3D PLGA Scaffolds Research Articles

Calreticulin silencing inhibits extracellular matrix synthesis of human gingival fibroblasts cultured on three-dimensional poly(lactic-co-glycolic acid) scaffolds by inhibiting the calcineurin/nuclear factor of activated T cells 3 signalling pathway

BackgroundThe retraction and compression of gingival tissue have a significant impact on the efficiency and stability of orthodontic treatment, but the underlying molecular mechanism has not been fully elucidated. The aim of the current study was to investigate the effects of mechanical forces on the expression level of calreticulin (CRT), the activity of the calcineurin (CaN)/nuclear factor of activated T cells (NFAT) 3 signalling pathway, and extracellular matrix (ECM) synthesis in human gingival fibroblasts (HGFs) cultured on three-dimensional (3D) poly(lactic-co-glycolic acid) (PLGA) scaffolds and to further explore the mechanical transduction pathways that may be involved. Materials and methodsA mechanical force of 25 g/cm2 was applied to HGFs for 0, 6, 24, 48, or 72 h. The expression of CRT, CaN, NFAT3, phosphorylated NFAT3 (p-NFAT3) and type I collagen (COL-I) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Subsequently, small interfering RNA (siRNA) was used to knock down the expression of CRT in HGFs, and the impacts of the applied force on the expression levels of CaN, NFAT3, p-NFAT3, and COL-I were also evaluated by RT-qPCR and western blotting. ResultsThe application of mechanical force on HGFs cultured on 3D PLGA scaffolds led to a significant increases in CRT, CaN, and COL-I expression as well as a decrease in p-NFAT3 expression. However, the effects of mechanical force on CaN, p-NFAT3, and COL-I expression were reversed following downregulation of CRT and displayed a significant decrease in CaN/NFAT3 activity and COL-I synthesis. ConclusionThis study showed that the CaN/NFAT3 signalling pathway and CRT appear to be involved in the mechanotransduction of HGFs, and downregulation of CRT inhibits COL-I synthesis potentially via the CaN/NFAT3 signalling pathway. Taken together, these findings ultimately provide novel insight into the mechanisms underlying mechanical force-induced ECM synthesis, which may be conducive to the development of targeted therapeutics to treat fibrotic diseases, including gingival fibrosis caused by orthodontic treatment.

Effects of Smad4 on the expression of caspase‑3 and Bcl‑2 in human gingival fibroblasts cultured on 3DPLGA scaffolds induced by compressive force.

Human gingival fibroblasts (HGFs) are the main cells that comprise gingival tissue, where they transfer mechanical signals under physiological and pathological conditions. The exact mechanism underlying gingival tissue reconstruction under compressive forces remains unclear. The present study aimed to explore the effects of Smad4, caspase-3 and Bcl-2 on the proliferation of HGFs induced by compressive force. HGFs were cultured on poly(lactide-co-glycolide) (PLGA) scaffolds under an optimal compressive force of 25 g/cm2. Cell viability was determined via Cell Counting Kit-8 assays at 0, 12, 24, 48 and 72 h. The expression levels of Smad4, caspase-3 and Bcl-2 were measured via reverse transcription-quantitative PCR and western blotting. The application of compressive force on HGFs for 24 h resulted in a significant increase in cell proliferation and Bcl-2 expression, but a significant decrease in the expression of Smad4 and caspase-3; however, inverse trends were observed by 72 h. Subsequently, a lentivirus was used to overexpress Smad4 in HGFs, which attenuated the effects of compressive force on HGF proliferation and Bcl-2 expression, but enhanced caspase-3 expression, suggesting that Smad4 may regulate compressive force-induced apoptosis in HGFs. In conclusion, these findings increased understanding regarding the mechanisms of compressive force-induced HGF proliferation and apoptosis, which may provide further insight for improving the efficacy and stability of orthodontic treatment.

Potential Role of Integrin α₅β₁/Focal Adhesion Kinase (FAK) and Actin Cytoskeleton in the Mechanotransduction and Response of Human Gingival Fibroblasts Cultured on a 3-Dimension Lactide-Co-Glycolide (3D PLGA) Scaffold.

BackgroundThe stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved.Material/MethodsA compressive force of 25 g/m2 was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin α5β1/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin α5β1 inhibitor (Ac-PHSCN-NH 2, ATN-161).ResultsMechanical force increased the expression of integrin α5β1, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin α5β1 reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited.ConclusionsThe integrin α5β1/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin α5β1 pathway.

Mechanical force promotes the proliferation and extracellular matrix synthesis of human gingival fibroblasts cultured on 3D PLGA scaffolds via TGF‑β expression.

Human gingival fibroblasts (HGFs) are responsible for connective tissue repair and scarring, and are exposed to mechanical forces under physiological and pathological conditions. The exact mechanisms underlying gingival tissue reconstruction under mechanical forces remain unclear. The present study aimfed to investigate the effects of mechanical forces on the proliferation and extracellular matrix synthesis in HGFs by establishing a 3-dimensional (3D) HGF culture model using poly(lactide-co-glycolide) (PLGA) scaffolds. HGFs were cultured in 3D PLGA scaffolds and a mechanical force of 0, 5, 15, 25 or 35 g/cm2 was applied to HGFs for 24 h. A mechanical force of 25 g/cm2 induced the highest proliferation rate, and thus was selected for subsequent experiments. Cell viability was determined using the MTT assay at 0, 24, 48 and 72 h. The expression levels of type I collagen (COL-1) and matrix metallopeptidase (MMP)-1 were examined by reverse transcription-quantitative polymerase chain reaction and ELISA, and transforming growth factor (TGF)-β expression was evaluated by ELISA. The application of mechanical force on HGFs cultured on the 3D PLGA scaffolds resulted in a significant increase in cell proliferation and COL-1 expression, as well as a decrease in MMP-1 expression. A TGF-β1 inhibitor was also applied, which attenuated the effects of mechanical force on HGF proliferation, and COL-1 and MMP-1 expression, thus suggesting that TGF-β signaling pathways may mediate the mechanical force-induced alterations observed in HGFs. In conclusion, these findings helped to clarify the mechanisms underlying mechanical force-induced HGF proliferation and ECM synthesis, which may promote the development of targeted therapeutics to treat various diseases, including gingival atrophy caused by orthodontic treatment.

Enhancement in sustained release of antimicrobial peptide and BMP-2 from degradable three dimensional-printed PLGA scaffold for bone regeneration.

One of the goals of bone tissue engineering is to create scaffolds with well-defined, inter-connected pores, excellent biocompatibility and osteoinductive ability. Three-dimensional (3D)-printed polymer scaffold coated with bioactive peptide are an effective approach to fabricating ideal bone tissue engineering scaffolds for bone defect repair. However, the current strategy of adding bioactive peptides generally cause degradation to the polymer materials or damage the bioactivity of the biomolecules. Thus, in this study, we used a biomimetic process via poly(dopamine) coating to prepare functional 3D PLGA porous scaffolds with immobilized BMP-2 and ponericin G1 that efficiently regulate the osteogenic differentiation of preosteoblasts (MC3T3-E1) and simultaneously inhibit of pathogenic microbes, thereby enhancing biological activity. In this study, we analysed a 3D PLGA porous scaffold by scanning electron microscopy, water contact angle measurements, and materials testing. Subsequently, we examined the adsorption, release and in vitro antimicrobial activity of the 3D PLGA. Surface characterization showed that poly(dopamine) surface modification could more efficiently mediate the immobilization of BMP-2 and ponericin G1 onto the scaffold surfaces than physical adsorption, and that ponericin G1-immobilized 3D PLGA scaffolds were able to maintain long-term antibacterial activity. We evaluated the influence on cell adhesion, proliferation and differentiation by culturing MC3T3-E1 cells on different modified 3D PLGA scaffolds in vitro. The biological results indicate that MC3T3-E1 cell attachment and proliferation on BMP-2/ponericin G1-immobilized 3D PLGA scaffolds were much higher than those on other groups. Calcium deposition, and gene expression results showed that the osteogenic differentiation of cells was effectively improved by loading the 3D PLGA scaffold with BMP-2 and ponericin G1. In summary, our findings indicated that the polydopamine-assisted surface modification method can be a useful tool for grafting biomolecules onto biodegradable implants, and the dual release of BMP-2 and ponericin G1 can enhance the osteointegration of bone implants and simultaneously inhibit of pathogenic microbes. Therefore, we conclude that the BMP-2/ponericin G1-loaded PLGA 3D scaffolds are versatile and biocompatible scaffolds for bone tissue engineering.

In vitro study of 3D PLGA/n-HAp/β-TCP composite scaffolds with etched oxygen plasma surface modification in bone tissue engineering

Three-dimensional (3D) scaffolds have many advantageous properties for bone tissue engineering application, due to its controllable properties such as pore size, structural shape and interconnectivity. In this study, effects on oxygen plasma surface modification and adding of nano-hydroxyapatite (n-HAp) and β-tricalcium phosphate (β-TCP) on the 3D PLGA/n-HAp/β-TCP scaffolds for improving preosteoblast cell (MC3T3-E1) adhesion, proliferation and differentiation were investigated. The 3D PLGA/n-HAp/β-TCP scaffolds were fabricated by 3D Bio-Extruder equipment. The 3D scaffolds were prepared with 0°/90° architecture and pore size of approximately 300μm. In addition 3D scaffolds surface were etched by oxygen plasma to enhance the hydrophilic property and surface roughness. After oxygen plasma treatment, the surface chemistry and morphology were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. And also hydrophilic property was measured by contact angle. The MC3T3-E1 cell proliferation and differentiation were investigated by MTT assay and ALP activity. In present work, the 3D PLGA/HAp/beta-TCP composite scaffold with suitable structure for the growth of osteoblast cells was successfully fabricated by 3D rapid prototyping technique. The surface hydrophilicity and roughness of 3D scaffold increased by oxygen plasma treatment had a positive effect on cell adhesion, proliferation, and differentiation. Furthermore, the differentiation of MC3T3-E1 cell was significantly enhanced by adding of n-HAp and β-TCP on 3D PLGA scaffold. As a result, combination of bioceramics and oxygen plasma treatment showed a synergistic effect on biocompatibility of 3D scaffolds. This result confirms that this technique was useful tool for improving the biocompatibility in bone tissue engineering application.

Promotion of spinal cord axon regeneration by 3D nanofibrous core–sheath scaffolds

Since spinal cord injury is a complicated problem, neural tissue repair, and regeneration strategies have received a great deal of attention. In this study, a three-dimensional (3D) nanofibrous core-sheath scaffold with nanorough sheath and aligned core were fabricated by a combined electrospinning method with water vortex and two-nozzle system. In vitro and in vivo biological tests were carried out on the poly(lactic-co-glycolic acid) (PLGA) scaffolds. The cell morphology and proliferation evaluation of nerve cells on 3D PLGA scaffolds were studied. Cells were properly orientated along the aligned fiber direction of the scaffold. In animal studies, adult rats received a complete lateral hemisection at the T9-T10 level. Scaffolds were engrafted to bridge 3 mm defects of 10 adult rat spinal cords; 10 rats were used as controls. For 8 weeks, motor and sensory recovery by open field locomotor scale, narrow beam and tail flick tests were assessed. Locomotor and sensory scores of grafted animals were significantly better than the control group. Histological findings demonstrated that the scaffold supports the axonal regeneration of injured spinal cords and regenerating axons were seen to enter the graft and extend along its length.

Hollow three-dimensional endothelialized microvessel networks based on femtosecond laser ablation

In this study, a novel method for the fabrication of hollow three-dimensional (3D) poly(lactic-co-glycolic acid) (PLGA) microvessel scaffolds is proposed. In this novel fabrication method, a salt ingot, which was used as a temporary frame to define the shape of the desired scaffold, was fabricated by extrusion molding. The salt ingot was immersed in a PLGA solution and the PGLA enveloped the ingot entirely. The femtosecond laser ablation technique was used for ablating the desired pattern on the PLGA layer and then the salt ingot was completely dissolved in distilled deionized water. A hollow 3D PLGA scaffold was obtained using this process on which bovine endothelial cells (BECs) were then cultured. Scanning electron microscopy (SEM) and fluorescent images of the cell seeding demonstrate that the BECs adhered and grew well on both the side-wall of the branches and the surroundings of each branch.

Highly porous three-dimensional poly(lactide-co-glycolide) (PLGA) microfibrous scaffold prepared by electrospinning method: A comparison study with other PLGA type scaffolds on its biological evaluation

In this study, a three-dimensional (3D) poly(lactide-co-glycolide) (PLGA) microfibrous scaffold with high porosity (ca. 90 % porosity) was developed for evaluating its performance in tissue engineering application. A dope solution of PLGA/polyethylene oxide (PEO) blend was electrospun into a methanol coagulation bath for fabricating highly porous 3D PLGA scaffold and a salt leaching method was used for making interconnected pores of 100–200 µm size inside the scaffold. The morphological structure, pore size and porosity of the microfibrous scaffold were determined, and compared with twodimensional (2D) mat-type and 3D sponge-type of PLGA scaffold. Also, swelling ratio, water uptake and compressive strength were compared in order to elucidate the structure-property relationships of different types of the scaffolds, especially in a wet condition. As a result of scanning electron microscopy (SEM) observation, normal human dermal fibroblasts (nHDF) were migrated, attached, and proliferated well inside the 3D scaffold. MTT assay confirmed that the highly porous 3D PLGA microfibrous scaffold had superior cell adhesion and proliferation abilities due to fibrous structure of large specific surface area, and interconnected pore structure. Therefore, this high performance 3D PLGA scaffold can have a high potentiality for application in tissue engineering in comparison with conventional PLGA scaffolds.

3D PLGA Scaffolds Improve Differentiation and Function of Bone Marrow Mesenchymal Stem Cell–Derived Hepatocytes

Liver tissue engineering with hepatic stem cells provides a promising alternative to liver transplantation in patients with acute and chronic hepatic failure. In this study, a three-dimensional (3D) bioscaffold was introduced for differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes. For hepatocyte differentiation, third passage BMSCs isolated from normal adult F344 rats were seeded into collagen-coated poly(lactic-co-glycolic acid) (C-PLGA) 3D scaffolds with hepatocyte differentiation medium for 3 weeks. Hepatogenesis in scaffolds was characterized by reverse transcript PCR, western blot, confocal laser scanning microscopy (CLSM), periodic acid-Schiff staining, histochemistry, and biochemical assays with hepatic-specific genes and markers. A monolayer culture system was used as a control differentiation group. The results showed that isolated cells possessed the basic features of BMSCs. Differentiated hepatocyte-like cells in C-PLGA scaffolds expressed hepatocyte-specific markers [eg, albumin (ALB), alpha-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4alpha, and cytochrome P450] at mRNA and protein levels. Most markers were expressed in C-PLGA group 1 week earlier than in the control group. Results of biocompatibility indicated that the differentiated hepatocyte-like cells grew more stably in C-PLGA scaffolds than that in controls during a 3-week differentiation period. The significantly higher metabolic functions in hepatocyte-like cells in the C-PLGA scaffold group further demonstrated the important role of the scaffold. As the phenomenon of transdifferentiation is uncommon, our successful transdifferentiation rates of BMSCs to mature hepatocytes prove the superiority of the C-PLGA scaffold in providing a suitable environment for such a differentiation. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.

THE EFFECT OF NAOH ON PROLIFERATION AND DIFFERENTIATION OF MC3T3-E1 PREOSTEOBLASTIC CELLS GROWN ON THREE-DIMENSIONAL POLY(L-LACTIDE-CO-GLYCOLIDE) SCAFFOLDS.

Background In creating a biosynthetic scaffold that promotes osteogenesis surface topography is an important variable. It has been demonstrated that chemical etching of poly(l-lactide-co-glycolide) (PLGA) with NaOH enhances adhesion and function of numerous cells types in two-dimensional systems (hepatocytes, endothelial cells, vascular and bladder smooth muscle cells) and three-dimensional (3-D) systems (chondrocytes). This study compares the biological behavior of osteoblasts grown on NaOH-treated PLGA scaffolds and conventional PLGA scaffolds by measuring osteoblast adhesion, differentiation, and proliferation of MC3T3-E1 cells cultured on NaOH-treated 3D PLGA scaffolds and untreated scaffolds. Methods MC3T3-E1 mouse preosteoblast cells were seeded onto 3-D PLGA scaffolds ± 1 M NaOH for 10 minutes. Quantitative real-time RT PCR was performed to measure mRNA expression of osteogenic marker genes. In addition, cell numbers were determined at varying time points. Results Pretreatment of 3D PLGA scaffolds with NaOH resulted in statistically significant up-regulation of mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF). A striking increase in mineralized cell matrix deposition in the NaOH group was also observed. Cell number was statistically higher in the NaOH group immediately after cell seeding, suggesting improved adhesion. During the first 24 hours of culture, cell proliferative rates were higher in the control group than the NaOH group. Conclusion Chemical etching of PLGA scaffolds with NaOH enhances adhesion and differentiation and slows proliferation of preosteoblast cells. NaOH treatment may represent an inexpensive way to improve scaffolds for use in bone tissue engineering.

Dissolution behavior of biomimetic minerals on 3D PLGA scaffold

The bone-like carbonate apatite (BLCA) coatings have a great potential to apply in orthopaedic and dental implants due to their excellent biocompatibility and biodegradability. The BLCA layer can be coated biomimetically in the surfaces of polymers or inorganic materials using simulated body plasma. We have prepared 3D poly(lactic-co-glycolic acid) (PLGA) porous scaffolds by solvent casting/particulate leaching method. The BLCA was coated on 3D PLGA scaffolds incubating them at 37 °C with modified simulated body fluid. We investigate the dissolution behavior of BLCA coated on PLGA scaffolds in two physiological medias such as Tris-buffered saline (pH = 7.4), 2-( N-morpholino)-ethanesulfonic acid (MES) (pH = 5.5), and cell culture media containing αMEM supplemented with 10% FBS and 1% penicillin–streptomycin. The BLCA dissolution and release ionic components were analyzed using FTIR, SEM and ICP. Approximately 70% of mineral was dissolved in Tris-buffered saline (8 ml) within 6 h relative to 48 h dissolution period. The investigation of mineral dissolution behavior is important when this BLCA use as a carrier for therapeutic agents in orthopaedic and dental implant applications.

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.