Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair

Preparation of nano-hydroxyapatite (nHA)

The nHA particles were synthesized, as described in our previous publication (Ramedani et al., 2014). Briefly, dipalmitoylphosphatidylcholine (DPPC) and cholesterol were dissolved in chloroform, and stirred for 30 min at 40°C. For preparation of the nHA particles, calcium nitrate tetrahydrate [Ca (NO₃)2.4H₂O] and diammonium hydrogen phosphate [(NH₄)2HPO₄] were used as the source of calcium and phosphate ions, respectively. An aqueous solution of calcium salt was prepared using deionized water and added to the dry film lipid and mixed at a temperature of 51°C for 2 h (the pH was adjusted at 4.5 using acetic acid). The prepared solution was then sonicated for 30 min to disperse the particles in the liquid media and form nano-sized structures. The aqueous solution of sodium phosphate prepared as deionized water drops was added to the solution of calcium and lipids. The milky suspension was placed inside an oven for 22 h at 120°C. At the end of the hydrothermal process, the suspension was centrifuged; the precipitate was washed with ethanol/deionized water, and dried at a temperature of 90°C. The dried precipitate was hand ground to prepare the nano-powder for the downstream experiments.

Preparation of nanocomposite scaffolds

The nHA/Gel scaffolds were fabricated according to a previously reported protocol (Ali Samadikuchaksaraei and Ghafouri, 2009) Briefly, a final weight composition of 60%-Gel and 40%-nHA + SiC was achieved by adding the appropriate amount of nHA powder to a 10% (wt/vol) solution of Gelatin (microbiology grade; Merck, Germany). The solution was cast in plastic Petri dishes after being completely homogenized by stirring. The solution was freezed at −20°C for 60 min and then freeze-dried to produce a porous structure through solvent sublimation. The nanocomposite layers were cut into the desired sizes and laminated by applying a gel solution of 10% wt/vol as a binding agent. The sample was then soaked in a 1% (wt/vol) glutaraldehyde (GA) solution for 24 h to cross-link the gel polymeric chains. Then, the nanocomposite was washed three times with deionized distilled water and dried using freeze drying. To prepare the SiC-containing nanocomposite scaffold, a similar method was applied. In this case, SiC particles were also added to the gel solution after nHA. The amount of SiC and nHA were adjusted versus the gel to finally obtain a weight composition of 30% nHA, 10%SiC, and 60% Gel. The next steps were carried out exactly the same as for the previously described scaffold lacking SiC.

Characterizations of nHA/Gel/SiC scaffold

Porosity and density

The porosity and density of the scaffolds were defined by the following formulae as described in our previously published study (Gholipourmalekabadi et al., 2015c):

Total volume of the scaffold (V):

(1)

The porosity (ε) was defined by:

(2)

The density (d) was defined by:

(3)

where W_d represents the dry weight of the scaffold when immersed in a known volume of hexane (V₁) for 10 min. The total volume of hexane plus the hexane-impregnated scaffold was recorded as V₂. After removing the hexane-impregnated scaffold,the residual hexane volume was recorded as V₃. The examinatione was repeated three times.

Cellular responses

The cellular response of both nHA/Gel and nHA/Gel/SiC nanocomposites was evaluated by culturing rBM-MSCs on the scaffolds. The short-term cell viability, cytotoxicity and cell adhesion property were studied by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assay and SEM micrographs, respectively.

rBM-MSCs isolation

rBM-MSCs were isolated from the rat bone marrow and expanded by using a protocol described in our previously published study (Gholipourmalekabadi et al., 2015d). In a sterile condition, both ends of the femur bone were excised and the bone marrow was flushed out into a cell culture flask of 25cc with a needle loaded with fetal albumin serum (10%), 100 ng/mL penicillin (Gibco, Carlsbad, CA), and 100 ng/ml streptomycin (Gibco). Then, the cell culture flask was incubated overnight at 37°C, 5% CO₂ and 95% humidity. Once in every 2 days, the medium containing non-adherent cells was removed and replaced with fresh media. Cell passage was carried out once the cultured cells reached 80–90% confluency.

Characterization of rBM-MSCs

Flow cytometry was used to verify the characterization of the rBM-MSCs. Cells from the third passage were counted and 4 × 10⁵ cells were incubated with monoclonal antibodies against surface antigens including CD90, CD105, CD34, and CD45.

To identify the differentiation ability of the rBM-MSCs in vitro, 1 × 10⁵ cells/well of rBM-MSCs were plated in 12-well plates and cultured using osteogenesis and adipogenesis differentiation kits (Gibco). The medium was replaced every 72 h for 3 weeks. For osteogenic and adipogenic differentiation, the induced cells were analyzed with Alizarin red S (ScienCell, Carlsbad, CA) and oil red O (Sigma, St. Louis, MO), respectively (Xue et al., 2017; Huang et al., 2018).

Cell viability and cytotoxicity

Short-term cell viability and cytotoxicity were measured by MTT (Gholipourmalekabadi et al., 2015b) and LDH-specific activity assay (Rostami et al., 2015), respectively. For this purpose, the cells were cultured on the scaffold for 24, 48, and 72 h as described in our previous studies (Johari et al., 2016a; Johari et al., 2016b; Kazemi et al., 2018) and their proliferation rate was compared with those of the cells cultured in a culture plate (as a positive control). After each period of time, the culture medium was removed and centrifuged for the following cytotoxicity assay with LDH. The cells were treated with MTT solution for 2 h at 37°C. The MTT solution was then replaced with dimethyl sulfoxide. The negative control was prepared with supplemented DMEM without scaffolds and cells in each well (ODnc). A blank optical density (OD) value was derived from each sample reading. The OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 590 nm with a reference filter of 620 nm (ODs). The absorbance value was defined by the following formula:

(4)

LDH-specific activity of cell culture medium was measured using an LDH kit (Zist Shimi kit, Co No: 10–503 and 10–533-1, Iran), based on the p-nitrophenyl phosphate conversion to p-nitrophenol. The UV absorbance of nicotinamide adenine dinucleotide (NADH), as an index of NADH concentration, was quantitated on a Biotek EL800 absorbance plate reader at 490 nm. At the same time, the cells were ruptured via freeze thawing (three times) and the total LDH activity in the medium was measured. LDH data were normalized for 10⁶ cells (Gholipourmalekabadi et al., 2015d).

Cell adhesion

Morphology of the rBM-MSCs cultured on the scaffold after 48 h was observed by SEM. The cell-nanocomposite complex was prepared for taking micrographs by SEM as described before (Mobini et al., 2013b; Gholipourmalekabadi et al., 2015a). The samples were fixed in 2.5% GA (Merck) for 2 h and then treated with 0.1% osmium tetroxide (Sigma-Aldrich) for 30 min at 25°C. After dehydrating in a graded concentration (30, 50, 70, and 100%) of acetone (Merck), the samples were dried under vacuum for 12 h. The samples were coated with gold by sputtering and observed by SEM (AIS2100; Seron Technology, Uiwang-si, Gyeonggi-do, South Korea) at an acceleration voltage of 25 kV.

Surgical procedure

Forty-five adult Wistar male rats (weighing 200–250 g) (Iran University of Medical Sciences), were acclimatized. The whole process was ratified by animal protocol (IR IR.IUMS.REC.1389.2014). The animals were placed under standard condition for 1 week prior to use. The rats were randomly divided into three groups: blank defect (defect without scaffold, n = 15), blank scaffold (defect with alone scaffold, n = 15), and rBM-MSCs/scaffold (defect with cell-seeded scaffold, n = 15). The animals were anesthetized by an injection of ketamine hydrochloride (85 mg/kg; Ketara, Yuhan) and xylazine (15 mg/kg, TM Rompun; Bayer). After shaving the hair of the desired area located on the upper surface of the skull, a skin incision was created by surgical blade 12 from the nasofrontal area to the external occipital protuberance along the midsagittal suture. A critical size bone defect with a diameter of 8 mm was made on the parietal area at an equal distance between the temporal muscle and the sagittal suture with a milling machine. The scaffolds with cultured rBM-MSCs on them were then fixed within the defect and the suture line was disinfected with tetracycline.

Histology and immunohistochemistry assessments

Calvarial bone specimens were prepared as previously described (Johari et al., 2016a). For each histological block, at least three sections were studied using optical microscopy (BH-2; Olympus). Briefly, calvarial specimens were fixed in 10% buffered formalin for 12 h and decalcified in 10% EDTA for 15 days. After washing the specimens with distilled water several times, they were dehydrated with ascending concentrations of ethyl alcohol, cleared in xylene (Sigma-Aldrich), and embedded in paraffin. Serial 5 µm thick sections were obtained from the central section of each defect site using a microtome and stained with hematoxylin and eosin (H&E), Masson's trichrome staining with previously described methods (Ohgushi et al., 1989; Mumford and Simpson, 1992).

Tissue samples obtained from defects at 12 weeks post-transplantation were studied by immunohistochemistry (IHC). For COL I, a DAB detection kit (Ventana medical system) was used. All tissue samples were rehydrated by phosphate-buffered saline (PBS; Sigma-Aldrich). Unmasking antigen was performed by incubating specimens with pepsin 0.5% for 15 min. After washing the sections with PBS, endogenous peroxidase was exposed to hydrogen peroxide in PBS for 10 min. Rat primary monoclonal antibodies (dilution 1:60 antibodies; Acris), transglutaminase (TG2) II (Labvision; dilution 1:40), and the rat nonessential antibody (IgG1) were used as negative controls and incubated for a full day in 88°C. Specimens were washed by PBS, and incubated with rat's biotinylated secondary antibody (density 1:50) for 90 min in room temperature. After washing with PBS, immune localization was carried out using 3,3’-diaminobenzidine. Finally, this process was completed using nuclear hematoxylin counterstaining to reveal the cores.

Histomorphometry analysis

First, we measured the area of new bone tissue with the ImageJ software (US National Institutes of Health, Bethesda, MD), using the photos obtained from different parts of H&E stained microscope slides tissue. Then by using the formula below (1 and 2), the bone formation percentage and biodegradation rate were measured in all three groups; blank defect, blank scaffold, and rBM-MSCs/scaffold, at three times 1, 4, and 12 weeks.

(5)

(6)

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6. The data rows were examined by t test and analysis of variance test, and expressed as mean ± SD. P value <0.05 was considered as the level of significance. All experiments were carried out with at least three times biological repetition with two times technical repetitions. P < 0.05(*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Characteristics of the nHA/GEL/SiC scaffold

Structural properties of the powders

XRD micrographs and TEM images of the nanocrystalline HA and SiC powders are presented in Figure 1A (a, b). The XRD analysis was performed using an X-ray diffractometer. The XRD pattern, shown in Figure 1A (a), indicated that nHA was formed in a semi-crystalline manner, and traces of other calcium phosphate impurities were not detected. The XRD pattern of the nHA samples can be completely indexed with apatite in the standard card (JCPDS No. 09–0432), the only phase found present. Similarly, the straight baseline and sharp peaks of the diffractogram confirmed that the SiC was well crystallized and perfectly matched with the SiC in the standard card (JCPDS No. 02–1048), shown in Figure 1A (b). No processing residue or secondary phases were found in the materials. Figure 1A also shows the typical TEM micrographs of the synthesized nHA and SiC. According to this figure, the average particle size of both powders was <100 nm. In addition, the particle size distribution for nHA and SiC powder was in the range of 80–120 nm and 40–80, respectively.

Characterization of rBM-MSCs

The cells were positive for CD90, CD105, and negative for CD34, CD45 (Figure 2A). The differentiation capacity of rBM-MSCs into osteogenic and adipogenic cells were investigated under in vitro conditions. In this analysis, calcium deposition by osteoblasts and oily vesicles in adipocytes was detected with alizarin red and Oil red staining, respectively. These results indicated that the isolated rBM-MSCs are MSCs (Figure 2B).

Biological behavior

The morphology of the cells cultured on the nHA/Gel and nHA/Gel/SiC nanocomposites are shown in Figure 1B (c, d), respectively. As shown in Figure 1B, the cells were actively growing, attached to both synthesized scaffolds, and migrated into the pores of the scaffolds. Expansion of the cytoplasm of the cells in all directions indicated the high biocompatibility, cell adhesion, and non-cytotoxicity properties of the scaffolds. Therefore, addition of SiC did not affect the cell adhesion property of the resulted scaffold.

The results obtained from MTT test and the LDH-specific assay are shown in Figures 3A and 3B, respectively. As can be seen, addition of 10% SiC did not change the rate of cell proliferation in all of the tested time points, when the positive control was considered as 100% cell viability. The nHA/Gel/SiC nanocomposite did not show any detectable cytotoxicity against the cells in comparison to the nHA/Gel and the positive control.

Histological and immunohistochemical evaluation

Rats were sacrificed at 1, 4, and 12 weeks post-implantation and the calvaria with a minimum amount of host bone were harvested for histological and immunohistochemical examination. Masson's Trichrome staining and immunohistochemistry COL I were used to observe formation of collagen.

After 1 week, histological results showed not significant new bone formation in the blank defect, blank scaffold and cell/scaffold groups and the scaffolds maintained their integrity. In all groups, thin connective tissue was observed with no substantial amount of collagen formation. Accumulation of multi-nucleated giant cells and inflammation cells were observed at the lesion site (Figure 4).

After 4 weeks, slight osteogenesis was observed on the edges of the defect and the connective tissue was more pronounced and thicker in comparison with 1 week in the blank defect group. In the blank scaffold group, bone tissue from the periphery of the defect and also the bony islands around the scaffold pores that were degraded were detectable. In the cell/scaffold group, a higher amount of bone formation was observed not only at the edges of defects but also around the degrading scaffold. Synthesis of collagen in varying densities could be observed in all three groups. At week 12, multi-nucleated giant cells or inflammatory cells were not observed around the scaffolds, indicating no cytotoxicity as a result of the scaffold destruction process (Figure 5).

After 12 weeks, in the blank defect group, a small amount of bone formation was observed, which was limited to the edges of scaffold. The thickness and amount of connective tissue had increased from 1 week until 12 weeks. In both blank scaffold and cell/scaffold groups, the remnants of scaffold were barely detectable in the graft area but the defects were largely filled with bone tissue around the degraded scaffold. In the blank scaffold group, woven bone formation increased at the edge of the host bone toward the center of the graft area. Over time, the scaffold's lamellas were replaced with woven bone. In the cell-scaffold group, lamellar bone formation increased from the woven bone toward the center of defect. Scaffold destruction continued until 12 weeks, but at the end of this time, no substantial amount of scaffold remained. According to the IHC result, production of COL I in the cell-seeded scaffold group was substantially elevated in comparison with the blank scaffold and blank defect groups (Figure 6).

Quantitative analysis

Histomorphometric analysis indicated a significant increase in new bone formation in the cell-seeded scaffold group compared with the blank defect and blank scaffold groups (Figure 7A). In addition, the data confirms the higher biodegradation rate of the scaffold in the cell-seeded group compared with the blank scaffold group (Figure 7B).