Phosphorylcholine materials, which have a molecular replica of the main zwitterionic phospholipid headgroup present in the cell lipid membrane, are bioinspired polymers that resemble the extracellular surface of red blood cells. The PC-polymer's phosphorylcholine side chain includes the phosphate moiety found in phosphatidylcholine and phosphatidylserine, the latter of which is a membrane lipid component involved in biomineralization and capable of binding calcium[1].

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Human osteoblasts (HOb) were cultured on non-coated, PC5 (5% CMA), and PC20 (20% CMA) coated surfaces. Phosphorylcholine with higher cationic charge (PC20) increased HOb cell adhesion compared to lower charge (PC5) and neutral (PC0) surfaces. At 6, 24, and 48 hours, tissue culture plastic controls exhibited the largest number of cells. Significant differences (P<0.001) were observed between control and PC0, and between PC20 and PC0 at all three time points. Significant differences (P<0.001) between PC20 and PC5 were seen at 24 and 48 hours. [1]
Over 28 days, the number of HOb cells increased on PC20 and control, while the number decreased on PC5 with increasing time up to 28 days. After day 1, significant differences (P<0.001) existed between PC5 and PC20 and control surfaces for all time points. Proliferation on control surfaces was significantly greater at all time points (P<0.001). [1]
Alkaline phosphatase (ALP) activity per thousand cells decreased on PC20 over the first 3 days and on control between day 1 and 2; thereafter ALP activity did not change significantly, and no significant difference was found between PC20 and control after day 2. ALP activity on PC5 was too low to give an accurate measure. [1]
Optical microscopy at 48 hours showed that PC20 and control surfaces had the largest number of HOb cells with fully spread morphologies and the presence of mineral deposits. PC0 supported minimal cell adhesion with rounded cells, and PC5 had more cells than PC0 but cells remained rounded. [1]
SEM with EDX at 48 hours and 28 days confirmed mineral deposits on PC20 and control surfaces containing calcium and phosphorus. The Ca:P ratio of these deposits was approximately 0.6, considerably lower than hydroxyapatite (1.5-1.67), indicating calcium-deficient mineral. The amount of mineral deposits increased with time up to 28 days. [1]

Stainless steel pins coated with PC0, PC6 (6% CMA), PC20, uncoated (negative control), or hydroxyapatite (HA, positive control) were implanted into the right tibia of rats. At 14 weeks post-implantation, histological analysis of the bone-implant interface and interfacial area was performed. HA showed significantly more bone and marrow apposition (P<0.05) than fibrous tissue and loosely associated matrix (LAM). The cationically-charged PC materials (PC6 and PC20) produced a similar response with no significant differences between tissue types. For PC0 and stainless steel (SS), there was significantly more fibrous tissue than bone/marrow and LAM (P<0.05). HA had the highest bone/marrow apposition and least fibrous tissue compared to the other four surfaces (P<0.05). PC0 had significantly more fibrous tissue than PC6 (P<0.05), but no significant differences between PC0 and PC20 or between PC0 and SS. There was a trend for a linear increase in LAM with increasing CMA content in the implant interfacial area (R²=0.9) and at the interface (R²=0.7). No significant bone bonding was observed for any PC-coated implants; instead, fibrous tissue and LAM were present. [1]

Alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation associated with skeletal mineralization, was measured. A substrate buffer of 0.5 M 2-amino-2-methyl-1-propanol (AMP) was prepared in distilled water (pH 10) and supplemented with 2 mM magnesium chloride and 9 mM p-nitrophenol phosphate (p-NPP). In an alkaline solution, ALP catalyzes p-NPP to p-nitrophenol, which appears yellow. Cells were lysed at various time points (1, 2, 4, 7, 14, and 28 days) using freeze-thaw cycles. 50 µL of each lysate (in duplicate) and 50 µL of a range of p-nitrophenol concentrations (for standard curve) were transferred to a 96-well plate. Then 50 µL of AMP buffer was added to each well, and the plate was incubated at 37°C for 15 minutes. Absorbance was read at 405 nm. A standard curve of absorbance as a function of p-nitrophenol concentration was generated to determine total ALP content. [1]

Human osteoblasts (HOb) isolated from hip bone were grown in T75 flasks in McCoy's 5a growth medium containing 10% foetal bovine serum (FBS), 1% glutamine, and 30 µg/mL vitamin C, at 37°C in 5% CO2 humidity. At 60-80% confluence, cells were washed with Hank's Balanced Salt Solution (HBSS), incubated with trypsin/EDTA to detach cells, neutralized with growth medium, centrifuged at 220×g for 4 min, and re-suspended for counting using a haemocytometer. [1]
For cell number determination, cells were cultured on non-coated, PC5, and PC20 coated T25 flasks for up to 28 days. At time points (6 h, 1,2,4,7,14,28 days), culture media was aspirated, cells washed with PBS, and 1 mL of 0.5% v/v Triton X-100 in PBS added. Two freeze-thaw cycles (15 min at -70°C and 37°C) were used to lyse cells. 180 µL of lysate was transferred in duplicate to a white-walled 96-well plate, 20 µL of ATP monitoring reagent added, and ATP concentration read immediately using a luminometer. A standard curve using predetermined HOb cell numbers (0, 2500, 5000, 10000, 20000, 40000) on uncoated plates was used to determine cell numbers. [1]
For optical imaging and SEM/EDX, HOb cells were seeded at 12×10³/cm² in T25 flasks coated with PC0, PC5, PC20, or non-coated control for 48 hours and 28 days. Phase-contrast microscopy was performed. For SEM, cells were washed with PBS, fixed in 4% paraformaldehyde, washed with distilled water, and a ~20×20 mm piece of each flask was mounted on an SEM stub, sputter-coated with carbon, and imaged at 5 kV. EDX was used to characterize elemental constituents of mineral exudates. [1]

Thirty mature Sprague Dawley rats (300-350 g) were used. Surgical grade stainless steel (316 L) pins were cleaned, sonicated in acetone and ethanol, and assigned to five groups (n=6 per group): uncoated (negative control), plasma-sprayed hydroxyapatite (60-100 µm thick, positive control), or dip-coated with PC0, PC6, or PC20 polymer solutions (5 mg/mL in ethanol) resulting in coatings ~30-50 nm thick, then cured at 70°C for 4 hours. Pins were sterilized by gamma irradiation (25 kGy). [1]
Anaesthesia was induced with a gaseous mixture of oxygen and halothane (4%) at 6 L/min in an anaesthetic chamber. Midazolam (3 mg/kg) was given intraperitoneally, and rats maintained with halothane 4% via mask. The right hind limb was shaved, peri-operative analgesia (carprofen, 5 mg/mL) given subcutaneously. A 1.5 cm incision was made lateral to the patella, a lateral capsulotomy performed allowing medial dislocation of the patella to expose the tibial plateau. A 1.5 mm diameter hand drill was used to drill through the centre of the tibial plateau to a depth of 10 mm and counter-sunk (4 mm counter-bore). The pin was press-fitted into the tibia, patella reduced, and incision closed. Immediately after surgery, rats received antibiotic (co-amoxyclavulanic acid, 150 mg/kg) intramuscularly and analgesia (buprenorphine, 0.15 mg/kg) subcutaneously. A second analgesia injection (buprenorphine, 0.1 mg/kg) was given after 5 hours. Rats were sacrificed at 14 weeks. Tibiae were removed, fixed in paraformaldehyde, washed, dehydrated in methylated spirits (50-100%), defatted under vacuum with acetone for 7 days, and embedded in poly(methyl methacrylate) (PMMA). Longitudinal sections (300-500 µm thick) were cut using a diamond saw, ground and polished to 100 µm thickness, stained with toluidine blue (pH 9, 56°C, 30 min), and five fields per slide were digitally photographed for histological analysis. [1]

The Phosphorylcholine polymers (PC0, PC6, PC20) were described as biotolerant in bone, stimulating the production of fibrous tissue and areas of loosely associated matrix (LAM) around the implant. No quantitative toxicity parameters (e.g., LD50, organ toxicity, protein binding) are reported. For PC0 and stainless steel, there was significantly more fibrous tissue than bone/marrow and LAM (P<0.05). Increasing cationic charge (CMA content) showed a linear increase in LAM formation (R²=0.7-0.9), suggesting a potential adverse tissue response. No other toxicity information is provided. [1]

[1]. The effect of cationically-modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo. J Mater Sci Mater Med. 2017 Aug 17;28(9):144.

Phosphorylcholine chloride is an organochloride composed of phosphoric acid cations and chloride ions. It contains phosphoric acid choline and chloride ions. Calcium and magnesium salts can be used to treat hepatobiliary dysfunction.

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Phosphorylcholine polymers have been used in a variety of medical devices to improve biocompatibility due to low protein adsorption, reduced complement activation, inflammatory response, and cell adhesion. However, for orthopaedic applications requiring bone-implant integration, unmodified PC is not suitable. Cationic modification (incorporation of choline methacrylate, CMA) was attempted to increase bioactivity. Despite in vitro evidence of increased osteoblast adhesion, differentiation, and calcium phosphate deposition on PC20, in vivo these materials did not promote bone bonding but rather induced fibrous tissue and LAM. The authors concluded that development of these cationically-modified PC polymers as bone-interfacing implant coatings is not warranted. Non-charged PC polymers were tolerated in the osseous environment and could potentially be used to coat orthopaedic devices for antibiotic delivery to reduce surgical site infection, where bone bonding is not required. [1]

C5H15CLNO4P

219.60400

219.042

107-73-3

7886

Colorless to light yellow solid-liquid Mixture

2

5

4

12

158

0

O=P(OCC[N+](C)(C)C)(O)O.[Cl-]

PYJNAPOPMIJKJZ-UHFFFAOYSA-N

InChI=1S/C5H14NO4P.ClH/c1-6(2,3)4-5-10-11(7,8)9;/h4-5H2,1-3H3,(H-,7,8,9);1H

trimethyl(2-phosphonooxyethyl)azanium;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~250 mg/mL (~1138.43 mM)

Solubility in Formulation 1: 100 mg/mL (455.37 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)