Elastic biodegradable starch/ethylene-co-vinyl alcohol fibre-mesh scaffolds for tissue engineering applications

The fabrication of a biomaterial scaffold, with adequate physical and structural properties for tissue engineering applications, is reported. A blend of starch with ethylene-vinyl alcohol (50/50 w/w, SEVA-C) is used to produce 3D fibre-mesh scaffolds by wet-spinning. The scaffolds are characterized in terms of morphology, porosity, interconnectivity, and pore size, using scanning electron microscopy (SEM) and microcomputed tomography (μCT). The degradation behavior, as well as the mechanical properties of the scaffolds, is investigated in presence of alpha-amylase enzyme at physiological concentration. Scaffolds with porosities ranging from 43 to 52%, interconnectivity of ∼70.5% and pore size between 118 and 159 μm, can be fabricated using the proposed methodology. The scaffolds exhibit an elastic behavior in the wet state with a compressive modulus of 7.96±0.32 MPa. Degradation studies show that SEVA-C scaffolds are susceptible to enzymatic degradation by alpha-amylase, confirmed by the increase of weight loss (40% of weight loss after 12 weeks) and presence of degradation products (reducing sugars) in solution. The diameter of SEVA-C scaffolds decreases with degradation time, increasing the overall porosity, interconnectivity and pore size. In vitro cell studies with human osteosarcoma cell line (SaOs-2) showed a nontoxic and cytocompatible behavior of the developed fibre mesh scaffolds. The positive cellular response, together with structural and degradable properties, suggests that 3D SEVA-C fibre-meshes may be good candidates as tissue engineering scaffolds. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40504. Copyright © 2014 Wiley Periodicals, Inc.This work was supported by national funds through the Portuguese Foundation for Science and Technology under the scope of the project PTDC/CTM/67560/2006 and by the European Regional Development Fund (ERDF) through the Operational Competitiveness Programme “COMPETE” (FCOMP-01-0124-FEDER-007148)

Cementum engineering with three-dimensional polymer scaffolds

Cloned cementoblasts (OCCMs), periodontal ligament fibroblasts (SV-PDLs), and dental follicle (SV-F) cells obtained from mice were used as a tool to study periodontal tissue engineering. OCCM, SV-PDL, and SV-F cells were seeded onto three-dimensional poly lactic- co -glycolic acid (PLGA) scaffolds and cultured with the use of bioreactors or implanted subcutaneously in severe combined immune deficiency (SCID) mice for up to 6 weeks. We explored the behavior of these cells in porous PLGA sponges by cell growth, expression of mineral-associated genes using reverse transcriptase polymerase chain reaction, and mineralization by histologic analysis in vitro and in vivo . Results indicated that cells attached to PLGA scaffolds under either static or dynamic conditions in vitro . Only OCCM implants, retrieved from both in vitro bioreactors and SCID mice at 3-and 6-weeks post–cell implantation exhibited mineral formation. Types I and XII collagens, osteocalcin, and bone sialoprotein genes were detected in all implants retrieved from SCID mice. These results suggest that delivery of selected cells via PLGA scaffolds may serve as a viable approach for promoting periodontal tissue regeneration. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 54–60, 2003Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34429/1/10058_ftp.pd

Depth-resolved cellular microrheology using HiLo microscopy

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties

Bacterial cellulose modified using recombinant proteins to improve neuronal and mesenchymal cell adhesion

A wide variety of biomaterials and bioactive molecules have been applied as scaffolds in neuronal tissue engineering. However, creating devices that enhance the regeneration of nervous system injuries is still a challenge, due the difficulty in providing an appropriate environment for cell growth and differentiation and active stimulation of nerve regeneration. In recent years, bacterial cellulose (BC) has emerged as a promising biomaterial for biomedical applications due its properties, such as high crystallinity, an ultrafine fiber network, high tensile strength and biocompatibility. The small signaling peptides found in the proteins of extracellular matrix are described in the literature as promoters of adhesion and proliferation for several cell lineages on different surfaces. In this work, the peptide IKVAV was fused to a carbohydrate-binding module (CBM3) and used to modify BC surfaces, with the goal of promoting neuronal and mesenchymal stem cell (MSC) adhesion. The recombinant proteins IKVAV-CBM3 and (19)IKVAV-CBM3 were successfully expressed in E. coli, purified through affinity chromatography and stably adsorbed to the BC membranes. The effect of these recombinant proteins, as well as RGD-CBM3, on cell adhesion was evaluated by MTS colorimetric assay. The results showed that the (19)IKVAV-CBM3 was able to significantly improve the adhesion of both neuronal and mesenchymal cells and had no effect on the other cell lineages tested. The MSC neurotrophin expression in cells grown on BC membranes modified with the recombinant proteins was also analyzed.Renata A. N. Pertile gratefully acknowledges support by the Programme Al beta an, the European Union Programme of High Level Scholarships for Latin America (Scholarship No. E07D401931BR). The author Susana Moreira is recipient of a SFRH/BPD/64726/2009 fellowship from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal). Fabia K. Andrade is the recipient of a fellowship from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil)

Effects of pH on human bone marrow stromal cells in vitro : Implications for tissue engineering of bone

The objective of this study was to address the hypothesis that changes in extracellular pH alter collagen gene expression, collagen synthesis, and alkaline phosphatase activity in bone marrow stromal cells (BMSCs). Potential effects of pH on cell function are of particular importance for tissue engineering because considerable effort is being placed on engineering biodegradable polymers that may generate a local acidic microenvironment on degradation. Human and murine single-cell marrow suspensions were plated at a density of 2 × 10 4 cells/cm 2 . After 7 days in culture, the pH of the culture medium was adjusted to one of six ranges: ≥7.8, 7.5.–7.7, 7.2–7.4, 6.9–7.1, 6.6–6.8, or ≤6.5. After 48 h of exposure to an altered pH, alkaline phosphatase activity and collagen synthesis decreased significantly with decreasing pH. This decrease was two-to threefold as pH decreased from 7.5 to 6.6. In contrast, Α1(I) procollagen mRNA levels increased two- to threefold as pH was decreased. The trend in osteocalcin mRNA expression was opposite to that of collagen. Small shifts in extracellular pH led to significant changes in the ability of BMSCs to express markers of the osteoblast phenotype. These pH effects potentially relate to the microenvironment supplied by a tissue-engineering scaffold and suggest that degrading polymer scaffolds may influence the biologic activity of the cells in the immediate environment. © 2002 John Wiley & Sons, Inc. J Biomed Mater Res 60: 292–299, 2002; DOI 10.1002/jbm.10050Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34423/1/10050_ftp.pd

Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.We would like to acknowledge Mariana T Cerqueira for the illustration in Figure 1. This study was supported by Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology 'Cell Sheet Tissue Engineering Center (CSTEC)' and the Global CUE program, the Multidisciplinary Education and Research Center for Regenerative Medicine (MERCREM), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Financial support to RP Pirraco by the Portuguese Foundation for Science and Technology (FCT) through the PhD Grant SFRH/BD/44893/2008 is also acknowledged

Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing

Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F‐127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed

Ectopic bone formation in cell-seeded poly(ethylene oxide)/poly(butylene terephthalate) copolymer scaffolds of varying porosity

Scaffolds from poly(ethylene oxide) and poly(butylene terephthalate), PEOT/PBT, with a PEO molecular weight of 1,000 and a PEOT content of 70 weight% (1000PEOT70PBT30) were prepared by leaching salt particles (425–500 μm). Scaffolds of 73.5, 80.6 and 85.0% porosity were treated with a CO2 gas plasma and seeded with rat bone marrow stromal cells (BMSCs). After in vitro culture for 7 days (d) in an osteogenic medium the scaffolds were subcutaneously implanted for 4 weeks in nude mice. Poly(d, l-lactide) (PDLLA) and biphasic calcium phosphate (BCP) scaffolds were included as references. After 4 weeks (wks) all scaffolds showed ectopic formation of bone and bone marrow. For the scaffolds of different porosities, no significant differences were observed in the relative amounts of bone (7–9%) and bone marrow (6–11%) formed, even though micro computed tomography (μ-CT) data showed considerable differences in accessible pore volume and surface area. 1000PEOT70PBT30 scaffolds with a porosity of 85% could not maintain their original shape in vivo. Surprisingly, 1000PEOT70PBT30 scaffolds with a porosity of 73.5% showed cartilage formation. This cartilage formation is most likely due to poorly accessible pores in the scaffolds, as was observed in histological sections. μ-CT data showed a considerably smaller accessible pore volume (as a fraction of the total volume) than in 1000PEOT70PBT30 scaffolds of 80.6 and 85.0% porosity. BMSC seeded PDLLA (83.5% porosity) and BCP scaffolds (29% porosity) always showed considerably more bone and bone marrow formation (bone marrow formation is approximately 40%) and less fibrous tissue ingrowth than the 1000PEOT70PBT30 scaffolds. The scaffold material itself can be of great influence. In more hydrophobic and rigid scaffolds like the PDLLA or BCP scaffolds, the accessibility of the pore structure is more likely to be preserved under the prevailing physiological conditions than in the case of hydrophilic 1000PEOT70PBT30 scaffolds. Scaffolds prepared from other PEOT/PBT polymer compositions, might prove to be more suited

Gap junction intercellular communication: A review of a potential platform to modulate craniofacial tissue engineering

Defects in craniofacial tissues, resulting from trauma, congenital abnormalities, oncologic resection or progressive deforming diseases, may result in aesthetic deformity, pain and reduced function. Restoring the structure, function and aesthetics of craniofacial tissues represents a substantial clinical problem in need of new solutions. More biologically-interactive biomaterials could potentially improve the treatment of craniofacial defects, and an understanding of developmental processes may help identify strategies and materials that can be used in tissue engineering. One such strategy that can potentially advance tissue engineering is cell–cell communication. Gap junction intercellular communication is the most direct way of achieving such signaling. Gap junction communication through connexin-mediated junctions, in particular connexin 43 (Cx43), plays a major role bone development. Given the important role of Cx43 in controlling development and differentiation, especially in bone cells, controlling the expression of Cx43 may provide control over cell-to-cell communication and may help overcome some of the challenges in craniofacial tissue engineering. Following a review of gap junctions in bone cells, the ability to enhance cell–cell communication and osteogenic differentiation via control of gap junctions is discussed, as is the potential utility of this approach in craniofacial tissue engineering. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61547/1/31127_ftp.pd