Construction of developmentally inspired periosteum-like tissue for bone regeneration

The periosteum, a highly vascularized thin tissue, has excellent osteogenic and bone regenerative abilities. The generation of periosteum-mimicking tissue has become a novel strategy for bone defect repair and regeneration, especially in critical-sized bone defects caused by trauma and bone tumor resection. Here, we utilized a bone morphogenetic protein-2(BMP-2)-loaded scaffold to create periosteum-like tissue(PT) in vivo, mimicking the mesenchymal condensation during native long bone development. We found that BMP-2-induced endochondral ossification plays an indispensable role in the construction of PTs. Moreover, we confirmed that BMP-2-induced PTs exhibit a similar architecture to the periosteum and harbor abundant functional periosteum-like tissue-derived cells(PTDCs), blood vessels, and osteochondral progenitor cells. Interestingly, we found that the addition of chondroitin sulfate(CS), an essential component of the extracellular matrix(ECM), could further increase the abundance and enhance the function of recruited PTDCs from the PTs and finally increase the regenerative capacity of the PTs in autologous transplantation assays, even in old mice. This novel biomimetic strategy for generating PT through in vivo endochondral ossification deserves further clinical translation.

Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

Bone marrow-derived mesenchymal stem cells(BM-MSCs)play a crucial role in stem cell therapy and are extensively used in regenerative medicine research.However,current methods for harvesting BM-MSCs present challenges,including a low yield of primary cells,long time of in vitro expansion,and diminished differentiation capability after passaging.Meanwhile mesenchymal stem cells(MSCs)recovered from cell banks also face issues like toxic effects of cryopreservation media.In this study,we provide a detailed protocol for the isolation and evaluation of MSCs derived from in vivo osteo-organoids,presenting an alternative to autologous MSCs.We used recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds to construct in vivo osteo-organoids,which were stable sources of MSCs with large quantity,high purity,and strong stemness.Compared with protocols using bone marrow,our protocol can obtain large numbers of high-purity MSCs in a shorter time(6 days vs.12 days for obtaining passage 1 MSCs)while maintaining higher stemness.Notably,we found that the in vivo osteo-organoid-derived MSCs exhibited stronger anti-replicative senescence capacity during passage and amplification,compared to BM-MSCs.The use of osteo-organoid-derived MSCs addresses the conflict between the limitations of autologous cells and the risks associated with allogeneic sources in stem cell transplantation.Consequently,our protocol emerges as a superior alternative for both stem cell research and tissue engineering.