Engineering 3D in vitro Models to Mimic Aging-associated Delayed Bone Healing and Inflammaging

Introduction: Aged populations suffer from a higher incidence of bone injury and delayed bone healing. Despite this, most research on bone regeneration to date is limited to the use of cells from young donors and young animal models. Previous patient data and animal studies suggest aging is associated with decreased stem cell differentiation and increased inflammation. However, how immune-stem cell crosstalk modulates bone healing during aging remains largely unknown. Given the short supply of aged animals, there remains a critical need for in vitro experimental models that can mimic key features of bone healing during aging. Our group has previously reported microribbon (µRB) hydrogels as 3D macroporous scaffolds that support robust bone regeneration in vitro and in vivo by promoting regenerative immune-stem cell crosstalk. Here, we seek to harness µRB hydrogels to develop 3D in vitro models that mimic key features of aging-associated delayed bone healing and immune cell-stem cell crosstalk. (Figure 1A) We hypothesize that culturing macrophages (Mφ) and mesenchymal stem cells (MSCs) from young or aged donors in µRB scaffolds will mimic aging-associated changes in inflammation and delayed bone healing.

Materials and

Methods: Mφ and MSCs were isolated from young (5 weeks) and aged mice (56 weeks). The effects of aging on Mφ phenotype and MSC osteogenesis were first evaluated using a 3D monoculture model. To determine the Mφ phenotype, qPCR of M1 or M2 markers was performed on young and aged Mφ in 3D culture. MSC metabolic activity was assessed using PrestoBlue over 18 days. Alizarin red S (ARS) staining was performed to evaluate the mineral deposition from MSCs. To mimic Mφ-MSC crosstalk, a conditioned medium (CM) treatment model was used. CM collected from young or aged Mφ was mixed with osteogenic medium and applied to MSCs in 3D culture. The effect of Mφ CM on MSC bone formation was assessed using ARS staining. Similarly, CM from young or aged MSCs was applied to Mφ, and the Mφ inflammatory response was evaluated using cytokine ELISA. Finally, serum was collected from young or aged mice and incorporated into MSC 3D culture to investigate the effects of aging-associated changes in serum on MSC mineral deposition.

Results, Conclusions, and Discussions: In 3D monoculture, MSCs from aged mice exhibited delayed proliferation, decreased metabolic activity, and mineralization (Figure 1B-1D), and Mφ from aged mice showed increased polarization towards the pro-inflammatory M1 phenotype (Figure 1E) compared to young cells. CM from M1-polarized Mφ significantly inhibited MSC osteogenesis for both young and aged cells. Interestingly, CM from aged MSCs triggered more inflammatory cytokine release from Mφ, suggesting that the senescence-associated secretory phenotype (SASP) from aged MSCs may promote inflammation. (Figure 1F) Furthermore, serum from aged mice significantly inhibited the mineralization of young MSCs, indicating that aging-associated changes in serum composition also contribute to delayed bone healing during aging. (Figure 1G)
In summary, in this study we report tissue engineered 3D models that mimic key features of aging-associated delayed bone healing, including reduced stem cell metabolism and osteogenesis, increased macrophage inflammation, and the inhibitory effects of blood serum. These 3D aging models hold potential for further mechanistic studies or drug screening to identify therapeutics for reversing aging effects and enhance bone regeneration in elderly populations.

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