Effect of hydroxyapatite microspheres, amoxicillin-hydroxyapatite and collagen-hydroxyapatite composites on human dental pulp-derived mesenchymal stem cells

The aim of this study was to evaluate the in vitro behavior of human dental pulp mesenchymal stem cells (hDPSCs) cultured on scaffolds of three hydroxyapatite-based materials: hydroxyapatite microspheres [HAp]; amoxicillin-hydroxyapatite composite [Amx-HAp]; and collagen-hydroxyapatite composite [Col-HAp]. These hydroxyapatites (HAps) were synthesized through three methods: microwave hydrothermal, hydrothermal reactor (teflon pouches), and precipitation, respectively. We performed an in vitro experimental study using dental pulp stem cells obtained from samples of third molars and characterized by immunophenotypic analysis. Cells were cultured on scaffolds with osteogenic differentiation medium and were maintained for 21 days. Cytotoxicity analysis and migration assay of hDPSCs were evaluated. Each experiment was performed in triplicate. Data analysis was performed using Kruskal-Wallis test and Dunn’s post-hoc test. After 21 days of induction, no differences in genes expression were observed. hDPSCs highly expressed the collagen IA and the osteonectin at the mRNA, which indicated these genes plays an important role in odontogenesis regardless of induction stimulus. Cytotoxicity assay using hDPSCs demonstrated that Col-HAp group presented a number of non-viable cells statistically lower than the control group (p=0.03). In the migration assay after 24h, biomaterials HAp, Amx-HAp, and Col-HAp revealed the same migration behavior for hDPSCs observed to the positive control. Col-HAp also provided a statistically significant higher migration of hDPSCs than HAp (p=0.02). The migration results in 48h for HAp, Amx-HAp, and Col-HAp was intermediate from those achieved by control groups. There was no statistical difference between positive control and Col-HAp (p>0.05). In general, Col-HAp scaffold showed better features for these dynamic parameters of cell viability and cell migration capacities for hDPSCs, leading to suitable adhesion, proliferation, and differentiation of this osteogenic lineage. These data present high clinical importance because Col-HAp can be used in a wide variety of therapeutic areas, including ridge preservation, minor bone augmentation, and periodontal regeneration.


Introduction
The developing of new biomaterials or their structural changing has been intensively proposed in order to enhance the final properties of novel biomedical devices. [1] Hydroxyapatite [HAp (Ca 10 (PO 4 ) 6 (OH) 2 )] is thermodynamically stable in body fluid due to its crystalline state and has a very similar composition to mineral bone. [2] Thus, numerous techniques to synthesize hydroxyapatite-based materials at elevated temperatures (750-1000 °C) have been used in order to achieve accurate control of their structure. These procedures may be divided into two major groups: wet-chemical In that sense, the preparation of modified HAps or its composites requires a careful biological investigation to guarantee the safe use. Cellular studies involving human dental pulp stem cells (hDPSCs) have been widely explored as an prior parameter to predict safety of dental material. These hDPSCs can be easily isolated from dental medical wastes, extracted teeth, and expanded ex vivo. hDPSCs are a kind of mesenchymal stem cell with the potential for cell-mediated therapy and tissue engineering applications because of their low morbidity after collection, easy surgical access, ability to be cryopreserved, ability to be recombined with many scaffolds and immune-privilege and anti-inflammatory abilities. [5][6][7] However, bone formation from hDPSCs requires a special structure provided by scaffolds which should provide an appropriate environment for cellular attachment, growth, and differentiation. [8] In that sense, novel hydroxyapatite-based materials need to be tested using hDPSCs in order to investigate their potential as a suitable environment for cell growth and that at the same time provide safety for clinical use.
In this study, the following HAP-based materials were obtained and characterized: and collagen-hydroxyapatite composite [Col-HAp]. These scaffolds could play a critical role in attachment, survival, migration, proliferation, and differentiation. However their effect on hDPSCs remains unknown. Therefore, we investigated the effects of HAP-based HAp, Amx-HAp, and Col-HAp were synthesized through three synthesis methods: microwave-hydrothermal, reactor-hydrothermal (teflon pouches), and precipitation, respectively.
Two aqueous solutions of 0.0835 mol/L calcium acetate and 0.0501 mol/L ammonium dihydrogen phosphate were prepared as precursors to obtain hydroxyapatite microspheres (HAp) by the microwave-hydrothermal method at 1.67 stoichiometric ratio of Ca/P. The complexing agent citric acid monohydrate was added after mixing the calcium and phosphorus precursors until a pH = 4 was achieved. Urea was then added at 0.016 mol/L concentration. The reaction mixture was transferred to reactors that were inserted into a microwave oven (Miliestone STAR D). The microwave oven was set for a gradual heating from room temperature (20ºC) to 180ºC with at 1000 W power. The synthesis time for reaching 180ºC was 5 min. The precipitate vacuum filtered, washed twice, and oven dried.
The amoxicillin-hydroxyapatite composite (Amx-HAp) was prepared by the reactor-hydrothermal method using teflon pouches. [9] Aqueous solutions of calcium acetate and ammonium dihydrogen phosphate at the same concentrations (0.0835 and 0.0501 mol/L, respectively) were used. Citric acid monohydrate was added to calcium acetate solution under magnetic stirring for 30 min to adjust pH at 4. This final solution was dripped under the ammonium dihydrogen phosphate solution and urea at 0.016 mol/L was added. The synthesis procedure was carried out into an autoclavable reactor at 180°C for 24 h. After drying, amoxicillin was blended by milling at a final ratio of 4.80 mg HAp:1 mg Amx. This method was performed into a high-density polypropylene flask using yttria stabilized zirconia spheres in ethanol as grinding agent for 3 h. After milling, Amx-HAp was oven dried for 24 h at 35°C.
The collagen-hydroxyapatite composite (Col-HAp) was obtained by the precipitation method. This reaction was performed by adding 1.2 mol/L phosphoric acid (85% pure) into an aqueous suspension of 2.0 mol/L calcium hydroxide at a Ca/P molar ratio of 1.67 under magnetic stirring at 40°C. The phosphoric acid dropping was controlled at 1 drop/s. The pH was then adjusted to 10.0 using ammonium hydroxide (28% pure). The precipitate was aged for 36 h, vacuum filtered and oven dried. Collagen was also blended by ball milling at a ratio of 4.80 mg HAp:1 mg Col.

Characterization of HAp-based materials
Hydroxyapatite-based materials were characterized by Fourier-transformed infrared (FTIR) spectra that were recorded from 4000 to 400 cm −1 on a Biorad Excalibur Series (FTS-3500 GX) IR spectrophotometer using KBr pellets with 32 scans and resolution of 2 cm −1 . [10] Morphological characterization was performed using fieldeffect emission gun scanning electron microscopy (FEG-SEM, TESCAN, model MIRA 3, Brno, Czech Republic) at various magnifications. The samples were previously deposited on polished stubs and then sputter-coated with gold. The average particle size of biomaterials were obtained by photon correlation spectroscopy (Zetasizer Nanoseries, Malvern Instruments, Malvern, UK) after diluting each sample in ultrapure water (1:500, v/v) with no previous filtration and sonicating for 30 min.

Collection and characterization of human dental pulp-derived mesenchymal stem cells (hDPSCs)
Previously to collect permanent teeth, the dental surgeon requested to the patient μg/mL streptomycin) (Gibco TM , Carlsbad, USA) and were stored in an incubator at 37°C with 5% CO 2 tension. The culture medium was replaced three times a week. When the cultures reached about 80-90% confluence, enzymatic dissociation was performed using trypsin/EDTA (0.25%) (Gibco TM , Carlsbad, USA). Inactivation of the enzyme was performed with FBS and IMDM. The cell suspension was centrifuged, the supernatant discarded and the cells counted and plated again. All experiments were performed between the third (P3) and fifth passage (P5) of the cells.

Osteogenic Differentiation
Positive control of reactions was performed by inducing the differentiation of hDPSCs using an osteogenic induction medium (Differentiation Basal Medium-Osteogenic, Lonza, Walkersville, MD, USA) 37°C with 5% CO 2 tension and were maintained for 21 days. The culture medium was replaced three times every seven days.
The differentiation was under the same conditions for each biomaterial tested: HAp, Amx-HAP and Col-HAp. Osteoblastic differentiation was confirmed by mineral deposition of the culture, which was assessed by Alizarin red S (Sigma-Aldrich, São Paulo, Brazil) staining using an optical microscope (NIKON Eclipse Ni, Tokyo, Japan).

Cytotoxicity Analysis
One hundred thousand hDPSCs were plated in two T25 culture flasks and cultured in incubator at 37°C with 5% CO 2 tension and 95% humidity for five days with IMDM

Migration Assay
To test whether biomaterials stimulate the chemotaxis of hDPSCs, the cell migration assay was performed using the Boyden chamber with 8.

Statistical Analysis
The

Synthesis and characterization of HAp-based materials
HAp-based materials were successfully obtained by three proposed methods and their characterization data are depicted in Fig 1. Considering the X-ray diffraction (XRD) analysis, the XRD patterns of HAp (Fig 1A), Amx-HAp (Fig 1B), and Col-HAp (Fig 1C) presented the typical crystalline peaks achieved for pure HAp (JCPDS n o 09-0432), in which the three most intense peaks were assigned at 2 of 31. HAp synthesized by the microwave-hydrothermal procedure (Fig 1G) showed broad bands at 3425 and 1620 cm −1 were attributed to adsorbed water, while the sharp peak at 3558 cm −1 was assigned to the stretching vibration of the lattice OH − ions and the medium sharp peak at 639 cm −1 was assigned to the OH deformation mode. Some typical bands for PO 4 3− were observed at 554, 873, 1026, and 1101 cm −1 . These bands confirmed that this biomaterial was consistent to HAp in accordance to literature. [13] Three more representative bands were recorded for Amx-HAp ( Fig 1H)  to HAp as proposed.

Collection and characterization of human dental pulp-derived mesenchymal stem cells (hDPSCs)
Dental pulp samples of permanent teeth were obtained from three patients with a mean age of 27 years. Visual observation under bright field microscopy showed fibroblastroid morphology and capacity to adhere to plastic and, after isolation, took on average 20 days to proliferate to exceed 80% confluence (Fig 2A).

Characterization of human dental pulp-derived mesenchymal stem cells (hDPSCs)
The  (Fig 2B). The cells had a mean viability of 99.55% and 0.4% of the cells were Annexin-V stained which was indicative of apoptosis.

Osteogenic Differentiation
The ability of osteogenic differentiation was compared between the positive control and each biomaterial (HAp, Amx-HAp, and Col-HAp). The cells cultured with commercial osteogenic differentiation medium (positive control) demonstrated the presence of calcium crystals after Alizarin red S staining as well as those cultured with HAp-based materials in study (Fig 3).
No difference in genes expression was observed after 21 days of induction.
hDPSCs with no induction and those under different conditions of induction highly expressed collagen IA and osteonectin at mRNA, which indicated these genes played an important role in odontogenesis regardless of induction stimulus (Fig 4).

Cytotoxicity Analysis
The cytotoxicity results from cell viability assay using 7-AAD dye showed that all groups had low mortality rate. The samples depicted the following mean ± SEM of

Migration Assay
The results obtained in the migration assay after 24 hours (Fig 5A) showed that the negative control was the only group with a statistically lower migration of hDPSCs when compared with the positive group (NC vs. PC, 0.3087 ± 0.2117 vs. 0.7840 ± 0.2493 nm, p=0.0008). In that sense, the biomaterials HAp, Amx-HAp, and Col-HAp revealed the same migration behavior for hDPSCs observed to the positive control. Col-HAp also provided a statistically significant higher migration of hDPSCs than HAp (Col-HAp vs. HAp, 0.9272 ± 0.3835 vs.0.5165 ± 0.1367 nm, p=0.0248).
In the 48-hour evaluation there was a significant difference between the negative control and the positive control (NC vs. PC, 0.3857 ± 0.1747 vs. 1.498 ± 0.6080 nm, p < 0.0001). The migration results obtained for the biomaterials HAp, Amx-HAp, and Col-HAp was intermediate from those achieved by control groups (Fig 5B). There was no statistical difference between positive control and Col-HAp (p>0.05).

Discussion
The studies concerning the synthesis of biomaterials such as hydroxyapatites with different particle sizes and forms are required to obtain suitable scaffolds in tissue engineering for achieving both osteoinductive and dentinogenic potential. Furthermore, advanced biomaterials inspired by drugs and biological molecules can broaden the prospects in terms of new technologies and variety of applications. [16] In recent years, various HAp-based biomaterials with different morphologies such as nanowires [17], nanoneedles [18], or nanoflowers [19], and hierarchically nanostructured porous microspheres [20][21][22][23] or microflowers [24] have been synthesized. In addition, HAp are adequate candidates as drug or protein delivery carriers [25][26][27]. The biomaterials obtained showed crystalline peaks attributed to pure HAp. In that sense, both Amx and Col were incorporated as non-crystalline (amorphous) materials in composites structure.
The microwave-hydrothermal HAp and Amx-HAp demonstrated high superficial area since these biomaterials were arranged as nanosheet-assembled microspheres as previously reported. analysis since this analysis can reveal the presence of calcium stores in hDPSCs extracellular matrix. [29] The biomaterials HAp, Amx-HAp, and Col-HAp did not avoid the osteogenic differentiation of hDPSCs comparing to the control groups. Hence, these data evidence that these biomaterials per se are able to induce hDPSC osteogenic differentiation in vitro; nevertheless, the presence of osteogenic differentiation promoting factors accelerates and strengthens this process, as resulted from the substantial augmentation of number and size of calcium content in the extracellular matrix. [29] Moreover, it has been already shown that scaffolds containing HAp improved cell proliferation and promoted production of mineralized extracellular matrix more than that observed for the scaffold without HAp. [30] Regarding to the cytotoxicity, no HAp-based biomaterial increased the percentage of non-viable cells when compared with the control group. Besides that, Col-HAp was able to preserve the cell viability significantly better than the control group. This is in line with data reported in literature, in fact, HAp appears to have great potential for bone tissue engineering as it showed no toxic effect on cell culture studies and also has good affinity of cellular attachment on the developed material surface. In addition, collagen is a natural component of bone tissue, where it stimulates mesenchymal stem cells to differentiate into osteoblasts, initiating new bone formation. [31] Collagen also increases water retention, which facilitates cell attachment. [32] Considering