Sulfated Polysaccharide Regulates the Homing of HSPCs in a BMP-2-Triggered In Vivo Osteo-Organoid.

Hematopoietic stem cell transplantation (HSCT) is a well-established method for a variety of acquired and congenital diseases. However, the limited number and sources of therapeutic hematopoietic stem/progenitor cells (HSPCs) hinder the further application of HSCT. A BMP-2 triggered in vivo osteo-organoid that is previously reported, serves as a kind of stem cell biogenerator, for obtaining therapeutic HSPCs via activating the residual regenerative capacity of mammals using bioactive biomaterials. Here, it is demonstrated that targeting the homing signaling of HSPCs elevates the proportions and biological functions of HSPCs in the in vivo osteo-organoid. Notably, it is identified that sulfonated chito-oligosaccharide, a degradation product of sulfonated chitosan, specifically elevates the expression of endothelial protein C receptor on HSPCs and vascular cell adhesion molecule-1 on macrophages in the in vivo osteo-organoid, ultimately leading to the production of adequate therapeutic HSPCs. This in vivo osteo-organoid approach has the potential to provide an alternative HSPCs source for HSCT and benefits more patients.

A BMP-2-triggered in vivo osteo-organoid for cell therapy.

Cell therapies and regenerative medicine interventions require an adequate source of therapeutic cells. Here, we demonstrate that constructing in vivo osteo-organoids by implanting bone morphogenetic protein-2-loaded scaffolds into the internal muscle pocket near the femur of mice supports the growth and subsequent harvest of therapeutically useful cells including hematopoietic stem/progenitor cells (HSPCs), mesenchymal stem cells (MSCs), lymphocytes, and myeloid cells. Profiling of the in vivo osteo-organoid maturation process delineated three stages-fibroproliferation, osteochondral differentiation, and marrow generation-each of which entailed obvious changes in the organoid structure and cell type distribution. The MSCs harvested from the osteochondral differentiation stage mitigated carbon tetrachloride (CCl4)-induced chronic liver fibrosis in mice, while HSPCs and immune cells harvested during the marrow generation stage rapidly and effectively reconstituted the impaired peripheral and solid immune organs of irradiated mice. These findings demonstrate the therapeutic potentials of in vivo osteo-organoid-derived cells in cell therapies.

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.

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.

Generation of rhBMP-2-induced juvenile ossicles in aged mice.

Critical-sized bone defects and nonunions following fracture are common among elderly patients and severely reduce the quality of life. Dysfunctional senescent endothelial and mesenchymal stromal cells (MSCs) inhibit bone defect repair. Here we provide a method to obtain surrogate vascularized juvenile bone by subcutaneous implantation of recombinant human bone morphogenic protein-2 (rhBMP-2)-loaded absorbable gelatin scaffolds. RhBMP-2-induced ossicles showed fewer senenscent MSCs whereas much more type H blood vessels (strongly positive for CD31 and endomucin (Emcn)) and osteoprogenitor cells than native bone (femur and tibiae) even in old mice. Treatment with this juvenile ossicles improved the regenerative capacity in critical-sized cranial defects versus standard treatment in both young and old mice. Furthermore, ossicles with custom size shape were obtained by 3D-printing for irregular bone defects repair. These customizable juvenile ossicles developed in aged individuals provide an alternative to resecting native bone in autologous bone transplantation, with superior regenerative efficacy in elderly patients due to their juvenile phenotype.