The periosteum, a membrane covering most bone surfaces, plays a vital role in bone repair due to its rich composition of osteogenic cells, fibroblasts, blood vessels, and nerve endings, and extracellular matrices (ECMs) like collagens and glycosaminoglycans. Periosteum-derived cells (PDCs), including multipotent mesenchymal stromal cells (MSCs), are crucial for bone, cartilage, and marrow generation. Specifically, leptin receptor-positive (LepR+) cells, expressing CD140α (PDGFR-α) and CD105, are key MSC subpopulations involved in bone and adipocyte formation. Type H blood vessels (CD31 and endomucin positive) are essential for coupling angiogenesis and osteogenesis during bone development and repair. Given the periosteum's remarkable bone regenerative capacity, creating periosteum-like tissue (PT) is a promising strategy for bone repair. Current methods for generating PT, such as the induced membrane (IM) technique, involve implantation of a cement spacer (e.g., polymethylmethacrylate) to induce tissue formation followed by autologous bone grafting, but are limited by long incubation times and complex procedures. Other methods using synthetic membranes with or without exogenous cells have limited regenerative capacity and require intensive manipulation. Previous studies showed that human periosteal cell-loaded scaffolds induce ectopic bone formation and that BMP-2 induces heterotopic ossification (HO) from recruited multipotent stromal cells and type H vessels. This study aimed to construct PT *in vivo* using a BMP-2-initiated endochondral ossification strategy, leveraging the body's natural regenerative capacity to create a multipotent stromal cell-rich and vascularized PT.

The literature extensively documents the periosteum's importance in bone regeneration. Studies highlight the various cell types within the periosteum and their contributions to bone repair. Research also underscores the role of specific blood vessel types in coordinating angiogenesis and osteogenesis. Existing methods for creating periosteum-like tissue are reviewed, revealing limitations in terms of efficiency, complexity, and regenerative capacity. Previous work exploring BMP-2's role in bone formation and heterotopic ossification provides the foundation for the current study's approach.

The study used a three-pronged approach involving *in vivo* tissue generation, characterization of the generated tissue, and functional assays to evaluate regenerative potential. First, PBS-, BMP-2-, and BMP-2/CS-loaded gelatin scaffolds were subcutaneously implanted into Lepr-cre; tdTomato mice (to label PDCs). The resulting tissues were analyzed histologically (H&E, Safranin O/Fast Green, TRAP staining) and immunofluorescence (periostin, LepR, Osterix, Aggrecan). Flow cytometry was used to quantify LepR+ cells and PTDCs (CD45-, Ter119-, CD31-, CD140a+, CD105+). CFU-F assays assessed PTDC self-renewal. qPCR analysis measured gene expression (Sox9, Acan, Runx2, OPN, CXCL12, SCF) in PTDCs. Allogeneic transplantation assays using critical-sized calvarial defects assessed the osteogenic capacity of the transplanted PTs, utilizing histological and immunofluorescence analysis. Autologous transplantation assays, conducted in both young and old mice, further assessed the PTs' regenerative capacity, employing µCT imaging (BV/TV, BMD) and histological analyses. The scaffolds' porous structures were characterized via FE-SEM.

The study demonstrated that BMP-2-loaded scaffolds successfully generated PTs *in vivo* through endochondral ossification. Histological and immunofluorescence analyses confirmed the PTs' periosteum-like architecture, rich in PTDCs, blood vessels (including type H vessels), and osteochondral progenitor cells. Flow cytometry revealed a significantly higher fraction of LepR+ cells in the BMP-2 and BMP-2/CS groups compared to the control group. The BMP-2/CS group showed a higher fraction of undifferentiated PTDCs and a greater number of CFU-F colonies, indicating enhanced self-renewal capacity. qPCR analysis demonstrated significantly higher expression of chondrogenic, osteogenic, and stem cell maintenance genes in the BMP-2/CS group. Allogeneic transplantation showed that transplanted PTs from the BMP-2 and BMP-2/CS groups differentiated into bone tissue. Autologous transplantation assays in young mice demonstrated enhanced osteogenesis and osseointegration in the BMP-2 and BMP-2/CS groups at 6 weeks, with the BMP-2/CS group showing superior BV/TV ratio compared to other groups. In older mice, BMP-2 and BMP-2/CS-induced PTs still effectively repaired critical-sized bone defects, with the BMP-2/CS group again outperforming others in BV/TV and BMD. In both young and old mice, BMP-2/CS PTs demonstrated complete integration with host bone, unlike the partial integration seen in the BMP-2 group. In both transplantation assays, the BMP-2/CS group demonstrated increased osteoprogenitors compared to the BMP-2 group.

This study introduces a novel approach to generating PTs *in vivo*, mimicking developmental processes. The successful generation of PTs rich in PTDCs and functional blood vessels addresses the limitations of previous methods. The use of BMP-2 and the synergistic effect of CS highlight the potential for modulating PT properties through biomaterial manipulation. The enhanced osteogenesis observed in the BMP-2/CS group is likely due to increased PTDC abundance and function, possibly resulting from prolonged BMP-2 activity and increased calcium binding facilitated by CS's negative charge. The increased expression of CXCL12 might also contribute to the recruitment of more progenitor cells. The study's success in older mice is particularly significant, suggesting the potential for treating age-related bone loss. Further investigation is needed to fully elucidate the underlying cellular and molecular mechanisms and optimize the approach.

This study demonstrates a promising novel method for creating functional periosteum-like tissue *in vivo* using BMP-2-loaded scaffolds, enhanced by CS. The generated PTs showed significant bone regenerative capacity, particularly in older mice. Further research should explore other biomaterials and growth factors, optimize the parameters, and delve deeper into the molecular mechanisms underlying the synergistic effect of BMP-2 and CS. This approach holds substantial promise for clinical translation in treating critical-sized bone defects, especially in elderly patients.

The study used a mouse model, and the findings may not directly translate to human applications. The long-term effects of the generated PTs and the potential for adverse effects require further investigation. The specific molecular mechanisms underlying the enhanced regenerative capacity of CS still need to be fully elucidated. The study focused on calvarial defects, and further research is necessary to determine the efficacy of this technique for other bone defects.