Preparation and Evaluation of Tibia- and Calvarium-Derived Decellularized Periosteum Scaffolds

Abstract

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.

Preparation and Characterization of Decellularized Caprine Periosteum Scaffolds for Fracture Gap Healing

“…Similar change in appearance was evidenced in aorta and esophagus after decellularization with 5% SPE (Goyal et al, 2021;2022 ). Zhang et al (2017) also found that the color of the NTP (native tibial periosteum) and NCP (native calvarial periosteum) samples changed from red to milky white after the decellularization treatement with Triton X 100 (1% v/v) for 12 h and with 10 g/L sodium dodecyl sulfate solution for 2 h Histology Microscopic evaluation of native tissues and 5% SPE processed caprine periosteum scaffolds at different time intervals is presented in fig. 2 and histological scoring of decellularized periosteal scaffolds is shown in table 1.…”

“…Decellularized layer of the periosteum had increased porosity as the gaps between the collagenous fiber bundles became a bit wider in fibrous layer of periosteum which was similar to the finding of He et al (2020). Dense massive bundles of collagen were noted in the fibrous layer of the tibial periosteum at native stage (Zhang et al, 2017). Increased porosity was due to washing out of most of cell components from their compartment leaving behind the collagen material.…”

“…Hematoxylin and eosin (H&E) staining of decellularized periosteum demonstrated a well reserving pink-staining collagen and a well reserving blue-staining collagen was noticed in Masson's trichrome stained decellularized periosteal tissues (Lin et al, 2018). The integrity and continuity of collagen fibers in the decellularized tibial periosteum (DTP) were well arranged (Zhang et al, 2017). There was no obvious difference in the ECM collagen structure and, distribution between FP (fresh periosteum) and AP (acellular periosteum) indicates that the decellularization process does not remarkably destroy the collagen structure (He et al, 2020).…”

“…An absence of staining indicated the absence of DNA and thus cells in decellularized periosteum (Chen et al, 2015). DAPI staining of decellularized tibial periosteum (DTP) also showed hardly no nuclear material remained after decellularization (Zhang et al, 2017)…”

Applications of decellularized extracellular matrix in bone and cartilage tissue engineering

“…ECM ornamentation thus primes the scaffold for better cell attachment and more robust cell growth/proliferation . This process has been demonstrated on multiple types of scaffolds, such as 3D‐printed scaffolds, electrospun scaffolds, and decellularized tissue …”