Effect of soluble zinc on differentiation of osteoprogenitor cells

Abstract

Amorphous calcium phosphates (ACPs) are attractive fillers for osseous defects and are stabilized through the incorporation of transition metals such as zirconium and zinc. As ACP converts in solution to hydroxyapatite (HAP) in a manner marked by a transient release of calcium and phosphate ions, it is capable of stimulating osteoblastic differentiation. Zinc is known to retard ACP conversion to HAP, and—when incorporated into ceramic biomaterials—has been shown to stimulate osteoblastic differentiation. Because zinc deficiency in vivo is marked by skeletal defects, we postulated that zinc ions released from ACP and other minerals could stimulate proliferation and osteoblastic differentiation of progenitor cells. To test this hypothesis, rat bone marrow stromal cells were cultured in osteogenic medium containing basal (3 × 10⁻⁶ M) or supplemented Zn²⁺ concentrations (1 × 10⁻⁵ and 4 × 10⁻⁵ M) for up to 3 weeks. No significant effects of zinc concentration on cell number, alkaline phosphatase activity, total protein content, collagen synthesis, or matrix mineralization were found.

INTRODUCTION

Calcium phosphate minerals such as hydroxyapatite (HAP) and β-tricalcium phosphate (β-TCP) have been studied extensively as fillers for bone defects and scaffolds for engineered bone tissue.¹ The advantage of these insoluble calcium phosphate ceramics in osseous reconstruction and regeneration is their biocompatibility and osteoconductivity.² However, amorphous calcium phosphates (ACPs)—which are soluble under aqueous conditions—may be attractive alternatives given that they can transiently stimulate osteoblastic differentiation as they release calcium and phosphate ions and raise the local pH.³ Although a limitation of ACPs has been their rapid dissolution, divalent cations such as Zn²⁺ and Cu²⁺—which are essential for normal cell function—retard the rate of ACP conversion.⁴ Interestingly, the process of ACP conversion may serve as means for delivering osteogenic divalent cations.

Zinc is a trace element necessary for mammalian growth,⁵ and zinc deficiency leads to skeletal defects, and retardation in bone growth.⁶ In vitro, zinc-containing biomaterials have been shown to enhance phenotypic markers of osteoblastic differentiation of osteoprogenitor cells. In particular, incorporating zinc into β-TCP stimulated alkaline phosphatase (ALP) activity of bone marrow stromal cells (BMSCs),⁷ while zinc-organoapatite films accelerated ALP activity and bone nodule formation by MC3T3-E1 cells.⁸ What is not clear from these studies is whether the reported osteoinductive effect is related to alteration of the mineral structure with incorporation of zinc, or a consequence of the release of zinc ions into the cell microenvironment. To test the latter mechanism, we examined the effect of soluble Zn²⁺ on proliferation and differentiation of rat BMSCs. Cells were cultured up to 3 weeks in an osteogenic medium with basal (3 × 10⁻⁶ M) or supplemented Zn²⁺ concentrations (1 × 10⁻⁵ and 4 × 10⁻⁵ M). The effect of zinc concentration on cell number, ALP activity, total protein content, collagen synthesis, and matrix mineralization was determined.

MATERIALS AND METHODS

Materials

Chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and materials were obtained from Fisher Scientific (Pittsburgh, PA) unless otherwise specified. Primary medium for cell culture was Minimum Essential Medium Alpha Modification (aMEM, Invitrogen, Gaithersburg, MD) with 10% fetal bovine serum (Gemini Biosciences, Calabasas, CA) and 1% antibiotic/antimycotic (Invitrogen). Osteogenic medium was primary medium supplemented with 2 mM β-glycerophosphate, 0.13 mM l-ascorbic acid-2-phosphate, and 0.01 μm dexamethasone.⁹ The concentration of Zn²⁺ in osteogenic medium was systematically varied by adding ZnCl₂ to the osteogenic medium and the resultant zinc concentrations—as determined by atomic absorption spectroscopy (Perkin-Elmer 5100-PC, Wellesley, MA)—were 3 × 10⁻⁶, 1 × 10⁻⁵, and 4 × 10⁻⁵ M. Here, the lowest concentration corresponds to the basal zinc concentration (no supplementation), while the latter two concentrations were chosen to stay below 9 × 10⁻⁵ M—a concentration that has been shown to inhibit normal osteoblast function.¹⁰

Cell culture

Studies were performed using rat BMSCs obtained from 125 to 150 g male Sprague-Dawley rats (Harlan, Dublin, VA) in accordance with the Animal Care Committee at Virginia Tech.⁹ Cells were expanded under subconfluent conditions and used at passages 2–4. Cells were seeded into 12-well plates at 10⁵ cells/well and allowed to attach overnight in primary medium. The following day, denoted as day 0, culture medium was replaced with osteogenic medium containing different concentrations of zinc. Cells were analyzed for cell number and ALP activity at days 7 and 14, for collagen synthesis and total protein synthesis at day 14, and for mineralization at day 21.

Cell number and ALP activity

Cell number was determined by fluorometric analysis of total DNA content using Hoechst 33258 dye. ALP activity was determined colorimetrically by the hydrolysis of p-nitrophenyl phosphate, and then normalized by cell number. These assays are described in detail elsewhere.¹¹

Total protein

To quantify total protein, cell layers were washed twice with phosphate buffered saline (PBS) and mechanically scraped in the presence of 0.1 mL 1× Laemmli Buffer with protease inhibitors (950 μL Laemmli stock solution (Biorad, Hercules, CA)), 50 μL β-mercaptoethanol (Fisher Scientific), 10 μL 100× protease inhibitors (0.2 mg/mL aprotinin, 0.2 mg/mL leupeptin, 0.1 mg/mL pepstatin (Calbiochem, La Jolla, CA), and 1 mL PBS). Total protein per well was determined by colorimetric assay (RC DC Protein Assay, Bio-Rad) according to the manufacturer’s instructions and normalized by cell number.

Collagen synthesis

Collagen synthesis was determined by incubating cell layers with 3 μCi/mL of ³H-proline (ICN, Irvine, CA) for 48 h. Cell layers were collected, separated into collagenase digestible and non-collagenase digestible fractions, and collagen content was determined by scintillation counting and reported as a percentage of total, as previously described.³

Mineralization

Mineralization of cell layers was assessed by Alizarin Red S staining of calcium deposits as described elsewhere.¹² Briefly, cell layers were stained with 1 mL of 40 mM Alizarin Red S, pH 4.1 for 20 min. The dye was extracted with 0.8 mL 10% (v/v) acetic acid and absorbance of the extract was measured at 405 nm. Cell layers in primary medium without zinc supplementation (3 × 10 ⁻⁶ M Zn²⁺) were used as a negative control.

Statistics

Values are presented as mean ± standard deviation. Statistical analyses were performed using Origin 6.1 (OriginLab Corporation, Northampton, MA). A one-way analysis of variance (ANOVA) procedure and Fisher’s protected least significant difference with a significance level of p = 0.05 was used to determine significance between groups.

RESULTS

To test the effects of zinc concentration on cell proliferation, cells were seeded at 10⁵ cells/well, cultured in osteogenic medium containing zinc for 7 and 14 days and assayed for cell number. Measurements of cell number are consistent with a 4 to 5-fold increase in cell number (Fig. 1) and the achievement of monolayer coverage. However, no stimulatory effect of Zn²⁺ on cell proliferation was noted. Concurrent analysis of ALP activity indicated the development of the osteoblastic phenotype, but did not indicate a stimulatory effect of Zn²⁺ (Fig. 2).

Figure 1.

Cell number as a function of Zn²⁺ concentration and time in culture. Bars correspond to the mean ± standard deviation for n = 8 samples. An asterisk denotes significant difference relative to the control.

Figure 2.

Alkaline phosphatase activity as a function of Zn²⁺ concentration and time in culture. Activity data is normalized by cell number. Bars correspond to the mean ± standard deviation for n = 8 samples.

Zinc ions have been shown to increase protein synthesis in osteoblasts.¹³ To test for the effects of zinc concentration on total protein content, cells were cultured with osteogenic medium containing 3 × 10⁻⁶, 1 × 10⁻⁵, and 4 × 10⁻⁵ M Zn²⁺ for a period of 14 days. At the end of this period, protein content was found to be the same for all treatment groups, indicating that zinc supplementation does not affect the protein content of osteoblasts in vitro [Fig. 3(a)]. Concurrently, collagen synthesis was determined by ³H-proline addition to culture on day 12 followed by a 48 h incubation to allow for incorporation of the radioactive proline into newly formed protein. Measurements of collagen synthesis, reported as percent collagenous protein per cell layer, were similar for all treatment groups [Fig. 3(b)].

Figure 3.

Protein and collagen synthesis as a function of Zn²⁺ concentration on Day 14. (a) Total protein in cell layers normalized by cell number. (b) Collagen as a percent of total protein. Bars correspond to the mean ± standard deviation for n = 8 and n = 4 samples for (a) and (b), respectively.

The effect of Zn²⁺ on deposition of a mineralized extracellular matrix was determined by Alizarin red staining of calcium depositions at day 21, followed by acid extraction and colorimetric analysis. Phase contrast micrographs revealed little mineralization for all treatment groups (data not shown), and analysis of the extracted dye indicated only slight differences in mineralization between groups (Fig. 4). This limited mineralization is consistent with other unpublished findings, and may be a consequence of initial seeding density, culture duration, or fetal bovine serum lot selection.

Figure 4.

Mineral deposition as a function of Zn²⁺ concentration on Day 21. Mineral deposition was quantified as the absorbance (at 405 nm) of Alizarin red extracted from cell layers. Bars correspond to the mean ± standard deviation for n = 6 samples. An asterisk denotes statistical significance between groups.

DISCUSSION

Zinc is a trace mineral, essential for the function of normal mammalian cell processes such as DNA^14,15 and protein synthesis.^13,16,17 Previous studies using serum-free medium without zinc supplementation demonstrated diminished ALP activity^18,19 and synthesis of collagen,^19,20 and bone sialoprotein.²¹ In contrast, this study found that addition of ZnCl₂ had no measurable effect on ALP activity, protein synthesis, or collagen content. A likely explanation for this difference is the use of 10% serum in this study, which provided the trace amounts of zinc (3 × 10⁻⁶ M) necessary to maintain normal cell function.

One mechanism by which zinc may regulate protein synthesis is through Class I aminoacyl-tRNA synthetase activity. Aminoacyl-tRNA synthetases are the family of enzymes responsible for covalently attaching amino acids to tRNAs, and zinc has been shown to be essential for the aminoacylation step.²² In particular, supplementation of serum-free medium with zinc sulfate resulted in a significant increase in aminoacyl-tRNA synthetase activity in MC3T3-E1 cells.¹³

Zinc also has been shown to affect cell proliferation. Studies have shown that addition of zinc to serum-free medium results in restoration of DNA synthesis in MC3T3-E1 osteoprogenitor cells and rat femoral tissue.^15,23 This stimulatory effect is likely regulated by new protein synthesis because pretreatment of cultures with cycloheximide—an inhibitor of protein synthesis—abolished the stimulatory effect of zinc on DNA synthesis.¹⁵ Further, one protein likely involved in the stimulatory effect of zinc on cell protein is insulin-like growth factor-I,^23,24 which is also known to stimulate osteoblastic cell proliferation.

In this study supplementation of culture medium with soluble zinc did not affect proliferation and osteoblastic differentiation of model osteoprogenitor cells. Although, the range of zinc concentration tested was narrow it was constrained by the level of zinc in 10% serum (3 × 10⁻⁶ M) and published data suggesting that 9 × 10⁻⁵ M inhibits normal osteoblast function.¹⁰ The results of this study indicate that sufficient zinc is provided in normal serum, and that zinc supplementation through controlled release from ceramic biomaterials is not anticipated to alter development of a bone-like tissue in vitro, or the healing of a bone defect in vivo.