Amorphous, Smart, and Bioinspired Polyphosphate Nano/Microparticles: A Biomaterial for Regeneration and Repair of Osteo-Articular Impairments In-Situ

Abstract

Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form of amorphous Ca-polyP nano/microparticles, polyP retains its function to act as both an intra- and extracellular metabolic fuel and a stimulus eliciting morphogenetic signals. The method for synthesis of the nano/microparticles with the polyanionic polyP also allowed the fabrication of hybrid particles with the bisphosphonate zoledronic acid, a drug used in therapy of bone metastases in cancer patients. The results revealed that the amorphous Ca-polyP particles promote the growth/viability of mesenchymal stem cells, as well as the osteogenic and chondrogenic differentiation of the bone marrow cells in rat femur explants, as revealed by an upregulation of the expression of the transcription factors SOX9 (differentiation towards osteoblasts) and RUNX2 (chondrocyte differentiation). In parallel to this bone anabolic effect, incubation of the femur explants with these particles significantly reduced the expression of the gene encoding the osteoclast bone-catabolic enzyme, cathepsin-K, while the expression of the tartrate-resistant acid phosphatase remained unaffected. The gene expression data were supported by the finding of an increased mineralization of the cells in the femur explants in response to the Ca-polyP particles. Finally, we show that the hybrid particles of polyP complexed with zoledronic acid exhibit both the cytotoxic effect of the bisphosphonate and the morphogenetic and mineralization inducing activity of polyP. Our results suggest that the Ca-polyP nano/microparticles are not only a promising scaffold material for repairing long bone osteo-articular damages but can also be applied, as a hybrid with zoledronic acid, as a drug delivery system for treatment of bone metastases. The polyP particles are highlighted as genuine, smart, bioinspired nano/micro biomaterials.

Keywords: Runx2; Sox9; bisphosphonates; bone marrow cells; cathepsin-K; human mesenchymal stem cells; inorganic polyphosphate; long bone defects; microparticles; tumor metastases.

Figure 1

Morphology of the polyP and ZOL particles; SEM. Images from (A–C) amorphous “Ca-polyP-MP”, (D–F) crystalline “Ca-ZOL-MP”, and (G–I) amorphous hybrid “Ca-polyP-ZOL-MP”. ZOL, zoledronic acid.

Figure 2

EDX spectra for (A) “Ca-polyP-MP”, (B) “Ca-ZOL-MP”, and (C) “Ca-polyP-ZOL-MP”. The respective signals for the different atoms are marked. EDX, energy dispersive X-ray.

Figure 3

XRD pattern obtained from (above) “Ca-polyP-MP”, (middle) “Ca-ZOL-MP”, and (below) “Ca-polyP-ZOL-MP”. The characteristic signals in the diffractogram from “Ca-ZOL-MP”, which match those of ZOL, are striking. XRD, X-ray diffraction.

Figure 4

Effect of the different concentrations of particles, either “Ca-polyP-MP”, “Ca-ZOL-MP”, or “Ca-polyP-ZOL-MP” the viability/growth of MSC. The number of viable cells was determined by the XTT assay (A₄₅₀ values). The incubation period was 36 h and 72 h, respectively. Ten parallel assays were performed, and the mean values (±SD) were determined. The significance correlations are determined with respect to the controls (no particles added); they are marked with rectangular brackets; (* p < 0.001) between the indicated two values. The concentrations of the particles for the respective test samples are given in the box.

Figure 5

Dissection and collection of mice femurs. (A) Immediately after sacrificing, the animals and the femurs were dissected and collected. In addition to the exposed femur (fe), the tibia (t), patella (p), and fibula (fi) are visible; (B) the explant, measuring up to 25 mm in length and 2 mm in diameter, shows the compact bone zone (c-b) and the diaphysis (d), and exposes the bone marrow (bm); (C) after incubation in DMEM medium/FBS for up to 7 days, the specimens were collected and processed for bone marrow cell isolation. On the pictured specimen the femoral head (f-h), the epiphysis (ep), the metaphysis (mp), and the diaphysis (dp) can be distinguished.

Figure 6

Effect of the compounds on the expression of osteogenic and chondrogenic differentiation markers in bone marrow cells. The cultures were supplemented with 30 µg/mL of “Ca-polyP-MP”, “Ca-ZOL-MP”, or “Ca-polyP-ZOL-MP” on bone marrow cells in mouse femurs; in the controls the explants received no additional compound. The cells were incubated for 3 days or 7 days, then the RNA was isolated from the samples and the steady-state-expression of the transcripts of (A) Sox9 and (B) Runx2 was quantified by qRT-PCR and correlated to the expression of the GAPDH house-keeping gene. Standard errors of the means are shown (n = 6 experiments per time point). The significant differences between the values in the controls and the respective treated samples are indicated with asterisks; * p < 0.01.

Figure 7

Alteration of the expression in bone marrow cells using (A) the established osteoclastic differentiation marker cathepsin-K and (B) the level of TRAP, which reflects both the osteogenic and the osteo-clastogenic proliferation state of the cells; * p < 0.001.

Figure 8

Influence of the particles (30 µg/mL of “Ca-polyP-MP”, “Ca-ZOL-MP”, or “Ca-polyP-ZOL-MP”) on bone marrow cells present in femurs on the extent of biomineralization during a 5 days incubation in DMEM medium/FBS. After termination of the experiments, the bone marrow cells were carefully removed from the femurs and quantitatively assayed for mineralization with Alizarin Red S. The reintroduced red color is measured at 405 nm applying the spectrophotometric assay. The amount of bound Alizarin Red S is given in nmol. Values were normalized to 1 µg of DNA in the samples; The significant differences are indicated with asterisks; * p < 0.01.

Figure 9

Polyphosphate as a smart genuine nano/micro biomaterial. The introduction of the technology to form amorphous nano-/microparticles under over-stoichiometric ratios between the different cations Mg²⁺ − Ca²⁺ or Sr²⁺ and the polyanion polyP disclosed that Mg²⁺-polyP accelerates wound healing and cartilage formation, Ca²⁺-polyP strongly induces bone formation, and Sr²⁺-polyP, besides inducing bone formation, also hardens the mature hydroxyapatite bone mineral. Furthermore, polyP can act as a basket/cage for encasing drugs, like zoledronic acid.