Scaffold porosity and oxygenation of printed hydrogel constructs affect functionality of embedded osteogenic progenitors.

Insufficient supply of oxygen and nutrients throughout the graft is considered one of the principal limitations in development of large, tissue-engineered bone grafts. Organ or tissue printing by means of three-dimensional (3D) fiber deposition is a novel modality in regenerative medicine that combines pore formation and defined cell placement, and is used here for development of cell-laden hydrogel structures with reproducible internal architecture to sustain oxygen supply and to support adequate tissue development. In this study we tested the effect of porosity on multipotent stromal cells (MSCs) embedded in hydrogel constructs printed with a 3D fiber deposition (3DF) machine. For this, porous and solid alginate hydrogel scaffolds, with MSCs homogeneously dispersed throughout the construct, were printed and analyzed in vitro for the presence of hypoxia markers, metabolism, survival, and osteogenic differentiation. We demonstrated that porosity promotes oxygenation of MSCs in printed hydrogel scaffolds and supported the viability and osteogenic differentiation of embedded cells. Porous and solid printed constructs were subsequently implanted subcutaneously in immunodeficient mice to analyze tissue formation in relation to hypoxia responses of embedded cells. Implantation of printed grafts resulted in ingrowth of vascularized tissue and significantly enhanced oxygenation of embedded MSCs. In conclusion, the introduction of pores significantly enhances the conductive properties of printed hydrogel constructs and contributes to the functionality of embedded osteogenic progenitors.

[1]  J. Jukes,et al.  Critical Steps toward a tissue-engineered cartilage implant using embryonic stem cells. , 2008, Tissue engineering. Part A.

[2]  Anthony Atala,et al.  Oxygen producing biomaterials for tissue regeneration. , 2007, Biomaterials.

[3]  Anthony Atala,et al.  Oxygen generating scaffolds for enhancing engineered tissue survival. , 2009, Biomaterials.

[4]  Johannes Gerdes,et al.  The Ki‐67 protein: From the known and the unknown , 2000, Journal of cellular physiology.

[5]  G. Vunjak‐Novakovic,et al.  Hypoxia and stem cell‐based engineering of mesenchymal tissues , 2009, Biotechnology progress.

[6]  M. Longaker,et al.  Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells. , 2006, American journal of physiology. Cell physiology.

[7]  D. Hutmacher,et al.  Scaffolds in tissue engineering bone and cartilage. , 2000, Biomaterials.

[8]  D. Pipeleers,et al.  Nutrient sensing in pancreatic beta cells suppresses mitochondrial superoxide generation and its contribution to apoptosis. , 2005, Biochemical Society transactions.

[9]  S. Bourne,et al.  The effect of surgically induced ischaemia on gene expression in a colorectal cancer xenograft model , 2005, British Journal of Cancer.

[10]  A. Harris,et al.  Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1alpha. , 1998, Blood.

[11]  L. Koch,et al.  Laser printing of cells into 3D scaffolds , 2010, Biofabrication.

[12]  S. Pfister,et al.  Secretion of angiogenic proteins by human multipotent mesenchymal stromal cells and their clinical potential in the treatment of avascular osteonecrosis , 2008, Leukemia.

[13]  Milica Radisic,et al.  Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue , 2006, Biotechnology and bioengineering.

[14]  W. Morrison,et al.  Implanted myoblast survival is dependent on the degree of vascularization in a novel delayed implantation/prevascularization tissue engineering model. , 2010, Tissue engineering. Part A.

[15]  Bernard A Roos,et al.  Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. , 2006, Bone.

[16]  Vladimir Mironov,et al.  Organ printing: promises and challenges. , 2008, Regenerative medicine.

[17]  B. Obradovic,et al.  Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue‐engineered cartilage , 1999, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[18]  Jueren Lou,et al.  Evaluation of different scaffolds for BMP-2 genetic orthopedic tissue engineering. , 2005, Journal of biomedical materials research. Part B, Applied biomaterials.

[19]  T. Boland,et al.  Inkjet printing of viable mammalian cells. , 2005, Biomaterials.

[20]  J. Elisseeff,et al.  Bioresponsive phosphoester hydrogels for bone tissue engineering. , 2005, Tissue engineering.

[21]  K. Lyons,et al.  BMP Signaling and Podocyte Markers are Decreased in Human Diabetic Nephropathy in Association with CTGF Overexpression , 2009, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[22]  Rakesh K Jain,et al.  Molecular regulation of vessel maturation , 2003, Nature Medicine.

[23]  Hod Lipson,et al.  Direct Freeform Fabrication of Seeded Hydrogels in Arbitrary Geometries , 2022 .

[24]  Wei Sun,et al.  Effects of dispensing pressure and nozzle diameter on cell survival from solid freeform fabrication-based direct cell writing. , 2008, Tissue engineering. Part A.

[25]  Jeroen Rouwkema,et al.  Analysis of the dynamics of bone formation, effect of cell seeding density, and potential of allogeneic cells in cell-based bone tissue engineering in goats. , 2008, Tissue engineering. Part A.

[26]  Feng Zhao,et al.  Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs , 2006, Journal of cellular physiology.

[27]  Richard Tuli,et al.  Human mesenchymal progenitor cell-based tissue engineering of a single-unit osteochondral construct. , 2004, Tissue engineering.

[28]  Moustapha Kassem,et al.  Induction of Adipocyte‐Like Phenotype in Human Mesenchymal Stem Cells by Hypoxia , 2004, Stem cells.

[29]  J. Urban,et al.  Functional replacement of oxygen by other oxidants in articular cartilage. , 2002, Arthritis and rheumatism.

[30]  Jos Malda,et al.  Heterogeneous proliferation within engineered cartilaginous tissue: the role of oxygen tension. , 2005, Biotechnology and bioengineering.

[31]  Eben Alsberg,et al.  Engineering growing tissues , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Gordana Vunjak-Novakovic,et al.  Perfusion improves tissue architecture of engineered cardiac muscle. , 2002, Tissue engineering.

[33]  D. Chan,et al.  In vitro chondrogenic differentiation of human mesenchymal stem cells in collagen microspheres: influence of cell seeding density and collagen concentration. , 2008, Biomaterials.

[34]  S. Hofer,et al.  The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber. , 2005, British journal of plastic surgery.

[35]  C. Please,et al.  Experimental characterization and computational modelling of two-dimensional cell spreading for skeletal regeneration , 2007, Journal of The Royal Society Interface.

[36]  A. Davis,et al.  Hypoxic adipocytes pattern early heterotopic bone formation. , 2007, The American journal of pathology.

[37]  M. Fujiwara,et al.  High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells. , 2007, Journal of bioscience and bioengineering.

[38]  Feng Zhao,et al.  Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. , 2007, Biochemical and biophysical research communications.

[39]  S. Nemoto,et al.  Nutrient Availability Regulates SIRT1 Through a Forkhead-Dependent Pathway , 2004, Science.

[40]  J. van den Dolder,et al.  Evaluation of various seeding techniques for culturing osteogenic cells on titanium fiber mesh. , 2003, Tissue engineering.

[41]  Bradley R. Ringeisen,et al.  Laser Printing of Single Cells: Statistical Analysis, Cell Viability, and Stress , 2005, Annals of Biomedical Engineering.

[42]  L. Griffith,et al.  Tissue Engineering--Current Challenges and Expanding Opportunities , 2002, Science.

[43]  A J Verbout,et al.  Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats. , 2003, Tissue engineering.

[44]  Robert L. Sah,et al.  Dependence of Cartilage Matrix Composition on Biosynthesis, Diffusion, and Reaction , 2003 .

[45]  Eli Weinberg,et al.  Comparison of hydrogels in the in vivo formation of tissue-engineered bone using mesenchymal stem cells and beta-tricalcium phosphate. , 2007, Tissue engineering.

[46]  Ingo Klimant,et al.  Determination of oxygen gradients in engineered tissue using a fluorescent sensor. , 2002, Biotechnology and bioengineering.

[47]  B. Aronow,et al.  Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9. , 2005, Bone.

[48]  Wayne A Morrison,et al.  An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue‐engineered construct , 2007, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[49]  Yao‐Hua Song,et al.  The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF. , 2007, Biochemical and biophysical research communications.

[50]  R Langer,et al.  Modulation of the mechanical properties of tissue engineered cartilage. , 2000, Biorheology.

[51]  Jacqueline Alblas,et al.  The role of endothelial progenitor cells in prevascularized bone tissue engineering: development of heterogeneous constructs. , 2010, Tissue engineering. Part A.

[52]  Mark Taylor,et al.  Computational modelling of cell spreading and tissue regeneration in porous scaffolds. , 2007, Biomaterials.

[53]  P. V. van Diest,et al.  Radiofrequency ablation of colorectal liver metastases induces an inflammatory response in distant hepatic metastases but not in local accelerated outgrowth , 2010, Journal of surgical oncology.

[54]  Karl R Edminster,et al.  Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. , 2009, Biomaterials.

[55]  Paolo A Netti,et al.  Oxygen consumption of chondrocytes in agarose and collagen gels: a comparative analysis. , 2008, Biomaterials.

[56]  J. Hubbell,et al.  Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering , 2005, Nature Biotechnology.

[57]  P. Prendergast,et al.  Effect of a degraded core on the mechanical behaviour of tissueengineered cartilage constructs: A poro-elastic finite element analysis , 2006, Medical and Biological Engineering and Computing.

[58]  J. Tramper,et al.  Oxygen gradients in tissue‐engineered Pegt/Pbt cartilaginous constructs: Measurement and modeling , 2004, Biotechnology and bioengineering.

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

[60]  Hideki Yoshikawa,et al.  Oxygen Tension Regulates Chondrocyte Differentiation and Function during Endochondral Ossification* , 2006, Journal of Biological Chemistry.

[61]  Patrick J Prendergast,et al.  Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: A role for AKT and hypoxia‐inducible factor (HIF)‐1α , 2008, Journal of cellular physiology.

[62]  K. Furukawa,et al.  Oscillatory perfusion seeding and culturing of osteoblast-like cells on porous beta-tricalcium phosphate scaffolds. , 2008, Journal of Biomedical Materials Research. Part A.

[63]  R. Mason,et al.  Bovine articular chondrocyte function in vitro depends upon oxygen tension. , 2000, Osteoarthritis and cartilage.

[64]  T. Arnett,et al.  Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts. , 2006, Experimental cell research.

[65]  G. Reilly,et al.  Vascularized adipose tissue grafts from human mesenchymal stem cells with bioactive cues and microchannel conduits. , 2007, Tissue engineering.

[66]  A. Mobasheri,et al.  Hypoxia inducible factor-1 and facilitative glucose transporters GLUT1 and GLUT3: putative molecular components of the oxygen and glucose sensing apparatus in articular chondrocytes. , 2005, Histology and histopathology.

[67]  Karim Oudina,et al.  Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression. , 2007, Bone.

[68]  W. Mutschler,et al.  Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone. , 2008, Tissue engineering. Part A.

[69]  Yong Wang,et al.  Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide). , 2005, Biomaterials.

[70]  Darwin J. Prockop,et al.  Short-Term Exposure of Multipotent Stromal Cells to Low Oxygen Increases Their Expression of CX3CR1 and CXCR4 and Their Engraftment In Vivo , 2007, PloS one.

[71]  R T Tranquillo,et al.  A fibrin-based arterial media equivalent. , 2003, Journal of biomedical materials research. Part A.

[72]  Milica Radisic,et al.  Mathematical model of oxygen distribution in engineered cardiac tissue with parallel channel array perfused with culture medium containing oxygen carriers. , 2005, American journal of physiology. Heart and circulatory physiology.

[73]  A. Harris,et al.  Hypoxia-Inducible Factor 1α Expression as an Intrinsic Marker of Hypoxia , 2004, Clinical Cancer Research.

[74]  Jos Malda,et al.  The roles of hypoxia in the in vitro engineering of tissues. , 2007, Tissue engineering.

[75]  G. Vunjak‐Novakovic,et al.  Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage. , 1999, Biotechnology and bioengineering.

[76]  A. Caplan,et al.  Cultivation of rat marrow‐derived mesenchymal stem cells in reduced oxygen tension: Effects on in vitro and in vivo osteochondrogenesis , 2001, Journal of cellular physiology.

[77]  E. Stanbridge,et al.  Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix , 2006, Cytometry. Part B, Clinical cytometry.

[78]  J. Blanco,et al.  Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. , 2008, Experimental hematology.

[79]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.