Immobilizing hydroxycholesterol with apatite on titanium surfaces to induce ossification
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 5 September 2014
Accepted: 5 October 2014
Published: 20 October 2014
Immobilizing bioactive molecules and osteoconductive apatite on titanium implants have investigated direct ossification. In this study, hydroxycholesterol (HC) was immobilized with apatite on titanium through simply adsorption or sandwich-like coating. Three kinds of hydroxycholesterol were chosen to induce ossification: 20α-hydroxycholesterol (20α- HC), 22(S)-hydroxycholesterol (22(S)-HC) and 25-hydroxycholesterol (25-HC).The effects of HC/apatite coating on ossification abilities were evaluated in vitro and in vivo.
At 6 d, adsorbed apatite/25-HC and apatite/22(S)-HC coating exhibited some cytotoxicity, while the cell viability of apatite/20α-HC coating was similar as apatite coating. Immobilizing HC with apatite significantly enhanced the ALP activities compared with apatite coating. There was no significant difference in ALP value between adsorbed apatite/HC coating and sandwich-like apatite/HC/apatite coating. When compared with apatite coating, the mineral deposition improved by adsorbed HC with apatite at higher concentration in vivo.
When compared with apatite coating, immobilizing HC with apatite coating induced the ossification in vitro and in vivo.
KeywordsHydroxycholesterol Apatite Titanium Ossification
Titanium (Ti) implants are widely used in orthopaedic surgery and dentistry because of favourable mechanical and biocompatible properties [1, 2]. Numerous studies have reported that the early events of bone healing around implants are strongly related to their long-term clinical success . In order to induce the direct ossification of implants, various surface treatments have been developed [4–6]. These treatments include modifications in their surface properties or coating various calcium phosphates (CaP) including a biomimetic apatite layer. The biomimetic precipitation whereby such apatite layers are produced, has profound consequences for their potential to serve carriers for bioactive molecules [7, 8], and control the release of loaded molecules .
Over the last decades, a variety of bioactive molecules have been immobilized with apatite layer to facilitate ossification [10–12]. Among all these researches, bone morphogenic proteins (BMPs) have drawn lots of attraction [13, 14]. While BMPs have exhibited clinical efficacy in early osseointegration, its potential for widespread application is limited by its high cost and its side effects .
Hydroxycholesterols (HC), also known oxysterols are oxidized derivatives of cholesterol found naturally in tissues and circulatory systems of mammal . The HCs are involved in different biological procedures, including cholesterol homeostasis, sphingolipid metabolism, platelet aggregation, and apoptosis . In particular, the naturally occurring 25-, 22(S)-, and 20α- hydroxycholesterol analogues have been demonstrated to induce osteogenic differentiation in primary murine mesenchymal stem cells , and also showed successful bone regeneration in a mouse spinal fusion when HC was delivered to the defect site .
There are many methods for loading bioactive molecules with biomimetically formed apatite layer such as physical adsorption, covalent binding, and biomimetic coprecipitation . As a derivative of cholesterol, HCs are hydrophobic, and as such when immobilized with apatite coating via coprecipitation, they are not efficient to induce osteogenic differentiation of fibroblast cells (C3H10T1/2) .
In the present study, we deposited a calcium phosphate layer on titanium discs by ion-beam assisted deposition (IBAD), and use such deposited layer as active substrates to biomimetically prepare apatite coating in Dulbecco’s phosphate buffered saline solution containing CaCl2. Then, HC was immobilized with apatite on Ti substrates through simply adsorption or a sandwich-like coating, the ossification of the apatite coated Ti with or without HC was investigated in vitro and in vivo.
Preparation of CaP deposited Ti substrate
Commercially pure titanium (grade IV) were obtained from Supra Alloys Inc. (Camarillo, CA, USA), and cut into discs (10 mm in diameter and 2 mm in thickness) in Dentium Co., Ltd. The surfaces of Ti discs were machined, and washed in acetone and distilled water ultrasonically to be used as substrates. Thin calcium phosphate films with a thickness of 500 nm were deposited by ion-beam assisted deposition (CaP-Ti). The details of CaP deposition through IBAD have been described elsewhere . Briefly, evaporants of CaP were prepared by sintering the powder mixtures of hydroxyapatite (Alfa, USA) and CaO (Cerac, USA) at 1200°C in air for 2 h. For CaP deposition, an electron beam evaporator (Telemark, USA) and an end-hall type ion gun (Commonwealth Scientific, USA) were employed. Heat treatments after the deposition were conducted at 350°C with the heating rate of 5°C/min and held for 1 h, and then cooled to room temperature in furnace.
Solutions and hydroxycholesterol used
Reagent grade CaCl2 (100 mg/L) was dissolved in Dulbecco’s phosphate buffered saline (calcium/magnesium free) to prepare the DPBS solution. Hydroxycholesterol was purchased from Sigma-Aldrich (St Louis, MO). Three kinds of hydroxycholesterol were chosen for direct ossification: 20α- hydroxycholesterol (20α-HC), 22(S)-hydroxycholesterol (22(S)-HC) and 25-hydroxycholesterol (25-HC). Hydroxycholesterol was dissolved in 100% ethanol (EtOH) (1 mg/mL) to prepare the working reagent. The DPBS was sterilized by filtration using a membrane with a pore size of 0.20 μm before use.
Immobilizing hydroxycholesterol with apatite on CaP deposited Ti discs
Mouse embryo fibroblast cells (C3H10T1/2) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in growth medium (Gibco-BRL) under a standard cell culture conditions (a sterile, 37°C, humidified, 5% CO2 environment). The growth medium was composed of Basal Medium Eagle (BME), 4.5 g/L of glucose, 10% fetal bovine serum (FBS) and antibiotic solutions (100 U/mL of penicillin-G and 100 μg/mL of streptomycin). The growth medium was changed every 3 d until the cells reached 80–100% confluence. For evaluation the effects of hydroxycholesterol in vitro, C3H10T1/2 cells were seeded on samples placed into 48-well culture plates (n =3 per group). For control experiments, cells were seeded on apatite coated CaP-Ti without HC.
C3H10T1/2 cells viability was quantitatively evaluated by measuring the total dsDNA amount described earlier . C3H10T1/2 cells were seeded on samples in growth medium for 6 d. At determined time, cells were washed twice with PBS, and lysed utilizing a buffer containing 0.5% Triton X-100. The total dsDNA in the lysate was measured using the quant-iT™ PicoGreen dsDNA reagent and kits (Molecular Probes, USA) according to the protocol from the manufacturer. The fluorescence at wavelengths of 480/520 nm was determined using a fluorescence microplate reader (Fluostar OPTIMA, Germany) and DNA quantity was determined by using standard DNA dilution series.
Alkaline phosphatase activity assay
The Alkaline phosphatase (ALP) activity of C3H10T1/2 cells on each sample was measured using a 4-nitrophenyl phosphate colorimetric assay . The cells were seeded on samples in growth medium or mineralizing medium (BME containing FBS, glucose, penicillin-streptomycin, ascorbate, and β-glycerophosphate), and the medium was changed every 3 d. After 6 d of cultivation, the cells on each sample were washed gently, and incubated in the mixture solution of 140 μL alkaline buffer, 10 μL of 1.5 M MgCl2 and 67 mM 4-nitrophenyl phosphate (Fluka, Buchs, Switzerland) at 37°C for 30 min. The reaction was stopped using 0.2 M NaOH. The absorbance was measured at 405 nm using a microplate reader. ALP activity was calculated from a standard curve after normalizing to the total protein content, which was measured using Micro-BCA protein assay kit (Pierce, USA). ALP activity was expressed as units per mg protein.
Mineralization on Ti in a rat model
All quantitative data were depicted as the mean ± standard deviation (n =3). Tests of significance were performed using Student’s t-test.
Results and discussion
Effects of hydroxycholesterol on osteogenic differentiation in vitro
To investigate the effects of hydroxycholesterol on osteogenic differentiation of C3H10T1/2, the cells were grown on Ti substrates with adsorbed apatite/25-HC, apatite/22(S)-HC, apatite/20α-HC coatings in growth medium for 6 days, and then the cell cytotoxicity together with ALP activity were evaluated.
As shown in Figure 3b, the ALP activities on apatite/HC coating layers significantly improved compared to apatite coating without HC (p <0.05), and there was no significant difference among apatite/25-HC, apatite/22(S)-HC and apatite/20α-HC coatings (p >0.05). As the derivative of cholesterol, hydroxycholesterols are hydrophobic, which are not easy to coprecipitate with apatite on Ti substrate  and also encounter with formulation-related issues for clinical application . In our study, although the HC was simply adsorbed on Ti substrates with apatite, the apatite/HC coating layer demonstrated higher ALP activity of C3H10T1/2 cells. This result shows that immobilizing HC onto apatite dos not interfere with its molecular properties. Based on the results of cytotoxicity and osteogenic differentiation ability, 20α-HC was chosen for the following investigation.
Effects of methods to immobilize hydroxycholesterol on ALP activities in vitro
The ALP activities were extremely enhanced by introducing 20α-HC to apatite coating (p <0.05), while no significant difference between adsorbed apatite/20α-HC coating and sandwich-like apatite/20α-HC/apatite coating (p >0.05). From the results of ALP activity, sandwich-like apatite/20α-HC/apatite demonstrated bioactivity similar to adsorbed apatite/20α-HC, which suggests that 20α-HC remained on the preformed first apatite layer during washing and re-mineralization of the second apatite layer. Interestingly, the second apatite layer could trap 20α-HC into two apatite layers for a more sustained release , but the ALP level of C3H10T1/2 was not affected much. ALP activities were enhanced by HC in a dose-dependent manner , while the effects of dose was less significant at higher concentration. At day 6, the released 20α-HC from both coating layers in culture medium might be higher than the threshold concentration, which made it difficult to relate release concentration to ALP activities.
Effects of hydroxycholesterol on mineral deposition in vivo
Adsorbed 25-HC with apatite presented higher cytotoxicity, followed by 22(S)-HC and 20α-HC. Immobilizing HC with apatite by simply adsorption and sandwich-like coating increased the ALP activity of C3H10T1/2, but there was no difference between apatite/HC and apatite/HC/apatite coatings. With immobilizing 20α-HC at the concentration of 4 mg/mL led to a higher and more uniform mineral deposition in rat model. Immobilizing hydroxycholesterol with apatite on titanium implants would have some positive effects on direct ossification.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Korea ( 2012R1A1A2040717).
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