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Traumatic brain injury (TBI) often leads to critical tissue damage and loss. Brain tissue engineering is a promising technique for cellular replacement and neurological functional recovery in animals. However, useful biomaterial scaffolds have not been found so far. In order to explore the feasibility of stem cell based brain tissue engineering, it is first necessary to evaluate the biocompatibility of biomaterials with stem cells and brain tissues. In the present study, two biomaterials, chondrocyte derived ECM (ECM) and silk fibroin (SF) scaffolds, were investigated for their biocompatibility in vitro and in acute TBI. We found that when the ECM scaffold was cultured with rat mesenchymal stem cells (rMSCs) or implanted directly into brain injury, had a good biocompatibility. rMSCs, seeded on the ECM scaffold, were evenly distributed with a spindle shape and attached well, while on SF scaffold attached with a round shape relatively weakly. In the TBI model, the SF scaffold resulted in a significant increase in the cavity volume at 6 weeks postimplantation, but not the ECM scaffold. In addition, the ECM scaffold induced a lower inflammatory response, with active microglia/macrophages at 1 week, than the SF scaffold and control. Interestingly, both scaffolds reduced glial fibrillary acidic protein (GFAP) and neurocan expression in the cavity boundary throughout the experimental periods. These results indicate that the scaffolds tested inhibit the glial scar formation in injured brain tissue. Moreover, there were no significant differences in the neurological outcome and weight body gain, when compared with the brain injury alone. Taken together, these findings reveal that the ECM scaffold showed good biocompatibility to the donor rMSCs and host brain tissue. Thus, the ECM scaffold seems to be beneficial as a biomaterial for brain tissue engineering.