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.

We investigated the effectiveness of the application of inbred adipose-derived stromal cells (IR-ASCs) in high inbred rat plasma (IRP) (6%)-Dulbecco modified Eagle medium (DMEM) gel with fragmin/protamine microparticles (F/P MPs) (IR-ASCs + IRP-DMEM gel + F/P MPs) on wound healing in streptozotocin-induced diabetic rats. F/P MPs have previously been used as a cell carrier for IR-ASCs in inbred Fisher 344 rats and for preservation and controlled release of various cytokines in IRP-DMEM gel. We applied IR-ASCs + IRP-DMEM gel + F/P MPs to full-thickness skin excisions on the backs of the diabetic rats. The statistical significance of wound closure was evaluated on postwounding days 3, 7, 10, and 14, and the skin area surrounding the wound was removed for histological examination on days 7 and 14. The wound closure rate and histological examination of wounds treated with IR-ASCs + IRP-DMEM gel + F/P MPs demonstrated significantly advanced epithelialization, capillary formation, and granulation tissue formation. When DiI-labeled IR-ASCs + IRP-DMEM gel + F/P MPs were applied to full-thickness skin wounds on the backs of the diabetic rats, histological observation at 2 weeks showed appearances of both DiI-labeled granulation tissue and CD31-immunostained microvessels in the transplant areas. A portion of the transplanted IR-ASCs + IRP-DMEM gel + F/P MPs had been taken up into the granulation tissues to promote wound healing. Thus, IR-ASCs + IRP-DMEM gel + F/P MPs were effective for repairing healing-impaired wounds such as those arising in the diabetic rats.

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Fragmin/protamine microparticles (F/P MPs) have been used as carriers for the preservation and activation of cytokines in human plasma (HP)-Dulbecco's modified Eagle's medium (DMEM) gels. This study investigated a three-dimensional (3D) culture system using an HP-DMEM gel with 0.1 mg/mL F/P MPs and 5 ng/mL FGF-2 for the proliferation of human dermal fibroblast cells (DFCs), human microvascular endothelial cells (MVECs) and human coronary smooth muscle cells (SMCs), or 5 ng/mL interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (GM-CSF) for a human hematopoietic cell line (TF-1 cells). DFCs, MVECs, SMCs and TF-1 cells grew rapidly under 3D culture conditions using a low-concentration HP (2 %)-DMEM gel with F/P MPs and FGF-2 (for DFCs, MVECs and SMCs) or IL-3/GM-CSF (for TF-1 cells) at doubling times of 22, 23, 25 and 18 h, respectively, without the use of animal serum, compared to under 2D culture conditions using low-concentration human serum (2 %)-DMEM with 5 ng/mL FGF-2 or IL-3/GM-CSF on F/P MP-coated plates at doubling times of approximately 26, 25, 40 and 20 h, respectively.

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