1. Gel/Glc Heat 24hCTS/Gel GTA 48h Gelatin GTA 48h CTS/Gel/Glc Heat 24h Severe Imflam Slight Imflam Rat subcutaneous test Combination use with FGF2 FGF2 (Fibrast spray) 50µg/piece Cell ingrowth Combination use with FGF2 FGF2 (Fibrast spray) 50µg/pieceBone augmentation Rat bone forming test Marked degradation Slight effect まとめ Gel/Glc Heat 24h CTS/Gel/Glc Heat 24h Gel/Glc Heat 24h CTS/Gel/Glc Heat 24h Cyto compatibility (SEM) Gelatin GTA 48h Gel/Glc Heat 24h CTS/Gel/Glc Heat 24h FGF2 stimulated Cells were attached to the surface of scaffolds CTS/Gel GTA 48h

2. Gel/Glc Heat 24h We found that cells were attached and spread on scaffold surface.

3. Scaffold; 8×8×6 mm CTS/Gel/GlcNAc scaffold + FGF2 ; 50 µg (Fiblast spray, Kaken Pharmaceutical, Tokyo, Japan) Following a skin incision, a flap was made in the scalp. Decortication of a 4 mm 2 area was performed in front of the coronal suture in the cranial bone using a rotating round bur under water irrigation. Subsequently, one of two types of sample was placed on the cranial bone with decortication (Fig): Gel/GlcNac and CTS/Gel/GlcNac scaffold. Scaffolds were loaded with FGF2 (50µg). As a control, no implantation was performed. Skin flaps were sutured and tetracycline hydrochloride ointment (Achromycin Ointment, POLA Pharma, Tokyo, Japan) was applied to the wound. Rats were euthanized 10 days after surgery using an overdose of sodium pentobarbital and specimens were collected from the wound. Six µm sections located every 300 µm, including the cranial bone and surrounding soft tissue, were prepared. Sections were stained with HE and examined using light microscopy. Wistar rats were used (N=4). The experimental protocol followed the institutional animal use and care regulations of Hokkaido University (Animal Research Committee of Hokkaido University, Approval No. 10-42).

4. Gel/Glc Heat 24h +FGF S NB S PB NB; new bone S; scaffold PB; preexisting bone Implantation of Gel/GlcNAc scaffold frequently promoted bone augmentation. Newly formed bone included osteoblastic cells, osteocyto- like cells and bone marrow. Residual scaffold was degraded by macrophage-like cells phagocytosis. 500µm 100µm

5. CTS/Gel/Glc Heat 24h +FGF NB; new bone PB; preexisting bone NB Marked inflammatory response was exhibited after implantation of CTS/Gel/GlcNAc scaffold. The result is similar to that of rat subcutaneous test. Newly formed bone was slightly formed. Scaffold was degraded and replaced by connective tissue. 500µm 100µm

6. Histomorphometric measurements from each scaffold. The newly formed bone area was measured in each stained section collected 10 days post-surgery using a software package (Image J 1.41, National Institute of Health, Bethesda, MD, USA). The means and standard deviations were calculated for each group. Statistical analysis was performed using the Scheffé test. P- values <0.05 were considered statistically significant. All statistical procedures were performed using a software package (SPSS Japan, DR. SPSS 11.0). * * * p<0.05 vs control ** p<0.05 vs CTS ** Bone area of the Gel/Glc scaffold with FGF2 was significantly greater than CTS/Gel/Glc with FGF2 and control.