Characteristics of macrophage aggregates prepared by rotation culture and their response to polymeric materials.

Understanding the interaction between macrophages and biomaterials is important for the creation of new biomaterials and the development of technologies to control macrophage function. Since macrophages are strongly adhesive, caution is required when performing in vitro evaluations. Similarly, when THP-1 cells, macrophage precursor cells, are differentiated into macrophages using phorbol-12-myristate-13-acetate (PMA), it becomes difficult to detach them from the adherent substrate, which has been a problem on investigation of immunological responses to biomaterials. In this study, the interaction of THP-1 cell-differentiated macrophages with biomaterials was analyzed based on a new method of seeding THP-1 cells. THP-1 cells were cultured in static and rotation culture without and with PMA. In undifferentiated THP-1 cells, there was no change in cellular function between static and rotation cultures. In rotation culture with PMA, THP-1 cells differentiated and formed macrophage aggregates. IL-1ß and MRC1 expression in macrophage aggregates was examined after differentiation and M1/M2 polarization. Macrophage aggregates in rotation culture tended to be polarized toward M2 macrophages compared with those in static culture. In the evaluation of the responses of macrophage aggregates to several kinds of polymeric materials, macrophage aggregates showed different changes in MRC1 expression over time at 30, 50, and 70 rpm. Rotation speed of 30 rpm was considered most appropriate condition in that it gave stable results with the same trend as obtained with static culture. The use of macrophage aggregates obtained by rotational culture is expected to provide new insights into the evaluation of inflammatory properties of biomaterials.

Therapeutic Effects of Albumin-Fused BMP7 on 2 Experimental Models of Liver Fibrosis.

Despite the fact that liver fibrosis is an intractable disease with a poor prognosis, effective therapeutic agents are not available. In this study, we focused on bone morphogenetic factor 7 (BMP7) that inhibits transforming growth factor (TGF)-ß signaling, which is involved in liver fibrosis. We prepared an albumin-fused BMP7 (HSA-BMP7) that is retained in the blood and evaluated its inhibitory effect on liver fibrosis. Bile duct ligated mice were used as an acute liver fibrosis model, and carbon tetrachloride-induced liver fibrosis mice were used as a chronic model. All mice were administered HSA-BMP7 once per week. In the mice with bile duct ligation, the administration of HSA-BMP7 significantly suppressed the infiltration of inflammatory cells, the area of fibrosis around the bile duct, and decreased in the level of hydroxyproline as compared with saline administration. The mRNA expression of TGF-ß and its downstream fibrosis-associated genes (α-SMA and Col1a2) were also suppressed by the administration of HSA-BMP7. In the carbon tetrachloride-induced liver fibrosis mice, the HSA-BMP7 administration significantly decreased the hepatic fibrosis area and the level of hydroxyproline. Based on these results, it appears that HSA-BMP7 has the potential for serving as a therapeutic agent for the treatment of liver fibrosis.

Engineering of a Long-Acting Bone Morphogenetic Protein-7 by Fusion with Albumin for the Treatment of Renal Injury.

The bone morphogenetic protein-7 (BMP7) is capable of inhibiting TGF-ß/Smad3 signaling, which subsequently results in protecting the kidney from renal fibrosis, but its lower blood retention and osteogenic activity are bottlenecks for its clinical application. We report herein on the fusion of carbohydrate-deficient human BMP7 and human serum albumin (HSA-BMP7) using albumin fusion technology and site-directed mutagenesis. When using mouse myoblast cells, no osteogenesis was observed in the glycosylated BMP7 derived from Chinese hamster ovary cells in the case of unglycosylated BMP7 derived from Escherichia coli and HSA-BMP7. On the contrary, the specific activity for the Smad1/5/8 phosphorylation of HSA-BMP7 was about 25~50-times lower than that for the glycosylated BMP7, but the phosphorylation activity of the HSA-BMP7 was retained. A pharmacokinetic profile showed that the plasma half-life of HSA-BMP7 was similar to that for HSA and was nearly 10 times longer than that of BMP7. In unilateral ureteral obstruction mice, weekly dosing of HSA-BMP7 significantly attenuated renal fibrosis, but the individual components, i.e., HSA or BMP7, did not. HSA-BMP7 also attenuated a cisplatin-induced acute kidney dysfunction model. The findings reported herein indicate that HSA-BMP7 has the potential for use in clinical applications for the treatment of renal injuries.

Optimization of Islet Microencapsulation with Thin Polymer Membranes for Long-Term Stability.

Microencapsulation of islets can protect against immune reactions from the host immune system after transplantation. However, sufficient numbers of islets cannot be transplanted due to the increase of the size and total volume. Therefore, thin and stable polymer membranes are required for the microencapsulation. Here, we undertook the cell microencapsulation using poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) and layer-by-layer membrane of multiple-arm PEG. In order to examine the membrane stability, we used different molecular weights of 4-arm PEG (10k, 20k and 40k)-Mal to examine the influence on the polymer membrane stability. We found that the polymer membrane made of 4-arm PEG(40k)-Mal showed the highest stability on the cell surface. Also, the polymer membrane did not disturb the insulin secretion from beta cells.