A new study in ACS Applied Materials and Interfaces used a combined geroscience and tissue engineering approach to regenerate bone in aged rats .
Replacement or rejuvenation?
Too often, tissue engineering and longevity therapeutics are viewed as competing strategies – two different paths to potentially combat aging. In tissue engineering, cells and biomaterials are used to form tissue that replaces aged or diseased tissues. Meanwhile, longevity therapeutics include drugs that target one or more aging pathways in order to rejuvenate existing aged or diseased tissue. When viewed in competition, the question becomes “Which strategy deserves our precious time, attention, and funding?”
In reality, these two fields are quite complementary. Current tissue engineering strategies rely heavily on the body’s innate ability to heal and self-regenerate – which declines considerably with age. Additionally, while evidence is mounting that various drugs can modulate aging, at least in animal models, their effects have been far from total reversal of disease. Eventually, tissue damage becomes too extensive to ever return to a healthy state. If drugs can improve the aged body’s regenerative potential, it would follow that tissue engineering approaches would be more successful in older adults if used in combination with those treatments.
While the two fields have been relatively separate, progressing at their own rates in parallel, the first studies that utilize both approaches are now beginning to be conducted. Most recently, scientists at Sichuan University have used the senolytic quercetin and a TG-18 hydrogel to regenerate a bone defect in aged rats .
Finding the best senolytic
Many senolytic drugs have shown cell type-specific effects. To focus their study on bone regeneration, the researchers used bone marrow-derived mesenchymal stem cells (BMSCs) from rats. Cellular senescence was induced in these cells in vitro by either hydrogen peroxide or d-galactose treatment. Both senescent and non-senescent cells were treated with 10 different known senolytics (dasatinib, quercetin, navitoclax, A-1331852, A-1155463, ABT-737, fisetin, geldanamycin, 17-AAG, and 17-DMAG) at a concentration of 10 µmol.
Each drug except navitoclax significantly reduced the ratio of senescent BMSCs (as measured by SA-ß-gal staining) after hydrogen peroxide or d-galactose treatment. Dasatinib, quercetin, geldanamycin, 17-AAG, and 17-DMAG notably reduced this ratio to a greater extent than the other drugs. However, only fisetin and quercetin showed no toxicity to non-senescent cells, with dasatinib, geldanamycin, 17-AAG, and 17-DMAG especially showing notable toxicity. Because of these results, the researchers chose to move forward with quercetin for the remainder of their experiments.
Quercetin was then optimized for concentration. In these dose optimization studies, the researchers investigated the induction of senescence and effectiveness of quercetin in their BMSCs beyond just SA-ß-gal, confirming their results with the DNA damage marker γH2AX, the SASP markers IL-1ß and IL-6, and the cell cycle regulators p16, p21, and p53. A 20 µmol concentration showed the greatest effectiveness at reducing senescence without toxicity.
At this concentration, quercetin treatment also improved the proliferation and osteogenic differentiation of the rat BMSCs exposed to hydrogen peroxide but not of non-senescent control cells. This suggested that eliminating senescent cells may be able to improve bone regeneration.
A drug release platform that responds to senescent cells
In order to deliver the drug directly to the site of injury and to release it more rapidly in the presence of senescent cells, a triglycerol monostearate (TG-18) hydrogel was utilized for this study.
Senescent cells are known to release matrix metalloproteinases (MMPs) as part of their SASP. In this study, elevated levels of MMPs were observed in the in vitro senescent BMSCs and in the bone tissue of aged rats in vivo. TG-18 is a hydrogel that can encapsulate drugs like quercetin and is disassembled by MMP enzymes. Therefore, the researchers hypothesized the quercetin would be released more rapidly in the presence of senescent cells.
A maximum loading concentration of quercetin was determined to be approximately 3% by weight. When the TG-18 hydrogel was immersed in cell culture media conditioned by senescent cells, quercetin was released more rapidly compared to media from control cells. Additionally, TG-18 degraded more rapidly in the skull defects created in old rats compared to young rats.
Bone regeneration was greater with the combination treatment
To investigate its ability to facilitate bone regeneration, TG-18 loaded with 0.2% quercetin, 2% quercetin, or no quercetin was implanted into aged rats. The hydrogel was also supplemented with 2% hydroxyapatite in all groups to further facilitate bone regeneration.
Bone defects were surgically created in either the femur or skull and filled with the TG-18 hydrogel. At 3 months, for both femur and skull defects, senescence was decreased with quercetin treatment as measured by p16, γH2AX, and MMP expression. Bone formation was also greater with quercetin treatment as measured by microCT, Masson trichrome staining, and OCN and OPN expression. For each of these measures, the 2% quercetin showed slightly better, although not statistically significant, results relative to the 0.2% group.
In this study, we screened out quercetin as the suitable senolytic drug for clearing senescent rBMSCs. According to the secretion of MMPs in senescent rBMSCs, a senescence-responsive hydrogel loading quercetin was prepared to eliminate the senescent rBMSCs in the bone defects (Scheme 1). In vivo, bone repair assay confirmed that the senescence-responsive hydrogel efficiently eliminated local senescent cells and promoted the repair of bone defects in aged rats. This work presents a promising strategy for local removal of the senescent rBMSCs to promote bone regeneration in aged individuals.
Many previous studies have shown similar success at regenerating bone in rats. However, these studies have almost always been conducted in younger rats, which regenerate quite well on their own. Future studies would do well to follow in the footsteps of this one, as these treatments will ultimately be used primarily in older adults.
We know that senescence is critical for wound healing, but in older organisms, the senescent response is typically prolonged and interferes with the healing process [2,3]. This study provides compelling evidence that senolytics can, in fact, be beneficial for bone regeneration in older organisms. With TG-18, the local delivery and controlled release of quercetin initiated by the presence of senescent cells is also an exciting strategy moving forward.
Notably, however, the defects treated in this study were very small. Larger defects are more difficult to heal, as fibrotic scar tissue infiltrates the area more quickly than bone can form. Whether this strategy would be successful in humans or in larger defects cannot be claimed from these results. Although, there are also many improvements to this strategy, such as the inclusion of other biomaterials, cells, and/or growth factors, that future studies could use when targeting the regeneration of larger bone defects.
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 Xing, X. et al. Local elimination of senescent cells promotes bone defect repair during aging. ACS Appl Mater Interfaces (2022). https://doi.org/10.1021/acsami.1c22138
 Demaria, M. et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Developmental Cell (2014). https://doi.org/10.1016/j.devcel.2014.11.012
 da Silva, P.F.L. et al. The bystander effect contributes to the accumulation of senescent cells in vivo. Aging Cell (2019). https://doi.org/10.1111/acel.12848