2.1

Fabrication of gas-foaming CPC with immobilized adhesive peptide

CPC powder consisted of an equimolar mixture of TTCP and DCPA. TTCP was synthesized from a solid-state reaction between CaHPO ₄ and CaCO ₃ (J.T. Baker, Phillipsburg, NJ) and then ground to obtain a median particle size of 17 μm. The DCPA powder was ground to obtain a median particle size of 1 μm. The TTCP and DCPA powders were mixed in a blender to form the CPC powder with a TTCP:DCPA molar ratio of 1:1. CPC liquid consisted of chitosan malate (Vanson, Redmond, WA) mixed with distilled water at a chitosan/(chitosan + water) mass fraction of 15%. Chitosan was used because it could cause fast-setting to CPC paste and strengthen the CPC . RGD was immobilized with chitosan by coupling G4RGDSP (Thermo Fisher, Waltham, MA) with chitosan. This was achieved by forming amide bonds between carboxyl groups in peptide and residual amine groups in chitosan, using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, Thermo Fisher) and sulfo- N -hydroxysuccinimide (Sulfo-NHS, Thermo Fisher) as coupling agents . After dissolving G4RGDSP peptide (12.4 mg, 16.32 × 10 ⁻⁶ mol) in 0.1 mol/L of 2-(N-morpholino) ethanesulfonic acid (MES) buffer (4 mL) (Thermo Fisher), EDC (3.76 mg, 19.6 × 10 ⁻⁶ mol) and Sulfo-NHS (0.28 mg, 2.44 × 10 ⁻⁶ mol) were added to the peptide solution (molar ratio of G4RGDSP:EDC:NHS = 1:1.2:0.6). The solution was incubated at room temperature for 30 min to activate the terminal carboxyl group of proline. Then, this solution was added to the chitosan solution dissolved in 0.1 mol/L of MES buffer (100 mL, 1 wt%). The coupling reaction was performed for 24 h at room temperature . The products were dialyzed against distilled water using a Dialysis Cassettes (MWCO = 3.5 kDa) (Thermo Fisher) for 3 d to remove the uncoupled peptides by changing water 3 times daily. Finally, the products were freeze-dried to yield RGD-immobilized chitosan .

A gas-foaming method was used to create macropores in CPC. Following a previous study , sodium bicarbonate (NaHCO ₃ ) and citric acid monohydrate (C ₆ H ₈ O ₇ ·H ₂ O) were added into CPC as the porogen. The acid–base reaction of C ₆ H ₈ O ₇ ·H ₂ O with NaHCO ₃ produced CO ₂ bubbles in CPC, resulting in macropores . NaHCO ₃ was added to the CPC powder at a NaHCO ₃ /(NaHCO ₃ + CPC powder) mass fraction of 15%. The corresponding amount of C ₆ H ₈ O ₇ ·H ₂ O was added to the CPC liquid, to maintain a NaHCO ₃ /(NaHCO ₃ + C ₆ H ₈ O ₇ ·H ₂ O) mass fraction of 54.52%, following a previous study .

CPC paste was formed by mixing CPC powder with CPC liquid at a powder to liquid mass ratio of 2 to 1. The paste was placed in Teflon molds to fabricate macroporous CPC disks with 12 mm in diameter and 1.5 mm in thickness. Two types of materials were fabricated: CPC control (in which the cement liquid used chitosan without RGD), and CPC-RGD (using chitosan with RGD). Specimens were set in a humidor with 100% relative humidity for 24 h at 37 °C, sterilized in an ethylene oxide sterilizer (Andersen, Haw River, NC) for 12 h and then degassed for 7 d prior to cell seeding.

2.2

Cell cultures

HUVEC (Lonza, Walkersville, MD) were cultured in Endothelial Cell Growth Medium-2 (EGM-2; Lonza) which consisted of Endothelial Cells Basal Medium-2 (EBM-2; Lonza) and a provided kit (Lonza). HOB (Lonza) were cultured in Osteoblast Growth Medium (OGM; Lonza) which consisted of Osbteoblasts Basal Medium (OBM; Lonza) and a provided kit (Lonza). Fourth passages of cells were used for the study. At 80–90% confluence, cells were detached by trypsin–EDTA (Invitrogen, Carlsbad, CA) and washed twice with phosphate-buffered saline (PBS). HUVEC were mixed with HOB at 4:1 ratio in EGM-2 for coculture . The macroporous CPC disks were pre-incubated with EGM-2 for 3 h in a humidified incubator prior to cell seeding. Then, they were placed individually in 12-well petri plate. The mixed HUVEC + HOB suspension (1.5 × 10 ⁵ cells/disk) was seeded drop-wise on the top surface of each disk and cultured with EGM-2 in a humidified incubator (5% CO ₂ , 37 °C) for up to 42 d. The medium was replaced every 2 d.

2.3

Quantitative real time-PCR

Quantitative real-time polymerase chain reaction (qRT-PCR, 7900HT, Applied Biosystems, Foster City, CA) was used to measure gene expression of cells on CPC at 14 d, 28 d, and 42 d. The total cellular RNA of the cells was extracted with TRIzol reagent (Invitrogen). RNA concentration was measured via a NanoDrop 2000 spectrophotometer (Thermo Fisher, Wilmington, DE) and diluted to 100 ng/μL if necessary. RNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). TaqMan gene expression assay kits (Applied Biosystems) were used to measure the transcript levels of the following genes: human alkaline phosphatase (ALP, Hs00758162_m1), osteocalcin (OC, Hs00609452_g1), collagen type I (Coll I, Hs00164004), vascular endothelial growth factor A (VEGF, HS00900055_ml), von-Willebrand factor (vWF, Hs00169795_m1), vascular endothelial cadherin (VE-cadherin, Hs00170986_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905). Relative expression level for each target gene was evaluated using the 2 ^−ΔΔCt method . The C _t values of target genes were normalized by the C _t of the TaqMan human housekeeping gene GAPDH to obtain the Δ C _t values. The C _t of HUVEC + HOB cocultured on tissue culture polystyrene for 1 d served as the calibrator.

2.4

Mineral synthesis by cocultured cells

The osteogenesis ability of cocultured cells on the scaffolds was evaluated by staining the minerals synthesized by the cells after 42 d of culture. The CPC disks with cells were washed with PBS, fixed with 10% formaldehyde, and stained with Alizarin Red S (ARS) (Millipore, Billerica, MA) for 20 min, which stained calcium-rich deposits by cells into a red color . An Osteogenesis Kit (Millipore) was used to extract the stained minerals and measure the ARS concentration, following the manufacturer’s protocol . An ARS standard curve was established with known concentrations of the dye. CPC control scaffolds with the same compositions and treatments, but without cell seeding, were also measured. The control’s ARS concentration was subtracted from the ARS concentration of the cell-seeded scaffolds, to yield the net mineral concentration synthesized by the cells .

2.5

Immunofluorescent staining of PECAM-1 (CD31)

To determine vascular development, cell-scaffold constructs were immunofluorescent stained for PECAM-1 (CD31, endothelial-specific) (Invitrogen) at 14 d, 28 d, and 42 d. The samples were briefly rinsed twice with PBS, fixed with 4% parformaldehyde for 20 min, washed twice with PBS, permeabilized with 0.5% Triton X-100 for 5 min and blocked with 0.1% bovine serum albumin (BSA) for 30 min. After washing twice with PBS, the samples were incubated with the primary mouse monoclonal antibody anti-human CD31 (1:200, Invitrogen) overnight at 4 °C. The samples were then washed twice with PBS and incubated with secondary antibody (1:1000, goat anti-mouse Alexa Fluor 488, green fluorescence, Invitrogen) for 1 h. This was followed by a brief rinse in PBS, staining of nuclei with DAPI (1 μg/mL, Sigma, St. Louis, MO) for 10 min at room temperature and washing with PBS. The samples were placed in Fluoromount Aqueous Mounting Medium (Sigma) and viewed under epifluorescent microscopy (TE2000S, Nikon, Melville, NY). The capillaries were stained green and showed as tube-like structures. Three random fields of view were imaged from each sample (five samples yielded 15 photos for each time point). The length of capillaries was measured and added together to obtain the cumulative length of capillaries for each image using Image-Pro Plus software (Media Cybernetics, Silver Springs, MD). The cumulative length of capillaries of the image was then divided by the area of that image, to yield the cumulative vessel length per scaffold surface area.

2.6

Scanning electron microscopy of cells on CPC

The cell-scaffold constructs were examined under scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR, SEM). After culturing for 14 d, the samples were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4, dehydrated with gradient ethanol, and rinsed with hexamethyldisilazane. The samples were dried overnight, sputter-coated with gold and then examined in SEM.

2.7

Statistical analysis

One-way and two-way ANOVA were performed to detect significant effects of the variables. Tukey’s multiple comparison was performed at p = 0.05 to analyze the measured values.

2.1

Fabrication of gas-foaming CPC with immobilized adhesive peptide

CPC powder consisted of an equimolar mixture of TTCP and DCPA. TTCP was synthesized from a solid-state reaction between CaHPO ₄ and CaCO ₃ (J.T. Baker, Phillipsburg, NJ) and then ground to obtain a median particle size of 17 μm. The DCPA powder was ground to obtain a median particle size of 1 μm. The TTCP and DCPA powders were mixed in a blender to form the CPC powder with a TTCP:DCPA molar ratio of 1:1. CPC liquid consisted of chitosan malate (Vanson, Redmond, WA) mixed with distilled water at a chitosan/(chitosan + water) mass fraction of 15%. Chitosan was used because it could cause fast-setting to CPC paste and strengthen the CPC . RGD was immobilized with chitosan by coupling G4RGDSP (Thermo Fisher, Waltham, MA) with chitosan. This was achieved by forming amide bonds between carboxyl groups in peptide and residual amine groups in chitosan, using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, Thermo Fisher) and sulfo- N -hydroxysuccinimide (Sulfo-NHS, Thermo Fisher) as coupling agents . After dissolving G4RGDSP peptide (12.4 mg, 16.32 × 10 ⁻⁶ mol) in 0.1 mol/L of 2-(N-morpholino) ethanesulfonic acid (MES) buffer (4 mL) (Thermo Fisher), EDC (3.76 mg, 19.6 × 10 ⁻⁶ mol) and Sulfo-NHS (0.28 mg, 2.44 × 10 ⁻⁶ mol) were added to the peptide solution (molar ratio of G4RGDSP:EDC:NHS = 1:1.2:0.6). The solution was incubated at room temperature for 30 min to activate the terminal carboxyl group of proline. Then, this solution was added to the chitosan solution dissolved in 0.1 mol/L of MES buffer (100 mL, 1 wt%). The coupling reaction was performed for 24 h at room temperature . The products were dialyzed against distilled water using a Dialysis Cassettes (MWCO = 3.5 kDa) (Thermo Fisher) for 3 d to remove the uncoupled peptides by changing water 3 times daily. Finally, the products were freeze-dried to yield RGD-immobilized chitosan .

A gas-foaming method was used to create macropores in CPC. Following a previous study , sodium bicarbonate (NaHCO ₃ ) and citric acid monohydrate (C ₆ H ₈ O ₇ ·H ₂ O) were added into CPC as the porogen. The acid–base reaction of C ₆ H ₈ O ₇ ·H ₂ O with NaHCO ₃ produced CO ₂ bubbles in CPC, resulting in macropores . NaHCO ₃ was added to the CPC powder at a NaHCO ₃ /(NaHCO ₃ + CPC powder) mass fraction of 15%. The corresponding amount of C ₆ H ₈ O ₇ ·H ₂ O was added to the CPC liquid, to maintain a NaHCO ₃ /(NaHCO ₃ + C ₆ H ₈ O ₇ ·H ₂ O) mass fraction of 54.52%, following a previous study .

CPC paste was formed by mixing CPC powder with CPC liquid at a powder to liquid mass ratio of 2 to 1. The paste was placed in Teflon molds to fabricate macroporous CPC disks with 12 mm in diameter and 1.5 mm in thickness. Two types of materials were fabricated: CPC control (in which the cement liquid used chitosan without RGD), and CPC-RGD (using chitosan with RGD). Specimens were set in a humidor with 100% relative humidity for 24 h at 37 °C, sterilized in an ethylene oxide sterilizer (Andersen, Haw River, NC) for 12 h and then degassed for 7 d prior to cell seeding.

2.2

Cell cultures

HUVEC (Lonza, Walkersville, MD) were cultured in Endothelial Cell Growth Medium-2 (EGM-2; Lonza) which consisted of Endothelial Cells Basal Medium-2 (EBM-2; Lonza) and a provided kit (Lonza). HOB (Lonza) were cultured in Osteoblast Growth Medium (OGM; Lonza) which consisted of Osbteoblasts Basal Medium (OBM; Lonza) and a provided kit (Lonza). Fourth passages of cells were used for the study. At 80–90% confluence, cells were detached by trypsin–EDTA (Invitrogen, Carlsbad, CA) and washed twice with phosphate-buffered saline (PBS). HUVEC were mixed with HOB at 4:1 ratio in EGM-2 for coculture . The macroporous CPC disks were pre-incubated with EGM-2 for 3 h in a humidified incubator prior to cell seeding. Then, they were placed individually in 12-well petri plate. The mixed HUVEC + HOB suspension (1.5 × 10 ⁵ cells/disk) was seeded drop-wise on the top surface of each disk and cultured with EGM-2 in a humidified incubator (5% CO ₂ , 37 °C) for up to 42 d. The medium was replaced every 2 d.

2.3

Quantitative real time-PCR

Quantitative real-time polymerase chain reaction (qRT-PCR, 7900HT, Applied Biosystems, Foster City, CA) was used to measure gene expression of cells on CPC at 14 d, 28 d, and 42 d. The total cellular RNA of the cells was extracted with TRIzol reagent (Invitrogen). RNA concentration was measured via a NanoDrop 2000 spectrophotometer (Thermo Fisher, Wilmington, DE) and diluted to 100 ng/μL if necessary. RNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). TaqMan gene expression assay kits (Applied Biosystems) were used to measure the transcript levels of the following genes: human alkaline phosphatase (ALP, Hs00758162_m1), osteocalcin (OC, Hs00609452_g1), collagen type I (Coll I, Hs00164004), vascular endothelial growth factor A (VEGF, HS00900055_ml), von-Willebrand factor (vWF, Hs00169795_m1), vascular endothelial cadherin (VE-cadherin, Hs00170986_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905). Relative expression level for each target gene was evaluated using the 2 ^−ΔΔCt method . The C _t values of target genes were normalized by the C _t of the TaqMan human housekeeping gene GAPDH to obtain the Δ C _t values. The C _t of HUVEC + HOB cocultured on tissue culture polystyrene for 1 d served as the calibrator.

2.4

Mineral synthesis by cocultured cells

The osteogenesis ability of cocultured cells on the scaffolds was evaluated by staining the minerals synthesized by the cells after 42 d of culture. The CPC disks with cells were washed with PBS, fixed with 10% formaldehyde, and stained with Alizarin Red S (ARS) (Millipore, Billerica, MA) for 20 min, which stained calcium-rich deposits by cells into a red color . An Osteogenesis Kit (Millipore) was used to extract the stained minerals and measure the ARS concentration, following the manufacturer’s protocol . An ARS standard curve was established with known concentrations of the dye. CPC control scaffolds with the same compositions and treatments, but without cell seeding, were also measured. The control’s ARS concentration was subtracted from the ARS concentration of the cell-seeded scaffolds, to yield the net mineral concentration synthesized by the cells .

2.5

Immunofluorescent staining of PECAM-1 (CD31)

To determine vascular development, cell-scaffold constructs were immunofluorescent stained for PECAM-1 (CD31, endothelial-specific) (Invitrogen) at 14 d, 28 d, and 42 d. The samples were briefly rinsed twice with PBS, fixed with 4% parformaldehyde for 20 min, washed twice with PBS, permeabilized with 0.5% Triton X-100 for 5 min and blocked with 0.1% bovine serum albumin (BSA) for 30 min. After washing twice with PBS, the samples were incubated with the primary mouse monoclonal antibody anti-human CD31 (1:200, Invitrogen) overnight at 4 °C. The samples were then washed twice with PBS and incubated with secondary antibody (1:1000, goat anti-mouse Alexa Fluor 488, green fluorescence, Invitrogen) for 1 h. This was followed by a brief rinse in PBS, staining of nuclei with DAPI (1 μg/mL, Sigma, St. Louis, MO) for 10 min at room temperature and washing with PBS. The samples were placed in Fluoromount Aqueous Mounting Medium (Sigma) and viewed under epifluorescent microscopy (TE2000S, Nikon, Melville, NY). The capillaries were stained green and showed as tube-like structures. Three random fields of view were imaged from each sample (five samples yielded 15 photos for each time point). The length of capillaries was measured and added together to obtain the cumulative length of capillaries for each image using Image-Pro Plus software (Media Cybernetics, Silver Springs, MD). The cumulative length of capillaries of the image was then divided by the area of that image, to yield the cumulative vessel length per scaffold surface area.

2.6

Scanning electron microscopy of cells on CPC

The cell-scaffold constructs were examined under scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR, SEM). After culturing for 14 d, the samples were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4, dehydrated with gradient ethanol, and rinsed with hexamethyldisilazane. The samples were dried overnight, sputter-coated with gold and then examined in SEM.

2.7

Statistical analysis

One-way and two-way ANOVA were performed to detect significant effects of the variables. Tukey’s multiple comparison was performed at p = 0.05 to analyze the measured values.