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

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

Tissue engineering: state of the art in oral rehabilitation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74998/1/j.1365-2842.2009.01939.x.pd

Commercial products for osteochondral tissue repair and regeneration

The osteochondral tissue represents a complex structure composed of four interconnected structures, namely hyaline cartilage, a thin layer of calcified cartilage, subchondral bone, and cancellous bone. Due to the several difficulties associated with its repair and regeneration, researchers have developed several studies aiming to restore the native tissue, some of which had led to tissue-engineered commercial products. In this sense, this chapter discusses the good manufacturing practices, regulatory medical conditions and challenges on clinical translations that should be fulfilled regarding the safety and efficacy of the new commercialized products. Furthermore, we review the current osteochondral products that are currently being marketed and applied in the clinical setting, emphasizing the advantages and difficulties of each one.FROnTHERA (NORTE-01-0145- FEDER-000023), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors would also like to acknowledge H2020-MSCA-RISE program, as this work is part of developments carried out in BAMOS project, funded by the European Union’s Horizon 2020 research and innovation program under grant agreement N° 734156. The financial support from the Portuguese Foundation for Science and Technology under the program Investigador FCT 2012 and 2015 (IF/00423/2012 and IF/01285/2015)info:eu-repo/semantics/publishedVersio

Bone formation by three-dimensional osteoblast culture in biodegradable poly(alpha-hydroxy ester) scaffolds

The feasibility of culturing osteoblasts on biodegradable poly($\alpha$-hydroxy esters) to form new bone was investigated through a series of five studies. The first study demonstrated that rat calvarial osteoblasts attached, proliferated, and functioned equally well on all the biodegradable polymer substrates studied (poly(L-lactic acid) (PLLA), 75:25 poly(DL-lactic-co-glycolic acid) (PLGA), 50:50 PLGA, and poly(glycolic acid) (PGA)) throughout the 14 day study, even though the polymer films were continuously degrading. The second study showed that osteoblast migration occurred as a monolayer of individual osteoblasts and not a calcified tissue front on poly($\alpha$-hydroxy ester) films. Copolymer ratio in the polymer films did not affect the rate of increase in culture area covered by the growing cell colony; however, the rate of increase in culture area was lower for cell colonies formed with a lower osteoblast seeding density. The proliferation rate for the osteoblasts arising from bone chips was lower than either of the isolated cell colonies. Bone formation in vitro was investigated in the third and fourth study by culturing stromal osteoblasts or rat calvarial osteoblasts in three-dimensional (3-D), biodegradable 75:25 PLGA foams. The polymer foams supported the growth of seeded osteoblasts as well as their differentiated function. Cell number, alkaline phosphatase activity, and mineralized tissue deposition increased significantly over time for all the polymer foams. Osteoblasts seeded at a lower cell density proliferated more rapidly, reaching comparable cell numbers at later culture times, but pore size over the range tested did not affect cell proliferation or function. In the final study, porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat stromal osteoblasts and implanted into the rat mesentery for up to 49 days to investigate in vivo bone formation using this osteoblast transplantation method. An organized and mineralized bone-like tissue was formed in all the constructs as early as 7 days post-implantation. Foam pore size did not affect the penetration depth of mineralized tissue or mineralized tissue volume per surface area found within the constructs at any time during the study