, 200, and 400 microm pore size). Regarding the cell proliferation, there was no significant difference among the three groups. On the other hand, glycosaminoglycan contents in the 400 microm group significantly increased during the culture period, compared with those in the other groups. The ratio of type II to type I collagen mRNA level was also significantly higher in the 400 microm group than in the other groups. These results indicate that our scaffold with 400 microm pore size significantly enhances the extracellular matrix synthesis by chondrocytes. Additionally, the current scaffolds showed high mechanical properties, compared with liquid and gel materials. The data derived from this study suggest great promise for the future of a novel fabricated material with relatively large pore size as a scaffold for cartilage regeneration. The biological and mechanical advantages presented here will make it possible to apply our scaffold to relatively wide cartilaginous lesions.     

The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds

Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.     

Mineralisation of chitosan scaffolds with nano-apatite formation by double diffusion technique

The study of inorganic crystal assembly in organic matrices has given rise to increasing interest in various fields of materials science to the natural process of biomineralisation. To mimic the formation of hydroxyapatite as natural bone, a double diffusion technique is utilised in this study to nucleate the hydroxyapatite crystals onto three-dimensional porous polymeric scaffolds. The porous polymer scaffolds were produced from chitosan by a thermally induced lyophilisation technique, which yields highly porous, well-controlled anisotropic open pore architecture. The nucleation of hydroxyapatite crystals was initiated at ambient conditions on the surface of the polymer scaffold, which was in contact with a calcium solution chamber, due to diffusion of phosphate ions through the scaffold. The morphology of the mineralised scaffold as analysed by scanning electron microscopy shows that apatite crystals were not only formed on the surface of the scaffold, but also in the pore channels and attached to the pore walls. The X-ray diffraction and Fourier transformed infrared analyses confirmed the phase purity of the formed apatite crystals. The transmission electron microscopy analysis reveals the microstructure of the entangled nano-apatite in the chitosan polymeric matrix. The in-vitro cytocompatibility tests with osteoblast-like cells (Saos-2) demonstrated that the biomineralised scaffold is a suitable substrate for cell attachment and migration in bone tissue engineering.