The periosteum plays a key role in bone regeneration and an artificial bionic material is urgently required. The periostea on the tibia and skull differ with respect to the types of cells, microstructure, and components, leading to different biological functions and biomechanical properties. We aimed to prepare decellularized periosteum scaffolds derived from different origins and evaluate their angiogenic and osteogenic activities. Histological assessment of α-smooth muscle actin, bone morphogenetic protein-2, and alkaline phosphatase in tibial and calvarial periosteum tissues provided preliminary information on their differing angiogenic and osteogenic properties. We developed decellularization protocols to completely remove the periosteum cellular components and for good maintenance of the hierarchical multilayer structures and components of the extracellular matrix (ECM) with no cytotoxicity. Moreover, using a chicken egg chorioallantoic membrane assay and a nude mouse implantation model, we found that tibia-derived periosteum ECM had superior osteogenic activity and calvarium-derived ECM had good angiogenic activity. The preliminary mechanisms of differing activities were then evaluated by osteogenesis- and angiogenesis-related gene expression in human umbilical vein endothelial cell- and MC-3T3 cell-seeded ECM scaffolds. Thus, this study provides periosteum biomaterials that are derived from specific tissues and have different functional properties and structures, for use in bone regeneration.

The increasing rate of injuries to the meniscus indicates the urgent need to develop effective repair strategies. Irreparably damaged menisci can be replaced and meniscus allografts represent the treatment of choice; however, they have several limitations, including availability and compatibility. Another approach is the use of artificial implants but their chondroprotective activities are still not proved clinically. In this situation, tissue engineering offers alternative natural decellularized extracellular matrix (ECM) scaffolds, which have shown biomechanical properties comparable to those of native menisci and are characterized by low immunogenicity and promising regenerative potential. In this article, we present an overview of meniscus decellularization methods and discuss their relative merits. In addition, we comparatively evaluate cell types used to repopulate decellularized scaffolds and analyze the biocompatibility of the existing experimental models. At present, acellular ECM hydrogels, as well as slices and powders, have been explored, which seems to be promising for partial meniscus regeneration. However, their inferior biomechanical properties (compressive and tensile stiffness) compared to natural menisci should be improved. Although an optimal decellularized meniscus scaffold still needs to be developed and thoroughly validated for its regenerative potential in vivo, we believe that decellularized ECM scaffolds are the future biomaterials for successful structural and functional replacement of menisci.

Objective To explore an optimal protocol for the fabrication of a decellularized tibia periosteum biological scaffold.Methods Seventy healthy New Zealand white rabbits were selected,and both proximal tibia periosteums,namely 140 specimens,were collected.Specimens were divided into three groups and they were processed using the following three methods respectively.Method 1,specimens were freeze-thawed (-80 ℃,24 h),2％ Triton-X100 mixed with 3.5x 10-5 mol/L PMSF acellular solution for 18 h,10 g/L SDS for 12 h and finally digested with enzyme (DNase and RNase).Method 2,it's similar with method 1,but the processing time of SDS was 6 h.Method 3,it's similar with method 1,but the process of 10 g/L SDS for 12 h is replaced by the process of 0.5 mol/L NaC1 for 12 h.Scaffolds fabricated using the three methods above,as well as the normal tibia periosteum,were characterized by HE staining,DAPI staining and quantitative analysis of genomic DNA to observe cellular structure and measure DNA content,which would be compared with standard concentration 50 ng/mg in decellularization; characterized using Safranin O staining,Masson staining and the hydroxyproline measurement to measure the retain of the main components (collagen and glycosaminoglycan) in the ECM of periosteum qualitatively and quantitatively; characterized using scanning electron microscope to observe the micro structure on the surface of the scaffolds; characterized histologically using subcutaneous embedding test to observe immunological rejection like the infiltration of inflammatory cells,and histological remodeling caused by scaffolds.Resuits After the treatment by method 1,2 and 3,HE staining showed the complete removal of cells in the scaffolds.DAPI staining showed the absence of nucleus or nucleic debris.Quantitative analysis of DNA indicated that all the acellular ratios reached 95％ and the contents were less than 50 ng/mg.Compared with method 1 and 2,the reserve of the main components (collagen 36.94±0.70 μg/mg and glycosaminoglycan) in acellular scaffolds processed with method 3 were more complete,and the collagen fibers were in good condition without breakage under the SEM.Furthermore,the infiltration of inflammatory cells after the allogenic embedding test was less,and the immunological rejection was not obvious with preferable degradation of scaffolds and histological remodeling.Conclusion An acellular tibia periosteum scaffold fabricated using method 3 showed more complete reservation of the structure and components of ECM,with minor immunological rejection and favorable biocompatibility.

目的制备兔骨膜去细胞生物支架,为骨缺损、骨不愈的组织工程研究提供天然的生物支架材料。方法取健康新西兰大白兔,游离双侧胫骨近端内侧骨膜,通过物理冻融（-80℃,24h）、去污剂洗脱（triton-X 100、SDS）和酶消化（DNA酶、RNA酶）获取骨膜去细胞生物支架。通过HE染色、DAPI染色、琼脂糖电泳和基因组DNA定量分析（n=5）测定细胞结构及DNA成分残留;Masson染色和羟脯氨酸测定法（n=6）定性定量检测骨膜细胞外基质的主要成分（胶原）的保留情况;扫描电子显微镜下观察骨膜去细胞生物支架的表面微结构;CCK8法检测支架浸提液毒性;皮下包埋实验（n=4）观察该支架的免疫排斥反应。结果 HE染色和4’,6-二脒基-2-苯基吲哚（DAPI）染色表明去细胞支架无残留细胞;琼脂糖电泳未见明显DNA条带;DNA定量检测显示组织去细胞率达95%以上;Masson染色及羟脯氨酸测定表明去细胞支架胶原成分被保留;扫描电子显微镜下细胞外基质呈现三维网状疏松结构;不同体积分数的浸提液对骨膜细胞的增殖与对照组（普通培养基）比较无明显抑制作用（P＞0.05）;异体皮下包埋实验显示,该去细胞支架免疫排斥反应不明显。结论运用物理冻融、去污剂洗脱和酶消化等方法所获取的骨膜去细胞生物支架细胞去除彻底,细胞外基质的结构及主要成分保留完好,生物相容性良好。

Bone grafting or bone substitute is typically used to bridge a bone defect that has been caused by trauma, tumor resection, pathological degeneration, or congenital deformations. However, bone graft healing and remodeling is always a major concern of orthopedic surgeons. Because the periosteum has a remarkable regenerative capacity and is widely recognized to be essential for the initiation of bone graft healing and remodeling, the present study aimed to produce a rabbit decellularized periosteum (D-periosteum) to be used as a biologic scaffold for future bone tissue engineering. We obtained the D-periosteum by employing a combination of commonly used decellularization processes, which include physical methods as well as chemical and enzymatic solutions. The cellular components were effectively removed, and this removal was demonstrated using current decellularization criteria (H&E staining, DAPI staining, DNA quantification and agarose gel electrophoresis); however, there were no significant alterations of the native extracellular matrix (ECM) properties (collagen, glycosaminoglycan (GAG), microarchitecture and mechanical properties). Periosteum-derived cells (PDCs) could adhere, proliferate and infiltrate into the D-periosteum in vitro. The allogenic D-periosteum was implanted subcutaneously into the backs of rabbits over 28 days to study the biocompatibility in vivo. The D-periosteum did not elicit a severe immunogenic response. In summary, a biologic scaffold composed of ECM from periosteum has been successfully developed. The D-periosteum maintains biocompatibility in vitro and in vivo and, therefore, can provide a naturally compatible scaffold for use in future bone tissue engineering.