The conventional tissue engineering is based on seeding of macroporous scaffold on its surface (“top–down” approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on “bottom–up” approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.
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Arealis G, Nikolaou VS. Bone printing: new frontiers in the treatment of bone defects. Injury. 2015;46(Suppl 8):S20–2.
O’Brien FJ. Biomaterials and scaffolds for tissue engineering,. Mater Today. 2011;14(3):88–95.
Oliveira H, et al. The proangiogenic potential of a novel calcium releasing biomaterial: impact on cell recruitment. Acta Biomater. 2016;29:435–45.
Feng T, Liu Y, Xu Q, Li X, Luo X, Chen Y. In vitro experimental study on influences of final degradation products of polyactic acid on proliferation and osteoblastic phenotype of osteoblast-like cells. J Repar Reconstr Surg. 2014;28(12):1525–9.
Saito E, Suarez-Gonzalez D, Murphy WL, Hollister SJ. Biomineral coating increases bone formation by ex vivo BMP-7 gene therapy in rapid prototyped poly(l-lactic acid) (PLLA) and poly(ε-caprolactone) (PCL) porous scaffolds. Adv Healthc Mater. 2015;4(4):621–32.
Ciocca L, De Crescenzio F, Fantini M, Scotti R. CAD/CAM and rapid prototyped scaffold construction for bone regenerative medicine and surgical transfer of virtual planning: a pilot study. Comput Med Imaging Graph. 2009;33(1):58–62.
Mangano F, et al. Maxillary ridge augmentation with custom-made CAD/CAM scaffolds. A 1-year prospective study on 10 patients. J Oral Implantol. 2014;40(5):561–9.
Nga NK, Hoai TT, Viet PH. Biomimetic scaffolds based on hydroxyapatite nanorod/poly(d,l) lactic acid with their corresponding apatite-forming capability and biocompatibility for bone-tissue engineering. Colloids Surf B. 2015;128:506–14.
Lou T, Wang X, Song G, Gu Z, Yang Z. Fabrication of PLLA/β-TCP nanocomposite scaffolds with hierarchical porosity for bone tissue engineering. Int J Biol Macromol. 2014;69:464–70.
D’Alessandro D, et al. Processing large-diameter poly(l-lactic acid) microfiber mesh/mesenchymal stromal cell constructs via resin embedding: an efficient histologic method. Biomed Mater Bristol Engl. 2014;9(4):045007
Zamparelli A, et al. Growth on poly(l-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells. J Mater Sci Mater Med. 2014;25(10):2421–36.
Kao C-T, Lin C-C, Chen Y-W, Yeh C-H, Fang H-Y, Shie M-Y. Poly(dopamine) coating of 3D printed poly(lactic acid) scaffolds for bone tissue engineering. Mater Sci Eng C. 2015;56:165–73.
Hu Y, Zou S, Chen W, Tong Z, Wang C. Mineralization and drug release of hydroxyapatite/poly(l-lactic acid) nanocomposite scaffolds prepared by pickering emulsion templating. Colloids Surf B Biointerfaces. 2014;122:559–65.
Ding M, Henriksen SS, Wendt D, Overgaard S. An automated perfusion bioreactor for the streamlined production of engineered osteogenic grafts. J Biomed Mater Res B. 2015;104:532–537.
Lian Q, Zhuang P, Li C, Jin Z, Li D. Mechanical properties of polylactic acid/beta-tricalcium phosphate composite scaffold with double channels based on three-dimensional printing technique. Chin J Repar Reconstr Surg. 2014;28(3):309–13.
Ronca A, et al. Large defect-tailored composite scaffolds for in vivo bone regeneration. J Biomater Appl. 2014;29(5):715–27.
Hamad K. Properties and medical applications of polylactic acid: a review. Express Polym Lett. 2015;9(5):435–55.
Vidyasekar P, Shyamsunder P, Sahoo SK, Verma RS. Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells. In Vitro Cell Dev Biol Anim. 2016;52(2):204–17.
Huang J, et al. Evaluation of the novel three-dimensional porous poly (l-lactic acid)/nano-hydroxyapatite composite scaffold. Biomed Mater Eng. 2015;26(Suppl 1):S197–205.
Giordano RA, Wu BM, Borland SW, Cima LG, Sachs EM, Cima MJ. Mechanical properties of dense polylactic acid structures fabricated by three dimensional printing. J Biomater Sci Polym Ed. 1996;8(1):63–75.
Almeida CR, Serra T, Oliveira MI, Planell JA, Barbosa MA, Navarro M. Impact of 3-D printed PLA- and chitosan-based scaffolds on human monocyte/macrophage responses: unraveling the effect of 3-D structures on inflammation. Acta Biomater. 2014;10(2):613–22.
Serra T, Mateos-Timoneda MA, Planell JA, Navarro M. 3D printed PLA-based scaffolds: a versatile tool in regenerative medicine. Organogenesis. 2013;9(4):239–44.
Schlaubitz S, et al. Pullulan/dextran/nHA macroporous composite beads for bone repair in a femoral condyle defect in rats. PLoS One. 2014;9(10):e110251
Groll J, et al. Biofabrication: reappraising the definition of an evolving field. Biofabrication. 2016;8(1):013001
Sathy BN, Mony U, Menon D, Baskaran VK, Mikos AG, Nair S. Bone tissue engineering with multilayered scaffolds-part I: an approach for vascularizing engineered constructs in vivo. Tissue Eng Part A. 2015;21(19–20):2480–94.
Ren L, et al. Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration. BioMed Res Int. 2014;2014:301279
Nishiguchi A, Matsusaki M, Asano Y, Shimoda H, Akashi M. Effects of angiogenic factors and 3D-microenvironments on vascularization within sandwich cultures. Biomaterials. 2014;35(17):4739–48.
Derda R, et al. Paper-supported 3D cell culture for tissue-based bioassays. Proc Natl Acad Sci USA. 2009;106(44):18457–62.
Wan W, et al. Layer-by-layer paper-stacking nanofibrous membranes to deliver adipose-derived stem cells for bone regeneration. Int J Nanomedicine. 2015;10:1273–90.
Catros S, et al. Layer-by-layer tissue microfabrication supports cell proliferation in vitro and in vivo. Tissue Eng Part C Methods. 2012;18(1):62–70.
Wen L, et al. Role of endothelial progenitor cells in maintaining stemness and enhancing differentiation of mesenchymal stem cells by indirect cell–cell interaction. Stem Cells Dev. 2016;25(2):123–38.
Eldesoqi K, et al. Safety evaluation of a bioglass-polylactic acid composite scaffold seeded with progenitor cells in a rat skull critical-size bone defect. PLoS One. 2014;9(2):e87642
Vilamitjana-Amedee J, Bareille R, Rouais F, Caplan AI, Harmand MF. Human bone marrow stromal cells express an osteoblastic phenotype in culture. In Vitro Cell Dev Biol Anim. 1993;29A(9):699–707.
Thebaud NB, Bareille R, Remy M, Bourget C, Daculsi R, Bordenave L. Human progenitor-derived endothelial cells vs. venous endothelial cells for vascular tissue engineering: an in vitro study. J Tissue Eng Regen Med. 2010;4(6):473–84.
Thébaud NB, et al. Labeling and qualification of endothelial progenitor cells for tracking in tissue engineering: an in vitro study. Int J Artif Organs. 2015;38(4):224–32.
Lau KR, Evans RL, Case RM. Intracellular Cl- concentration in striated intralobular ducts from rabbit mandibular salivary glands. Pflüg Arch Eur J Physiol. 1994;427(1–2):24–32.
Poole CA, Brookes NH, Clover GM. Keratocyte networks visualised in the living cornea using vital dyes. J Cell Sci. 1993;106(Pt 2):685–91.
Vaughan PJ, Pike CJ, Cotman CW, Cunningham DD. Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults. J Neurosci. 1995;15(7):5389–401. Pt 2
Metcalf DJ, Nightingale TD, Zenner HL, Lui-Roberts WW, Cutler DF. Formation and function of Weibel-Palade bodies. J Cell Sci. 2008;121(Pt 1):19–27.
Szczurek AT, et al. Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes. Nucl Austin Tex. 2014;5(4):331–40.
Serra T, Ortiz-Hernandez M, Engel E, Planell JA, Navarro M. Relevance of PEG in PLA-based blends for tissue engineering 3D-printed scaffolds. Mater Sci Eng C. 2014;38:55–62.
Ahn S, Lee H, Kim G. Functional cell-laden alginate scaffolds consisting of core/shell struts for tissue regeneration. Carbohydr Polym. 2013;98(1):936–42.
Aguirre A, Planell JA, Engel E. Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis. Biochem Biophys Res Commun. 2010;400(2):284–91.
Grellier M, Bordenave L, Amédée J. Cell-to-cell communication between osteogenic and endothelial lineages: implications for tissue engineering. Trends Biotechnol. 2009;27(10):562–71.
The authors wish to thank the French Institute in Belgrade, Serbia, via Campus France agency. 2-photon observations were done at Bordeaux Imaging Center, France.
Conflict of interest
The authors declare that they have no conflict of interest.
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Guduric, V., Metz, C., Siadous, R. et al. Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering. J Mater Sci: Mater Med 28, 78 (2017). https://doi.org/10.1007/s10856-017-5887-6