Amide-linkage formed between ammonia plasma treated poly(D,L-lactide acid) scaffolds and bio-peptides: enhancement of cell adhesion and osteogenic differentiation in vitro

The surface characteristics of scaffolds for bone tissue engineering must support cell adhesion, migration, proliferation, and osteogenic differentiation. In the study, poly(D,L ‐lactide acid) (PDLLA) scaffolds were modified by combing ammonia (NH₃) plasma pretreatment with Gly‐Arg‐Gly‐Asp‐Ser (GRGDS)‐peptides coupling technologies. The x‐ray photoelectron spectroscopy (XPS) survey spectra showed the peak of N1s at the surface of NH₃ plasma pretreated PDLLA, which was further raised after GRGDS conjugation. Furthermore, N1s and C1s in the high‐resolution XPS spectra revealed the presence of ? C?N (imine), ? C? NH? (amine), and ? C?O? NH? (amide) groups. The GRGDS conjugation increased amide groups and decreased amine groups in the plasma‐treated PDLLA. Confocal microscope and high performance liquid chromatography verified the anchored peptides after the conjugation process. Bone marrow mesenchymal stem cells were co‐cultured with scaffolds. Fluorescent microscope and scanning electron microscope photographs revealed the best cell adhesion in NH₃ plasma pretreated and GRGDS conjugated scaffolds, and the least attachment in unmodified scaffolds. Real‐time PCR demonstrated that expression of osteogenesis‐related genes, such as osteocalcin, alkaline phosphatase, type I collagen, bone morphogenetic protein‐2 and osteopontin, was upregulated in the single NH₃ plasma treated and NH₃ plasma pretreated scaffolds following GRGDS conjugation. The results show that NH₃ plasma treatment promotes the conjugation of GRGDS peptides to the PDLLA scaffolds via the formation of amide linkage, and combination of NH₃ plasma treatment and peptides conjugation may enhance the cell adhesion and osteogenic differentiation in the PDLLA scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 682–694, 2011.     

Liquid perfluorochemical-supported hybrid cell culture system for proliferation of chondrocytes on fibrous polylactide scaffolds

CP5 bovine chondrocytes were cultured on biodegradable electrospun fibrous polylactide (PLA) scaffolds placed on a flexible interface formed between two immiscible liquid phases: (1) hydrophobic perfluorochemical (PFC) and (2) aqueous culture medium, as a new way of cartilage implant development. Robust and intensive growth of CP5 cells was achieved in our hybrid liquid–solid–liquid culture system consisting of the fibrous PLA scaffolds in contrast to limited growth of the CP5 cells in traditional culture system with PLA scaffold placed on solid surface. The multicellular aggregates of CP5 cells covered the surface of PLA scaffolds and the chondrocytes migrated through and overgrew internal fibers of the scaffolds. Our hybrid culture system simultaneously allows the adhesion of adherent CP5 cells to fibers of PLA scaffolds as well as, due to use of phase of PFC, enhances the mass transfer in the case of supplying/removing of respiratory gases, i.e., O₂ and CO₂. Our flexible (independent of vessel shape) system is simple, ready-to-use and may utilize a variety of polymer-based scaffolds traditionally proposed for implant development.     

Composite 3D printed scaffold with structured electrospun nanofibers promotes chondrocyte adhesion and infiltration

Additive manufacturing, also called 3D printing, is an effective method for preparing scaffolds with defined structure and porosity. The disadvantage of the technique is the excessive smoothness of the printed fibers, which does not support cell adhesion. In the present study, a 3D printed scaffold was combined with electrospun classic or structured nanofibers to promote cell adhesion. Structured nanofibers were used to improve the infiltration of cells into the scaffold. Electrospun layers were connected to 3D printed fibers by gluing, thus enabling the fabrication of scaffolds with unlimited thickness. The composite 3D printed/nanofibrous scaffolds were seeded with primary chondrocytes and tested in vitro for cell adhesion, proliferation and differentiation. The experiment showed excellent cell infiltration, viability, and good cell proliferation. On the other hand, partial chondrocyte dedifferentiation was shown. Other materials supporting chondrogenic differentiation will be investigated in future studies.     

Lanthanum phosphate/chitosan scaffolds enhance cytocompatibility and osteogenic efficiency via the Wnt/β-catenin pathway

Background

Fabrication of porous scaffolds with great biocompatibility and osteoinductivity to promote bone defect healing has attracted extensive attention.

Methods

In a previous study, novel lanthanum phosphate (LaPO₄)/chitosan (CS) scaffolds were prepared by distributing 40- to 60-nm LaPO₄ nanoparticles throughout plate-like CS films.

Results

Interconnected three dimensional (3D) macropores within the scaffolds increased the scaffold osteoconductivity, thereby promoting cell adhesion and bone tissue in-growth. The LaPO₄/CS scaffolds showed no obvious toxicity and accelerated bone generation in a rat cranial defect model. Notably, the element La in the scaffolds was found to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the Wnt/β-catenin signalling pathway and induced high expression of the osteogenesis-related genes alkaline phosphatase, osteocalcin and Collagen I (Col-I). Moreover, the LaPO₄/CS scaffolds enhanced bone regeneration and collagen fibre deposition in rat critical-sized calvarial defect sites.

Conclusion

The novel LaPO₄/CS scaffolds provide an admirable and promising platform for the repair of bone defects.

     

Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways

Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.     

Improvement of biomaterials used in tissue engineering by an ageing treatment

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60 % (scaffold without ageing treatment) to 80 %. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.     

Sensing scaffolds to monitor cellular activity using impedance measurements

Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density.     

Bioactive/Natural Polymeric Scaffolds Loaded with Ciprofloxacin for Treatment of Osteomyelitis

Local delivery of antibiotic into injured bone is a demand. In this work, different scaffolds of chitosan (C) with or without bioactive glass (G) were prepared using the freeze-drying technique in 2:1, 1:1, and 1:2 weight ratios. Chitosan scaffolds and selected formulas of chitosan to bioglass were loaded with ciprofloxacin in 5%, 10%, and 20% w/w. Scaffold morphology showed an interconnected porous structure, where the glass particles were homogeneously dispersed in the chitosan matrix. The kinetic study confirmed that the scaffold containing 1:2 weight ratio of chitosan to glass (CG12) showed optimal bioactivity with good compromise between Ca and P uptake capacities and Si release rate. Chitosan/bioactive glass scaffolds showed larger t ₅₀ values indicating less burst drug release followed by a sustained drug release profile compared to that of chitosan scaffolds. The cell growth, migration, adhesion, and invasion were enhanced onto CG12 scaffold surfaces. Samples of CG12 scaffolds with or without 5% drug induced vascular endothelial growth factor (VEGF), while those containing 10% drug diminished VEGF level. Only CG12 induced the cell differentiation (alkaline phosphatase activity). In conclusion, CG12 containing 5% drug can be considered a biocompatible carrier which would help in the localized osteomyelitis treatment.     

Structural and Surface Compatibility Study of Modified Electrospun Poly(ε-caprolactone) (PCL) Composites for Skin Tissue Engineering

In this study, biodegradable poly(ε-caprolactone) (PCL) nanofibers (PCL-NF), collagen-coated PCL nanofibers (Col-c-PCL), and titanium dioxide-incorporated PCL (TiO₂-i-PCL) nanofibers were prepared by electrospinning technique to study the surface and structural compatibility of these scaffolds for skin tisuue engineering. Collagen coating over the PCL nanofibers was done by electrospinning process. Morphology of PCL nanofibers in electrospinning was investigated at different voltages and at different concentrations of PCL. The morphology, interaction between different materials, surface property, and presence of TiO₂ were studied by scanning electron microscopy (SEM), Fourier transform IR spectroscopy (FTIR), contact angle measurement, energy dispersion X-ray spectroscopy (EDX), and X-ray photoelectron spectroscopy (XPS). MTT assay and cell adhesion study were done to check biocompatibilty of these scaffolds. SEM study confirmed the formation of nanofibers without beads. FTIR proved presence of collagen on PCL scaffold, and contact angle study showed increment of hydrophilicity of Col-c-PCL and TiO₂-i-PCL due to collagen coating and incorporation of TiO₂, respectively. EDX and XPS studies revealed distribution of entrapped TiO₂ at molecular level. MTT assay and cell adhesion study using L929 fibroblast cell line proved viability of cells with attachment of fibroblasts over the scaffold. Thus, in a nutshell, we can conclude from the outcomes of our investigational works that such composite can be considered as a tissue engineered construct for skin wound healing.     

Application of polyethyleneimine‐modified scaffolds to the regeneration of cartilaginous tissue

In this study, we analyzed the physicochemical and biophysical properties of three‐dimensional scaffolds modified using polyethyleneimine (PEI) and applied these scaffolds to the cultivation of bovine knee chondrocytes (BKCs). PEI was crosslinked in the bulk or on the surface of the ternary scaffolds comprising polyethylene oxide, chitin and chitosan. The results revealed that when the concentration of PEI was less than 300 μg/mL, the cytotoxicity of a scaffold was on the same order in the two method of modification. An increase in the concentration of PEI favored the adhesion of BKCs. When the amount of PEI in scaffolds is fixed, the surface‐modified scaffolds exhibited a higher adhesion efficiency of BKCs than the bulk‐modified scaffolds. For the regeneration of cartilaginous components, a higher amount of PEI in a scaffold yielded larger amounts of proliferated BKCs, secreted glycosaminoglycans, and produced collagen. In addition, the formation of neocartilage in the surface‐modified scaffolds was more effective than that in the bulk‐modified scaffolds. These tissue‐engineered scaffolds, modified by an appropriate concentration of PEI, can be potentially applied to cartilage repair in clinical trials. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Fabrication of chitin-chitosan/nano ZrO(2) composite scaffolds for tissue engineering applications

The urge to repair and regenerate natural tissues can now be satisfactorily fulfilled by various tissue engineering approaches. Chitin and chitosan are the most widely accepted biodegradable and biocompatible materials subsequent to cellulose. The incorporation of nano ZrO₂ onto the chitin-chitosan scaffold is thought to enhance osteogenesis. Hence a nanocomposite scaffold was fabricated by lyophilization technique using chitin-chitosan with nano ZrO₂. The prepared nanocomposite scaffolds were characterized using SEM, FTIR, XRD and TGA. In addition, the swelling, degradation, biomineralization, cell viability and cell attachment of the composite scaffolds were also evaluated. The results demonstrated better swelling and controlled degradation in comparison to the control scaffold. Cell viability studies proved the non toxic nature of the nanocomposite scaffolds. Cells were found to be attached to the pore walls and spread uniformly throughout the scaffolds. All these results suggested that the developed nanocomposite scaffolds possess the prerequisites for tissue engineering scaffolds and could be used for various tissue engineering applications.     

BMSCs在PLGA-[ASP-PEG]基质材料表面粘附及增殖的研究

目的：探讨大鼠骨髓间充质干细胞BMSCs在聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段多元共聚物 PLGA-[ASP-PEG]表面粘附、增殖的情况,为组织工程学体外诱导种子细胞生长提供新的生物材料。方法：在PLGA支架材料中引入聚乙二醇（PEG）和含有多个功能位点的天冬氨酸（ASP）,制成PLGA-[ASP-PEG]高分子支架材料。 将PLGA-[ASP-PEG]支架材料与BMSCs复合培养,以未改性的PLGA支架材料作对照,通过沉淀法、MTT法和考马斯亮蓝法分别检测BMSCs的粘附和增殖变化;扫描电镜观察黏附细胞的形态。结果 BMSCs在PLGA-[ASP-PEG]材料表面帖壁生长,细胞数目明显多于单纯PLGA组。细胞粘附率检测显示：改性后的PLGA-[ASP-PEG]表面BMSCs的粘附性能和增殖能力明显高于对照组,P＜0.05。MTT比色试验,BMSCs在三嵌段材料上培养20d后,吸光值A=1.336,约为对照组0.780的两倍。细胞内蛋白总量间接反映细胞黏附及增殖情况。培养12d时,在PLGA-[ASP-PEG]材料组细胞的蛋白含量为66.44μg/孔,单纯PLGA组为41.23μg/孔,间接说明了三嵌段材料生物相容性好,细胞黏附力强的特点。结论PLGA-[ASP-PEG]能促进组织工程种子细胞在骨基质材料表面的黏附、增殖并能较好地保持细胞的形态。     

A keratin scaffold regulates epidermal barrier formation,mitochondrial lipid composition,and activity

Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI^−/− and KtyII^−/−_K8 mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation.     

Significant Type I and Type III Collagen Production from Human Periodontal Ligament Fibroblasts in 3D Peptide Scaffolds without Extra Growth Factors

We here report the development of two peptide scaffolds designed for periodontal ligament fibroblasts. The scaffolds consist of one of the pure self-assembling peptide scaffolds RADA16 through direct coupling to short biologically active motifs. The motifs are 2-unit RGD binding sequence PRG (PRGDSGYRGDS) and laminin cell adhesion motif PDS (PDSGR). RGD and laminin have been previously shown to promote specific biological activities including periodontal ligament fibroblasts adhesion, proliferation and protein production. Compared to the pure RADA16 peptide scaffold, we here show that these designer peptide scaffolds significantly promote human periodontal ligament fibroblasts to proliferate and migrate into the scaffolds (for ∼300 µm/two weeks). Moreover these peptide scaffolds significantly stimulated periodontal ligament fibroblasts to produce extracellular matrix proteins without using extra additional growth factors. Immunofluorescent images clearly demonstrated that the peptide scaffolds were almost completely covered with type I and type III collagens which were main protein components of periodontal ligament. Our results suggest that these designer self-assembling peptide nanofiber scaffolds may be useful for promoting wound healing and especially periodontal ligament tissue regeneration.     

Effect of carbon nanotube coating of aligned nanofibrous polymer scaffolds on the neurite outgrowth of PC-12 cells

Neurite outgrowth from endogenous or transplanted cells is important for neural regeneration following nerve tissue injury. Modified substrates often provide better environments for cell adhesion and neurite outgrowth. This study was conducted to determine if MWCNT (multiwalled carbon nanotube)-coated electrospun PLCL [poly (l-lactic acid-co-3-caprolactone)] nanofibres improved the neurite outgrowth of PC-12 cells. To accomplish this, two groups, PC-12 cells in either uncoated PLCL scaffolds or MWCNT-coated PLCL scaffolds were cultured for 9 days. MWCNT-coated PLCL scaffolds showed improved adhesion, proliferation and neurite outgrowth of PC-12 cells. These findings suggest that MWCNT-coated nanofibrous scaffolds may be an attractive platform for cell transplantation application in neural tissue engineering.     

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate–polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na₂CO₃-degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.     

Poly(3-hydroxubutyrate-co-4-hydroxubutyrate)/bacterial cellulose composite porous scaffold: Preparation, characterization and biocompatibility evaluation

Bio-composite scaffolds were prepared by freeze-drying using poly(3-hydroxubutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) and bacterial cellulose (BC) as raw materials and trifluoroacetic acid (TFA) as co-solvent. The characteristics of the composite scaffold were investigated by field emission scanning electron microscopy (FESEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), water contact angle measurement and tensile testing. Preliminary biodegradation test was performed for P(3HB-co-4HB) and P(3HB-co-4HB)/BC composite scaffold in buffer solution and enzyme solution. The biocompatibility of the composite scaffold was preliminarily evaluated by cell adhesion studies using Chinese Hamster Lung (CHL) fibroblast cells. The cells incubated with composite scaffold for 48 h were capable of forming cell adhesion and proliferation, which showed better biocompatibility than pure P(3HB-co-4HB) scaffold. Thus, the prepared P(3HB-co-4HB)/BC composite scaffold was bioactive and may be suitable for cell adhesion/attachment suggesting that these scaffolds can be used for wound dressing or tissue-engineering scaffolds.     

Tissue-Engineered Regeneration of Completely Transected Spinal Cord Using Induced Neural Stem Cells and Gelatin-Electrospun Poly (Lactide-Co-Glycolide)/Polyethylene Glycol Scaffolds

Tissue engineering has brought new possibilities for the treatment of spinal cord injury. Two important components for tissue engineering of the spinal cord include a suitable cell source and scaffold. In our study, we investigated induced mouse embryonic fibroblasts (MEFs) directly reprogrammed into neural stem cells (iNSCs), as a cell source. Three-dimensional (3D) electrospun poly (lactide-co-glycolide)/polyethylene glycol (PLGA-PEG) nanofiber scaffolds were used for iNSCs adhesion and growth. Cell growth, survival and proliferation on the scaffolds were investigated. Scanning electron microcopy (SEM) and nuclei staining were used to assess cell growth on the scaffolds. Scaffolds with iNSCs were then transplanted into transected rat spinal cords. Two or 8 weeks following transplantation, immunofluorescence was performed to determine iNSC survival and differentiation within the scaffolds. Functional recovery was assessed using the Basso, Beattie, Bresnahan (BBB) Scale. Results indicated that iNSCs showed similar morphological features with wild-type neural stem cells (wt-NSCs), and expressed a variety of neural stem cell marker genes. Furthermore, iNSCs were shown to survive, with the ability to self-renew and undergo neural differentiation into neurons and glial cells within the 3D scaffolds in vivo. The iNSC-seeded scaffolds restored the continuity of the spinal cord and reduced cavity formation. Additionally, iNSC-seeded scaffolds contributed to functional recovery of the spinal cord. Therefore, PLGA-PEG scaffolds seeded with iNSCs may serve as promising supporting transplants for repairing spinal cord injury (SCI).