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

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.

Therapeutic angiogenesis induced by controlled release of fibroblast growth factor-2 from injectable chitosan/non-anticoagulant heparin hydrogel in a rat hindlimb ischemia model.

The addition of non-anticoagulant heparin [periodate-oxidized (IO4) heparin] and fibroblast growth factor (FGF)-2 to a viscous water-soluble chitosan (CH-LA) aqueous solution produces an injectable FGF-2/CH-LA/IO4-heparin hydrogel. The purpose of this study was to examine the ability of the injected FGF-2/CH-LA/IO4-heparin hydrogel to induce vascularization and fibrous tissue formation. FGF-2/CH-LA/IO4-heparin hydrogels (100 microL of hydrogel consisting of 20 mg/mL of CH-LA, 2 mg/mL of IO4-heparin, and 50 microg/mL of FGF-2) were subcutaneously injected into the backs of wound healing-impaired diabetic (db/db) mice. Furthermore, the effect of percutaneous injection of FGF-2/CH-LA/IO4-heparin hydrogel at eight sites (25 microL/site) into ischemic left lower limbs of rats was examined from day 4 to at least day 28 postinjection. The injection of FGF-2/CH-LA/IO4-heparin hydrogels into the backs of db/db mice resulted in significant increases in blood vessel formation, significant vascularization, and fibrous tissue formation near the injection site. Injection of FGF-2/CH-LA/IO4-heparin hydrogel into ischemic left lower limbs of rats also significantly recovered and increased blood flow and blood oxygen in the calf and thigh. These results indicate that the controlled release of biologically active FGF-2 molecules from FGF-2/CH-LA/IO4-heparin induces angiogenesis and possibly collateral circulation in db/db mice and the ischemic limbs of rats.

Inhibition of neointimal proliferation in balloon-injured arteries using non-anticoagulant heparin-carrying polystyrene.

Non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) has a higher activity to inhibit proliferation and migration of smooth muscle cells (SMCs) than heparin (Hep), periodate-oxidized (IO4-) Hep, and periodate-oxidized alkaline-degraded low molecular weight (IO4-LMW-) Hep. Less than 10 microg/ml of NAC-HCPS significantly inhibited the proliferation and migration of SMCs in vitro, while over 10-fold higher concentrations of Hep, IO4-Hep, and IO4-LMW-Hep were required to obtain the same inhibition. On the other hand, neointimal growth (intimal cross-section area and intimal cross-section area/medial cross-section area ratio) in vivo following vascular injury 28 days after balloon denudation in a rat carotid artery was substantially inhibited with high dose of intravenous administration (total 30 mg) of respectively IO4-Hep, IO4-LMW-Hep, and NAC-HCPS. A low-dose (total 10 mg) administration of IO4-Hep and IO4-LMW-Hep did not prevent the neointimal growth when compared with the control; only NAC-HCPS (total 10 mg) was able to significantly inhibit the neointimal. Thus, NAC-HCPS has a more-than 10-fold larger activity to inhibit SMC activities such as proliferation and migration in vitro, when comparing with Hep, IO4-Hep, and IO4-LMW-Hep; NAC-HCPS also prevents neointimal growth in vivo at lower doses.

Adsorption of inflammatory cytokines using a heparin-coated extracorporeal circuit.

Cardiopulmonary bypass (CPB) surgeries cause an increase in plasma inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) along with whole-body inflammatory responses. The inflammatory responses during a CPB treatment are reduced when using a heparin-coated extracorporeal circuit. Because many cytokines, growth factors, and complements are known to interact with heparin, the reduction of inflammatory responses by a heparin-coated circuit is likely to depend on this heparin-binding nature of the inflammatory cytokines. In this study, the inflammatory cytokines, TNF-alpha and IL-6, in fetal bovine serum (FBS) bound to a heparin-agarose beads (heparin beads)-column and the adsorptions were competitively inhibited on addition of heparin in a concentration-dependent manner. TNF-alpha in FBS required a higher concentration of heparin (50% concentration inhibition [IC50] > 20microg/ml) to inhibit adsorption to the heparin beads-column compared with IL-6, probably because of a stronger interaction between TNF-alpha and heparin-beads. TNF-alpha and IL-6 concentrations in human heparinized blood significantly increased after a CPB treatment. Although the adsorbed amount of IL-6 onto the heparin-coated circuit was low (less than 6% of free circulating IL-6), a significant amount of TNF-alpha adsorbed onto the circuit (23.9-755% of free circulating TNF-alpha). Therefore, the adsorption of inflammatory cytokines, especially TNF-alpha, onto the inner heparin-coated surface of an extracorporeal circuit may partly account for a reduction in inflammatory responses.