Effect of hydroxyapatite nanocrystals functionalized with lactoferrin in osteogenic differentiation of mesenchymal stem cells Monica Montesi, Silvia Panseri, Michele Iafisco, Alessio Adamiano, Anna Tampieri Laboratory of Bioceramics and Bio-Hybrid Composites, Institute of Science and Technology for Ceramics (ISTEC), National Research Council (CNR), 48018 Faenza, Italy Received 14 January 2014; revised 4 March 2014; accepted 13 March 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35170 Abstract: Lactoferrin (LF) is a bioactive glycoprotein that became recently interesting in the field of bone regeneration for its modulatory effect on bone cells. On the basis of this evidence this work aims to functionalize biomimetic hydroxyapatite (HA) nanocrystals with LF to study their effect on osteogenic differentiation of mesenchymal stem cells (MSCs). The orientation of LF on the HA surface was analyzed by spectroscopic and thermal techniques. Three samples with different amounts of LF attached to HA nanocrystals were tested in vitro. The combined effect of HA and LF on MSC proliferation and morphology, alkaline phosphatase (ALP) activity, and gene expression were evaluated at different time points. The sample with the lowest LF amount showed the best bioactivity probably due to the for-
mation of a single layer of protein with a better molecular orientation. Coupling of HA–LF did not affect cell proliferation and morphology, while analysis of HA–LF on ALP activity and messenger RNA expression of the selected genes, demonstrated the role of HA–LF in the induction of osteogenic markers. HA–LF represents a promising system to be used to manufacture bioactive functional materials in tissue engineering (as scaffolds, injectable cements, or coatings for metallic implants) with enhanced anabolic activity to treat bone disC 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: eases. V 00A:000–000, 2014.
Key Words: hydroxyapatite, lactoferrin, mesenchymal stem cells, osteogenic differentiation, nanocrystals
How to cite this article: Montesi M, Panseri S, Iafisco M, Adamiano A, Tampieri A. 2014. Effect of hydroxyapatite nanocrystals functionalized with lactoferrin in osteogenic differentiation of mesenchymal stem cells. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Bone remodeling is a fundamental mechanism for removing and replacing bone during adaptation of the skeleton that occurs throughout life, but in some diseases this anabolic/ catabolic balance is compromised.1 In particular in osteoporosis, a common systemic bone metabolic disorder, there is an imbalanced bone formation in favor of bone resorption causing low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture.1,2 Osteoporosis develops with age and it is more prevalent in women aged above 50 years old.3 Therefore, with the aging of the population, the complications of osteoporosis represent a growing medical and socioeconomic threat, especially in industrialized countries. The recent demographic scenarios, projecting the aging of the population in Europe, showed an increasing trend that will not level-off before year 2040, leading to 526 million people older than 65 years of age by that date.4 For these reasons, there is an intensive search for potential therapeutic strategies aimed to reduce the clinical complications of osteoporosis. Generally, patients diagnosed with osteoporosis are treated sys-
temically via oral administration of bisphosphonates to reduce osteoclast activity and hence decrease bone resorption.5 Conventional, systemic delivery of bisphosphonates is often associated with serious side effects, which have raised a negative attitude by patients and the medical community toward their use. Particularly, controversies arise concerning their oral administration that is usually associated to osteonecrosis of the jaw6 and gastric-digestive associated pathologies.7 To circumvent these disadvantages of conventional bisphosphonate-based treatment, a therapy based on local (rather than systemic) delivery of bisphosphonate or other biomolecules with similar biological effects is particularly appealing. In this domain, the study of the interaction between biomolecules, with anabolic effects in bone, and different kinds of bone biomimetic materials is a growing topic in the bioengineering field.8 Lactoferrin (LF) is a 80-kDa iron-binding glycoprotein belonging to transferrin family, produced by mucosal epithelial cells in various mammalian species, including humans, cows, goats, horses, dogs, and several rodents; and it is present in exocrine secretions.9 LF seems to be involved in different biological processes, such as regulation of the
Correspondence to: M. Montesi; e-mail: [email protected] Contract grant sponsors: PNR-CNR Aging Program 2012–2014 and Fondazione Banca del Monte e Cassa di Risparmio Faenza
C 2014 WILEY PERIODICALS, INC. V
1
levels of free iron in the body fluids, immunomodulation, anticancer activity, antibacterial, and antiviral functions.10,11 Recently, LF became interesting in the field of bone regenerative medicine for its modulatory effect on bone tissue.12 Due to its effect on the osteogenic differentiation of pluripotent mesenchymal cells,13 its proliferative and antiapoptotic actions on osteoblasts and inhibition of osteoclastogenesis,14 LF represents a promising anabolic factor for the antiosteoporotic treatment. Moreover, it is important to consider that milk-derived LF is considerably less expensive than drugs or growth factors. Therefore, an innovative strategy to improve the efficacy of the biomedical devices presently used to treat several bone diseases such as osteoporosis, could be the introduction of LF to provide further osteogenic stimuli to the native cells. LF could either be directly injected or attached to a biomaterial surface. In contrast to injected biomolecules, which are usually rapidly cleared from the body, locally attached proteins can be addressed only to specific part of the body enhancing the concentration and release in the site of interest and minimizing possible unwanted side effects. In this perspective, the use of functionalized biomimetic hydroxyapatite (HA) nanocrystals with LF could represent a promising approach. In fact, HA is a well-know material used to manufacture biomedical devices for bone tissue engineering due to its high biocompatibility, osteoconductivity, and biodegradability and the ability to bind several biomolecules, without affecting their biological function.15–18 To this aim, in the present study LF was attached to biomimetic HA nanocrystals, and for the first time the effect of the HA–LF complex on mesenchymal stem cells (MSCs) culture was investigated. In order to verify any combined effect of LF and HA, we analyzed MSCs behavior in response to different quantities of LF attached on HA nanocrystals looking at cell proliferation, morphology and osteogenic differentiation.
from Life Technologies Corporation (Carlsbad, CA); LF, a 97% pure protein fraction from cow’s milk was obtained from DMW International (Veghel, The Netherlands). Preparation of HA and functionalization with LF HA nanocrystals were synthesized by a precipitation method, described in previous articles.15,19,20 Briefly, a solution of H3PO4 (0.21 M) was dropped into a solution of Ca(CH3COO)2 (0.35 M) maintaining the pH at the constant value of 10 by addition of NH4OH. The reaction mixture was kept under stirring at room temperature for 24 h, then the stirring was suspended and the mixture was left standing for 2 h to allow the deposition of the inorganic phase. This latter was isolated by centrifugation of the reaction mixture, repeatedly washed with water, and freezedried at 260 C under vacuum (3 mbar) overnight. Further details of the synthesis procedure and the physical–chemical properties of the HA are reported elsewhere.19,20 HA nanocrystals were functionalized with LF by mixing at 37 C for 24 h, 50 mg of HA with 7.5 mL of LF solution at different concentrations ranging from 0.5 to 10 mg mL21 in HEPES buffer at pH 7.4 (0.01 M HEPES, 0.15 M NaCl). The solid was washed twice with 1 mL of ultrapure water, recovered by centrifugation and then freeze dried at 260 C under vacuum (3 mbar) overnight. Protein content was assessed by ultraviolet–visible (UV–Vis) spectroscopy (k 5 280 nm, e 5 92,000 M21 cm21) using a Cary Bio spectrophotometer (Varian, Palo Alto, CA). The amount of adsorbed protein was calculated from the difference between the protein concentration in solution before and after the adsorption on HA. To determine the LF released from HA, an aliquot (about 10 mg) of the HA functionalized with LF (hereafter HA–LF) was mixed with 10 mL of HEPES buffer at pH 7.4 (0.01 M HEPES, 0.15 M NaCl). The suspension was maintained at 37 C and at scheduled times ranging from 1 to 14 days the supernatant (that was well separated from the solid phase by centrifugation) was analyzed by UV–Vis spectroscopy to evaluate the concentration of LF.
MATERIALS AND METHODS
Materials Ultrapure water (0.22 mS, 25 C) was used in all the experiments. Minimum essential medium alpha (a-MEM) and fluorescein isothiocyanate (FITC)-conjugated phalloidin antibody were purchased from Invitrogen Corporation (Carlsbad, CA); fetal bovine serum, penicillin–streptomycin, trypsin–EDTA, and Dulbecco’s PBS (13) without Ca and Mg from PAA (Pasching, Austria); trypan-blue, MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), phosphoric acid (85 wt%), calcium acetate hydrate (99%), ammonium hydroxide solution (99%), dimethyl sulfoxide, p-nitrophenyl phosphate (pNP-PO4), TritonV X-100, HEPES buffer, and hematoxilin and eosin Y from Sigma-Aldrich (St Louis, MO); Purelink RNA Mini Kit, Tri Reagent, THE RNA storage solution, agarose, QubitV RNA BR assay kit, highcapacity complementary DNA (cDNA) reverse transcription kit, and Taqman gene expression assays: Runt-related transcription factor 2 (RUNX2): Oc02386741_m1, bone morphogenetic protein 2 (BMP2): Oc03824113_s1, osteonectin (SPARC): Oc03395842_m1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Oc03823402_g1 were purchased
Characterization of HA functionalized with LF In order to investigate the amount of protein chemically attached onto HA after washing, thermogravimetric analysis (TGA) investigations were carried out on freeze dried samples using a Thermal Analysis SDT Q 600 (TA Instruments, New Castle, DE). Heating was performed under a nitrogen flow (100 mL min21) using an alumina sample holder. The temperature was increased to 1200 C using a heating rate of 10 C min21. The weight of the samples was approximately 10 mg. The percentage amount of adsorbed LF was calculated by using the following equation15:
R
LF % 5 1002ðDW%LF 2DW%HA –LF Þ 3 100=ðDW%LF 2DW%HA Þ
(1)
R
2
MONTESI ET AL.
where DW%LF, DW%HA, and DW%HA–LF are the percentage weight losses in the range Troom—600 C of pure LF, HA, and HA–LF conjugates, respectively. The infrared spectra of the HA and HA–LF were recorded in the wavelength range from 4000 to 400 cm21 with 2 cm21 resolution using a Nicolet 380 spectrometer (Thermo
HA NANOCRYSTALS FUNCTIONALIZED WITH LF
ORIGINAL ARTICLE
TABLE I. Samples Identification Samples HA–LF (a) 10 mg mL21 HA (a) 10 mg mL21 HA–LF (b) 10 mg mL21 HA (b) 10 mg mL21 HA–LF (c) 10 mg mL21 HA (c) 10 mg mL21 HA–LF (c) 100 mg mL21 HA (c) 100 mg mL21 LF 10 mg mL21 LF 100 mg mL21
% LF Loaded
LF Tested (mg mL21)
HA Tested (mg mL21)
15.1 – 9.7 – 6.6
10 – 10 – 10
6.6 – – –
100 – 10 100
56.2 56.2 93.1 93.1 141.5 141.5 1415 1415 – –
Labeling of the samples, weight percentage of LF loaded onto HA, and LF and HA concentration (mg mL21) of the samples tested in the study.
Fisher Scientific, Waltham, MA). A powdered sample (approximately 1 mg) was mixed with about 100 mg of anhydrous KBr. The mixture was pressed at 10 t pressure into 7 mm diameter discs. A pure KBr disk was used as blank. Size and f-potential distributions of freeze-dried HA and HA–LF redispersed in HEPES buffer at pH 7.4 and in other buffer solutions at different pH values were measured by dynamic light scattering with a Zetasizer Nano ZS (Malvern, Worcestershire, UK). For size distribution measurements, low-volume quartz cuvettes (105.251-QS; Hellma, M€ ullheim, Germany) were used. Ten runs of 30 s each were performed for each measurement and four measurements were carried out for each sample. f-Potential was quantified by laser Doppler velocimetry as electrophoretic mobility using disposable electrophoretic cells (DTS1061; Malvern). Twenty runs of 3 s each were collected in each measurement. Cell culture MSCs isolated from the rabbit bone marrow were cultured in a-MEM plus 10% Fetal Bovine Serum and 1% penicillin– streptomycin (100 U mL21 to 100 mg mL21) and kept at 37 C in an atmosphere of 5% CO2. Cells were detached from culture flasks by trypsinization, centrifuged and resuspended. Cell number and viability were assessed with the trypan-blue dye exclusion test. Cells were plated at a density of 2.5 3 103 cells cm22 in 6- and 24-well plates and cultured in presence of HA with three different amounts of surface loaded LF (15.1; 9.7 and 6.6 wt%; hereafter labeled HA–LF (a), HA–LF (b), and HA–LF (c), respectively) at 10 and 100 mg mL21 concentration of attached LF (Table I). Moreover, the equivalent amounts of HA used for each groups and LF only were tested (Table I). A group of cells only was used as control group. Cells were incubated under standard conditions (37 C, 5% CO2) for up to 14 days without any osteogenic supplements. Culture media was partially changed every three days. All cell-handling procedures were performed in a sterile laminar flow hood. Cell viability and proliferation assay The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was prepared at 5 mg mL21 in 13 PBS. Cell were incubated with the MTT reagent 1:10 for 2 h
at 37 C. Medium was collected and cells incubated with 1 mL of dimethyl sulfoxide for 15 min. In this assay, the metabolically active cells react with the tetrazolium salt in the MTT reagent to produce a formazan dye that can be observed at kmax of 570 nm, using a Multiskan FC Microplate Photometer (Thermo Scientific). This absorbance is directly proportional to the number of metabolically active cells. Mean values of absorbance were determined. The following samples were analyzed in triplicate at day 1, 4, 7, and 14: HA–LF (a) 10 mg mL21, HA–LF (b) 10 mg mL21 and HA–LF (c) 10 mg mL21, HA (a) 10 mg mL21, HA (b) 10 mg mL21, HA (c) 10 mg mL21, LF 10 mg mL21, and cells only.
Alkaline phosphatase assay Cell alkaline phosphatase (ALP) activity was quantified using an enzymatic assay based on the hydrolysis of pNPPO4 to p-nitrophenol (pNP).21 Briefly, 50 mL of cell lysate, obtained after cell incubation with 100 mL 13 PBS with 0.1% (v/v) Triton-X, was added to pNP-PO4 solution and allowed to react at 37 C. Absorbance was read at 0, 30, 60, and 90 min at kmax of 405 nm, using a microplate reader (Tecan, Research Triangle Park, NC) and ALP activity calculated by cross-reference to a standard curve of picomoles of p-nitrophenol liberated per cell. ALP activity was normalized to total cell number, as measured by MTT assay. HA–LF (a) 10 mg mL21, HA–LF (b) 10 mg mL21 and HA–LF (c) 10 mg mL21, HA (a) 10 mg mL21, HA (b) 10 mg mL21, HA (c) 10 mg mL21, LF 10 mg mL21, and cells only were analyzed in triplicate at day 7 and 14.
Hematoxylin and eosin staining After 7 days cells were washed with 13 PBS for 5 min, fixed with 4% (w/v) formaldehyde for 15 min, washed twice with 13 PBS for 5 min and stained with hematoxylin and eosin (H&E). Briefly, the cells were incubated in Mayer’s hematoxylin for 10 min and differentiated with tap water for 10 min. Cells were then stained with eosin Y (0.25%) for 1–2 min, washed with distillated water and mounted. Images were acquired by an Inverted Ti-E fluorescence microscope (Nikon). The samples HA–LF (c) 10 mg mL21, HA–LF (c)
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technical replicates for each experiment. Data was collected using the OneStep Software (v.2.2.2) and relative quantification was performed using the comparative threshold (Ct) method (DDCt), where relative gene expression level equals 22DDCt.22 In detail, changes in gene expression level of the target genes RUNX2, BMP, and SPARC were calculated by normalization to the reference GAPDH and by normalization to the cells only, used as calibrator, within each sample set.
FIGURE 1. Adsorption isotherm of LF on HA nanocrystals at pH 7.4. The points labeled as low, medium, and high correspond to the samples analyzed in this work.
100 mg mL21, HA (c) 10 mg mL21, HA (c) 100 mg mL21, LF 10 mg mL21, LF 100 mg mL21, and cells only were analyzed. Phalloidin immunofluorescence staining Cells were washed with 13 PBS for 5 min, fixed with 4% (w/v) paraformaldehyde for 15 min and washed with 13 PBS for 5 min. Permeabilization was performed with 13 PBS with 0.1% (v/v) Triton X-100 for 5 min. FITCconjugated phalloidin antibody (Invitrogen) 1:500 in 13 PBS was added for 20 min at room temperature in the dark. Cells were washed with 13 PBS for 5 min and incubated with DAPI (Invitrogen) in 13 PBS for 5 min. Images were acquired by an Inverted Ti-E fluorescence microscope (Nikon). The samples HA–LF (c) 10 mg mL21, HA–LF (c) 100 mg mL21, HA (c) 10 mg mL21, HA (c) 100 mg mL21, LF 10 mg mL21, LF 100 mg mL21, and cells only were analyzed at day 7. Quantitative real-time polymerase chain reaction At days 7 and 14, cells grown in presence of HA–LF (c) 10 mg mL21, HA–LF (c) 100 mg mL21, HA (c) 10 mg mL21, HA (c) 100 mg mL21, LF 10 mg mL21, LF 100 mg mL21, and cells only, used as a calibrator, were homogenized and total RNA extraction was performed by use of the Tri Reagent, followed by the Purelink RNA Mini kit according to manufacturer’s instructions. Purified total RNA was eluted with THE RNA storage solution and kept at 280 C until reverse transcription. RNA integrity was analyzed by native agarose gel electrophoresis and quantification performed by the QubitV 2.0 Fluorometer together with the QubitV RNA BR assay kit, following manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed to cDNA using the HighCapacity cDNA Reverse Transcription Kit, according to manufacturer’s instructions. Quantification of gene expression for RUNX2, BMP2, SPARC, and GAPDH (used as housekeeping gene) was performed by use of the StepOneTM RealTime polymerase chain reaction (PCR) System (Applied Biosystems). Experiment was done in triplicate, using three R
4
MONTESI ET AL.
R
Statistical analysis Results were expressed as mean 6 standard error of the mean (SEM) plotted on graph. Analysis of the effect of the biomaterials on cell culture was made by two-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test for MTT and ALP activity results. Analysis of the effect of the biomaterials on gene expression was made by one-way ANOVA, followed by Tukey’s Multiple Comparison Test. Statistical analyses were performed by the GraphPad Prism software (version 5.0), with statistical significance set at p 0.05. RESULTS
Functionalization of HA nanocrystals with LF Plate-shaped HA nanocrystals with chemical–physical features (in terms of size, crystallinity degree, Ca/P ratio, and carbonate substitution) very close to those of bone and dentin apatite were synthesized according to a well-developed procedure.19,20 Quantification of the total adsorbed LF onto HA (physically adsorbed and chemically bounded) is reported as an isotherm in Figure 1, where the LF amount (mg of LF on mg of HA) is plotted as a function of the LF concentration remaining in solution after adsorption. The presence of the adsorption plateau at low equilibrium concentration is an indicator of the high affinity of LF for the surface of HA. The increase in LF concentration in contact with the nanocrystals increased the surface coverage, reaching a maximum amount of adsorbed LF of about 200 mg g21 of HA (calculated as plateau value of the isotherm curve). The curve profile followed a Langmuir isotherm, as already pointed out in a previous work.15 In order to investigate the amount of LF remained attached onto HA after washings and thus to discriminate between the LF physically adsorbed (protein removed by washings not chemically bounded and commonly responsible for the fast burst release) and chemically bounded, TGA analyses were carried out on freeze-dried samples. The TGA curves of HA, LF and HA–LF at high (point in the plateau of the isotherm curve), medium (point in the middle of the isotherm curve), and low (point in the initial part of the isotherm curve) surface coverage corresponding to a protein starting concentration of 2.5 mg mL21, 1 mg mL21, and 0.5 mg mL21 are reported in Figure 2. These samples were used for the following analyses and hereafter were labeled as HA–LF (a), HA–LF (b), and HA–LF (c), respectively (Table I). According to equation 1, the percentage amount of attached LF was calculated to be 15.1, 9.7 and 6.6 wt% for the HA–LF (a), HA–LF (b), and HA–LF (c), corresponding to 183, 118, and 80 mg of LF g21 of HA, respectively. These
HA NANOCRYSTALS FUNCTIONALIZED WITH LF
ORIGINAL ARTICLE
TABLE II. Size Distribution and f-Potential of LF, HA, and HA–LF Conjugates With Different LF Surface Coverage Amount at pH 7.4
FIGURE 2. TGA curves of (1) HA, (2) HA–LF (c), (3) HA–LF (b), (4) HA– LF (a), and (5) LF.
values are lower to those calculated by UV–Vis in the isotherm curve (198, 161, and 111 mg of LF g21 of HA) indicating that part of the initial LF used for the functionalization was only physically adsorbed and was removed by washings. The main aim of this work is the preparation of stable HA–LF conjugates, thus the samples used for the following investigations were extensively washed prior to use. FT-IR spectra of the HA and HA–LF conjugates are presented in Figure 3. The HA–LF samples showed the typical bands of nanocrystalline apatites;23 i.e., 1032 and 1075 cm21 due to the triply degenerated asymmetric stretching mode of the apatitic PO4 groups; 605 and 565 cm21 corresponding to the triply degenerated bending mode of the PO4 groups; 873 cm21 related to the B-type carbonate substitutions (CO3 occupying PO4 sites) derived from dissolved CO2 in the preparation media and from CO2 adsorbed onto the materials surface during storage and the band at 1650 and 1550 cm21 corresponding to the amide I and amide II of LF.24,25 The relative intensity of these latter bands increased as a function of the LF concentration in solution, confirming the effective increase in the protein amount attached on the HA surface. The dimension of the HA nanocrystals was in the range of 115 nm and their f-potential was negative due to their poor crystallinity degree and presence of the hydrate surface layer composed of nonstructured Ca, PO4 and HPO4 ionic groups which caused unbalancing on the surface charges.20,26 LF dimension was in the range of 8.7 nm and its surface charge was calculated to be 21.5 mV. These values are in agreement with the literature data describing that LF has a bilobal shape and size in the range of 100 Å,9 moreover the isoelectric point was reported to be about 8.09 in agreement with our experimental value close to 0 at pH 7.4. The size distribution of the HA–LF decreased as a function of the amount of protein, while the surface charge moved to more negative values (Table II). The decrease in size is strictly correlated with the surface charge; in fact, the HA–LF conjugates with f-potential close to the neutrality have higher tendency to form aggregates in solution, respect to those with surface charge farther from 0.
Samples
Size Distribution (nm)
f-Potential (mV)
LF HA HA–LF (a) HA–LF (b) HA–LF (c)
8.7 6 0.1 115.7 6 11.2 123.8 6 10.5 249.0 6 15.3 269.7 6 13.5
21.5 6 0.2 215.7 6 1.6 237.5 6 0.5 212.2 6 0.4 27.6 6 0.4
The examination of the f-potential of LF at different pH and the graphical representation of the distribution of the electrostatic surface potential at pH 7.4 (Fig. 4) allowed the study of the interaction of LF with HA in terms of electrostatic interactions. The surface of LF at pH 7.4 is a mix of zones with negative and positive charge contributing to a total value close to the neutrality. When HA nanocrystals were conjugated with low amount of LF the f-potential shifted to 27.6 mV toward a value close to that of pure LF, indicating that probably a single layer of the protein was exposed on the HA surface and it is attached through the interaction of its positive group with the negative surface ionic groups of HA (PO4 and HPO4). Increasing the amount of LF on the HA surface the f-potential moved to more negative values demonstrating that, probably due to the steric hindrance, strong protein–protein interactions can occur, resulting in multiple protein layers with different molecular orientations exposing a high net negative charge. No LF released has been found up to 14 days confirming a strong bind between the protein and HA surface. Evaluation of cell viability and ALP activity Focusing on human physiological concentration of LF (10 mg mL21), the cell proliferation and the ALP activity at several time points up to 14 days were evaluated.
FIGURE 3. FT-IR spectra of (1) HA, (2) HA–LF (c), (3) HA–LF (b), and (4) HA–LF (a).
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
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FIGURE 4. (a) GRASP (graphical representation and analysis of structural properties) representation of the distribution of the electrostatic surface potential of the LF (PDB ID2BJJ) at pH 7.4. The values of surface potentials are expressed as a spectrum ranging from 110 kT e21 (deep blue) through 0 kT e21 (white) to 210 kT e21 (deep red), at a temperature of 298.15 K, kT e21 5 25.7 mV. (b) f-Potential of LF as a function of pH. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
No significant differences were detected in cellular proliferation among all the groups after 1 and 4 days of culture [Fig. 5(a)]. At day 7, the cells grown in presence of LF 10 mg mL21 showed a significant increase in proliferation compared to the remaining samples [Fig. 5(a)]. Significant increase in cell proliferation exists also in the group cells only, except with HA–LF (b) 10 mg mL21 and HA (b) 10 mg mL21 [Fig. 5(a)]. Moreover, after 7 days of culture the sample HA–LF (b) 10 mg mL21 showed a significant increase in cellular proliferation compared to HA–LF (c) 10 mg mL21 and HA (c) 10 mg mL21 (p value
mation of a single layer of protein with a better molecular orientation. Coupling of HA–LF did not affect cell proliferation and morphology, while analysis of HA–LF on ALP activity and messenger RNA expression of the selected genes, demonstrated the role of HA–LF in the induction of osteogenic markers. HA–LF represents a promising system to be used to manufacture bioactive functional materials in tissue engineering (as scaffolds, injectable cements, or coatings for metallic implants) with enhanced anabolic activity to treat bone disC 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: eases. V 00A:000–000, 2014.
Key Words: hydroxyapatite, lactoferrin, mesenchymal stem cells, osteogenic differentiation, nanocrystals
How to cite this article: Montesi M, Panseri S, Iafisco M, Adamiano A, Tampieri A. 2014. Effect of hydroxyapatite nanocrystals functionalized with lactoferrin in osteogenic differentiation of mesenchymal stem cells. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Bone remodeling is a fundamental mechanism for removing and replacing bone during adaptation of the skeleton that occurs throughout life, but in some diseases this anabolic/ catabolic balance is compromised.1 In particular in osteoporosis, a common systemic bone metabolic disorder, there is an imbalanced bone formation in favor of bone resorption causing low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture.1,2 Osteoporosis develops with age and it is more prevalent in women aged above 50 years old.3 Therefore, with the aging of the population, the complications of osteoporosis represent a growing medical and socioeconomic threat, especially in industrialized countries. The recent demographic scenarios, projecting the aging of the population in Europe, showed an increasing trend that will not level-off before year 2040, leading to 526 million people older than 65 years of age by that date.4 For these reasons, there is an intensive search for potential therapeutic strategies aimed to reduce the clinical complications of osteoporosis. Generally, patients diagnosed with osteoporosis are treated sys-
temically via oral administration of bisphosphonates to reduce osteoclast activity and hence decrease bone resorption.5 Conventional, systemic delivery of bisphosphonates is often associated with serious side effects, which have raised a negative attitude by patients and the medical community toward their use. Particularly, controversies arise concerning their oral administration that is usually associated to osteonecrosis of the jaw6 and gastric-digestive associated pathologies.7 To circumvent these disadvantages of conventional bisphosphonate-based treatment, a therapy based on local (rather than systemic) delivery of bisphosphonate or other biomolecules with similar biological effects is particularly appealing. In this domain, the study of the interaction between biomolecules, with anabolic effects in bone, and different kinds of bone biomimetic materials is a growing topic in the bioengineering field.8 Lactoferrin (LF) is a 80-kDa iron-binding glycoprotein belonging to transferrin family, produced by mucosal epithelial cells in various mammalian species, including humans, cows, goats, horses, dogs, and several rodents; and it is present in exocrine secretions.9 LF seems to be involved in different biological processes, such as regulation of the
Correspondence to: M. Montesi; e-mail: [email protected] Contract grant sponsors: PNR-CNR Aging Program 2012–2014 and Fondazione Banca del Monte e Cassa di Risparmio Faenza
C 2014 WILEY PERIODICALS, INC. V
1
levels of free iron in the body fluids, immunomodulation, anticancer activity, antibacterial, and antiviral functions.10,11 Recently, LF became interesting in the field of bone regenerative medicine for its modulatory effect on bone tissue.12 Due to its effect on the osteogenic differentiation of pluripotent mesenchymal cells,13 its proliferative and antiapoptotic actions on osteoblasts and inhibition of osteoclastogenesis,14 LF represents a promising anabolic factor for the antiosteoporotic treatment. Moreover, it is important to consider that milk-derived LF is considerably less expensive than drugs or growth factors. Therefore, an innovative strategy to improve the efficacy of the biomedical devices presently used to treat several bone diseases such as osteoporosis, could be the introduction of LF to provide further osteogenic stimuli to the native cells. LF could either be directly injected or attached to a biomaterial surface. In contrast to injected biomolecules, which are usually rapidly cleared from the body, locally attached proteins can be addressed only to specific part of the body enhancing the concentration and release in the site of interest and minimizing possible unwanted side effects. In this perspective, the use of functionalized biomimetic hydroxyapatite (HA) nanocrystals with LF could represent a promising approach. In fact, HA is a well-know material used to manufacture biomedical devices for bone tissue engineering due to its high biocompatibility, osteoconductivity, and biodegradability and the ability to bind several biomolecules, without affecting their biological function.15–18 To this aim, in the present study LF was attached to biomimetic HA nanocrystals, and for the first time the effect of the HA–LF complex on mesenchymal stem cells (MSCs) culture was investigated. In order to verify any combined effect of LF and HA, we analyzed MSCs behavior in response to different quantities of LF attached on HA nanocrystals looking at cell proliferation, morphology and osteogenic differentiation.
from Life Technologies Corporation (Carlsbad, CA); LF, a 97% pure protein fraction from cow’s milk was obtained from DMW International (Veghel, The Netherlands). Preparation of HA and functionalization with LF HA nanocrystals were synthesized by a precipitation method, described in previous articles.15,19,20 Briefly, a solution of H3PO4 (0.21 M) was dropped into a solution of Ca(CH3COO)2 (0.35 M) maintaining the pH at the constant value of 10 by addition of NH4OH. The reaction mixture was kept under stirring at room temperature for 24 h, then the stirring was suspended and the mixture was left standing for 2 h to allow the deposition of the inorganic phase. This latter was isolated by centrifugation of the reaction mixture, repeatedly washed with water, and freezedried at 260 C under vacuum (3 mbar) overnight. Further details of the synthesis procedure and the physical–chemical properties of the HA are reported elsewhere.19,20 HA nanocrystals were functionalized with LF by mixing at 37 C for 24 h, 50 mg of HA with 7.5 mL of LF solution at different concentrations ranging from 0.5 to 10 mg mL21 in HEPES buffer at pH 7.4 (0.01 M HEPES, 0.15 M NaCl). The solid was washed twice with 1 mL of ultrapure water, recovered by centrifugation and then freeze dried at 260 C under vacuum (3 mbar) overnight. Protein content was assessed by ultraviolet–visible (UV–Vis) spectroscopy (k 5 280 nm, e 5 92,000 M21 cm21) using a Cary Bio spectrophotometer (Varian, Palo Alto, CA). The amount of adsorbed protein was calculated from the difference between the protein concentration in solution before and after the adsorption on HA. To determine the LF released from HA, an aliquot (about 10 mg) of the HA functionalized with LF (hereafter HA–LF) was mixed with 10 mL of HEPES buffer at pH 7.4 (0.01 M HEPES, 0.15 M NaCl). The suspension was maintained at 37 C and at scheduled times ranging from 1 to 14 days the supernatant (that was well separated from the solid phase by centrifugation) was analyzed by UV–Vis spectroscopy to evaluate the concentration of LF.
MATERIALS AND METHODS
Materials Ultrapure water (0.22 mS, 25 C) was used in all the experiments. Minimum essential medium alpha (a-MEM) and fluorescein isothiocyanate (FITC)-conjugated phalloidin antibody were purchased from Invitrogen Corporation (Carlsbad, CA); fetal bovine serum, penicillin–streptomycin, trypsin–EDTA, and Dulbecco’s PBS (13) without Ca and Mg from PAA (Pasching, Austria); trypan-blue, MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), phosphoric acid (85 wt%), calcium acetate hydrate (99%), ammonium hydroxide solution (99%), dimethyl sulfoxide, p-nitrophenyl phosphate (pNP-PO4), TritonV X-100, HEPES buffer, and hematoxilin and eosin Y from Sigma-Aldrich (St Louis, MO); Purelink RNA Mini Kit, Tri Reagent, THE RNA storage solution, agarose, QubitV RNA BR assay kit, highcapacity complementary DNA (cDNA) reverse transcription kit, and Taqman gene expression assays: Runt-related transcription factor 2 (RUNX2): Oc02386741_m1, bone morphogenetic protein 2 (BMP2): Oc03824113_s1, osteonectin (SPARC): Oc03395842_m1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Oc03823402_g1 were purchased
Characterization of HA functionalized with LF In order to investigate the amount of protein chemically attached onto HA after washing, thermogravimetric analysis (TGA) investigations were carried out on freeze dried samples using a Thermal Analysis SDT Q 600 (TA Instruments, New Castle, DE). Heating was performed under a nitrogen flow (100 mL min21) using an alumina sample holder. The temperature was increased to 1200 C using a heating rate of 10 C min21. The weight of the samples was approximately 10 mg. The percentage amount of adsorbed LF was calculated by using the following equation15:
R
LF % 5 1002ðDW%LF 2DW%HA –LF Þ 3 100=ðDW%LF 2DW%HA Þ
(1)
R
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where DW%LF, DW%HA, and DW%HA–LF are the percentage weight losses in the range Troom—600 C of pure LF, HA, and HA–LF conjugates, respectively. The infrared spectra of the HA and HA–LF were recorded in the wavelength range from 4000 to 400 cm21 with 2 cm21 resolution using a Nicolet 380 spectrometer (Thermo
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ORIGINAL ARTICLE
TABLE I. Samples Identification Samples HA–LF (a) 10 mg mL21 HA (a) 10 mg mL21 HA–LF (b) 10 mg mL21 HA (b) 10 mg mL21 HA–LF (c) 10 mg mL21 HA (c) 10 mg mL21 HA–LF (c) 100 mg mL21 HA (c) 100 mg mL21 LF 10 mg mL21 LF 100 mg mL21
% LF Loaded
LF Tested (mg mL21)
HA Tested (mg mL21)
15.1 – 9.7 – 6.6
10 – 10 – 10
6.6 – – –
100 – 10 100
56.2 56.2 93.1 93.1 141.5 141.5 1415 1415 – –
Labeling of the samples, weight percentage of LF loaded onto HA, and LF and HA concentration (mg mL21) of the samples tested in the study.
Fisher Scientific, Waltham, MA). A powdered sample (approximately 1 mg) was mixed with about 100 mg of anhydrous KBr. The mixture was pressed at 10 t pressure into 7 mm diameter discs. A pure KBr disk was used as blank. Size and f-potential distributions of freeze-dried HA and HA–LF redispersed in HEPES buffer at pH 7.4 and in other buffer solutions at different pH values were measured by dynamic light scattering with a Zetasizer Nano ZS (Malvern, Worcestershire, UK). For size distribution measurements, low-volume quartz cuvettes (105.251-QS; Hellma, M€ ullheim, Germany) were used. Ten runs of 30 s each were performed for each measurement and four measurements were carried out for each sample. f-Potential was quantified by laser Doppler velocimetry as electrophoretic mobility using disposable electrophoretic cells (DTS1061; Malvern). Twenty runs of 3 s each were collected in each measurement. Cell culture MSCs isolated from the rabbit bone marrow were cultured in a-MEM plus 10% Fetal Bovine Serum and 1% penicillin– streptomycin (100 U mL21 to 100 mg mL21) and kept at 37 C in an atmosphere of 5% CO2. Cells were detached from culture flasks by trypsinization, centrifuged and resuspended. Cell number and viability were assessed with the trypan-blue dye exclusion test. Cells were plated at a density of 2.5 3 103 cells cm22 in 6- and 24-well plates and cultured in presence of HA with three different amounts of surface loaded LF (15.1; 9.7 and 6.6 wt%; hereafter labeled HA–LF (a), HA–LF (b), and HA–LF (c), respectively) at 10 and 100 mg mL21 concentration of attached LF (Table I). Moreover, the equivalent amounts of HA used for each groups and LF only were tested (Table I). A group of cells only was used as control group. Cells were incubated under standard conditions (37 C, 5% CO2) for up to 14 days without any osteogenic supplements. Culture media was partially changed every three days. All cell-handling procedures were performed in a sterile laminar flow hood. Cell viability and proliferation assay The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was prepared at 5 mg mL21 in 13 PBS. Cell were incubated with the MTT reagent 1:10 for 2 h
at 37 C. Medium was collected and cells incubated with 1 mL of dimethyl sulfoxide for 15 min. In this assay, the metabolically active cells react with the tetrazolium salt in the MTT reagent to produce a formazan dye that can be observed at kmax of 570 nm, using a Multiskan FC Microplate Photometer (Thermo Scientific). This absorbance is directly proportional to the number of metabolically active cells. Mean values of absorbance were determined. The following samples were analyzed in triplicate at day 1, 4, 7, and 14: HA–LF (a) 10 mg mL21, HA–LF (b) 10 mg mL21 and HA–LF (c) 10 mg mL21, HA (a) 10 mg mL21, HA (b) 10 mg mL21, HA (c) 10 mg mL21, LF 10 mg mL21, and cells only.
Alkaline phosphatase assay Cell alkaline phosphatase (ALP) activity was quantified using an enzymatic assay based on the hydrolysis of pNPPO4 to p-nitrophenol (pNP).21 Briefly, 50 mL of cell lysate, obtained after cell incubation with 100 mL 13 PBS with 0.1% (v/v) Triton-X, was added to pNP-PO4 solution and allowed to react at 37 C. Absorbance was read at 0, 30, 60, and 90 min at kmax of 405 nm, using a microplate reader (Tecan, Research Triangle Park, NC) and ALP activity calculated by cross-reference to a standard curve of picomoles of p-nitrophenol liberated per cell. ALP activity was normalized to total cell number, as measured by MTT assay. HA–LF (a) 10 mg mL21, HA–LF (b) 10 mg mL21 and HA–LF (c) 10 mg mL21, HA (a) 10 mg mL21, HA (b) 10 mg mL21, HA (c) 10 mg mL21, LF 10 mg mL21, and cells only were analyzed in triplicate at day 7 and 14.
Hematoxylin and eosin staining After 7 days cells were washed with 13 PBS for 5 min, fixed with 4% (w/v) formaldehyde for 15 min, washed twice with 13 PBS for 5 min and stained with hematoxylin and eosin (H&E). Briefly, the cells were incubated in Mayer’s hematoxylin for 10 min and differentiated with tap water for 10 min. Cells were then stained with eosin Y (0.25%) for 1–2 min, washed with distillated water and mounted. Images were acquired by an Inverted Ti-E fluorescence microscope (Nikon). The samples HA–LF (c) 10 mg mL21, HA–LF (c)
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technical replicates for each experiment. Data was collected using the OneStep Software (v.2.2.2) and relative quantification was performed using the comparative threshold (Ct) method (DDCt), where relative gene expression level equals 22DDCt.22 In detail, changes in gene expression level of the target genes RUNX2, BMP, and SPARC were calculated by normalization to the reference GAPDH and by normalization to the cells only, used as calibrator, within each sample set.
FIGURE 1. Adsorption isotherm of LF on HA nanocrystals at pH 7.4. The points labeled as low, medium, and high correspond to the samples analyzed in this work.
100 mg mL21, HA (c) 10 mg mL21, HA (c) 100 mg mL21, LF 10 mg mL21, LF 100 mg mL21, and cells only were analyzed. Phalloidin immunofluorescence staining Cells were washed with 13 PBS for 5 min, fixed with 4% (w/v) paraformaldehyde for 15 min and washed with 13 PBS for 5 min. Permeabilization was performed with 13 PBS with 0.1% (v/v) Triton X-100 for 5 min. FITCconjugated phalloidin antibody (Invitrogen) 1:500 in 13 PBS was added for 20 min at room temperature in the dark. Cells were washed with 13 PBS for 5 min and incubated with DAPI (Invitrogen) in 13 PBS for 5 min. Images were acquired by an Inverted Ti-E fluorescence microscope (Nikon). The samples HA–LF (c) 10 mg mL21, HA–LF (c) 100 mg mL21, HA (c) 10 mg mL21, HA (c) 100 mg mL21, LF 10 mg mL21, LF 100 mg mL21, and cells only were analyzed at day 7. Quantitative real-time polymerase chain reaction At days 7 and 14, cells grown in presence of HA–LF (c) 10 mg mL21, HA–LF (c) 100 mg mL21, HA (c) 10 mg mL21, HA (c) 100 mg mL21, LF 10 mg mL21, LF 100 mg mL21, and cells only, used as a calibrator, were homogenized and total RNA extraction was performed by use of the Tri Reagent, followed by the Purelink RNA Mini kit according to manufacturer’s instructions. Purified total RNA was eluted with THE RNA storage solution and kept at 280 C until reverse transcription. RNA integrity was analyzed by native agarose gel electrophoresis and quantification performed by the QubitV 2.0 Fluorometer together with the QubitV RNA BR assay kit, following manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed to cDNA using the HighCapacity cDNA Reverse Transcription Kit, according to manufacturer’s instructions. Quantification of gene expression for RUNX2, BMP2, SPARC, and GAPDH (used as housekeeping gene) was performed by use of the StepOneTM RealTime polymerase chain reaction (PCR) System (Applied Biosystems). Experiment was done in triplicate, using three R
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R
Statistical analysis Results were expressed as mean 6 standard error of the mean (SEM) plotted on graph. Analysis of the effect of the biomaterials on cell culture was made by two-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test for MTT and ALP activity results. Analysis of the effect of the biomaterials on gene expression was made by one-way ANOVA, followed by Tukey’s Multiple Comparison Test. Statistical analyses were performed by the GraphPad Prism software (version 5.0), with statistical significance set at p 0.05. RESULTS
Functionalization of HA nanocrystals with LF Plate-shaped HA nanocrystals with chemical–physical features (in terms of size, crystallinity degree, Ca/P ratio, and carbonate substitution) very close to those of bone and dentin apatite were synthesized according to a well-developed procedure.19,20 Quantification of the total adsorbed LF onto HA (physically adsorbed and chemically bounded) is reported as an isotherm in Figure 1, where the LF amount (mg of LF on mg of HA) is plotted as a function of the LF concentration remaining in solution after adsorption. The presence of the adsorption plateau at low equilibrium concentration is an indicator of the high affinity of LF for the surface of HA. The increase in LF concentration in contact with the nanocrystals increased the surface coverage, reaching a maximum amount of adsorbed LF of about 200 mg g21 of HA (calculated as plateau value of the isotherm curve). The curve profile followed a Langmuir isotherm, as already pointed out in a previous work.15 In order to investigate the amount of LF remained attached onto HA after washings and thus to discriminate between the LF physically adsorbed (protein removed by washings not chemically bounded and commonly responsible for the fast burst release) and chemically bounded, TGA analyses were carried out on freeze-dried samples. The TGA curves of HA, LF and HA–LF at high (point in the plateau of the isotherm curve), medium (point in the middle of the isotherm curve), and low (point in the initial part of the isotherm curve) surface coverage corresponding to a protein starting concentration of 2.5 mg mL21, 1 mg mL21, and 0.5 mg mL21 are reported in Figure 2. These samples were used for the following analyses and hereafter were labeled as HA–LF (a), HA–LF (b), and HA–LF (c), respectively (Table I). According to equation 1, the percentage amount of attached LF was calculated to be 15.1, 9.7 and 6.6 wt% for the HA–LF (a), HA–LF (b), and HA–LF (c), corresponding to 183, 118, and 80 mg of LF g21 of HA, respectively. These
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ORIGINAL ARTICLE
TABLE II. Size Distribution and f-Potential of LF, HA, and HA–LF Conjugates With Different LF Surface Coverage Amount at pH 7.4
FIGURE 2. TGA curves of (1) HA, (2) HA–LF (c), (3) HA–LF (b), (4) HA– LF (a), and (5) LF.
values are lower to those calculated by UV–Vis in the isotherm curve (198, 161, and 111 mg of LF g21 of HA) indicating that part of the initial LF used for the functionalization was only physically adsorbed and was removed by washings. The main aim of this work is the preparation of stable HA–LF conjugates, thus the samples used for the following investigations were extensively washed prior to use. FT-IR spectra of the HA and HA–LF conjugates are presented in Figure 3. The HA–LF samples showed the typical bands of nanocrystalline apatites;23 i.e., 1032 and 1075 cm21 due to the triply degenerated asymmetric stretching mode of the apatitic PO4 groups; 605 and 565 cm21 corresponding to the triply degenerated bending mode of the PO4 groups; 873 cm21 related to the B-type carbonate substitutions (CO3 occupying PO4 sites) derived from dissolved CO2 in the preparation media and from CO2 adsorbed onto the materials surface during storage and the band at 1650 and 1550 cm21 corresponding to the amide I and amide II of LF.24,25 The relative intensity of these latter bands increased as a function of the LF concentration in solution, confirming the effective increase in the protein amount attached on the HA surface. The dimension of the HA nanocrystals was in the range of 115 nm and their f-potential was negative due to their poor crystallinity degree and presence of the hydrate surface layer composed of nonstructured Ca, PO4 and HPO4 ionic groups which caused unbalancing on the surface charges.20,26 LF dimension was in the range of 8.7 nm and its surface charge was calculated to be 21.5 mV. These values are in agreement with the literature data describing that LF has a bilobal shape and size in the range of 100 Å,9 moreover the isoelectric point was reported to be about 8.09 in agreement with our experimental value close to 0 at pH 7.4. The size distribution of the HA–LF decreased as a function of the amount of protein, while the surface charge moved to more negative values (Table II). The decrease in size is strictly correlated with the surface charge; in fact, the HA–LF conjugates with f-potential close to the neutrality have higher tendency to form aggregates in solution, respect to those with surface charge farther from 0.
Samples
Size Distribution (nm)
f-Potential (mV)
LF HA HA–LF (a) HA–LF (b) HA–LF (c)
8.7 6 0.1 115.7 6 11.2 123.8 6 10.5 249.0 6 15.3 269.7 6 13.5
21.5 6 0.2 215.7 6 1.6 237.5 6 0.5 212.2 6 0.4 27.6 6 0.4
The examination of the f-potential of LF at different pH and the graphical representation of the distribution of the electrostatic surface potential at pH 7.4 (Fig. 4) allowed the study of the interaction of LF with HA in terms of electrostatic interactions. The surface of LF at pH 7.4 is a mix of zones with negative and positive charge contributing to a total value close to the neutrality. When HA nanocrystals were conjugated with low amount of LF the f-potential shifted to 27.6 mV toward a value close to that of pure LF, indicating that probably a single layer of the protein was exposed on the HA surface and it is attached through the interaction of its positive group with the negative surface ionic groups of HA (PO4 and HPO4). Increasing the amount of LF on the HA surface the f-potential moved to more negative values demonstrating that, probably due to the steric hindrance, strong protein–protein interactions can occur, resulting in multiple protein layers with different molecular orientations exposing a high net negative charge. No LF released has been found up to 14 days confirming a strong bind between the protein and HA surface. Evaluation of cell viability and ALP activity Focusing on human physiological concentration of LF (10 mg mL21), the cell proliferation and the ALP activity at several time points up to 14 days were evaluated.
FIGURE 3. FT-IR spectra of (1) HA, (2) HA–LF (c), (3) HA–LF (b), and (4) HA–LF (a).
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FIGURE 4. (a) GRASP (graphical representation and analysis of structural properties) representation of the distribution of the electrostatic surface potential of the LF (PDB ID2BJJ) at pH 7.4. The values of surface potentials are expressed as a spectrum ranging from 110 kT e21 (deep blue) through 0 kT e21 (white) to 210 kT e21 (deep red), at a temperature of 298.15 K, kT e21 5 25.7 mV. (b) f-Potential of LF as a function of pH. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
No significant differences were detected in cellular proliferation among all the groups after 1 and 4 days of culture [Fig. 5(a)]. At day 7, the cells grown in presence of LF 10 mg mL21 showed a significant increase in proliferation compared to the remaining samples [Fig. 5(a)]. Significant increase in cell proliferation exists also in the group cells only, except with HA–LF (b) 10 mg mL21 and HA (b) 10 mg mL21 [Fig. 5(a)]. Moreover, after 7 days of culture the sample HA–LF (b) 10 mg mL21 showed a significant increase in cellular proliferation compared to HA–LF (c) 10 mg mL21 and HA (c) 10 mg mL21 (p value
