Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

[1]  Rui L. Reis,et al.  3D Plotted PCL Scaffolds for Stem Cell Based Bone Tissue Engineering , 2008 .

[2]  VLADIMIR MIRONOV,et al.  Bioprinting : A Beginning , 2022 .

[3]  Vladimir Mironov,et al.  Organ printing: computer-aided jet-based 3D tissue engineering. , 2003, Trends in biotechnology.

[4]  G. Evans,et al.  In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. , 2007, Tissue engineering.

[5]  Yunsong Liu,et al.  Injectable tissue-engineered bone composed of human adipose-derived stromal cells and platelet-rich plasma. , 2008, Biomaterials.

[6]  Liliang Ouyang,et al.  3D printing of HEK 293FT cell-laden hydrogel into macroporous constructs with high cell viability and normal biological functions , 2015, Biofabrication.

[7]  P. Dubruel,et al.  The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. , 2014, Biomaterials.

[8]  F. Guillemot,et al.  High-throughput laser printing of cells and biomaterials for tissue engineering. , 2010, Acta biomaterialia.

[9]  Vladimir Mironov,et al.  Review: bioprinting: a beginning. , 2006, Tissue engineering.

[10]  Anthony Atala,et al.  Evaluation of hydrogels for bio-printing applications. , 2013, Journal of biomedical materials research. Part A.

[11]  C A van Blitterswijk,et al.  3D fiber-deposited scaffolds for tissue engineering: influence of pores geometry and architecture on dynamic mechanical properties. , 2006, Biomaterials.

[12]  Peng Shang,et al.  Three-dimensional printed multiphase scaffolds for regeneration of periodontium complex. , 2014, Tissue engineering. Part A.

[13]  Jos Malda,et al.  Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydrogel scaffolds. , 2012, Tissue engineering. Part C, Methods.

[14]  Barry J. Spargo,et al.  Biological laser printing of three dimensional cellular structures , 2004 .

[15]  Carolyn M. Scott,et al.  A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells. , 2015, Tissue engineering. Part A.

[16]  Jason A Inzana,et al.  3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration. , 2014, Biomaterials.

[17]  B N Chichkov,et al.  Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells , 2011, Biofabrication.

[18]  Keekyoung Kim,et al.  3D bioprinting for engineering complex tissues. , 2016, Biotechnology advances.

[19]  W. Dhert,et al.  Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing. , 2008, Tissue engineering. Part A.

[20]  Suwan N Jayasinghe,et al.  Bio-electrospraying embryonic stem cells: interrogating cellular viability and pluripotency. , 2009, Integrative biology : quantitative biosciences from nano to macro.

[21]  U. Demirci,et al.  Bioprinting for stem cell research. , 2013, Trends in biotechnology.

[22]  Vladimir Mironov,et al.  Bioprinting living structures , 2007 .

[23]  R. Landers,et al.  Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering. , 2002, Biomaterials.

[24]  J. K. Leach,et al.  Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing , 2014, Cell and Tissue Research.

[25]  Yunsong Liu,et al.  The effect of simvastatin on chemotactic capability of SDF-1α and the promotion of bone regeneration. , 2014, Biomaterials.

[26]  A. Kolk,et al.  Current and future options of regeneration methods and reconstructive surgery of the facial skeleton. , 2015, Oral surgery, oral medicine, oral pathology and oral radiology.

[27]  Gang Wu,et al.  Bi-Functionalization of a Calcium Phosphate-Coated Titanium Surface with Slow-Release Simvastatin and Metronidazole to Provide Antibacterial Activities and Pro-Osteodifferentiation Capabilities , 2014, PloS one.

[28]  Gordon G. Wallace,et al.  Biofabrication: an overview of the approaches used for printing of living cells , 2013, Applied Microbiology and Biotechnology.

[29]  Meik Neufurth,et al.  Engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells. , 2014, Biomaterials.

[30]  David J Mooney,et al.  Alginate hydrogels as biomaterials. , 2006, Macromolecular bioscience.

[31]  Ying Mei,et al.  Engineering alginate as bioink for bioprinting. , 2014, Acta biomaterialia.

[32]  Alexander K. Nguyen,et al.  Osteogenic Differentiation of Human Mesenchymal Stem Cells in 3-D Zr-Si Organic-Inorganic Scaffolds Produced by Two-Photon Polymerization Technique , 2015, PloS one.

[33]  Guifang Gao,et al.  Bioactive nanoparticles stimulate bone tissue formation in bioprinted three-dimensional scaffold and human mesenchymal stem cells. , 2014, Biotechnology journal.

[34]  Eric D. Miller,et al.  Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle‐ and Bone‐Like Subpopulations , 2008, Stem cells.

[35]  Antonios G Mikos,et al.  Dual growth factor delivery from bilayered, biodegradable hydrogel composites for spatially-guided osteochondral tissue repair. , 2014, Biomaterials.

[36]  I. Correia,et al.  Bioactive polymeric-ceramic hybrid 3D scaffold for application in bone tissue regeneration. , 2013, Materials science & engineering. C, Materials for biological applications.

[37]  A Daducci,et al.  3D Printing of Rat Salivary Glands: The Submandibular–Sublingual Complex , 2014, Anatomia, histologia, embryologia.