Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and Cell Photoencapsulation.

Three-dimensional (3D) printing offers versatile possibilities for adapting the structural parameters of tissue engineering scaffolds. However, it is also essential to develop procedures allowing efficient cell seeding independent of scaffold geometry and pore size. The aim of this study was to establish a method for seeding the scaffolds using photopolymerizable cell-laden hydrogels. The latter facilitates convenient preparation, and handling of cell suspension, while distributing the hydrogel precursor throughout the pores, before it is cross-linked with light. In addition, encapsulation of living cells within hydrogels can produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural extra-cellular matrix (ECM). Three dimensional scaffolds were produced from poly(lactic) acid (PLA) by means of fused deposition modeling. A solution of methacrylamide-modified gelatin (Gel-MOD) in cell culture medium containing photoinitiator Li-TPO-L was used as a hydrogel precursor. Being an enzymatically degradable derivative of natural collagen, gelatin-based matrices are biomimetic and potentially support the process of cell-induced remodeling. Preosteoblast cells MC3T3-E1 at a density of 10 × 106 cells per 1 mL were used for testing the seeding procedure and cell proliferation studies. Obtained results indicate that produced constructs support cell survival and proliferation over extended duration of our experiment. The established two-step approach for scaffold seeding with the cells is simple, rapid, and is shown to be highly reproducible. Furthermore, it enables precise control of the initial cell density, while yielding their uniform distribution throughout the scaffold. Such hybrid tissue engineering constructs merge the advantages of rigid 3D printed constructs with the soft hydrogel matrix, potentially mimicking the process of ECM remodeling.

[1]  C. V. van Blitterswijk,et al.  Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique. , 2004, Biomaterials.

[2]  Magdi H. Yacoub,et al.  Hydrogel scaffolds for tissue engineering: Progress and challenges , 2013, Global cardiology science & practice.

[3]  S. Bryant,et al.  Cell encapsulation in biodegradable hydrogels for tissue engineering applications. , 2008, Tissue engineering. Part B, Reviews.

[4]  Christopher S. Chen,et al.  How cells sense extracellular matrix stiffness: a material's perspective. , 2013, Current opinion in biotechnology.

[5]  Jyh-Ping Chen,et al.  Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: in vitro and in vivo studies. , 2013, Acta biomaterialia.

[6]  Delivering MC3T3-E1 cells into injectable calcium phosphate cement through alginate-chitosan microcapsules for bone tissue engineering , 2014, Journal of Zhejiang University SCIENCE B.

[7]  Paolo A. Netti,et al.  Dynamic-mechanical properties of a novel composite intervertebral disc prosthesis , 2007, Journal of materials science. Materials in medicine.

[8]  S. Guelcher,et al.  Gel microstructure regulates proliferation and differentiation of MC3T3-E1 cells encapsulated in alginate beads. , 2012, Acta biomaterialia.

[9]  Gordana Vunjak-Novakovic,et al.  Bioreactors mediate the effectiveness of tissue engineering scaffolds , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  Y. Inoue,et al.  Polymorphic Transition in Disordered Poly(l-lactide) Crystals Induced by Annealing at Elevated Temperatures , 2008 .

[11]  Kristi S Anseth,et al.  Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. , 2009, Biomaterials.

[12]  D. Hutmacher,et al.  Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices. , 2003, Tissue engineering.

[13]  Paul N Manson,et al.  Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation. , 2005, Biomaterials.

[14]  Matthew S. Rehmann,et al.  Tunable and dynamic soft materials for three-dimensional cell culture , 2013, Soft matter.

[15]  P. Carreau,et al.  Control of thermal degradation of polylactide/clay nanocomposites during melt processing by chain extension reaction , 2012 .

[16]  M. H. Fernandes,et al.  Preparation and characterization of collagen-nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications. , 2013, Journal of biomedical materials research. Part A.

[17]  J. Xue,et al.  Time-resolved spectroscopic and density functional theory study of the photochemistry of Irgacure-2959 in an aqueous solution. , 2014, The journal of physical chemistry. A.

[18]  T. He,et al.  Layer-by-layer assembly to modify poly(l-lactic acid) surface toward improving its cytocompatibility to human endothelial cells. , 2003, Biomacromolecules.

[19]  YangFan,et al.  A facile method to fabricate hydrogels with microchannel-like porosity for tissue engineering. , 2014 .

[20]  S. Mikhalovsky,et al.  Gelatin-fibrinogen cryogel dermal matrices for wound repair: preparation, optimisation and in vitro study. , 2010, Biomaterials.

[21]  Jason A. Burdick,et al.  Spatially controlled hydrogel mechanics to modulate stem cell interactions , 2010 .

[22]  R Langer,et al.  In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. , 2000, Biomaterials.

[23]  Jan Feijen,et al.  A poly(D,L-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography. , 2009, Biomaterials.

[24]  Ann L. Johnson,et al.  Chitosan scaffolds: interconnective pore size and cartilage engineering. , 2006, Acta biomaterialia.

[25]  J Malda,et al.  Bioprinting of hybrid tissue constructs with tailorable mechanical properties , 2011, Biofabrication.

[26]  Peter Dubruel,et al.  A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. , 2012, Biomaterials.

[27]  Maryam Tabrizian,et al.  Three-dimensional growth of differentiating MC3T3-E1 pre-osteoblasts on porous titanium scaffolds. , 2005, Biomaterials.

[28]  Lee Tin Sin,et al.  Polylactic Acid: PLA Biopolymer Technology and Applications , 2012 .

[29]  X. Sun,et al.  Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells , 2013, PloS one.

[30]  P. Janmey,et al.  Biomechanics and Mechanotransduction in Cells and Tissues Cell type-specific response to growth on soft materials , 2005 .

[31]  K. Anseth,et al.  Osteogenic differentiation of human mesenchymal stem cells on α5 integrin binding peptide hydrogels is dependent on substrate elasticity. , 2014, Biomaterials science.

[32]  P. Dubruel,et al.  Development of Mechanically Tailored Gelatin‐Chondroitin Sulphate Hydrogel Films , 2011 .

[33]  Ashok Kumar,et al.  Conducting cryogel scaffold as a potential biomaterial for cell stimulation and proliferation , 2013, Journal of Materials Science: Materials in Medicine.

[34]  Yiqi Yang,et al.  Molecular modeling study of the resistance of PLA to hydrolysis based on the blending of PLLA and PDLA , 2006 .

[35]  Judith M Curran,et al.  The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds. , 2006, Biomaterials.

[36]  J. Cadet,et al.  Ultraviolet radiation-mediated damage to cellular DNA. , 2005, Mutation research.

[37]  P. Janmey,et al.  Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. , 2005, Cell motility and the cytoskeleton.

[38]  J. West,et al.  Visible light photoinitiation of mesenchymal stem cell-laden bioresponsive hydrogels. , 2011, European cells & materials.

[39]  K. Schulmeister,et al.  Short- and long-wave UV light (UVB and UVA) induce similar mutations in human skin cells. , 2006, The Journal of investigative dermatology.

[40]  Matthew P. Brennan,et al.  Centrifugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds. , 2007, Tissue engineering.

[41]  A. Gefen,et al.  In situ forming hydrogels composed of oxidized high molecular weight hyaluronic acid and gelatin for nucleus pulposus regeneration. , 2013, Acta biomaterialia.

[42]  G. Palmese,et al.  Mechanical evaluation of poly(vinyl alcohol)-based fibrous composites as biomaterials for meniscal tissue replacement. , 2010, Acta biomaterialia.

[43]  Yilin Cao,et al.  Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture. , 2002, Journal of biomedical materials research.

[44]  Benjamin M. Wu,et al.  In vitro response of MC3T3-E1 pre-osteoblasts within three-dimensional apatite-coated PLGA scaffolds. , 2005, Journal of biomedical materials research. Part B, Applied biomaterials.

[45]  B. Müller,et al.  The stiffness of bone marrow cell-knit composites is increased during mechanical load. , 2001, Biomaterials.

[46]  G. Reinholz,et al.  Poly-epsilon-caprolactone/gel hybrid scaffolds for cartilage tissue engineering. , 2009, Journal of biomedical materials research. Part A.

[47]  Wim E Hennink,et al.  The effect of photopolymerization on stem cells embedded in hydrogels. , 2009, Biomaterials.

[48]  J. Seppälä,et al.  Photocrosslinkable polyesters and poly(ester anhydride)s for biomedical applications. , 2011, Macromolecular bioscience.

[49]  F. Sun,et al.  Tuning gradient property and initiating gradient photopolymerization of acrylamide aqueous solution of a hydrosoluble photocleavage polysiloxane-based photoinitiator , 2014 .

[50]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[51]  Harihara Baskaran,et al.  A rapid seeding technique for the assembly of large cell/scaffold composite constructs. , 2006, Tissue engineering.

[52]  Aleksandr Ovsianikov,et al.  Laser fabrication of three-dimensional CAD scaffolds from photosensitive gelatin for applications in tissue engineering. , 2011, Biomacromolecules.

[53]  J. Kehrer.  Free radicals as mediators of tissue injury and disease. , 1993, Critical reviews in toxicology.

[54]  Dan L. Sackett,et al.  Fabrication of Hydrogels with Steep Stiffness Gradients for Studying Cell Mechanical Response , 2012, PloS one.