Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering

Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

Porous ceramic bone scaffolds for vascularized bone tissue regeneration

Hydroxyapatite scaffolds with a multi modal porosity designed for use in tissue engineering of vascularized bone graft substitutes were prepared by three dimensional printing. Depending on the ratio of coarse (mean particle size 50 microm) to fine powder (mean particle size 4 microm) in the powder granulate and the sintering temperature total porosity was varied from 30% to 64%. While macroscopic pore channels with a diameter of 1 mm were created by CAD design, porosity structure in the sintered solid phase was governed by the granulate structure of the printing powder. Scaffolds sintered at 1,250 degrees C were characterized by a bimodal pore structure with intragranular pores of 0.3-0.4 microm and intergranular pores of 20 microm whereas scaffolds sintered at 1,400 degrees C exhibit a monomodal porosity with a maximum of pore size distribution at 10-20 microm. For in-vivo testing, matrices were implanted subcutaneously in four male Lewis rats. Scaffolds with 50% porosity and an average pore size of approximately 18 microm were successfully transferred to rats and vascularized within 4 weeks.     

Hybrid bilayered chitosan-xanthan/PCL scaffolds as artificial periosteum substitutes for bone tissue regeneration

Journal of Materials Science - The periosteum, a bilayered membrane that covers bone surfaces, acts as a source of bone-forming cells and plays a pivotal role in bone homeostasis and defect...     

Fibrous hydrogel scaffolds with cells embedded in the fibers as a potential tissue scaffold for skin repair

A novel approach was undertaken to create a potential skin wound dressing. L929 fibroblast cells and alginate solution were simultaneously dispensed into a calcium chloride solution using a three-dimensional plotting system to manufacture a fibrous alginate scaffold with interconnected pores. These cells were then embedded in the alginate hydrogel fibers of the scaffold. A conventional scaffold with cells directly seeded on the fiber surface was used as a control. The encapsulated fibroblasts made using the co-dispensing method distributed homogeneously within the scaffold and showed the delayed formation of large cell aggregates compared to the control. The cells embedded in the hydrogel fibers also deposited more type I collagen in the extracellular matrix and expressed higher levels of fgf11 and fn1 than the control, indicating increased cellular proliferation and attachment. The results indicate that the novel co-dispensing alginate scaffold may promote skin regeneration better than the conventional directly-seeded scaffold.     

Poly(lactide-co-glycolide)/hydroxyapatite nanofibrous scaffolds fabricated by electrospinning for bone tissue engineering

Poly(lactide-co-glycolide) (PLGA) nanofibrous composite scaffolds having nano-hydroxyapatite particles (HAp) in the fibers were prepared by electrospinning of PLGA and HAp with an average diameter of 266.6 ± 7.3 nm. Microscopy and spectroscopy characterizations confirmed integration of the crystalline HAp in the scaffolds. Agglomerates gradually appeared and increased on the fiber surface along with increase of the HAp concentration. In vitro mineralization in a 5 × simulated body fluid (SBF) revealed that the PLGA/HAp nanofibrous scaffolds had a stronger biomineralization ability than the control PLGA scaffolds. Biological performance of the nanofibrous scaffolds of the control PLGA and PLGA with 5 wt% HAp (PLGA/5HAp) was assessed by in vitro culture of neonatal mouse calvaria-derived MC3T3-E1 osteoblasts. Both types of the scaffolds could support cell proliferation and showed sharp increase of viability until 7 days, but the cells cultured on the PLGA/5HAp nanofibers showed a more spreading morphology. Despite the similar level of the cell viability and cell number at each time interval, the alkaline phosphatase secretion was significantly enhanced on the PLGA/5HAp scaffolds, indicating the higher bioactivity of the as-prepared nano-HAp and the success of the present method for preparing biomimetic scaffold for bone regeneration.     

Poly(L-lactic acid) nanocylinders as nanofibrous structures for macroporous gelatin scaffolds

Electrospun Nanofiber sheets have been shown to mimic the structure of extracellular matrix (ECM). Although these nanofibers have shown great potential for use as tissue engineering scaffolds, it is difficult for the electrospun nanofiber based sheets to be shaped into the desired three-dimensional structure. In this study, poly(L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, was electrospun to produce nanofibers that were treated with an amino group containing base in order to fabricate polymeric nanocylinders. The aspect ratio of the PLLA nanocylinders was tunable by varying the aminolysis time and density of the amino group containing base. The effects of changes in nanofibrous morphology of the PLLA nanocylinders/macro-porous gelatin scaffolds on cell adhesion and proliferation were evaluated. The results revealed different cell morphology, adhesion, and proliferation in the nanocylinders composite gelatin scaffold versus gelatin scaffold alone. Confocal laser scanning microscopy observation showed more spreading and a more flattened cell morphology after NIH3T3 cells were cultured on PLLA nanocylinders/gelatin scaffolds for 10 hours and 4 days. These results indicate that the gelatin/PLLA nanocylinder composite is a promising way to fabricate 3D nanofibrous scaffolds that accelerates cell adhesion and proliferation for tissue engineering.     

Fabrication and characterization of gold nanoparticle-doped electrospun PCL/chitosan nanofibrous scaffolds for nerve tissue engineering

In the field of nerve tissue engineering, nanofibrous scaffolds could be a promising candidate when they are incorporated with electrical cues. Unique physico-chemical properties of gold nanoparticles (AuNPs) make them an appropriate component for increasing the conductivity of scaffolds to enhance the electrical signal transfer between neural cells. The aim of this study was fabrication of AuNPs-doped nanofibrous scaffolds for peripheral nerve tissue engineering. Polycaprolactone (PCL)/chitosan mixtures with different concentrations of chitosan (0.5, 1 and 1.5) were electrospun to obtain nanofibrous scaffolds. AuNPs were synthesized by the reduction of HAuCl₄ using chitosan as a reducing/stabilizing agent. A uniform distribution of AuNPs with spherical shape was achieved throughout the PCL/chitosan matrix. The UV–Vis spectrum revealed that the amount of gold ions absorbed by nanofibrous scaffolds is in direct relationship with their chitosan content. Evaluation of electrical property showed that inclusion of AuNPs significantly enhanced the conductivity of scaffolds. Finally, after 5 days of culture, biological response of Schwann cells on the AuNPs-doped scaffolds was superior to that on as-prepared scaffolds in terms of improved cell attachment and higher proliferation. It can be concluded that the prepared AuNPs-doped scaffolds can be used to promote peripheral nerve regeneration.

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Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering

Successful tissue engineering with the aid of a polymer scaffold offers the possibility to produce a larger construct and to mould the shape after the defect. We investigated the use of cryogelation to form protein-based scaffolds through different types of formation mechanisms; enzymatic crosslinking, chemical crosslinking, and non-covalent interactions. Casein was found to best suited for enzymatic crosslinking, gelatin for chemical crosslinking, and ovalbumin for non-covalent interactions. Fibroblasts and myoblasts were used to evaluate the cryogels for tissue engineering purposes. The stability of the cryogels over time in culture differed depending on formation mechanism. Casein cryogels showed best potential to be used in skeletal tissue engineering, whereas gelatin cryogels would be more suitable for compliable soft tissues even though it also seemed to support a myogenic phenotype. Ovalbumin cryogels would be better suited for elastic tissues with faster regeneration properties due to its faster degradation time. Overall, the cryogelation technique offers a fast, cheap and reproducible way of creating porous scaffolds from proteins without the use of toxic compounds.     

Cellular materials as porous scaffolds for tissue engineering

A major goal of tissue engineering is to synthesize or regenerate tissues and organs. Today, this is done by providing a synthetic porous scaffold, or matrix, which mimics the body's own extracellular matrix, onto which cells attach, multiply, migrate and function. Porous scaffolds are currently being developed for regeneration of skin, cartilage, bone, nerve and liver. The microstructures of many porous scaffolds ressemble that of an engineering foam. In this paper, we describe the microstructural requirements for porous scaffolds, review several processes for making them and show typical microstructures. Clinical studies have found that a collagen-based scaffold for skin regeneration reduces wound contraction during the healing process, reducing scar formation. The process of wound contraction is not well understood. Here, we describe the measurement of contraction of collagen-based scaffolds by fibroblasts in vitro using a cell force monitor.     

Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds for bone tissue regeneration

Hydroxyapatite/alginate nanocomposite fibrous scaffolds were fabricated via electrospinning and a novel in situ synthesis of hydroxyapatite (HAp) that mimics mineralized collagen fibrils in bone tissue. Poorly crystalline HAp nanocrystals, as confirmed by X-ray diffractometer peak approximately at 2θ = 32° and Fourier transform infrared spectroscopy spectrum with double split bands of PO₄(v ₄) at 564 and 602 cm^?1, were induced to nucleate and grow at the [–COO^?]–Ca²⁺–[–COO^?] linkage sites on electrospun alginate nanofibers impregnated with PO₄ ^3? ions. This novel process resulted in a uniform deposition of HAp nanocrystals on the nanofibers, overcoming the severe agglomeration of HAp nanoparticles processed by the conventional mechanical blending/electrospinning method. Preliminary in vitro cell study showed that rat calvarial osteoblasts attached more stably on the surface of the HAp/alginate scaffolds than on the pure alginate scaffold. In general, the osteoblasts were stretched and elongated into a spindle-shape on the HAp/alginate scaffolds, whereas the cells had a round-shaped morphology on the alginate scaffold. The unique nanofibrous topography combined with the hybridization of HAp and alginate can be advantageous in bone tissue regenerative medicine applications.     

Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration

The cytocompatibility, in vivo biodegradation and wound healing of keratin biomaterials were investigated. For the purposes, three groups of keratin scaffolds were fabricated by freeze-drying reduced solutions at 2 wt.%, 4 wt.% and 8 wt.% keratins extracted from human hairs. These scaffolds exhibited evenly distributed high porous structures with pore size of 120–220 μm and the porosity > 90%. NIH3T3 cells proliferated well on these scaffolds in culture lasting up to 22 days. Confocal micrographs stained with AO visually revealed cell attachment and infiltration as well as scaffold architectural stability. In vivo animal experiments were conducted with 4 wt.% keratin scaffolds. Early degradation of subcutaneously implanted scaffolds occurred at 3 weeks in the outermost surface, in concomitant with inflammatory response. At 5 weeks, the overall porous structure of scaffolds severely deteriorated while the early inflammatory response in the outermost surface obviously subsided. A faster keratin biodegradation was observed in repairing full-thickness skin defects. Compared with the blank control, keratin scaffolds gave rise to more blood vessels at 2 weeks and better complete wound repair at 3 weeks with a thicker epidermis, less contraction and newly formed hair follicles. These preliminary results suggest that human hair keratin scaffolds are promising dermal substitutes for skin regeneration.     

In vitro anti-bacterial and cytotoxic properties of silver-containing poly(L-lactide-co-glycolide) nanofibrous scaffolds

Electrospinning has recently emerged as a leading technique for the formation of nanofibrous structures made of organic and inorganic components. In this study, nanofibrous scaffolds were prepared by electrospining a bend solution of poly(L-lactide-co-glycolide) (PLGA) and silver nanoparticles in 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP). The resulting fibers ranged from 420 to 590 nm in diameter. To evaluate the possibility of using silver-containing PLGA as a tissue engineering scaffold, experiments on cell viability and antibacterial activity were carried out. As a result, PLGA nanofibrous scaffolds having silver nanoparticles of more than 0.5 wt% showed antibacterial effect against Staphylococcus aureus and Klebsiella pneumonia. Furthermore, silver-containing PLGA nanofibrous scaffolds showed viability, indicating their possible application in the field of tissue engineering.     

Multifunctional nanofibrous scaffold for tissue engineering

In tissue engineering, scaffolds with multiscale functionality, especially with the ability to release locally multiple or specific bioactive molecules to targeted cell types, are highly desired in regulating appropriate cell phenotypes. In this study, poly (epsilon-caprolactone) (PCL) solutions (8% w/v) containing different amounts of bovine serum albumin (BSA) with or without collagen were electrospun into nanofibres. As verified by protein release assay and fluorescent labelling, BSA and collagen were successfully incorporated into electrospun nanofibres. The biological activity of functionalised fibres was proven in the cell culture experiments using human dermal fibroblasts. By controlling the sequential deposition and fibre alignment, 3D scaffolds with spatial distribution of collagen or BSA were assembled using fluorescently labelled nanofibres. Human dermal fibroblasts showed preferential adhesion to PCL nanofibres containing collagen than PCL alone. Taken together, multiscale scaffolds with diverse functionality and tunable distribution of biomolecules across the nanofibrous scaffold can be fabricated using electrospun nanofibres.     

Production of new 3D scaffolds for bone tissue regeneration by rapid prototyping

The incidence of bone disorders, whether due to trauma or pathology, has been trending upward with the aging of the worldwide population. The currently available treatments for bone injuries are rather limited, involving mainly bone grafts and implants. A particularly promising approach for bone regeneration uses rapid prototyping (RP) technologies to produce 3D scaffolds with highly controlled structure and orientation, based on computer-aided design models or medical data. Herein, tricalcium phosphate (TCP)/alginate scaffolds were produced using RP and subsequently their physicochemical, mechanical and biological properties were characterized. The results showed that 60/40 of TCP and alginate formulation was able to match the compression and present a similar Young modulus to that of trabecular bone while presenting an adequate biocompatibility. Moreover, the biomineralization ability, roughness and macro and microporosity of scaffolds allowed cell anchoring and proliferation at their surface, as well as cell migration to its interior, processes that are fundamental for osteointegration and bone regeneration.     

In vivo performance of bilayer hydroxyapatite scaffolds for bone tissue regeneration in the rabbit radius

The objective of this study was to investigate the in vivo biomechanical performance of bone defects implanted with novel bilayer hydroxyapatite (HAp) scaffolds that mimic the cortical and cancellous organization of bone. The scaffolds maintained architectural continuity in a rabbit radius segmental defect model and were compared to an untreated defect group (negative control) and autologous bone grafts (positive control). Micro-CT evaluations indicated total bone and scaffold volume in the experimental group was significantly greater than the defect group but lesser than the autologous bone graft treatment. The flexural toughness of the scaffold and the autograft groups was significantly greater than the flexural toughness of the defect group. Interestingly, the absolute density of the bone mineral as well as calcium to phosphorus (Ca/P) ratio in that mineral for the scaffold and autograft contralateral bones was significantly higher than those for the defect contralaterals suggesting that the scaffolds contributed to calcium homeostasis. It was concluded from this study that new bone regenerated in the bilayer HAp scaffolds was comparable to the empty defects and while the HAp scaffolds provided significant increase in modulus when compared to empty defect and their flexural toughness was comparable to autografts after 8 weeks of implantation.     

Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips)

The CellChip is a microstructured polymer scaffold, which favours a three-dimensional cultivation of cells within an array of cubic microcontainers. The manufacturing process used so far is microinjection moulding combined with laser-based perforation. In a first attempt to simplify the process, costly perforation was avoided by using commercially available, inexpensive microfiltration membranes for the bottom of the microcavities. Microthermoforming is a promising novel technique which allows the CellChip to be produced from thin film. Working pressures of approximately 4000 kPa were required for the adequate moulding of 50 microm thick films from three different polymers (polystyrene, polycarbonate, cyclo-olefin polymer). Integrating drafts and chamfers in micromoulds is not going to eliminate an uneven thickness profile, but reduces demoulding forces. Microthermoformed CellChips of polycarbonate were perforated by an ion track technique to guarantee a sufficient supply of medium and gases to the cells. The prestructured CellChips were irradiated with 1460 MeV xenon ions at a fluence of a few 10(6) ions/cm2. The tracks were etched in an aqueous solution of 5 N NaOH at 30 degrees C, which resulted in cylindrical pores approximately 2 microm in diameter. Microinjection-moulded, membrane-bonded and thermoformed CellChips were subjected to comparative examination for viability in a cell culture experiment with parenchymal liver cells (HepG2). The cells stayed viable over a period of more than 20 days. No significant differences in viability between injection-moulded, membrane-bonded, and thermoformed CellChips were observed.     

皮肤组织工程支架材料的研究现状及其发展

支架材料在人工皮肤的构建中起关键作用,按化学性质可分为合成类和生物类.前者主要研究通过表面仿生技术开发具有生物活性的聚酯类支架材料;后者主要研究用交联或与聚合物膜复合的方法来提高胶原的力学性能,用化学方法对壳聚糖进行改性,提高脱细胞真皮基质的渗透性和血管化速度.新的研究重点为支架材料表面与细胞的相互作用机理、制备结构仿生支架材料及复合型支架材料.     

In vitro aging of mineralized collagen-based composite as guided tissue regeneration membrane

The technique of guided tissue regeneration (GTR) has been developed for the regeneration of periodontal tissues, bone around natural teeth and dental implants. The aim of this study is to investigate the biodegradability and mechanic behavior of a novel mineralized nano-hydroxyapatite/collagen/poly (lactic acid) (nHAC/PLA) composite as GTR membrane in vitro. The elastic modulus and maximum tensile strength of GTR film samples with different nHAC/PLA ratio were measured to get an optimal nHAC/PLA ratio. Thermogravimetric analysis was conducted to evaluate the change of the inorganic component in the samples during the process of in vitro aging. Morphology of samples was checked by using scanning electron microscopy. On the basis of the above results, it can be concluded that the GTR membranes maintained integrity and the original appearance throughout the 1-month in vitro aging. There is an active dissolution and deposition process of crystals which is propitious to the bone formation on the surface of the composite membrane. The optimal nHAC/PLA ratio of the novel membrane is 0.4:1. For a longer period of bone repair, PLA with higher molecular weight should be chosen as the scaffold for the GTR membrane.