Rejuvenating Age-impaired Fracture Healing

Skeletal aging and disease are associated with a misbalance in the opposing actions of osteoblasts and osteoclasts that are responsible for maintaining the integrity of bone tissues. Here, we show through detailed functional and single-cell genomic studies that intrinsic aging of bona fide mouse skeletal stem cells (SSCs) alters bone marrow niche signaling and skews bone and blood lineage differentiation leading to fragile bones that regenerate poorly. Aged SSCs have diminished bone and cartilage forming potential but produce higher frequencies of stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell transcriptomic studies reveal a distinct population of SSCs in aged mice that gradually outcompete their younger counterparts in the bone marrow niche. While systemic exposure to a youthful circulation through heterochronic parabiosis reduced local expression of inflammatory cytokines, it did not reverse the diminished osteochondrogenic activity of aged SSCs and was insufficient to improve bone mass and skeletal-healing parameters in aged mice. Hematopoietic reconstitution of aged mice with young hematopoietic stem cells (HSC) also did not improve bone integrity and repair. We find that deficient bone regeneration in aged mice could only be reversed by the local application of a combinatorial treatment that re-activates aged SSCs and simultaneously abates crosstalk to hematopoietic cells favoring an inflammatory milieu. This treatment expanded aged SSC pools, reduced osteoclast activity, and enhanced bone healing to youthful levels. Our findings provide mechanistic insight into the complex, multifactorial mechanisms underlying skeletal aging and offer new prospects for rejuvenating the aged skeletal system. Overall design: 10X Genomics single cell RNA-sequencing of fracture calluses from 24-months old mice at day-10 after stabilized bi-cortical fracture induction. Fracture callus tissue was harvested from 24-mo mice at day-10 after injury. Three fracture calluses treated with hydrogels containing PBS at day of injury and three fractures with hydrogels containing anti-Csf1/BMP2 were processed, digested, and prepared for FACS as described above. Single cell solutions of each treatment group were then pooled (n=3 per group) and 2x105 PI-Ter119- cells were sorted into collection tubes containing FACS buffer. Cells were then processed with 10X Chromium Next GEM Single Cell 3' GEM kit (10X Genomics Inc, v3.1) according to manufacturer's instruction.