Comparative cell behavior on carbon-coated TiO2 nanotube surfaces for osteoblasts vs. osteo-progenitor cells

Surface engineering approaches that alter the topological chemistry of a substrate could be used as an effective tool for directing cell interactions and their subsequent function. It is well known that the physical environment of nanotopography has positive effects on cell behavior, yet direct comparisons of nanotopographic surface chemistry have not been fully explored. Here we compare TiO(2) nanotubes with carbon-coated TiO(2) nanotubes, probing osteogenic cell behavior, including osteoblast (bone cells) and mesenchymal stem cell (MSC) (osteo-progenitor cells) interactions with the different surface chemistries (TiO(2) vs. carbon). The roles played by the material surface chemistry of the nanotubes did not have an effect on the adhesion, growth or morphology, but had a major influence on the alkaline phosphatase (ALP) activity of osteoblast cells, with the original TiO(2) chemistry having higher ALP levels. In addition, the different chemistries caused different levels of osteogenic differentiation in MSCs; however, it was the carbon-coated TiO(2) nanotubes that had the greater advantage, with higher levels of osteo-differentiation. It was observed in this study that: (a) chemistry plays a role in cell functionality, such as ALP activity and osteogenic protein gene expression (PCR); (b) different cell types may have different chemical preferences for optimal function. The ability to optimize cell behavior using surface chemistry factors has a profound effect on both orthopedic and tissue engineering in general. This study aims to highlight the importance of the chemistry of the carrier material in osteogenic tissue engineering schemes.     

Laminin nanofiber meshes that mimic morphological properties and bioactivity of basement membranes

The basement membrane protein, laminin I, has been used broadly as a planar two-dimensional film or in a three-dimensional form as a reconstituted basement membrane gel such as Matrigel to support cellular attachment, growth, and differentiation in vitro. In basement membranes in vivo, laminin exhibits a fibrillar morphology, highlighting the electrospinning process as an ideal method to recreate such fibrous substrates in vitro. Electrospinning was employed to fabricate meshes of murine laminin I nanofibers (LNFs) with fiber size, geometry, and porosity of authentic basement membranes. Purified laminin I was solubilized and electrospun in parametric studies of fiber diameters as a function of polymer solution concentration, collecting distance, and flow rate. Resulting fiber diameters ranged from 90 to 300 nm with mesh morphologies containing beads. Unlike previously described nanofibers (NFs) synthesized from proteins such as collagen, meshes of LNFs retain their structural features when wetted and do not require fixation by chemical crosslinking, which often destroys cell attachment and other biological activity. The LNF meshes maintained their geometry for at least 2 days in culture without chemical crosslinking. PC12 cells extended neurites without nerve growth factor stimulation on LNF substrates. Additionally, LNFs significantly enhance both the rate and quantity of attachment of human adipose stem cells (ASCs) compared to laminin films. ASCs were viable and maintained attachment to LNF meshes in serum-free media for at least 3 days in culture and extended neurite-like processes after 24 h in serum-free media conditions without media additives to induce differentiation. LNF meshes are a novel substrate for cell studies in vitro, whose properties may be an excellent scaffold material for delivering cells in tissue engineering applications in vivo.     

Surface controlled biomimetic coating of polycaprolactone nanofiber meshes to be used as bone extracellular matrix analogues

The aim of this work was to develop novel electrospun nanofiber meshes coated with a biomimetic calcium phosphate (BCP) layer that mimics the extracellular microenvironment found in the human bone structure. Poly (epsilon-caprolactone) (PCL) was selected because of its well-known medical applications, its biodegradability, biocompatibility and its susceptibility to partial hydrolysis by a straightforward alkaline treatment. The deposition of a calcium phosphate layer, similar to the inorganic phase of bone, on PCL nanofiber meshes was achieved by means of a surface modification. This initial surface modification was followed by treatment with solutions containing calcium and phosphate ions. The process was finished by a posterior immersion in a simulated body fluid (SBF) with nearly 1.5 x the inorganic concentration of the human blood plasma ions. After some optimization work, the best conditions were chosen to perform the biological assays. The influence of the bone-like BCP layer on the viability and adhesion, as well as on the proliferation of human osteoblast-like cells, was assessed. It was shown that PCL nanofiber meshes coated with a BCP layer support and enhance the proliferation of osteoblasts for long culture periods. The attractive properties of the coated structures produced in the present work demonstrated that those materials have potential to be used for applications in bone tissue engineering. This is the first time that nanofiber meshes could be coated with a biomimetic bone-like calcium phosphate layer produced in a way that the original mesh architecture can be fully maintained.     

Osteoblast function on nanophase alumina materials: Influence of chemistry, phase, and topography

Alumina is a material that has been used in both dental and orthopedic applications. It is with these uses in mind that osteoblast (bone-forming cell) function on alumina of varying particulate size, chemistry, and phase was tested in order to determine what formulation might be the most beneficial for bone regeneration. Specifically, in vitro osteoblast adhesion, proliferation, intracellular alkaline phosphatase activity, and calcium deposition was observed on delta-phase nanospherical, alpha-phase conventional spherical, and boehmite nanofiber alumina. Results showed for the first time increased osteoblast functions on the nanofiber alumina. Specifically, a 16% increase in osteoblast adhesion over nanophase spherical alumina and a 97% increase over conventional spherical alumina were found for nanofiber alumina after 2 h. A 29% increase in cell number after 5 days and up to a 57% greater amount of calcium was found on the surface of the nanofiber alumina compared with other alumina surfaces. Some of the possible explanations for such enhanced osteoblast behavior on nanofiber alumina may be attributed to chemistry, crystalline phase, and topography. Increased osteoblast function on nanofiber alumina suggests that it may be an ideal material for use in orthopedic and dental applications.     

Preparation,characterization and in vitro analysis of novel structured nanofibrous scaffolds for bone tissue engineering

In a previous study, a three-dimensional nanofibrous spiral scaffold for bone tissue engineering was developed, which showed enhanced human osteoblast cell attachment, proliferation and differentiation compared with traditional cylinder scaffolds, owing to the incorporation of spiral structures and nanofiber. However, the application of these scaffolds to bone tissue engineering was limited by their weak mechanical strength. This limitation triggered the design for novel structured scaffolds with reinforced physical characteristics. In this study, spiral polycaprolactone (PCL) nanofibrous scaffolds were inserted into poly(lactide-co-glycolide) (PLGA) microsphere sintered tubular scaffolds to form integrated scaffolds to provide mechanical properties and bioactivity appropriate for bone tissue engineering. Four experiment groups were designed: PLGA cylinder scaffold; PLGA tubular scaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubular scaffold with PCL nanofiber containing spiral structured inner core. The morphology, porosity and mechanical properties of the scaffolds were characterized. Furthermore, human osteoblastic cells were seeded on these scaffolds, and the cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds were evaluated. The integrated scaffolds had Young’s modulus 250–300 MPa, and compressive strength 8–11 MPa under uniaxial compression. With the addition of an inner highly porous insert to the tubular shell, human osteoblast cells seeded on the integrated scaffolds showed slightly higher cell proliferation, 20–25% more alkaline phosphatase expression and twofold higher calcium deposition than those on the cylinder and tubular scaffolds. Furthermore, compared with sintered PLGA cylinder scaffolds, the integrated scaffolds allowed better cellular infiltration Therefore, this design demonstrates great potential for integrated scaffolds in bone tissue engineering applications.     

Biomimetic surfaces for control of cell adhesion to facilitate bone formation

Cell adhesion to extracellular matrix ligands through integrin receptors plays a central role in bone formation and maintenance by anchoring cells and triggering signals that direct osteoblast proliferation and differentiation. Moreover, osteoblast adhesion to adsorbed, synthesized, or engineered extracellular ligands on synthetic surfaces is critical to numerous biomedical and biotechnological applications. Considerable research efforts have concentrated on the development of surfaces that promote osteoblast differentiation and bone formation. Emerging surface engineering approaches have focused on creating biomimetic substrates that target integrins to activate signaling pathways directing the osteoblast differentiation program. These initiatives generally rely on controlling the adsorption of extracellular matrix ligands or engineering synthetic supports presenting bioadhesive motifs from extracellular matrix proteins. These biomolecular approaches provide promising strategies for the engineering of robust biofunctional matrices that control cell adhesion and signaling and promote osteoblast proliferation, differentiation, and matrix mineralization.     

Collagen–PCL Sheath–Core Bicomponent Electrospun Scaffolds Increase Osteogenic Differentiation and Calcium Accretion of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hASCs) are an abundant cell source capable of osteogenic differentiation, and have been investigated as an autologous stem cell source for bone tissue engineering applications. The objective of this study was to determine if the addition of a type-I collagen sheath to the surface of poly(ε-caprolactone) (PCL) nanofibers would enhance viability, proliferation and osteogenesis of hASCs. This is the first study to examine the differentiation behavior of hASCs on collagen–PCL sheath–core bicomponent nanofiber scaffolds developed using a co-axial electrospinning technique. The use of a sheath–core configuration ensured a uniform coating of collagen on the PCL nanofibers. PCL nanofiber scaffolds prepared using a conventional electrospinning technique served as controls. hASCs were seeded at a density of 20 000 cells/cm² on 1 cm² electrospun nanofiber (pure PCL or collagen–PCL sheath–core) sheets. Confocal microscopy and hASC proliferation data confirmed the presence of viable cells after 2 weeks in culture on all scaffolds. Greater cell spreading occurred on bicomponent collagen–PCL scaffolds at earlier time points. hASCs were osteogenically differentiated by addition of soluble osteogenic inductive factors. Calcium quantification indicated cell-mediated calcium accretion was approx. 5-times higher on bicomponent collagen–PCL sheath–core scaffolds compared to PCL controls, indicating collagen–PCL bicomponent scaffolds promoted greater hASC osteogenesis after two weeks of culture in osteogenic medium. This is the first study to examine the effects of collagen–PCL sheath–core composite nanofibers on hASC viability, proliferation and osteogenesis. The sheath–core composite fibers significantly increased calcium accretion of hASCs, indicating that collagen–PCL sheath–core bicomponent structures have potential for bone tissue engineering applications using hASCs.     

Osteoblast differentiation and disinfection induced by nitrogen plasma-treated surfaces

Plasma technology is widely employed to tailor the surface chemistry of polymeric biomaterials. In this work, nitrogen-containing functional groups were generated on a polymer surface by N? plasma immersion ion implantation (PIII). We evaluated the abilities of the resulting surface to inhibit bacterial growth and to enhance osteoblast differentiation from the perspective of bone tissue engineering. Our results demonstrate that the N? PIII-treated polymer surface exhibits antibacterial properties against Escherichia coli. Moreover, the N? PIII-treated polymer surface has the ability to enhance differentiation of osteoblasts. N? PIII-treated polymer surface may therefore be useful in bone tissue engineering.     

Differential regulation of osteoblasts by substrate microstructural features

Microtextured titanium implant surfaces enhance bone formation in vivo and osteoblast phenotypic expression in vitro, but the mechanisms are not understood. To determine the roles of specific microarchitectural features in modulating osteoblast behavior, we used Ti surfaces prepared by electrochemical micromachining as substrates for MG63 osteoblast-like cell culture. Cell response was compared to tissue culture plastic, a sand-blasted with large grit and acid-etched surface with defined mixed microtopography (SLA), polished Ti surfaces, and polished surfaces electrochemically machined through a photoresist pattern to produce cavities with 100, 30 and 10 microm diameters arranged so that the ratio of the microscopic-scale area of the cavities versus the microscopic-scale area of the flat region between the cavities was equal to 1 or 6. Microstructured disks were acid-etched, producing overall sub-micron-scale roughness (Ra=0.7 microm). Cell number, differentiation (alkaline phosphatase; osteocalcin) and local factor levels (TGF-beta1; PGE(2)) varied with microarchitecture. 100 microm cavities favored osteoblast attachment and growth, the sub-micron-scale etch enhanced differentiation and TGF-beta1 production, whereas PGE(2) depended on cavity dimensions but not the sub-micron-scale roughness.     

Increased osteoblast functions among nanophase titania/poly(lactide-co-glycolide) composites of the highest nanometer surface roughness

Currently, the scientific challenges for bone tissue engineering lie in the development of suitable scaffold materials that can improve bone cell adhesion, proliferation, and differentiation. The design of nanophase titania/poly(lactide-co-glycolide) (PLGA) composites offers an exciting approach to combine the advantages of a degradable polymer with nanosize ceramic particles to optimize the physical and biological properties necessary for bone regeneration. Moreover, because of the presence of nanosized ceramics, such composites can be formulated to match the surface roughness of bone. For these reasons, the objective of the present in vitro study was to investigate osteoblast (bone-forming cell) adhesion and long-term functions on nanophase titania/PLGA composites that mimic the surface roughness of bone. Various sonication powers were applied in this study to manipulate titania dispersions in PLGA and consequently control their surface roughness. Most importantly, results correlated better osteoblast adhesion and long-term functions (such as collagen, alkaline phosphatase activity, and calcium-containing mineral deposition) among nanophase titania/PLGA composites that had surface roughness values closer to natural bone. In this manner, this present study demonstrated that the nanophase titania/PLGA composites sonicated to have nanometer surface roughness values can improve osteoblast functions necessary for enhanced bone tissue engineering applications.     

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering

The collagen–chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen–chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen–chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen–chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young’s modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen–chitosan nanofiber reinforced by TPU, both the break strength and the Young’s modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen–chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.     

Degradation of electrospun nanofiber scaffold by short wave length ultraviolet radiation treatment and its potential applications in tissue engineering

Development in the field of tissue engineering has brought much attention in the fabrication and preparation of scaffold with biodegradable synthetic polymer nanofibers. Electrospun biodegradable polymeric nanofibers are increasingly being used to fabricate scaffolds for tissue engineering applications as they provide high surface area-to-volume ratio and possess high porosity. One common way to sterilize polymeric nanofiber scaffolds is 254-nm ultraviolet (UV) irradiation. In this study, we aim to evaluate the effects of UV radiation on the degradation in polymeric nanofibers, and then capitalize on UV-induced degradation and UV photolithography in polymeric nanofiber scaffolds for tissue engineering applications. Poly(D,L-lactic-co-glycolic) acid (PLGA, 75:25) and poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL), 70:30] nanofibrous meshes were produced by electrospinning. The nanofibers were irradiated by commercial germicide UV (lambda=254 nm) lamp for different intervals. We found that UV sterilization induced significant degradation of nanofiber. At 1 h UV irradiation, the average molecular weight of PLGA and P(LLA-CL) nanofibers were reduced by 46% and 35%, respectively, with corresponding reduction in the tensile strength of 26% for PLGA and 28% for P(LLA-CL). Hence, precautions may have to be taken into consideration when sterilizing polymeric nanofibers by UV treatment. UV-induced degradation on nanofibers was applied to fabrication of a three-dimensional (3D) tissue engineering scaffold by UV photolithography. Masked exposure to UV could generate patterned holes (d=100 microm) on the nanofibrous mesh. Cell culture study showed that smooth muscle cells were able to migrate into the holes. This method can be used to fabricate a 3D nanofibrous scaffold with micropores.     

Covalent functionalization of NiTi surfaces with bioactive peptide amphiphile nanofibers

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium (NiTi) shape memory alloy in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO(2) layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO(2) with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM techniques. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers.     

The response of osteoblast-like cells towards collagen type I coating immobilized by p-nitrophenylchloroformate to titanium

The scaffold surface composition can be altered by the use of surface coatings. The use of thin coatings will give special surface properties, while the bulk properties of the scaffold are preserved. Collagen type I is known to play an important role during cell adhesion as well as osteoblast differentiation. A common way to coat surfaces is the adsorption method. An alternative way is the use of a protein immobilization method like p-nitrophenyl chloroformate. In this study, we investigated the effect of a collagen type I coating and p-nitrophenyl chloroformate as a protein immobilization method on osteoblast adhesion, proliferation, and differentiation. Titanium fiber meshes were treated with sodium hydroxide (NaOH), followed by p-nitrophenyl chloroformate, and coated with collagen type I. Osteoblast-like cells were seeded into the meshes and cultured for 24 days. The cell attachment, proliferation, and differentiation were measured by using Live and Dead assay, cell counting, DNA analysis, alkaline phosphatase activity assay, calcium content measurement, Real Time PCR (QPCR), and scanning electron microscopy (SEM). Results demonstrated that initially less cells were attached to the covalently bounded collagen meshes (NPC-Col) compared with titanium as control (Ti) and adsorbed collagen meshes (ABS-Col). Further, a decreased growth curve of cells cultured on the NPC-Col meshes was observed in comparison with Ti and ABS-Col meshes. The calcium measurements and SEM pictures revealed that all three surfaces showed differentiation of osteoblast-like cells after 8-24 days. On the basis of our results, we conclude that initially less cells were attached to the NPC-Col meshes and that they had a decreased proliferation rate. Further, we conclude that an adsorbed collagen type I coating stimulated the osteoblastic differentiation of rat bone marrow cells.     

High surface energy enhances cell response to titanium substrate microstructure

Titanium (Ti) is used for implantable devices because of its biocompatible oxide surface layer. TiO2 surfaces that have a complex microtopography increase bone-to-implant contact and removal torque forces in vivo and induce osteoblast differentiation in vitro. Studies examining osteoblast response to controlled surface chemistries indicate that hydrophilic surfaces are osteogenic, but TiO2 surfaces produced until now exhibit low surface energy because of adsorbed hydrocarbons and carbonates from the ambient atmosphere or roughness induced hydrophobicity. Novel hydroxylated/hydrated Ti surfaces were used to retain high surface energy of TiO2. Osteoblasts grown on this modified surface exhibited a more differentiated phenotype characterized by increased alkaline phosphatase activity and osteocalcin and generated an osteogenic microenvironment through higher production of PGE2 and TGF-beta1. Moreover, 1alpha,25OH2D3 increased these effects in a manner that was synergistic with high surface energy. This suggests that increased bone formation observed on modified Ti surfaces in vivo is due in part to stimulatory effects of high surface energy on osteoblasts.     

中药在干细胞增殖和组织工程中的应用

BACKGROUND:Some Chinese herbal ingredients produce the analogical and pharmacological effects equivalent to growth factors, which can promote cell proliferation as well as tissue and organ recovery and regeneration. Additionally, they possess low price and relative stable physicochemical property. Thus, the applications of traditional Chinese medicine in tissue engineering has attracted more attentions of researchers all over the world. OBJECTIVE:To review the application of the traditional Chinese medicine in stem cell proliferation and tissue engineering. METHODS:A computer-based retrieval of PubMed and CNKI databases was performed for articles concerning the application of traditional Chinese medicine in stem cell proliferation and tissue engineering published from 1978 to 2015. The keywords were “the traditional Chinese medicine, tissue engineering, application, stem cell, anti-inflammatory, cross linking, surface modification, osteoblast, blood vessel” in English and Chinese, respectively. RESULTS AND CONCLUSION: Natural medicine and the traditional Chinese medicine are expected to be applied in tissue engineering as a surrogate of growth factor and antibiotics, which exert anti-inflammatory, angiogenesis, surface modification, anticoagulation and cross-linking roles as well as promote proliferation and differentiation of stem cells in tissue engineering.     

Fabrication and evaluation of nanoporous alumina membranes for osteoblast culture

An understanding of osteoblast response to surface topography is essential for successful bone tissue engineering applications. Alumina has been extensively used as a substrate for bone tissue constructs. However, current techniques do not allow precise surface topography and orientation of the material. In this research, a two-step anodization process was optimized for the fabrication of nanoporous alumina membranes with uniform pore dimension and distribution. The anodization voltage can be varied to create nanoporous alumina membranes with pore sizes ranging from 30 to 80 nm in diameter. The impact of the nanoscale pores on osteoblast response was studied by evaluating cell adhesion, morphology, and matrix production. Scanning electron microscopy and atomic force microscopy were used to characterize the nanoporous alumina membranes. Osteoblast adhesion and morphology were investigated using scanning electron microscopy images and matrix production was characterized using energy dispersive spectroscopy. This research combined the advantages of using alumina, a material with proven biocompatibility and current orthopedic clinical applications, and incorporated porous features on the nanoscale which have been reported to improve osteoblast response.     

Biocompatible nanofiber matrices for the engineering of a dermal substitute for skin regeneration

Natural and synthetic biodegradable nanofibers are extensively used for biomedical applications and tissue engineering. Biocompatibility and a well-established safety profile for polycaprolactone (PCL) and collagen represent a favorable matrix for preparing a dermal substitute for engineering skin. Collagen synthesized by fibroblasts is a good surface active agent and demonstrates its ability to penetrate a lipid-free interface. During granulation tissue formation, fibronectin provides a temporary substratum for migration and proliferation of cells and provides a template for collagen deposition, which increases stiffness and tensile strength of this healing tissues. The objective of this study was to fabricate nanofiber matrices from novel biodegradable PCL and collagen to mimic natural extracellular matrix (ECM) and to examine the cell behavior, cell attachment, and interaction between cells and nanofiber matrices. Collagen nanofiber matrices show a significant (p < 0.001) level of fibroblast proliferation and increase up to 54% compared with control tissue culture plate (TCP) after 72 h. The present investigation shows that PCL-coated collagen matrices are suitable for fibroblast growth, proliferation, and migration inside the matrices. This novel biodegradable PCL and collagen nanofiber matrices support the attachment and proliferation of human dermal fibroblasts and might have potential in tissue engineering as a dermal substitute for skin regeneration.