Exploring cellular adhesion and differentiation in a micro‐/nano‐hybrid polymer scaffold

Polymer scaffolds play an important role in three dimensional (3‐D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano‐fibrous and micro‐porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro‐scale pores and nano‐scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Electrospinning of highly porous scaffolds for cartilage regeneration

This study presents a new innovative method where electrospinning is used to coat single microfibers with nanofibers. The nanofiber-coated microfibers can be formed into scaffolds with the combined benefits of tailored porosity for cellular infiltration and nanostructured surface morphology for cell growth. The nanofiber coating is obtained by using a grounded collector rotating around the microfiber, to establish an electrical field yet allow collection of nanofibers on the microfiber. A Teflon tube surrounding the fibers and collector is used to force the nanofibers to the microfiber. Polycaprolactone nanofibers were electrospun onto polylactic acid microfibers and scaffolds of 95 and 97% porosities were made. Human chondrocytes were seeded on these scaffolds and on reference scaffolds of purely nanofibers and microfibers. Thereafter, cellular infiltration was investigated. The results indicated that scaffold porosity had great effects on cellular infiltration, with higher porosity resulting in increased infiltration, thereby confirming the advantage of the presented method.     

3D-printed PLA/Gel hybrid in liver tissue engineering: Effects of architecture on biological functions

The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific three-dimensional (3D) structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, poly-lactic acid (PLA) polymer was used as the input of the fused deposition modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-sectional designs were printed. One percent and 2% of gelatin coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. Scanning electron microscopy pictures were used to analyze the structural properties of PLA–Gel hybrid scaffolds, energy dispersive spectroscopy to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ± 0.02-fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

Fabrication of conducting electrospun nanofibers scaffold for three-dimensional cells culture

Electrospinning is a versatile method to fabricate nanofibers of a range of polymeric and composite materials suitable as scaffolds for tissue engineering applications. In this study, we report the fabrication and characterization of polyaniline-carbon nanotube/poly(N-isopropyl acrylamide-co-methacrylic acid) (PANI-CNT/PNIPAm-co-MAA) composite nanofibers and PNIPAm-co-MAA nanofibers suitable as a three-dimensional (3D) conducting smart tissue scaffold using electrospinning. The chemical structure of the resulting nanofibers was characterized with FTIR and (1)H NMR spectroscopy. The surface morphology and average diameter of the nanofibers were observed by SEM. Cellular response of the nanofibers was studied with mice L929 fibroblasts. Cell viability was checked on 7th day of cell culture by double staining the cells with calcein-AM and PI dye. PANI-CNT/PNIPAm-co-MAA composite nanofibers were shown the highest cell growth and cell viability as compared to PNIPAm-co-MAA nanofibers. Cell viability in the composite nanofibers was obtained in order of 98% that indicates the composite nanofibers provide a better environment as a 3D scaffold for the cell proliferation and attachment suitable for tissue engineering applications.     

Improved infiltration of stem cells on electrospun nanofibers

Nanofibrous scaffolds have been recently used in the field of tissue engineering because of their nano-size structure which promotes cell attachment, function, proliferation and infiltration. In this study, nanofibrous polyethersulfone (PES) scaffolds was prepared via electrospinning. The scaffolds were surface modified by plasma treatment and collagen grafting. The surface changes then investigated by contact angle measurements and FTIR-ATR. The results proved grafting of the collagen on nanofibers surface and increased hydrophilicity after plasma treatment and collagen grafting. The cell interaction study was done using stem cells because of their ability to differentiate to different kinds of cell lines. The cells had normal morphology on nanofibers and showed very high infiltration through collagen grafted PES nanofibers. This infiltration capability is very useful and needed to make 3D scaffolds in tissue engineering.     

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) on four commercially available scaffold biomaterials.METHODS: hDPSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. hDPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureOss (Allograft), Cerabone (Xenograft), PLLA (Synthetic), and OSTEON II Collagen (Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy (SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase (ALP) activity assay, respectively.RESULTS: All scaffold biomaterials except SureOss (Allograft) supported hDPSC adhesion, proliferation and differentiation. hDPSCs seeded on PLLA (Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hDPSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA (Synthetic) and OSTEON II Collagen (Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone (Xenograft) and OSTEON II Collagen (Composite) scaffolds, the hDPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape.CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hDPSCs. Hence, it may be useful in combination with hDPSCs for cell-based reconstructive therapy.     

Electrospinning of carboxymethyl chitin/poly(vinyl alcohol) nanofibrous scaffolds for tissue engineering applications

A novel fibrous membrane of carboxymethyl chitin (CMC)/poly(vinyl alcohol) (PVA) blend was successfully prepared by electrospinning technique. The concentration of CMC (7%) with PVA (8%) was optimized, blended in different ratios (0–100%) and electrospun to get nanofibers. Fibers were made water insoluble by chemical followed by thermal cross-linking. In vitro mineralization studies identified the ability of formation of hydroxyapatite deposits on the nanofibrous surfaces. Cytotoxicity of the nanofibrous scaffold was evaluated using human mesenchymal stem cells (hMSCs) by the MTT assays. The cell viability was not altered when these nanofibrous scaffolds were pre-washed with phosphate buffer containing saline (PBS) before seeding the cells. The SEM images also revealed that cells were able to attach and spread in the nanofibrous scaffolds. Thus our results indicate that the nanofibrous CMC/PVA scaffold supports cell adhesion/attachment and proliferation and hence this scaffold will be a promising candidate for tissue engineering applications.     

A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.     

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.     

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).     

Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.     

Combining electrospun scaffolds with electrosprayed hydrogels leads to three-dimensional cellularization of hybrid constructs

A common problem in the design of tissue engineered scaffolds using electrospun scaffolds is the poor cellular infiltration into the structure. To tackle this issue, three approaches to scaffold design using electrospinning were investigated: selective leaching of a water-soluble fiber phase (poly ethylene oxide (PEO) or gelatin), the use of micron-sized fibers as the scaffold, and a combination of micron-sized fibers with codeposition of a hyaluronic acid-derivative hydrogel, Heprasil. These designs were achieved by modifying a conventional electrospinning system with two charged capillaries and a rotating mandrel collector. Three types of scaffolds were fabricated: medical grade poly(epsilon-caprolactone)/collagen (mPCL/Col) cospun with PEO or gelatin, mPCL/Col meshes with micron-sized fibers, and mPCL/Col microfibers cosprayed with Heprasil. All three scaffold types supported attachment and proliferation of human fetal osteoblasts. However, selective leaching only marginally improved cellular infiltration when compared to meshes obtained by conventional electrospinning. Better cell penetration was seen in mPCL/Col microfibers, and this effect was more pronounced when Heprasil regions were present in the structure. Thus, such techniques could be further exploited for the design of cell permeable fibrous meshes for tissue engineering applications.     

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.     

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.     

Development of a 3D cell culture system for investigating cell interactions with electrospun fibers

There are many variables to be considered in studying how cells interact with 3D scaffolds used in tissue engineering. In this study we investigated the influence of the fiber diameter and interfiber spaces of 3D electrospun fiber scaffolds on the behavior of human dermal fibroblasts. Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid, with a broad range of diameters were constructed in a specifically developed 3D cell culture system. When fibroblasts were introduced to freestanding fibers, and encouraged to "walk the plank," a minimum fiber diameter of 10 microm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 microm was also observed to be the maximum gap that could be bridged by cell aggregates--a behavior not seen in conventional 2D culture. This approach has identified some basic micro-architectural parameters for electrospun scaffold design and some key differences in fibroblast growth in 3D. We suggest the findings will be of value for optimizing the integration of cells in these scaffolds for skin tissue engineering.     

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm⁻¹ can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.