The objective of this study was to fabricate an acellular sheep periosteum and explore its potential application in guided bone regeneration. Sheep periosteum was collected and decellularized by a modified decellularization protocol. The effectiveness of cell removal was proved by hematoxylin and eosin and 4 0 ,6-diamidino-2-phenylindole staining, DNA quantitative test, and agarose gel electrophoresis. After decellularization, its microstructure was found to become more porous while the integrality of collagen fibers remained undamaged, and the contents of collagen and glycosaminoglycan were not decreased significantly. Biomechanical analysis showed that the elastic modulus was significantly declined, while the yield stress was not affected, probably due to the collagen integrality. In vitro study of CCK-8 assay demonstrated that the acellular periosteum not only had no toxic effect to the MC3T3-E1 cells, but benefited the cell proliferation to some degree. In vivo experiment of guided bone regeneration was performed using a rabbit cranial model.Micro-CT and histological results revealed that the acellular periosteum not only effectively prevented the ingrowth of fibrous connective tissues, but also potentially facilitated bone regeneration. In conclusion, acellular sheep periostea, with wider sources, less costs, and more convenient fabrication process, would have great potential in the employment for guided bone regeneration. K E Y W O R D S acellular periosteum, animal experiment, barrier membrane, extracellular matrix, guided bone regeneration

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This study addresses the fabrication of an extracellular matrix material of the acellular sheep periosteum and the systematic evaluation of its biocompatibility to explore its potential application in guided bone regeneration. Sheep periosteum was harvested and decellularized by a combined decellularization protocol. The effectiveness of cell removal was proved and residual α-Gal antigen was also quantitatively detected. Then, mouse MC3T3-E1 cells were seeded onto the acellular periosteum. A scanning electron microscope (SEM) was used to record the whole process of cell adhesion. The CCK-8 assay suggested that the acellular periosteum not only had zero toxic effect on pre-osteoblasts, but played a positive role in cell proliferation. We also tested whether the acellular periosteum possesses favorable osteogenesis induction activity using an alkaline phosphatase (ALP) assay and a quantitative real-time PCR (Col I, Runx2, OCN) assay. An in vivo study of a subcutaneous implantation test using Sprague Dawley (SD) rats was performed to detect the changes in IL-2, IFN-γ and IL-4 in serum and elucidate the host’s local response to acellular periosteum through hematoxylin and eosin (HE) and immunohistochemical staining. The results show that acellular sheep periosteum did not elicit a severe immunogenic response via the Th1 pathway, unlike fresh sheep periosteum. In conclusion, acellular sheep periosteum possesses favorable biocompatibility to be employed for guided bone regeneration.

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Nerve regeneration can be promoted using nerve guide conduits (NGCs). Carbon nanotubes (CNTs) are often used to prepare conductive NGCs, however, the major concern for their applications is the final location of the implanted CNTs in vivo. Herein, photoluminescent multiwalled CNTs (MWCNTs) were prepared and electrospun with poly(lactide-co-glycolide) (PLGA), followed by shaping into multichannel NGCs for repairing of injured rat sciatic nerve, thereby the distribution of CNTs in vivo could be detected via bioimaging. Photoluminescent MWCNTs (MWCNT-FITC) were prepared by functionalization with poly(glycidyl methacrylate) (PGMA) and fluoresceinisothiocyanate-isomer I (FITC) subsequently. The conductivity of the PLGA/ MWCNT-FITC fibers was approx. 10 −4 S/cm at 3 wt % MWCNTs. Compared with PLGA fibers, Schwann cells on PLGA/MWCNT-FITC fibers matured at a faster rate, accordingly, nerve regeneration was promoted by the PLGA/ MWCNT-FITC NGC. With a confocal laser scanning microscope and small-animal imaging system, the location of MWCNTs was detected. Alongside the degradation of PLGA, MWCNTs intended to aggregate and were entrapped in the regenerated nerve tissue without migrating into surrounding tissues and other organs (liver, kidneys, and spleen). This study provides a useful characterization method for MWCNTs and the guidance for in vivo applications of MWCNTs in tissue engineering.