2.1

Fabrication of biofunctionalized CPC

Tetracalcium phosphate [TTCP: Ca ₄ (PO ₄ ) ₂ O] was synthesized using dicalcium phosphate anhydrous (DCPA: CaHPO ₄ ) and calcium carbonate (J.T. Baker, Philipsburg, NJ). TTCP was ground to obtain particles of 1–80 μm, with a median of 17 μm . DCPA was ground to obtain a median particle size of 1 μm. TTCP and DCPA powders were mixed at 1:1 molar ratio to form the CPC powder. Chitosan lactate (Vanson, Redmond, WA) was mixed with water at a chitosan/(chitosan + water) mass fraction of 15% to form the liquid, which could cause CPC to set fast . For mechanical reinforcement, a resorbable suture fiber (Vicryl, polyglactin 910, Ethicon, NJ) was cut to filaments of a length of 3 mm and mixed with CPC paste at a fiber volume fraction 20%, following a previous study . The CPC powder to liquid mass ratio of 2:1 was used to form a flowable paste. This CPC is referred to as “CPC control”.

Five biofunctionalized CPCs were prepared by incorporating the following biofunctional agents: RGD, Fn, FEPP, Geltrex, and platelet concentrate. Each biofunctional agent was mixed with the chitosan liquid, which was then mixed with the CPC powder. The concentration of RGD (Sigma, St. Louis, MO) was 50 μg RGD per 1 g of CPC paste (0.005% by mass), following a previous study . For Fn (human plasma Fn, Invitrogen, Carlsbad, CA) and FEPP (Sigma), the same 0.005% concentration was used in CPC. Geltrex (Invitrogen) was added to CPC at 100 μL Geltrex per 1 g of CPC paste (0.1% by mass). This percentage was selected because preliminary study showed that it did not compromise the CPC setting time and mechanical property, while greatly improving cell function. Similarly, human platelet concentrate (1.2 × 10 ⁶ platelets per μL, Biological Specialty, Colmar, PA) was added to CPC at 100 μL of platelet concentrate per 1 g of CPC paste (0.1% by mass). CPC containing these agents are referred to as CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets, respectively.

2.2

Setting time and mechanical properties of biofunctionalized CPC

Setting time of CPC was measured using a previous method . Briefly, CPC paste was filled into a mold of 3 mm × 4 mm × 25 mm and placed in a humidor at 37 °C. At 1 min intervals, the specimen was scrubbed gently with figures until the powder component did not come off, indicating that the setting reaction had occurred sufficiently to hold the specimen together. The time measured from the powder–liquid mixing to this point was used as the setting time .

To measure mechanical properties, the paste was placed into a mold of 3 mm × 4 mm × 25 mm. The specimens were incubated at 37 °C for 4 h in a humidor, and then demolded and immersed in water at 37 °C for 20 h. The specimens were then fractured in three-point flexure with a span of 20 mm at a crosshead speed of 1 mm/min on a Universal Testing Machine (5500R, MTS, Cary, NC). Flexural strength and elastic modulus were measured ( n = 6) . Specimens were tested within a few minutes after being taking out of the water, and fractured while still being wet.

2.3

hUCMSC culture and proliferation

hUCMSCs (ScienCell, Carlsbad, CA) were derived from the Wharton’s Jelly in umbilical cords of healthy babies and harvested as previously described . The use of hUCMSCs was approved by the University of Maryland. Cells were cultured in a low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen), which is referred to as the control media. Passage 4 cells were used. The osteogenic media had 100 nM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and 10 nM 1α,25-dihydroxyvitamin (Sigma) .

The materials for the preparation of CPC were sterilized in an ethylene oxide sterilizer (Andersen, Haw River, NC). Each CPC paste was filled into a disk mold with a diameter of 12 mm and a thickness of 1.5 mm. Each disk was incubated in the mold at 37 °C for 4 h in a humidor, and then demolded and immersed in water at 37 °C for 20 h. Each disk was placed in a well of a 24-well plate, and 50,000 cells in osteogenic medium were added to each well. After 1, 4, and 8 d, the constructs were washed in Tyrode’s Hepes buffer, live/dead stained and viewed by epifluorescence microscopy (TE2000S, Nikon, Melville, NY) . Images were taken at a magnification of 4×. Three randomly chosen fields of view were photographed for each disk. Five disks yielded 15 photos for each material at each time point. Live cells (stained green) and dead cells (stained red) were counted. The live cell density, D , is the number of live cells ( N _L ) attaching to the specimen divided by the surface area A : D = N _L / A .

2.4

Fluorescence of actin fibers in hUCMSCs on biofunctionalized CPC

Actin fibers in the cell cytoskeleton were examined to determine if the biofunctional agents in CPC would enhance cell attachment and increase the amount of actin stress fibers. hUCMSCs were seeded at a relatively low density of 50,000 cells per well, the same as that for live/dead assay, in order to clearly see the stained cells and their actin fibers. The hUCMSC constructs after 1-d culture were washed with PBS, fixed with 4% parformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 0.1% bovine serum albumin (BSA) for 30 min . An actin cytoskeleton and focal adhesion staining kit (Chemicon, Temecula, CA) was used, which stained actin fibers into a red color. After incubating the construct with diluted (1:400) TRITC-conjugated phalloidin, cell nuclei were labeled with 4′-6-diamidino-2-phenylindole (DAPI), which revealed the nuclei in blue color. Fluorescence microscopy (Nikon) was used to examine the specimens. The fluorescence of actin fibers in hUCMSCs was measured via a NIS-Elements BR software (Nikon) .

2.5

Osteogenic differentiation of hUCMSCs on biofunctionalized CPC

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR, 7900HT, Applied Biosystems, Foster City, CA) was used. Each disk was placed in a well of a 24-well plate. A seeding density of 150,000 cells per well was used following previous studies . The constructs were cultured in osteogenic media for 1, 4, and 8 d . The total cellular RNA on the scaffolds was extracted with TRIzol reagent (Invitrogen). RNA (50 ng/μL) was reverse-transcribed into cDNA. TaqMan gene expression kits were used to measure the transcript levels of the proposed genes on human alkaline phosphatase (ALP, Hs00758162_m1), osteocalcin (OC, Hs00609452_g1), collagen type I (Coll I, Hs00164004), Runx2 (Hs00231692_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905). Relative expression for each target gene was evaluated using the <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='2−ΔΔCt’>2−ΔΔCt2−ΔΔCt
2 − Δ Δ C t
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. These values were subtracted by the C _t value of the hUCMSCs cultured on tissue culture polystyrene in the control media for 1 d (the calibrator) to obtain the ΔΔ C _t values .

2.6

hUCMSC mineralization on biofunctionalized CPC

Alizarin Red S (ARS) staining was used to examine mineralization by hUCMSCs . hUCMSCs were seeded on CPC disks and cultured in osteogenic media. A seeding density of 150,000 cells per well was used following previous studies . After 4, 14 and 21 d, the constructs were stained with ARS. An osteogenesis assay (Millipore, Billerica, MA) was used to measure the ARS concentration at OD ₄₀₅ , following the manufacturer’s instructions. ARS standard curve was done with known concentration of the dye. CPC disks with the same composition and treatment, but without hUCMSC seeding, were also measured as control, and the control’s ARS concentration was subtracted from the ARS concentration of the CPC scaffold with hUCMSCs . This method yielded the net mineral concentration synthesized by the cells . The time points of 14 d and 21 d were selected because previous studies found a large increase in calcium content from 12 d to 21 d .

2.7

Statistical analyses

One-way and two-way ANOVA were performed to detect significant effects of the variables. Tukey’s multiple comparison procedures were used to group and rank the measured values, and Dunn’s multiple comparison tests were used on data with non-normal distribution or unequal variance, both at a family confidence coefficient of 0.95.

2.1

Fabrication of biofunctionalized CPC

Tetracalcium phosphate [TTCP: Ca ₄ (PO ₄ ) ₂ O] was synthesized using dicalcium phosphate anhydrous (DCPA: CaHPO ₄ ) and calcium carbonate (J.T. Baker, Philipsburg, NJ). TTCP was ground to obtain particles of 1–80 μm, with a median of 17 μm . DCPA was ground to obtain a median particle size of 1 μm. TTCP and DCPA powders were mixed at 1:1 molar ratio to form the CPC powder. Chitosan lactate (Vanson, Redmond, WA) was mixed with water at a chitosan/(chitosan + water) mass fraction of 15% to form the liquid, which could cause CPC to set fast . For mechanical reinforcement, a resorbable suture fiber (Vicryl, polyglactin 910, Ethicon, NJ) was cut to filaments of a length of 3 mm and mixed with CPC paste at a fiber volume fraction 20%, following a previous study . The CPC powder to liquid mass ratio of 2:1 was used to form a flowable paste. This CPC is referred to as “CPC control”.

Five biofunctionalized CPCs were prepared by incorporating the following biofunctional agents: RGD, Fn, FEPP, Geltrex, and platelet concentrate. Each biofunctional agent was mixed with the chitosan liquid, which was then mixed with the CPC powder. The concentration of RGD (Sigma, St. Louis, MO) was 50 μg RGD per 1 g of CPC paste (0.005% by mass), following a previous study . For Fn (human plasma Fn, Invitrogen, Carlsbad, CA) and FEPP (Sigma), the same 0.005% concentration was used in CPC. Geltrex (Invitrogen) was added to CPC at 100 μL Geltrex per 1 g of CPC paste (0.1% by mass). This percentage was selected because preliminary study showed that it did not compromise the CPC setting time and mechanical property, while greatly improving cell function. Similarly, human platelet concentrate (1.2 × 10 ⁶ platelets per μL, Biological Specialty, Colmar, PA) was added to CPC at 100 μL of platelet concentrate per 1 g of CPC paste (0.1% by mass). CPC containing these agents are referred to as CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets, respectively.

2.2

Setting time and mechanical properties of biofunctionalized CPC

Setting time of CPC was measured using a previous method . Briefly, CPC paste was filled into a mold of 3 mm × 4 mm × 25 mm and placed in a humidor at 37 °C. At 1 min intervals, the specimen was scrubbed gently with figures until the powder component did not come off, indicating that the setting reaction had occurred sufficiently to hold the specimen together. The time measured from the powder–liquid mixing to this point was used as the setting time .

To measure mechanical properties, the paste was placed into a mold of 3 mm × 4 mm × 25 mm. The specimens were incubated at 37 °C for 4 h in a humidor, and then demolded and immersed in water at 37 °C for 20 h. The specimens were then fractured in three-point flexure with a span of 20 mm at a crosshead speed of 1 mm/min on a Universal Testing Machine (5500R, MTS, Cary, NC). Flexural strength and elastic modulus were measured ( n = 6) . Specimens were tested within a few minutes after being taking out of the water, and fractured while still being wet.

2.3

hUCMSC culture and proliferation

hUCMSCs (ScienCell, Carlsbad, CA) were derived from the Wharton’s Jelly in umbilical cords of healthy babies and harvested as previously described . The use of hUCMSCs was approved by the University of Maryland. Cells were cultured in a low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen), which is referred to as the control media. Passage 4 cells were used. The osteogenic media had 100 nM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and 10 nM 1α,25-dihydroxyvitamin (Sigma) .

The materials for the preparation of CPC were sterilized in an ethylene oxide sterilizer (Andersen, Haw River, NC). Each CPC paste was filled into a disk mold with a diameter of 12 mm and a thickness of 1.5 mm. Each disk was incubated in the mold at 37 °C for 4 h in a humidor, and then demolded and immersed in water at 37 °C for 20 h. Each disk was placed in a well of a 24-well plate, and 50,000 cells in osteogenic medium were added to each well. After 1, 4, and 8 d, the constructs were washed in Tyrode’s Hepes buffer, live/dead stained and viewed by epifluorescence microscopy (TE2000S, Nikon, Melville, NY) . Images were taken at a magnification of 4×. Three randomly chosen fields of view were photographed for each disk. Five disks yielded 15 photos for each material at each time point. Live cells (stained green) and dead cells (stained red) were counted. The live cell density, D , is the number of live cells ( N _L ) attaching to the specimen divided by the surface area A : D = N _L / A .

2.4

Fluorescence of actin fibers in hUCMSCs on biofunctionalized CPC

Actin fibers in the cell cytoskeleton were examined to determine if the biofunctional agents in CPC would enhance cell attachment and increase the amount of actin stress fibers. hUCMSCs were seeded at a relatively low density of 50,000 cells per well, the same as that for live/dead assay, in order to clearly see the stained cells and their actin fibers. The hUCMSC constructs after 1-d culture were washed with PBS, fixed with 4% parformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 0.1% bovine serum albumin (BSA) for 30 min . An actin cytoskeleton and focal adhesion staining kit (Chemicon, Temecula, CA) was used, which stained actin fibers into a red color. After incubating the construct with diluted (1:400) TRITC-conjugated phalloidin, cell nuclei were labeled with 4′-6-diamidino-2-phenylindole (DAPI), which revealed the nuclei in blue color. Fluorescence microscopy (Nikon) was used to examine the specimens. The fluorescence of actin fibers in hUCMSCs was measured via a NIS-Elements BR software (Nikon) .

2.5

Osteogenic differentiation of hUCMSCs on biofunctionalized CPC

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR, 7900HT, Applied Biosystems, Foster City, CA) was used. Each disk was placed in a well of a 24-well plate. A seeding density of 150,000 cells per well was used following previous studies . The constructs were cultured in osteogenic media for 1, 4, and 8 d . The total cellular RNA on the scaffolds was extracted with TRIzol reagent (Invitrogen). RNA (50 ng/μL) was reverse-transcribed into cDNA. TaqMan gene expression kits were used to measure the transcript levels of the proposed genes on human alkaline phosphatase (ALP, Hs00758162_m1), osteocalcin (OC, Hs00609452_g1), collagen type I (Coll I, Hs00164004), Runx2 (Hs00231692_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905). Relative expression for each target gene was evaluated using the 2 − Δ Δ C t 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. These values were subtracted by the C _t value of the hUCMSCs cultured on tissue culture polystyrene in the control media for 1 d (the calibrator) to obtain the ΔΔ C _t values .

2.6

hUCMSC mineralization on biofunctionalized CPC

Alizarin Red S (ARS) staining was used to examine mineralization by hUCMSCs . hUCMSCs were seeded on CPC disks and cultured in osteogenic media. A seeding density of 150,000 cells per well was used following previous studies . After 4, 14 and 21 d, the constructs were stained with ARS. An osteogenesis assay (Millipore, Billerica, MA) was used to measure the ARS concentration at OD ₄₀₅ , following the manufacturer’s instructions. ARS standard curve was done with known concentration of the dye. CPC disks with the same composition and treatment, but without hUCMSC seeding, were also measured as control, and the control’s ARS concentration was subtracted from the ARS concentration of the CPC scaffold with hUCMSCs . This method yielded the net mineral concentration synthesized by the cells . The time points of 14 d and 21 d were selected because previous studies found a large increase in calcium content from 12 d to 21 d .

2.7

Statistical analyses

One-way and two-way ANOVA were performed to detect significant effects of the variables. Tukey’s multiple comparison procedures were used to group and rank the measured values, and Dunn’s multiple comparison tests were used on data with non-normal distribution or unequal variance, both at a family confidence coefficient of 0.95.