Age-related osteoporosis is characterized by the deterioration in bone volume and strength, partly due to the dysfunction of bone marrow mesenchymal stromal/stem cells (MSCs) during aging. Alpha-ketoglutarate (αKG) is an essential intermediate in the tricarboxylic acid (TCA) cycle. Studies have revealed that αKG extends the lifespan of worms and maintains the pluripotency of embryonic stem cells (ESCs). Here, we show that the administration of αKG increases the bone mass of aged mice, attenuates age-related bone loss, and accelerates bone regeneration of aged rodents. αKG ameliorates the senescence-associated (SA) phenotypes of bone marrow MSCs derived from aged mice, as well as promoting their proliferation, colony formation, migration, and osteogenic potential. Mechanistically, αKG decreases the accumulations of H3K9me3 and H3K27me3, and subsequently upregulates BMP signaling and Nanog expression. Collectively, our findings illuminate the role of αKG in rejuvenating MSCs and ameliorating age-related osteoporosis, with a promising therapeutic potential in age-related diseases.

Age-dependent bone loss has been well documented in both human and animal models. Although the underlying causal mechanisms are probably multifactorial, it has been hypothesized that alterations in progenitor cell number or function are important. Little is known regarding the properties of bone marrow stromal cells (BMSCs) or bone progenitor cells during the aging process, so the question of whether aging alters BMSC/progenitor osteogenic differentiation remains unanswered. In this study, we examined agedependent changes in bone marrow progenitor cell number and differentiation potential between mature (3 and 6 mo old), middle-aged (12 and 18 mo old), and aged (24 mo old) C57BL/6 mice. BMSCs or progenitors were isolated from five age groups of C57BL/6 mice using negative immunodepletion and positive immunoselection approaches. The osteogenic differentiation potential of multipotent BMSCs was determined using standard osteogenic differentiation procedures. Our results show that both BMSC/progenitor number and differentiation potential increase between the ages of 3 and 18 mo and decrease rapidly thereafter with advancing age. These results are consistent with the changes of the mRNA levels of osteoblast lineageassociated genes. Our data suggest that the decline in BMSC number and osteogenic differentiation capacity are important factors contributing to age-related bone loss.

Combined regimens of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) were investigated to stimulate osteogenic differentiation. In young mouse calvaria-derived cells, FGF-2 (0.16ng/mL) in combination with BMP-2 (50ng/mL) did not enhance mineralization, but in old mouse cells it resulted in more mineralization than BMP-2 alone. In young long bone mouse cultures, FGF-2 enhanced mineralization relative to BMP-2 alone, but in old cultures, lower dose of FGF-2 (0.016ng/mL) was necessary. In neonatal mouse calvarial cells, sequential delivery of low-dose FGF-2 and low-dose BMP-2 (5ng/mL) was more stimulatory than co-delivery. In young human cultures, 0.016ng/mL of FGF-2 did not enhance mineralization, in combination with 5ng/mL of BMP-2, but in older cultures, codelivery of FGF-2 and BMP-2 was superior to BMP-2 alone. In conclusion, BMP-2 treatment alone was sufficient for maximal mineralization in young osteoblast cultures. However, coadministration of FGF-2 and BMP-2 increases mineralization more than BMP-2 alone in cultures from old and young mouse long bones and old humans but not in young mouse calvarial cultures.

In orthopedics, the regeneration and repair of cartilage or bone defects after trauma, cancer, or metabolic disorders is still a major clinical challenge. Through developmental plasticity, bone marrow mesenchymal stem cells (BMSSCs) are important seed cells for the musculoskeletal tissue engineering approach. The present study sought to determine the ectopic osteogenic and chondrogenic ability of BMSSCs in combination with a scaffolding material made from alginate gel. After isolation from the bone marrow of BALB/C mice, BMSSCs were expanded in vitro and induced to chondrogenesis or osteogenesis for 14 days, respectively. Subsequently, these induced cells were seeded into alginate gel, and the constructs implanted into BALB/C nude mice subcutaneously for up to 8 weeks. In the histological analysis, the transmission electron microscopy of the retrieved specimens at various intervals showed obvious trends of ectopic cartilage or bone formation along with the alteration of the cellular phenotype. Simultaneously, the results of the immunohistochemical staining and RT-PCR both confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), SOX9, and aggrecan, or alternatively, markers for osteoid tissue, such as osteopontin (OPN), osteocalcin (OCN), and collagen type I (Col-I). During subcutaneous implantation, the elevating production of ECM and the initiation of the characteristic structure were closely correlated with the increase of time. In contrast, there was an apparent degradation and resorption of the scaffolding material in blank controls, but with no newly formed tissues. Finally, the constructs that were made of non-induced BMSSCs nearly disappeared during the 8 weeks after implantation. Therefore, it is suggested that alginate gel, which is combined with BMSSCs undergoing differentiation into skeletal lineages, may represent a useful strategy for the clinical reconstruction of bone and cartilage defects.

The potential of fibroblast growth factor-2 (FGF-2) to stimulate osteoprogenitors in aging bone was investigated. Previous work showed a decrease in bone formation in cell cultures derived from bone of elderly female patients, but not in cells from age-matched male or younger female patients, with transforming growth factor β increasing bone formation but not increasing osteoprogenitors. In the present study, FGF-2 was shown to significantly stimulate, in a dose-dependent manner, proliferation of mesenchyme-derived progenitor cells from bones of young and old mouse and humans. In proliferation assays, human cells were more responsive to lower concentrations (0.0016 ng/mL) of FGF-2 than mouse cells, but proliferation was less in cells from older bone. Immunofluorescence microscopy revealed that FGF-2 increased and prevented the decline in cells expressing activated leukocyte cell adhesion molecule, a novel marker for early lineage osteoblasts, but not α-smooth muscle actin. FGF-2 may have therapeutic potential for stimulating osteoblast progenitors in aging.