title: Visualizing osteogenesis in vivo within a cell-scaffold construct for bone tissue engineering using two-photon microscopy.
abstract: Tissue-engineering therapies have shown early success in the clinic, however, the cell-biomaterial interactions that result in successful outcomes are not yet well understood and are difficult to observe. Here we describe a method for visualizing bone formation within a tissue-engineered construct in vivo, at a single-cell resolution, and in situ in three dimensions using two-photon microscopy. First, two-photon microscopy and histological perspectives were spatially linked using fluorescent reporters for cells in the skeletal lineage. In the process, the tissue microenvironment that precedes a repair-focused study was described. The distribution and organization of type I collagen in the calvarial microenvironment was also described using its second harmonic signal. Second, this platform was used to observe in vivo, for the first time, host cells, donor cells, scaffold, and new bone formation within cell-seeded constructs in a bone defect. We examined constructs during bone repair 4 and 6 weeks after implantation. New bone formed on scaffolds, primarily by donor cells. Host cells formed a new periosteal layer that covered the implant. Scaffold resorption appeared to be site specific, where areas near the top were removed and deeper areas were completely embedded in new mineral. Visualizing the in vivo progression of the cell and scaffold microenvironment will contribute to our understanding of tissue-engineered regeneration and should lead to the development of more streamlined and therapeutically powerful approaches.
authors: 1 Department of Materials Science and Engineering, University of Connecticut , Storrs, Connecticut.
authors: Max M
authors: MM
authors: Villa
authors: Liping
authors: L
authors: Wang
authors: Jianping
authors: J
authors: Huang
authors: David W
authors: DW
authors: Rowe
authors: Mei
authors: M
authors: Wei
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000502:physiology*; D002113:Calcification, Physiologic; D019070:Cell Lineage; D060833:Cellular Microenvironment; D003094:Collagen / Q000378:metabolism; D017930:Genes, Reporter; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton; D010006:Osteoblasts / Q000166:cytology; D010011:Osteocytes / Q000166:cytology; D010012:Osteogenesis; D012886:Skull / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*; D014945:Wound Healing
chemical_list: D003094:Collagen
references: Science. 2007 Oct 5;318(5847):116-20
references: 17916739
references: Science. 2002 Feb 8;295(5557):1009-14
references: 11834815
references: Nat Rev Mol Cell Biol. 2007 Mar;8(3):221-33
references: 17318226
references: Lancet. 2008 Dec 13;372(9655):2023-30
references: 19022496
references: J Vis Exp. 2008 Feb 25;(12):
references: 19066563
references: Tissue Eng Part B Rev. 2010 Feb;16(1):123-45
references: 19891542
references: J Vis Exp. 2008 Feb 15;(12):
references: 19066562
references: Science. 2003 Apr 4;300(5616):76-7
references: 12677053
references: Nat Biotechnol. 2004 May;22(5):560-7
references: 15077117
references: Lancet. 2006 Apr 15;367(9518):1241-6
references: 16631879
references: Proc Natl Acad Sci U S A. 2012 May 29;109(22):8434-9
references: 22586078
references: Nat Med. 2011 Jun;17(6):744-9
references: 21572426
references: J Cell Biochem. 1994 Nov;56(3):283-94
references: 7876320
references: Nat Med. 2003 Jun;9(6):796-800
references: 12754503
references: Biophys J. 2005 Feb;88(2):1377-86
references: 15533922
references: Science. 2010 Jul 30;329(5991):538-41
references: 20576850
references: Nat Protoc. 2011 Jan;6(1):1-14
references: 21212779
references: Methods Enzymol. 2009;461:349-78
references: 19480927
references: Mol Ther. 2009 Nov;17(11):1967-78
references: 19603005
references: J Bone Miner Res. 2002 Jan;17(1):15-25
references: 11771662
references: Nature. 2008 May 15;453(7193):345-51
references: 18480816
references: Curr Opin Immunol. 2006 Aug;18(4):476-82
references: 16765574
references: Nat Med. 2010 Aug;16(8):927-33
references: 20628374
references: Cell Stem Cell. 2012 Mar 2;10(3):259-72
references: 22385654
references: Bone. 2002 Dec;31(6):654-60
references: 12531558
references: J Struct Biol. 2003 Jan;141(1):53-62
references: 12576020
references: J Oral Maxillofac Surg. 2010 Feb;68(2):300-8
references: 20116699
references: Science. 1990 Apr 6;248(4951):73-6
references: 2321027
references: Nat Methods. 2012 Jun 28;9(7):676-82
references: 22743772
references: Cell Host Microbe. 2009 Jan 22;5(1):13-21
references: 19154984
references: J Clin Invest. 2012 Jul;122(7):2499-508
references: 22706307
references: Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3311-6
references: 20133604
references: Nat Protoc. 2009;4(8):1128-44
references: 19617885
references: J Cell Biol. 1997 Jun 16;137(6):1445-57
references: 9182674
references: Bone. 2008 Sep;43(3):501-10
references: 18571490
references: BMC Bioinformatics. 2010 May 21;11:274
references: 20492697
references: Front Endocrinol (Lausanne). 2012 Aug 09;3:98
references: 22908007
references: Mol Ther. 2010 Apr;18(4):835-42
references: 20125125
references: Science. 2011 Aug 26;333(6046):1088-9
references: 21868648
references: Nature. 2011 Mar 10;471(7337):170-2
references: 21390119
references: J Tissue Eng Regen Med. 2013 Dec;7(12):974-83
references: 22473786
references: Nat Rev Immunol. 2002 Nov;2(11):872-80
references: 12415310
references: Nature. 2009 Jan 1;457(7225):92-6
references: 19052546