Biomedical application of low molecular weight heparin/protamine nano/micro particles as cell- and growth factor-carriers and coating matrix

Abstract

Low molecular weight heparin (LMWH)/protamine (P) nano/micro particles (N/MPs) (LMWH/P N/MPs) were applied as carriers for heparin-binding growth factors (GFs) and for adhesive cells including adipose-derived stromal cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs). A mixture of LMWH and P yields a dispersion of N/MPs (100 nm-3 μm in diameter). LMWH/P N/MPs can be immobilized onto cell surfaces or extracellular matrix, control the release, activate GFs and protect various GFs. Furthermore, LMWH/P N/MPs can also bind to adhesive cell surfaces, inducing cells and LMWH/P N/MPs-aggregate formation. Those aggregates substantially promoted cellular viability, and induced vascularization and fibrous tissue formation in vivo. The LMWH/P N/MPs, in combination with ADSCs or BMSCs, are effective cell-carriers and are potential promising novel therapeutic agents for inducing vascularization and fibrous tissue formation in ischemic disease by transplantation of the ADSCs and LMWH/P N/MPs-aggregates. LMWH/P N/MPs can also bind to tissue culture plates and adsorb exogenous GFs or GFs from those cells. The LMWH/P N/MPs-coated matrix in the presence of GFs may provide novel biomaterials that can control cellular activity such as growth and differentiation. Furthermore, three-dimensional (3D) cultures of cells including ADSCs and BMSCs using plasma-medium gel with LMWH/P N/MPs exhibited efficient cell proliferation. Thus, LMWH/P N/MPs are an adequate carrier both for GFs and for stromal cells such as ADSCs and BMSCs, and are a functional coating matrix for their cultures.

Keywords: adipose-derived stromal cells; bone marrow-derived mesenchymal stem cells; cell delivery systems; coating matrix; nano/micro particles.

Figure 1

(A) Preparation and appearance of low molecular weight heparin/protamine nano/micro particles (LMWH/P N/MPs); (B) Electron microscopic appearance of generated LMWH/P N/MPs.

Figure 2

(A) Distribution of diameter of LMWH/P N/MPs; (B) Characterization of LMWH/P N/MPs and their stabilities. Stabilities of fibroblast growth factor (FGF)-2 and LMWH/P N/MPs were examined by measuring diameters of N/MPs and their zeta-electric charges.

Figure 3

(A) Growth factors (GFs) from platelet-rich plasma (PRP) and LMWH/P N/MPs significantly induce neovascularization and granulation tissue formations. (B) Histological examinations of the base of wounds treated with saline (control), GFs from PRP and LMWH/P N/MPs-treated skin. Each photograph for wounds on day 7 was representative of eight rats. The vertical arrows show the length of formed granulation tissue, and black triangles show generated small blood vassels.

Figure 4

GFs from PRP and LMWH/P N/MPs treatment for ordinary baldness. (A) An appearances of hair before and after GFs from PRP and LMWH/P N/MPs-treatment (five injections for 12 weeks); (B) Hair cross-sections after administration of GFs from PRP and LMWH/P N/MPs (■), GFs from PRP alone (□), and saline alone (control: ○); (C) Binding of GFs onto LMWH/P N/MPs.

Figure 5

Mechanism of formation of cells and LMWH/P N/MPs-aggregates and in vivo activities. (A) Formation of ADSCs&LMWH/P N/MPs; (B) Immunostained and Dil-labeled tissue.

Figure 6

Expansion of adipose-derived stromal cells (ADSCs) with LMWH/P NPs-coated plates and human serum (HS). ADSCs effectively expanded with low HS (2%) and FGF-2 (5 ng/mL) instead of high FBS (10%) on LMWH/P N/MP-coated plates.

Figure 7

Three dimensional culture of ADSCs with HP-DMEM gel and LMWH/P NPs. (A) Three dimensional culture of ADSCs; (B) Two dimensional culture of ADSCs.

Figure 8

Two methods (A,B) for ADSCs transplantations using IRP-DMEM gel with LMWH/P N/MPs/GF.