To evaluate chondrogenecity of the complex biohybrid, the following experimental groups were designed and analyzed:

Calcite (control) group—MSCs were seeded on the calcite scaffold and cultured in a basic DMEM medium;

Calcite-HA group—MSCs were seeded on the calcite scaffold and cultured in a basic DMEM medium supplemented with HA at the concentration described in the section below on cell culturing;

Gold calcite group—the calcite scaffold was coated by a nano-scale layer of gold prior to MSCs seeding, and the cells were cultured in a basic DMEM medium. Gold coating is used to neutralize a calcite chemical effect on the cultured MSCs, thereby preserving complex 3D topography of the scaffold;

Gold calcite-HA group—the calcite scaffold was coated by a nano-scale layer of gold prior to MSC seeding, and the cells were cultured in a basic DMEM medium supplemented with HA at a concentration of 0.5 mg/mL. Gold coating is designed to neutralize a calcite chemical effect on the cultured MSCs, thereby preserving complex 3D topography of the scaffold.

Specimens of T. rufotincta were collected from the upper belt of the shallow seawater zones adjacent to the Interuniversity Institute for Marine Science (IUI) of the Red Sea, Eilat, Israel. The average overall diameter of the shells included in this study was 4.5–5.0 cm. The animals were removed from the shells, which were subsequently bleached in 2% sodium hypochlorite solution for 30 min. Hypochlorite solution residue was washed away with distilled water (DW). The shells were then cut into 5 mm thick samples that were polished to a thickness of approximately 0.5 mm and an area of 0.5 cm² using an 8'' grinder machine (model SPT 900). To produce the gold-covered scaffolds, polished samples were placed in a Veeco VE, VP-776 evaporator and coated with a 300 Å gold layer under a constant chamber pressure of 10⁻⁶ Torr at a rate of 3 Å/s. The samples were then rinsed with 70% ethanol. Prior to cell seeding, the natural and gold-coated scaffolds were sterilized by autoclave (121 °C, 30 min) and then dried overnight at 60 °C.

The mesenchymal mouse cell line ATCC/CRL 12424 (passage 4) was seeded at a concentration of 10,000 cells per scaffold for further study. Cells were allowed to adhere for 15 min, and then culture medium was added. The basic culture medium contains Dulbecco modified Eagle’s medium (DMEM) (Biological Industries, Israel) supplemented with 4.5 g/L D-glucose, 1.5 g/L sodium bicarbonate (Sigma), 1 mM sodium pyruvate (Biological Industries, Israel), 10% (v/v) fetal calf serum (Biological Industries, Israel), 1% L-glutamine (Biological Industries, Israel), and 1% PSN antibiotic solution (Gibco, USA). Unless noted otherwise, the conditions for added HA comprised supplementing the basic medium with HA (MW 3000kD, BTG LTD, Israel) at a concentration of 0.5 mg/mL. Cells were cultured at 37 °C in a humidified 5% CO₂ incubator. The medium was replaced every two days.

MSC cultures were rinsed with saline and fixed with fixative containing 2% paraformaldehyde in DW and 2.5% glutaraldehyde in 0.1 M buffer phosphate. After fixation, the samples were washed and subjected to graded ethanol dehydration, followed by final dehydration in hexamethyldisilazane (HMDS) (Electron Microscopy Sciences, PA). Then the samples were gold coated with a sputter-coater at 15 mA for 60 s. Samples morphologies were visualized using scanning electron microscopy (SEM) (JEOL, JSM-5610-LV).

Cell proliferation was measured by an XTT cell proliferation assay kit (Biological Industries, Israel) according to the manufacturer's instructions. A calibration curve was plotted using counted MSCs. Tests were performed in triplicate and the average cell number on each scaffold was calculated using the calibration curve.

For GAG quantification after 7, 14, and 21 d, scaffolds containing MSCs were first digested with a papain solution comprising 0.01 M L-cysteine and 0.05% papain dissolved in phosphate buffered EDTA adjusted to a pH of 6.5, for 24 h in a 60 °C water bath. Following digestion, 50 µL from each sample was added to a 96-well plate and 200 µL of dimethylmethilene blue (DMMB) solution was added to each well. Absorbance was read at 525 nm. Chondroitin sulfate (C-8529, Sigma-Aldrich Israel) at concentrations of 100–0.75 µg/mL was used to build a calibration curve. GAG quantities were normalized to cell number as measured using the Alamar Blue™ Cell Proliferation Indicator prior to digestion by papain.

An assay based on a GAG distribution analysis was developed to visualize the effect of the addition of HA on MSCs cultured on a calcite scaffold. About 5000 cells were seeded on the calcite samples in Petri dishes, one sample per dish, and cultured for six weeks. The GAG accumulation process in the cell culture was visualized by safranin O/fast green staining, which is specific for GAGs. In the safranin O/fast green combination, the balance between the two stains reveals the chondrogenic character of the culture: the fast green stained the cell culture pale green, and safranin O, which is specific for the cartilage tissue, stained proteoglycans (PG) and glycosaminoglycans (GAGs) a pink-red color. All photographs were taken at the same light intensity and exposure time to ensure fixed conditions and therefore to enable subsequent color image analyses. The images were analyzed for the size of the area colored by safranin O and for its digital color intensity (i.e., the overall number of colored pixels for the image). For the analysis, the area colored by safranin O was defined manually, after which the image was stacked to RGB colors, and the red color intensity was automatically calculated within the defined area. The analysis was performed in at least six duplicates using the NIH-developed open source software ImageJ.

Samples were rinsed gently with PBS and fixed with buffered 4% glutaraldehyde solution for 10 min at 4 °C. Fixed samples were rinsed three times with PBS (5 min per wash) and incubated in blocking solution containing 10% normal serum in PBS for 1 h. The samples were placed in a humidified chamber (overnight) and stained with goat polyclonal Immunoglobulin G (IgG) against type I collagen (Santa Cruz Biotechnology, USA), or goat polyclonal IgG against type II collagen (Santa Cruz Biotechnology, USA), diluted to a 1:300 ratio with PBS at 4 °C. The samples were washed three times (5 min per wash) with PBS and then incubated in a 1:200 diluted solution of donkey anti-goat IgG fluorescein isothiocyanate (FITC) conjugated affinity purified antibody (Santa Cruz Biotechnology, USA) or rhodamine red-X-conjugated affiniPure donkey anti goat IgG (H + L) (Jackson Immunoresearch Laboratories, INC., USA) in PBS for 60 min at room temperature. After the secondary antibodies were removed, the samples were again washed three times with PBS and photographed using an inverted fluorescence microscope (Nikon, Eclipse TE300) with FITC or rhodamine filters.

On days 7, 14, and 21, biolattices (n = 2–3) were transferred to 1.5 mL centrifuge tubes and washed with cold PBS. Total RNA was isolated using a power SYBR green GEx Cells-to-Ct kit (Invitrogen Life Science, USA) according to the manufacturer's instructions. First-strand cDNA was subsequently synthesized in a 50 µL reaction mixture from 1 µg total RNA using the same kit. The sample that did not contain transcription enzyme was used as the negative control to test for genomic contaminations.

The PCR reaction was carried out in a final volume of 20 µL, which contained 2 µL of cDNA template, gene-specific primer sets (Table 1), and SYBR Green (Invitrogen Life Science, USA) according to the manufacturer's protocol. The primer sets were designed using primer express software (Applied Biosystems). Thermal cycling was carried out using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA) and mRNA expression levels of target genes were analyzed using the ΔΔCT method [40], with glyceraldehide-3-phosphate dehydrogenaze (GAPDH) as the housekeeping gene. The results are presented as the mean of triplicates experiments from two independent experiments (error bars represent SD).

Quantitative data were presented as means ± standard deviation. The independent sample t-test was used to detect statistically significant differences in two groups; a two-way repeated measure of ANOVA was used to compare more than two groups over time. Statistical significance was defined as * p < 0.05, ** p < 0.01, and ^# p < 0.001.

The XTT cell proliferation test was performed to evaluate the effect of HA concentrations on MSC proliferation in the 3D system. Five different concentrations of HA (0.1, 0.5, 1, 2, and 4 mg/mL) were selected for this study, in which the control cell culture was grown without the addition of HA. As shown in Figure 1a, the cells cultured in the presence of HA in the medium show dose–dependent responses compared to control cell cultures. At all concentrations, the presence of HA increased cell proliferation at days 4–7. After day 7, the addition of HA had an inhibitory effect on cell proliferation, while concentrations above 2 mg/mL dramatically reduced the number of cells. The HA concentration of 0.5 mg/mL was chosen as optimal for further analyses due to its stable effect on the proliferation of MSCs: at this concentration, total cell numbers rapidly increased after seeding and peaked at day 7, after which the number of cells remained statistically the same (Figure 1b). Our evaluation of the effect of HA concentrations on MSCs indicates that HA affected MSC proliferation in a dose-dependent manner. Data in the literature shows that the addition of small concentrations of low molecular weight HA stimulates MSC proliferation [42,43], while the same concentration of high molecular weight HA appears to inhibit the cells [44,45]. Our experiments with high molecular weight HA in a 2D system did not show any significant effect on MSC proliferation (data not shown) while cells cultured on a calcite scaffold in a 3D system in the presence of HA of various concentrations displayed a reduction in cell number. Indeed, concentrations of HA above 2 mg/mL significantly inhibited MSCs cultured on calcite, and in fact, HA seems to be cytotoxic at high concentrations. In contrast, low concentrations of HA (0.1–0.5 mg/mL), after the initial (during the first week) accelerated proliferation of MSCs, appear to maintain the cells at the same level of proliferation activity for up to three weeks, while MSCs cultured on calcite without HA continued to proliferate. The HA concentration of 0.5 mg/mL has thus been recommended as optimal for studying its effect on MSC differentiation. We suggest that at this concentration, HA accelerates MSC proliferative activity subsequent to MSC chondrogenic differentiation.

Figure 1B presents cell proliferation kinetics in all groups during the test period as measured by the XXT method. The graph shows how cell numbers changed from an initial (at day 0) density of 10,000 cells per scaffold on both the calcite and gold calcite scaffolds with and without HA, thus revealing the effects of HA and calcite independently on cell proliferation rates. The proliferation kinetics of the cells cultured on calcite were significantly affected by the addition of HA. The number of the cells cultured in the presence of HA reached a maximum at 7 d, showing no statistical difference at either 14 or 21 d. The cell count for cultures on calcite in the absence of HA, however, increased linearly during the entire experiment. In the gold calcite and the gold calcite-HA cultures, cell numbers gradually increased during the test period.

MSC adhesion, morphology and distribution were observed using SEM. Figure 2 shows SEM micrographs taken at days 1 (a, c), 4 (b, d), and 21 (e, f). The attachment and morphology of MSCs on calcite were affected by the presence or absence of HA in the medium. Compared with those containing only calcite, cultures of calcite with HA promoted initial cell attachment (Figure 2a,c). As growth continued, the cells cultured on the calcite-HA complex had spherical morphologies characteristic of chondrocytes [41] and well-developed extracellular matrices (Figure 2g,h). SEM evaluation demonstrated that the MSCs cultured in the presence of HA underwent morphological changes into chondrogenic-like phenotypes, about 2 weeks after the culture was begun. Cells became round and exhibited extensive, highly organized extracellular matrix while the cells cultured in the absence of HA on the calcite scaffold assumed more flattened, fibroblast-like morphologies, were embedded in the fine extracellular matrix, and they became over-confluent after three weeks (Figure 2e,f).

Quantification of sulfated GAGs for the cells cultured in the calcite and calcite-HA cultures reveals that GAG accumulation substantially increased in the presence of HA compared with the cells cultured on calcite alone (Figure 3). For the calcite group, GAG accumulation in the ECM increased during the first two weeks, after which it remained at the same level. In the calcite-HA group, three weeks after beginning the culture, the GAG content was more than twice that of the calcite group. For the gold-coated groups, the addition of HA to the medium had very little effect on GAG accumulation, in either group (gold calcite without or with HA), such that the cells produced scant amounts of the GAGs compared with the calcite group.

Long-term (up to six weeks) GAG distribution in the ECM of the cultured cells was visualized using a newly developed assay (Figure 4). Digital analysis of the images (Figure 4b,c) revealed that cells grown on the calcite scaffold had the potential to differentiate toward the chondrogenic lineage, but in the long-term, the cultured cells lost their chondrogenic character. Maintaining a steady supply of HA to the medium increased the tendency for GAG molecules to be synthesized. Cells grown on the gold coated calcite without added HA showed no indications of chondrogenecity during the entire test period. Results comparable to those of the control (calcite) group were obtained for cells grown on the gold coated calcite with added HA.

The results in supplement with the sulfated GAGs quantification and SEM analysis clearly indicate that the tissue underwent chondrogenic differentiation in the calcite-HA culture at day 14. GAG expression in the calcite, gold, and gold-HA groups was significantly lower than in the calcite-HA culture for the entire length of the experiment, suggesting that the chondrogenic potential exists in the combined calcite-HA complex and that it is not restricted to the calcite or the gold-HA cultures. Likewise, the results of the chondrogenecity assay (Figure 4) demonstrate that the cells grown on the calcite scaffold have the potential to chondrogenically differentiate. The data showed that cells cultured in the calcite-HA tissue displayed strong chondrogenic tendencies as they underwent chondrogenic differentiation, and they maintained the chondrogenic phenotype in long-term cultures. Thus, in the case of calcite, the addition of HA supported and promoted the chondrogenic potential of the calcite biomaterial.

A fold change analysis of gene expression in the calcite-HA, gold calcite, and gold calcite-HA was compared to that in the calcite (control) culture. A primary HA receptor CD44 was expressed at significantly higher levels in the calcite-HA culture, where initially its expression increased approximately eightfold by about day 7 but then gradually decreased fourfold by day 21 (Figure 5a). CD44 gene expression in the gold and gold-HA cultures was markedly lower than in the calcite culture for the first two weeks, but at 21 day it increased slightly. There were no significant differences between the gold calcite and gold calcite-HA groups, apart from the slightly higher expression of CD44 on day 21.

Expression of the chondrogenic genes collagen type II Col2a1 and Sox9 in the calcite-HA culture was higher at day 7 and exhibited a decreasing trend over the test period (Figure 5b,c). By day 7, expression of the collagen type II gene was strongly upregulated (23-fold) while Sox9 gene expression increased approximately threefold. For the Col2a1 and Sox9 genes, we did not find any significant difference between the gold calcite and gold calcite-HA cultures. The expression of both genes increased over the test period and at day 21 for Sox9 no significant differences were found between all the groups.

Expression of the osteogenic genes RunX2 and collagen type I increased significantly during the test period in the gold calcite-HA culture with the exception of day 21 for the RunX2 gene, for which we did not find any significant differences relative to the control group (Figure 5d,e). For the calcite-HA culture, the expression of RunX2 and collagen type I genes did not differ statistically from the calcite culture. For the gold group, collagen type I increased approximately twofold on days 7 and 14 and showed no difference on day 21. RunX2 gene expression for this group decreased significantly in the early stage (day 7), but by days 14 and 21, it had increased twofold.

Gene expression analysis by relative RT-PCR revealed that expression of the primary HA receptor CD44 was affected by the presence of HA in calcite-HA culture but not in the gold calcite-HA culture (Figure 5a). The CD44 receptor is involved in multiple cellular mechanisms related to ECM-activated signaling and its expression increased with increasing cell proliferation rate [26,27,46]. Indeed, from the proliferation test (Figure 1) it is clear that during the first week, the cells in the calcite-HA culture exhibited an enhanced proliferation rate relative to those of the other groups. After the first week, however, both proliferation rate and CD44 gene level decreased in the calcite-HA culture. Activation of the receptor by HA is achieved via precise spatial folding of the HA chains [47,48], and we hypothesize that at the calcite-HA interface, the HA chains adopt a spatially orientated molecular conformation that is necessary to activate the CD44 receptor, a hypothesis supported by our previous investigation [20].

The expression of Sox9, which reaches high levels after mesenchymal condensation and is completely downregulated in hypertrophic chondrocytes, is important for chondrogenic differentiation [49,50,51]. The collagen type II Col2a1 gene is a direct target of Sox9 [52]. The expressions of these two cartilage-specific factors in the calcite-HA culture were amplified at day 7 (23-fold for Col2a1 and 2.8-fold for Sox9), suggesting that during the first week of culture, the MSCs committed mainly to a chondrocytic phenotype (Figure 5b, c). In contrast, the expression of RunX2, which is required for osteoblastic differentiation and expressed in the final stages of hypertrophic chondrocytes [53], reached high levels in the gold calcite-HA culture, and the data correlated well with expression of the osteoblast-specific marker collagen type I.

Immunohistochemistry analyses of N-Cadherin (Figure 6) for calcite and calcite-HA cultures at days 7 (Figure 6a) and 14 (Figure 6b) revealed that the expression of N-cadherin in the calcite-HA culture increased on both days relative to the expression of the protein in the calcite group.

The results of immunohistochemistry staining with monoclonal antibodies against collagen I and collagen II of MCSs at 7, 14, and 21 day showed that the cells grown in the calcite culture expressed both collagen I and collagen II, indicating the heterogenic character of the tissue formed (Figure 7). Collagen I expression on aragonite tended to increase during the culture period, however, and on day 21 its expression exceeded that of collagen II, indicating that osteogenesis predominated. The addition of HA to the aragonite did not significantly change cell differentiation fates. When the MCSs were cultured on the calcite, however, collagen I expression increased while that of collagen II decreased. In contrast, cells cultured on the calcite-HA biocomplex showed much stronger collagen II than collagen I expression, a trend that increased over time, thus providing evidence that the calcite-HA biocomplex promoted MSC chondrogenic differentiation.

Immunohistochemistry analysis of the expressions of the proteins collagen type I and type II indicated that the calcite culture had a heterogenic character, such that the expressions of both proteins were displayed. In contrast, in the calcite-HA culture, expression of the collagen type II protein prevailed (Figure 7). Because sulfated GAGs and collagen type II are the major indicators of tissue culture chondrogenecity, our findings reveal that the calcite-HA environment promotes the chondrogenic differentiation of MSCs. The initially enhanced expression of N-cadherin in the calcite-HA culture is also indicative of the dominance of chondrogenic over osteogenic differentiation (Figure 6). It could be concluded that, immobilized on the calcite surface, HA activates its receptors, particularly CD44, thus triggering their signal transduction pathways and leading to the chondrogenic differentiation of MSCs while concurrently preventing the dedifferentiation process. Previous studies reported that differentiated MSCs were able to dedifferentiate, meaning that they lost their chondrogenic phenotypes when grown in long-term culture [54]. If the chondrogenic phenotype of MSCs cultured on the calcite-HA composite is maintained in long-term culture, that implies that interactions between HA and its receptors are a major determinant in preventing MSC dedifferentiation.

The combination of HA with synthetic polymers, such as polylactic/polyglycolic acids was intended to improve the poor biological properties of the latter [55,56]. Mixtures of HA with hydroxyapatite and calcium phosphate ceramics for developing composite biomaterials [57,58,59,60,61] resulted in prolonged scaffold stability and reduced degradation rates, but the composite exhibited impaired biological activity. Described here is a novel combination of non-modified HA with natural calcite that produces a biohybrid with unique biological activities.