Spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of biomimetic nanofiber-enabled bone tissue engineering.

2.1. Mouse strains. The Col 1 (2.3) GFP transgenic mice, which specifically label mature osteoblasts with GFP [ 23 ], and the immunodeficient NOD.CB17- Prkdc scid /J (NOD/SCID) mice, which are deficient in functional T cells and B cells, were purchased from the Jackson Laboratory (Bar Harbor, Maine). The Osterix-RFP Cherry reporter mice [ 24 ], which label all osteoblastic lineages with cherry red fluorescence protein (RFP cherry ) were kindly provided by Dr. Peter Maye at the University of Connecticut Heath Science Center. The immunodeficient NOD/SCID;OSX-REP cherry mice were established by backcrossing OSX-RFP cherry mice with NOD/SCID mice for more than eight generations. In these mice, RFP could only be detected in mature osteoblasts and osteocytes in bone tissue. The immunodeficiency of NOD/SCID-OSX-RFP cherry mice was confirmed by the lack of T and B cells in peripheral blood via fluorescence-activated cell sorting (FACS). All in vivo experiments were performed using adult 8–12 week old animals housed in pathogen-free, temperature and humidity controlled facilities with a 12-hour day-night cycle in the vivarium at the University of Rochester Medical Center. All cages contained wood shavings, bedding and a cardboard tube for environmental enrichment. All experimental procedures were reviewed and approved by the University Committee on Animal Resources. General anesthesia, and analgesia procedures were performed based on the mouse formulary provided by the University Committee on Animal Resources at the University of Rochester. The animals’ health status was monitored throughout the experiments by experienced veterinarians according to the Guide for the Care and Use of Laboratory Animals outlined by the National Institute of Health. 2.2. Fabrication of PCL/Collagen/HAp composite nanofibrous mesh. Polycaprolactone (8% w/v) (PCL, SigmaAldrich, St. Louis, MO) and Collagen type I (8% w/v) (Elastin Products, Owensville, MI) solution were prepared by dissolving in 1,1,1,3,3,3,-isopropanal (HFIP, Oakwood Products, New Orleans, Louisiana) respectively and then mixed at a volume ratio of 3:1. Nano hydroxyapatite (HAp, M K Impex Corp. Missisauga, ON, Canada) was suspended in HFIP (8% w/v) by stirring overnight and treated with ultrasound bath to achieve better suspension prior mixing with the PCL/Collagen (3:1). The ratio of PCL:Collagen:HAp is 3:1:2. Electrospinning was performed at a feeding speed of 10 μL/min by a syringe pump (Chemyx Incorporation, Houston, TX) and a 15 kV voltage power supply (Gamma High Voltage Research, Ormond Beach, FL) with a distance of 10 cm between needle and collector. Composite nanofibrous meshes were collected onto circular stainless-steel wire loops for further cell seeding and handling. The composite fiber mesh was examined by Scanning Electron Microscope (SEM) and show randomly oriented non-woven fibers with a mean diameter of ~600 nm [ 20 ]. 2.3. Assembly of 3D cell/scaffold constructs. Bone marrow cells were isolated as previously described [ 20 , 21 ] from 8-12 weeks old Col 1 (2.3) GFP transgenic mice. Briefly, cells were flushed from marrow cavity by slow injection of α-MEM at one end of the bone using a sterile 21-gauge needle. The marrow suspension was dispersed gently by pipetting several times to obtain a single cell suspension. The cell suspension was further filtered through a 70μm cell strainer (Falcon) to remove debris. About 5x10 6 freshly isolated bone marrow cells were seeded on each sterile electrospun fibrous sheet in 12-well plates and cultured in alpha-MEM media containing 15% fetal bovine serum (SigmaAldrich, St. Louis, MO) for 10 days with change of media every two days. Osteogenic differentiation media containing 50 μg/mL ascorbic acid (SigmaAldrich, St. Louis, MO), 5mM β-glycerophosphate (SigmaAldrich, St. Louis, MO), and 10% FBS in alpha-MEM was added at day 10 and cultured for an additional 11 days with medium change every two days. BMSC-seeded fiber sheets (12 layers) were stacked layer-by-layer via a custom-made metal clip to form a flexible membranous tissue construct (~200 μm in thickness). 2.4. The cranial bone defect repair model in mice. Procedures for creating a cranial defect and mounting a glass window for imaging in mice have been previously described [ 21 , 25 ]. Briefly, experimental mice were anesthetized with a mixture of Ketamine and Xylazine. Under anesthesia, hairs on the skull were removed and skin at the surgical site was sterilized with alcohol and iodide solution. A stereotaxic instrument (Stoelting Inc., Wood Dale, IL) was used to stabilize mouse head for surgery under a dissection microscope. A 2-mm in diameter full thickness defect was created in the parietal bone of mouse calvarium using a same-sized Busch inverted cone bur (Armstrong Tool & Supply Company, Livonia, MI). A circular graft of the same size was harvested from the nanofibrous construct via a biopsy punch and used to repair the defect. The skin was closed using absorbable 3-0 Ethilon sutures. Samples were harvested at the indicated time points for histology, MicroCT as well as MPLSM imaging. To perform intravital imaging during cranial defect healing, a custom-made 0.5-mm thick spacer made of poly (aryl-ether-ether-ketone) (PEEK) was glued onto the skull using cyanoacrylate glue (Loctite; Cat #45404, Düsseldorf, Germany). A glass window was mounted on top of the wound for intravital imaging as previously described [ 21 , 25 ]. 2.5. Multiphoton Laser Scanning Microscopy (MPLSM). An Olympus FVMPE-RS system equipped with two 2-photon lasers: Spectra-Physics InSightX3 (680nm-1300nm) and Spectra-Physics MaiTai DeepSee Ti:Sapphire laser (690nm-1040nm), and a 25X water objective (XLPLN25XWMP2, 1.05NA), was used for high resolution imaging. With the laser tuned to 780nm, images were acquired from resonant scanners at a resolution of 512x512 pixels with the z-step size of 5 μm. The fluorescence of GFP, RFP, far-red RFP and Second Harmonic Generation (SHG) signals were collected with a 517/23-nm, a 605/25-nm, a 665/20nm, and a 390/20-nm bandpass filters (Semrock), respectively. The 2D slice viewing and 3D reconstruction of the defect were performed in Imaris (Bitplane Inc., Concord, MA) and Amira (Visage Imaging, Berlin, Germany) image analysis software. Red blood cell (RBC) velocity analyses were performed using a water-immersion objective (×25, NA 1.05) for line-scan measurements. These measurements utilized 640x640 pixel images with a pixel dwell of 10us/pixel. RBC velocity was calculated based on Radon transformation and an automated image-processing algorithm provided by MATLAB [ 26 ]. Vessel diameters were measured manually using ImageJ (National Institutes of Health). 2.6. Measurement of partial oxygen pressure (pO 2 ) in blood vessels via 2-photon phosphorescence lifetime microscopy (2PLM). To examine the oxygen content in various blood vessels, 2-photon phosphorescence lifetime imaging (2PLM) was performed in cranial defect window chamber model, which allows realtime interrogation of pO 2 within each vessel at high spatial resolution [ 27 , 28 ]. To perform 2PLM, 0.5μmol oxygen sensitive phosphorescence probe PtP-C343 mixed with 0.1 μmol Rhodamine Dextran (2,000,000 MW) was administered into circulation via retro-orbital injection. A two-photon microscope with a tunable Mai Tai laser (100 fs, 80 MHz; Spectra-physics, Santa Clara, CA) for excitation and a modified Olympus Fluoview 300 confocal unit was used for imaging. Excitation of PtP-C343 was performed at 900 nm. The light transmitted through the dichroic was passed through a 706/167 nm band-pass filter and directed onto a PMT (Hamamatsu R10699 , Shizuoka, Japan) and a photon counting system (SR 400, Stanford Research Systems, Sunnyvale, CA) for quantification of PtP-C343 phosphorescence (λ max 680 nm). Raw phosphorescence decay data were fit to a single exponential function after subtraction of the offset, to determine the decay time constant, τ. Using an independently measured calibration curve, τ was converted into oxygen tension (pO 2 ) as previously described [ 22 ]. Point scans were performed in randomly selected vessels within the defect. A mean from at least two measurements was used to determine pO 2 within each vessel. Following intravital imaging, samples were stained with CD31 antibody (Biolegend, San Diego, CA) and EMCN (Santa Cruz Biotechnology, Santa Cruz, CA) to determine the identity of the vessels. 2.7. Evaluation of cranial defect repair via histology and MicroCT. The cranial defect samples were scanned by Viva μCT 40 system (Scanco Medical AG, Bassersdorf, Switzerland) at indicated time points post-surgery. The imaging data were anonymized and exported as DICOM files for the evaluation of graft bone formation using Amira (FEI Visualization Sciences Group, Hillsboro, OR, USA). The 2-mm circular defect region was contoured via VolumeEdit in Amira. Bone volumes within the defect were read from the Amira. For histologic analyses, samples were harvested and decalcified in 10% EDTA. Mid-sagittal frozen sections (20μm thick) were prepared via cryosectioning and stained with Hematoxylin & Eosin (H&E). High resolution digital images of the histologic sections were obtained via Olympus VS110™ Virtual Slide Scanning System (Olympus, Tokyo, Japan). Histomorphometric analyses to evaluate bone, scaffolds and fibrotic tissues in the tissue sections were performed using the VisioPharm Image Analysis Software (Hørsholm, Denmark) as previously described [ 29 ]. 2.8. Immunofluorescent staining of blood vessels and microscopy. At the end of the experiments, mice were perfused systemically with freshly made 4% paraformaldehyde via cardiac puncture followed by additional overnight fixation with the same solution. The cranial samples were fully decalcified in 10% EDTA and then treated with blocking solution containing 3% bovine albumin and 0.3% Triton X-100 in PBS overnight. All samples were incubated with CD31 (1:100 dilution, Biolegend, San Diego, CA) and EMCN (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies labeled with respective fluorescent conjugates for 4-7 days at 4°C. The samples were mounted in anti-fade mounting medium and imaged via MPLSM as described above. 2.9. Quantitative and histomorphometric analyses of neovascularization at the site of cranial bone defect repair. All samples were scanned from superior and dura side of the cranial bone for analyses. Multichannel z-series image stacks were used for visualization, 3D reconstruction, and quantitative analyses. A detailed method for quantitative analyses of blood vessels at the site of cranial defect repair has been previously described [ 21 , 25 ]. Evaluation of bone accruement via SHG, an intrinsic signal from the collagen matrix in bone tissue, was performed alongside vascular analyses in Figs. 4 and 5 using image stacks obtained from multiphoton microscopy. A schematic to illustrate our analysis is shown in supplemental Fig. S2 . Briefly, CD31 + EMCN − or CD31 + EMCN + vessels along with SHG and Col 1 (2.3) GFP cells were reconstructed in a 3D format using a multichannel z-series stack. To analyze nanofibrous membrane-mediated defect repair, a circular region consisting of the 2-mm defect up to 200-350 μm in depth (36 tiles of 512x512 z-series stack at 5 μm z-step) was contoured and specified as the region of interest (ROI). Based on unique SHG signals from bone tissue, bone forming and non-bone forming regions were contoured and specified within the circular region of the defect. Vessels within each region were isolated in Amira Segmentation Editor, followed by volumetric and length analyses using Autoskeleton Module combined with Filamental Editor as previously described [ 21 , 25 ]. Based on the unique morphology of the vessel type, CD31 + (total vessels) and CD31 + EMCN + vessels were subjected for volumetric analyses whereas CD31 + EMCN − vessels were subjected for length analyses. Based on the segmented vessel network, vessel volume fractions (Vol. Fract.) (i.e., ratio of vessel volume to total volume) were read directly from the Amira. For length analyses, we used Amira’s CenterTree algorithm to generate a line-based network that was topologically equivalent to the original network. The skeleton was superimposed on the original image to assess the relative accuracy of this method. The final skeletonized vessel network was obtained by manually retracing of the skeletons using Amira’s Filamental Editor to remove false segments. Based on the skeletonized network, vessel Length Fractions (L.Fract.) (i.e., ratio of vessel length to total volume) were read from the Amira software. Quantitative and histomorphometric analyses of neovasculature were performed simultaneously with volumetric quantification of Col 1 (2.3) GFP cells and SHG using Amira Segmentation Editor and volumetric analysis protocol. Analyses were performed in a group of 4 mice, covering the entire defect regions. 2.10. Microarray analyses. Nanofibrous constructs with or without BMSCs were used to repair the 2-mm cranial defects as described above. The implants were punched out at week 3 post-implantation and immediately immersed in liquid nitrogen for RNA isolation. The tissues were pulverized using a nitrogen-cooled mortar and pestle apparatus (Bel-Art, Scienceware, Pequannock, NJ, USA), and purified for total RNA isolation using the TRIzol system (Invitrogen, Carlsbad, CA, USA). A total of 6 samples in two groups (n=3) were prepared for analyses. Total RNA from each sample was isolated using an RNeasy Mini extraction kit. RNA quality and purity were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was determined by the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Whole mouse gene expression microarrays were performed using Clariom S mouse assay (ThermoFisher Scientific) that allows accurately measurements of gene-level expression from >20,000 well-annotated genes. The microarray assays were performed by Center for Functional Genomics at the SUNY University at Albany. The data were analyzed using Transcriptomic Analyses Console (TAC) (ThermoFisher) which provides log2 transformed expression values for statistical processing and hierarchical clustering analyses. Differentially expressed genes were selected with a p value less than 0.01, FDR (false discovery rate) p value less than 0.05 and a fold of change of more than 1.5 when comparing between groups. Heat maps were generated by TAC. Biological processes, functional classifications and gene annotations were analyzed using Gene Set Enrichment Analyses (GSEA) with Hallmark and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases as well as Database for Annotation, Visualization and Integrated Discovery (DAVID) ( http://david.abcc.ncifcrf.gov ). To identify biological processes with significant enrichment, the distribution of genes from our data was compared with a reference annotation gene list for each gene ontology (GO) category. Normalized Enrichment Score (NES) were used for gene enrichment analysis. A nominal p value (NOM) less than 0.05 is considered significantly enriched in the annotation categories. 2.11. Quantitative PCR analyses. Quantitative RT-PCR reaction was performed using SyberGreen (ABgene, Rochester, NY) in a RotorGene real time PCR machine (Corbett Research, Carlsbad, CA). All genes were compared to a standard β-actin control. Data were assessed quantitatively using analysis of variance, comparing relative levels of transcript expression as a function of time. All primers used for the assessment can be found in previous publications [ 30 , 31 ]. Additional primer sequences can be found in supplemental data Table S1 . Data are expressed as the means ± SEM. 2.12. Statistical analyses. All data are shown as the mean ± standard error. Statistical analysis was analyzed by one-way ANOVA in GraphPad Prism (GraphPad Prism, San Diego, CA). A p value