Biomimetic Therapeutics for Bone Regeneration: A Perspective on Antiaging Strategies

Abstract

Advances in modern medicine and the significant reduction in infant mortality have steadily increased the population’s lifespan. As more and more people in the world grow older, incidence of chronic, noncommunicable disease is anticipated to drastically increase. Recent studies have shown that improving the health of the aging population is anticipated to provide the most cost-effective and impactful improvement in quality of life during aging-driven disease. In bone, aging is tightly linked to increased risk of fracture, and markedly decreased regenerative potential, deeming it critical to develop therapeutics to improve aging-driven bone regeneration. Biomimetics offer a cost-effective method in regenerative therapeutics for bone, where there are numerous innovations improving outcomes in young models, but adapting biomimetics to aged models is still a challenge. Chronic inflammation, accumulation of reactive oxygen species, and cellular senescence are among three of the more unique challenges facing aging-induced defect repair. This review dissects many of the innovative biomimetic approaches research groups have taken to tackle these challenges, and discusses the further uncertainties that need to be addressed to push the field further. Through these research innovations, it can be noted that biomimetic therapeutics hold great potential for the future of aging-complicated defect repair.

1. Introduction

Orthopedic trauma is highly prevalent worldwide, with new injuries affecting over an estimated 175 million persons annually.^[1] Skeletal tissue has exceptional ability to repair minor fractures and injuries with minimal to no scarring in many cases, however this capability is notably diminished by comorbidities such as obesity or osteoporosis, and additionally by intrinsic factors such as aging. These factors drive a significant increase in health care costs, and complicate the clinical management of skeletal injury and chronic disease. The world’s population continues to shift toward a more progressively aging society, as evident by the population growth of individuals 65 years and older being the most rapid growing cohort worldwide, where already more than 125 million persons are over 80 years old.^[2] With this shift in population demographics, it is ever imperative to develop more effective interventions for treatment of skeletal injury and disease that are adapted to an increasing-age society.

Therapeutic strategies for repair of bone injury due to trauma, tumor resection, or simple fractures aim to provide complete restoration of normal function of bone in affected patients. In many cases, bone-grafting strategies can restore shape and function to damaged tissue, but these strategies are accompanied by several definite limitations.^[3-6] Autografts are the “gold standard” treatment, with patient harvested tissue repairing defects without risk of immune rejection and limited risk of disease transmission. However, autograft procedures increase the risk of infection and infer donor site morbidity and pain/discomfort to patients, and generally have a very limited supply. Allografts are relatively abundant and avoid morbidity concerns, but require heavy processing and sterilization due to disease screening and preparation, in addition to immune rejection concerns. A rising alternative strategy to grafts is biomaterials-based substitute materials, which have the advantages of low relative cost, unlimited supply, and high degree of customizability in their shape and chemistry.^[7-10] For restoration of host tissue shape and function, biomaterials must be biocompatible, biodegradable, and provide adequate support for the recruitment and proliferation of progenitor cells and vascularization to repair the injury site. Regeneration of skeletal tissue is especially challenging, due to the lengthy repair timeline encompassing numerous transient changes in cellular makeup and bioactive signaling environment.

One particularly effective strategy for biomaterial design for bone defect repair is to mimic the morphology and signaling environment of the endogenous bone matrix, also known as “biomimetics.”^[11-13] Biomimetic strategies aim to emulate the natural environment of bone in order to provide a conductive environment for progenitor cell recruitment, differentiation, and proliferation to form new functional tissue. For the purpose of biomaterial scaffolds, this typically involves mimicking the nanofibrous morphology of the extracellular matrix of bone, which consists of collagen fibers in the size of 50–200 nm. To date, several types of strategies to synthesize biomimetic materials with these dimensions have seen use in regenerative therapies in vivo, including techniques such as phase separation, electrospinning,^[14] and self-assembly, among others.^[15] Over time, the meaning of biomimetics has grown to encompass many more natural-mimicking factors in regenerative medicine for bone, including but not limited to synthetic apatites,^[16] bio-electrical stimulation,^[17] and controlled bioactive factor delivery methods mediated through various techniques such as material conjugation^[18] or via natural methods like exosomes.^[19]

Aging is an intrinsic phenomenon that drives additional complications to numerous types of disease and injuries, and bone repair is no exception. In bone, regeneration is impaired by a decreased stem cell population and impaired differentiation capacity.^[20] In addition to this, changes in the signaling environment, chronic inflammation, and cellular senescence contribute to produce additional complications on the road to repair of bone tissue. This review will discuss recent and current advances in antiaging therapies for bone repair through the window of biomimetic approaches, and highlight challenges and areas of need for advancement in the near future of the field.

2. Basic Pathology of Aging Bone and Its Reduced Regenerative Potential

Healthy bone tissue has incredible ability for self-healing through a robust remodeling process. Aging is an intrinsic factor to all humans where regenerative potential of all tissue types is reduced as age advances, and bone is no exception. In aging bone, remodeling homeostasis begins to imbalance as it shifts toward favoring resorption over new bone formation, leading to an overall reduction in total bone mineral density. A significant enough imbalance can lead to diagnosis with osteoporosis, where age is one of the highest risk factors for developing this condition. Osteoporosis is one of the most common bone diseases worldwide, affecting an estimated over 200 million elderly persons in the world.^[21] Characterized by a reduction in bone mineral density, osteoporosis brings increased risk of fracture to those affected, directly affecting mortality rates and catapulting healthcare costs worldwide.^[22,23] Pathologically, osteoporosis is described as a disruption in remodeling homeostasis where the rate of bone resorption eclipses the rate of new bone deposition. Most commonly, it is well understood that osteoclast activity is increased, while osteoblast building capacity is simultaneously lowered. Recent evidence has suggested that these two phenomena are not directly coupled,^[24,25] leading us to face an increasingly complex therapeutic challenge that must be approached via a more holistic manner for successful intervention.

Attempting to look under the hood of osteoporosis and other general intrinsic changes during aging in bone has uncovered a wide variety of independent and interconnected mechanisms driving these changes. The ultimate bone-building cells, osteoblasts, are reduced in number with advancing age.^[26] Osteoblast precursor stem cells and potentially osteoblast lifespan are theorized to also be decreased, although more rigorous research is needed to clarify these points.^[27] Osteoclast activity is generally increased during advancing age—older bone is preferentially removed by the osteoclasts in vitro, leading to overall increase in activity in old specimens.^[28] In women, it is also suggested that osteoclast precursor monocytes are preprogrammed to remember age, further aggressing resorption activity post-menopause.^[29] Bone marrow stromal cells acquire aging-related increase in senescence markers, and population doubling time was directly correlated with age, demonstrating an age-dependent decrease in activity and differentiation capacity.^[30] Mechanistically, the stem cell niche appears to be directly impacted by aging via these observations.

2.1. Aging and Chronic Inflammation

Looking into other adjacent, chronic conditions during aging help shed light on other factors contributing to aging-induced degeneration of bone. Aging is generally associated with chronic and systemic low level subclinical inflammation, characterized by elevated baseline of pro-inflammatory cytokines, as evidenced by aged mouse models.^[31] Fracture healing is a partially inflammation-mediated process, where inflammatory factors play integral supporting roles in driving remodeling and healing.^[32] In fracture healing, inflammatory genes are normally reduced after a period of time in young specimens but remain at higher levels in aged animals after a similar time period.^[31] Elevated chronic inflammation can cause disruptive activity with macrophage polarization, leading to increased bone resorption and decreased formation, and drives further increases in osteoclastogenesis from persistent levels of TNF-α.^[33,34]

2.2. Increasing Oxidative Stress

Another persistent intrinsic factor in aging is systemic increase in oxidative stress. Increased oxidative stress can be triggered by overloading, diet, predisposed factors, among others; however aging is common to all animals, driving an increase in oxidative stress that contributes to bone destruction.^[35] Increased reactive oxygen species (ROS) levels can be caused by age, other exogenous factors, or a combination of the two. ROS can drive and play significant roles in many disease pathologies, including cardiovascular disease, cancer, and bone-related ones such as periodontitis^[36] and osteoporosis.^[37] Similar to typical remodeling homeostasis, free radical oxygen species production and endogenous antioxidant mechanisms maintain a balance throughout normal lifetime, and begin dysfunction during aging, injury, or other exceptional circumstances. As one mechanism of ROS accumulation causing increased bone resorption, ROS contributes to promotion of osteoclast activity by way of increased RANKL (receptor activator of nuclear factor kappa-B ligand) production to help drive bone resorption.^[38]

2.3. Cellular Senescence

Perhaps a more holistic marker of aging in bone could be described by cellular senescence—irreversible cell cycle arrest upon damage to cell’s DNA or other causes that can be driven by inflammation, ROS, among other factors. Many roads can lead to a senescent phenotype of cells in tissue; where senescence markers in cells have been noted to be present in the progression of many chronic noncommunicable diseases.^[39] In bone, the causal factors and direct consequences of cellular senescence are still relatively under-researched. Accelerated aging models frequently demonstrate skeletal changes, e.g., reduced mineral density and increased rate of fracture, typical hallmarks of aged bone.^[40] Bone marrow cells isolated from accelerated aged animal models show typical markers of DNA damage (γH2AX) and cellular senescence (p16^Ink4a).^[41] Translation to human models has remained difficult, and even in animal models due to high incidence of premature death. Despite this, there is growing evidence that targeting senescent cell populations for removal (senolytics) could be a promising therapeutic target for prevention of aging-induced bone diseases^[42] (Figure 1).

Figure 1.

Schematic describing basic pathology of impaired bone regeneration due to advancing age and three potential main causes. Various biomimetic strategies to address each challenge predominately driven by advancing age to be discussed in this review are described in the table.

3. Biomimetic Approaches to Modulate Chronic Inflammation

Immune system regulation plays a critical role in bone healing, as evident through macrophage activity playing a key role in the orchestration of pro-inflammatory and pro-reparative roles during scaffold-mediated repair of skeletal tissue.^[33,43,44] Macrophages elastically polarize into broadly defined pro-inflammatory (M1) or pro-healing (M2) phenotypes through various conditions, each producing a distinct profile of cytokines.^[45,46] In an ideal bone fracture healing model, macrophages undergo a transient polarization change from M1 in early wound healing during the acute inflammatory response to M2 during later stage healing to accelerate regeneration. In aged patients, this M1 to M2 polarization transition can be adversely impaired and slowed, leading to poor outcomes in healing.^[47,48] This dysregulation of polarization commonly leads to chronic inflammation, further compounding clinical treatment in addition to already existing injury or trauma.^[49] Strategies aimed at assisting this M1–M2 transition have arisen in order to shed light on the mechanisms governing this shift, with a goal of defining potential targets for therapeutic interventions in the future to improve outcomes in aged bone repair.

3.1. Modulating Surface Chemistry and Topography

Surface topography of biomaterial scaffolds plays a demonstrable role in macrophage polarization. It is well-studied phenomena that matrix stiffness and nanostructure can induce osteogenic differentiation of progenitor cells,^[50] however the interaction on macrophage polarity control is less clear.^[51] One recent study demonstrated macrophage polarity could be influenced with a hierarchical biomimetic nanoapatite assembly, provoking the M2 polarization of recruited endogenous macrophages to scaffolds in vivo (Figure 2).^[52] The authors noted that almost no macrophages arriving at the early stages during repair were polarized, suggesting that macrophage interaction with biomaterial is a key aspect to polarization. The hierarchical apatite nanostructure guided M2 polarization more effectively than a randomly mineralized nanostructure; where the authors showed macrophages evenly spreading across the hierarchically structured scaffolds more often took on M2 polarization markers than not. Transmission electron microscope analysis showed improved vacuole/lysosome activity promoting mineralized collagen turnover in the macrophages present in the hierarchically arranged scaffolds, which suggests M2 polarization is favored by the biomimetic hierarchical structure. Like described in this work, nanoscale roughness control of calcium phosphate is often used to improve osteointegration of implants.^[53] However, recently its physical and chemical interaction with macrophages has been unclear. One study found that a material surface coating of calcium phosphate could modulate the transition of M1 to M2 phenotype of RAW 264.7 cells through a combination of surface topography and ionic microenvironment changes.^[54] This study found that surface-coated calcium phosphate layers could be converted into ions more effectively in the presence of macrophages, independently of the presence of BMP2. Through this, supernatant calcium ions likely enhanced RAW 264.7 expression of anti-inflammatory genes via the calcium sensing receptor pathway. Transient increase in medium pH followed by decrease in pH after several days also may have contributed to the M1–M2 polarization, but this effect was not independently confirmed. Another group deposited calcium phosphate via a simulated body fluid as a carrier to sequentially deliver M1- and M2-promoting stimuli to transiently guide macrophage polarization in aged mice.^[55] Traditional M2 activating factor IL-4 was previously shown to minimally improve osteoblastic differentiation when delivered immediately to macrophages in injury models, however this study demonstrated a delayed administration showed a much more potent effect on osteoblast differentiation after initial M1 activation.

Figure 2.

a) 3D HIMC scaffolds morphology and b) immunofluorescent staining. h) Immunohistochemistry of macrophage polarization in defect areas. i) Quantification of positively stained macrophages per each scaffold group. Adapted with permission.^[52] Copyright 2019, American Chemical Society.

As a highly prevalent and destructive disease, periodontitis models are another effective tool in characterizing macrophage activity. Ding et al. fabricated a core/shell fibrous scaffold to sequentially release basic fibroblast growth factor and BMP2 to promote angiogenesis and late stage M2 macrophage polarization, leading to improved periodontal bone regeneration.^[56] By using demonstrating equally comparable morphology in scaffold groups, the authors demonstrate timed release of growth factors could induce or aid the M1–M2 switch in macrophages. Interestingly, without the presence of calcium phosphates or hydroxyapatite (HA), the scaffold by itself did not impact macrophage polarization, suggesting the chemical presence of mineral in absence of other factors may be essential to polarization change. Our group has recently demonstrated controlled release of the metabolite di-methyl alpha-Ketoglutarate (DMAKG) from gelatin scaffolds can downregulate inflammatory response of macrophages, rescuing alkaline phosphatase (ALP) activity of pre-osteoblast cells and significantly improving new bone formation in aged mice.^[57] Naturally circulating AKG is reduced with age, where prior research showed dietary administration of AKG attenuated age-related bone loss and improved new bone formation in aged mice.^[58] However, the mechanism by which AKG supported bone regeneration remained unclear. In this work, one of the uncovered mechanisms of DMAKG local administration was found to be suppression of pro-inflammatory response of J774A.1 macrophage cells, which contributed to increased osteoblastic differentiation and reduced adipogenic differentiation in aged mice.

3.2. Controlled Electrical Microenvironmental Cues

Electrical microenvironment is another very understudied niche in bone regeneration, as development of appropriate conductive materials has been limited by high cost and poor biocompatibility.^[59] Recent developments in electroactive biomaterials have begun to show promise in promoting bone regeneration through simulating native tissue microenvironment cues.^[60,61] Electrical stimulation has seen more study in neuromuscular applications, and it is recently becoming clear that it can play a role in guiding polarization of macrophages as well. Dai et al. utilized ferroelectric nanocomposites to induce M2 polarization in human cells and rat model by mimicking the electrical microenvironment, ultimately restoring more bone in diabetic compromised critical-sized rat calvarial defects.^[62] Their study found that nanocomposite membrane polarization induced M2 polarization of macrophages under high glucose conditions via the AKT2-IRF5 signaling pathway, compared to nonpolarized membranes which favored M1 polarization. Hu and co-workers reported an electroactive nanocomposite hydrogel based on regenerated silk fibroin that improved bone regeneration in vivo partly by promoting the polarization of M2 macrophages among other benefits.^[63] This scaffold serves as a piezoresistive pressure transducer, coupling the matrix stiffness and electrical microenvironment together to promote new bone formation via both direct osteogenic and indirect immunomodulatory mechanisms, although their interaction was unclear.

3.3. Author Insights

While there are several well-studied inflammation-activated models in vitro, the in vivo macrophage polarization stimuli are much more in-depth, nuanced, and complex. It is in fact difficult to completely isolate the numerous factors contributing to macrophage polarization, from the aforementioned matrix topography and chemistry and electrical microenvironment, but also other influences beyond the scope of this review such as osteoblast/clast-macrophage cross-talk, soluble factor and hormone levels, and more.^[64,65] Like with mechanical stresses playing a guiding role in osteoblastic differentiation via energy metabolism, it is highly possible and suggested there are other unexplored intrinsic factors playing a hand in managing macrophage polarization. Additionally, the unique role of macrophage intertissue mobility could play a role in understanding systemic effects of therapeutics, especially in various metabolic conditions, demanding further studies to isolate these effects. This suggests a focus on localized delivery and administration of therapeutics in in vivo models is becoming ever essential to uncovering new mechanisms by which macrophage polarization stimuli are controlled.

4. Biomimetic Strategies to Combat Oxidative Stress

ROS are key physiological signaling molecules that are responsible for transmission of cell signals and other normal functions. Under abnormal circumstances, ROS can build up in tissue, causing damage to protein, DNA, and other cellular functions. ROS accumulation is heavily associated with many diseases and incidence is strongly linked to aging.^[66] Accumulation of ROS has been linked to dysfunction of bone remodeling homeostasis, contributing to a number of metabolic bone diseases such as osteoporosis.^[67] In many studies, ROS accumulation is shown to be linked to increased osteoclast activity and downstream bone resorption, mechanistically contributing to disease such as osteoporosis.^[68,69] Through these findings, there is growing interest in targeting ROS as an antioxidant therapeutic method for treatment of bone disease and age-induced fracture repair, while a deep understanding of ROS modulation on disease pathology still remains elusive.

4.1. ROS Scavenging Materials

Bone defects are often accompanied by higher than normal levels of ROS during healing, and this is also exacerbated with age. Vasculature damage associated with bone defects often induces hypoxic conditions at the injury site. Hypoxia-induced factor (HIF) can induce excessive production of ROS, hindering repair, and exacerbating local inflammation.^[70] In aged models, ROS accumulation is significantly enhanced, although the mechanism during bone healing is unclear, whether it is through further increased HIF levels, aging-related mitochondrial damage, or perhaps other reasons.^[66] One strategy to limit damage caused by these elevated levels is to functionalize materials to locally scavenge endogenously produced ROS.^[71] Early efforts to develop ROS scavenging materials for bone leveraged antioxidant drugs such as N-acetylcysteine^[72] or ceria-based nanoparticles via mesoporous silica.^[73] However, these strategies were limited due to systemic administration, poor therapeutic effect, and cytotoxicity of the ceria nanoparticles. Zhou and co-workers developed a biocompatible nanofibrous polypyrrole (ppy)-based scaffold functionalized with polydopamine (PDA) and hydroxyapatite that protected cells from ROS damage in vitro and significantly enhanced new bone formation in rabbits (Figures 3 and 4).^[74] Redox behavior of ppy films eliminate ROS via catechol conversion into quinones, which contributed to numerous benefits to bone regeneration, including improved M2 macrophage polarization and increased angiogenesis, eventually leading to stronger bone formation via the Ca²⁺/CALM signaling pathway in bone marrow mesenchymal stromal cells (BMSCs). As the conductive polypyrrole is nonbiodegradable, Huang et al. fabricated biodegradable microspheres from polyorganophosphazene that demonstrated ROS scavenging potential in cell culture and bone defects and improved bone regeneration versus poly(lactic-co-glycolic acid) (PLGA) microspheres in rat calvarial defects, although the total amount of new bone after 16 weeks was still insufficiently able to bridge a critical sized defect.^[75] Synthesized microspheres significantly lowered intracellular ROS levels in freshly punched rat calvarial defects via their surface charges, also allowing for acceleration of mineral deposition and ion attraction for new bone formation. Chitosan-derived nitrogen-doped nanodots were synthesized by Chen et al. to scavenge ROS in order to inhibit osteoclast overactivation in vitro and protect against lipopolysaccharide-induced bone destruction in mice.^[76] These nanocarbon dots attenuated RANKL-induced osteoclastogenesis by reducing intracellular ROS generation from bone marrow macrophages via elevating expression of antioxidant enzymes heme oxygenase-1 and catalase. In other experiments, previously described natural-inspired PDA has been known for its capability of free-radical scavenging,^[77] where PDA nanoparticles were for the first time shown to protect against ROS-induced inflammation in a periodontitis model in mice by Bao et al.^[78] Synthesized PDA nanoparticles held broad removal capacity of hydroxyl and superoxide radicals in solution, however the particular scavenging mechanism was not probed in-depth.

Figure 3.

Ppy-PDA-HA films morphology. a) Close up, b) top view, and c) side view. Adapted with permission.^[74] Copyright 2019, John Wiley and Sons.

Figure 4.

Ppy-PDA-HA protects cells from ROS in vitro. a) Schematic of protection. b) Typical fluorescence of ROS formation. c) Quantitative measurement of fluorescence. Adapted with permission.^[74] Copyright 2019, John Wiley and Sons.

4.2. ROS-Responsive Materials

While the consequences of excess accumulation of ROS in bone repair have become abundantly clear, it is still well acknowledged that some minimum physiological level of ROS is still required for progression of regenerative processes. Thus, striking a delicate balance is essential when applying engineering strategies aimed at reducing ROS levels in tissue. Several research projects have demonstrated novel techniques to capitalize on the excess ROS, by using high ROS concentration as a trigger for drug release, or even as a fuel source for oxygen production to combat hypoxia during tissue repair. Hammond et al. utilized excess ROS production as a tool to develop an oxidation-responsive thioketal-based polymer scaffold to trigger response of BMP2 upon oxidation, enhancing bone regeneration in critically sized rat calvarial defects and significantly improving the BMP2 delivery half-life in vivo.^[79] Scaffolds could release bioactive payload via oxidation-specific degradation mechanisms when ROS accumulation increased to trigger degradation of scaffolds. This allowed for retention of drug under insignificant levels of ROS, significantly reducing waste and also lowering cost. Qiu and co-workers also utilized thioketal bonds in their designed ROS-responsive nanoparticles, releasing N-acetylcysteine to eliminate ROS in a periodontitis model, significantly alleviating tissue destruction in periodontal bone.^[80] Sun et al. took the responsivity concept a step further, demonstrating ROS-responsive perfluorocarbon-nanoparticle-loaded hydrogels to combat hypoxic conditions in mouse cranial defect model by degrading hydrogen peroxide to replenish oxygen.^[81] These ROS-responsive hydrogels could improve angiogenesis, reduce osteoclastic activity, and significantly improve new bone formation in mouse cranial defect model.

4.3. Author Insights

While numerous studies have shown reduction of ROS leads to improved outcomes in bone regeneration, the mechanisms linking advancing age and change in capacity for endogenous ROS production is still unclear—most studies do not utilize aged animal models and thus the efficacy of many approaches is uncertain in these cases. Without a deeper understanding of ROS levels in advancing age, and without knowing whether transient changes are more or less frequent and in what magnitudes, strategies responsive to ROS levels may be inefficient. It is also poorly known if aging tissue has an enhanced or impaired response to accumulation of local ROS. ROS plays a role in many processes interlinked with bone regeneration, such as angiogenesis, where we still have limited knowledge of the effects of advancing age and its interaction with bone repair. Additionally, the direct role of ROS in osteoblastic differentiation is still undefined, and more research into the underlying mechanisms of oxidative stress in differentiation is warranted. It is certainly essential to develop advanced age models of increased ROS environments to better understand the mechanisms of therapeutic intervention strategies to combat the problems that arise with significant ROS accumulation.

5. Modulating Cellular Senescence in Bone

Cellular senescence refers to a cellular phenotype characterized by irreversible cell cycle arrest, changes in gene expression and metabolism, and development of a pro-inflammatory secretome, among other factors.^[82,83] Senescent cells have demonstrated acute beneficial functions in scenarios such as wound healing response,^[84] but excess accumulation has been heavily linked toward progression of many age-associated chronic diseases.^[85] In bone, accumulation of age-related molecular damage, telomere shortening, epigenetic alterations, and other traditional hallmarks of aging contribute to functional decline of bone tissue to maintain healthy mineral density. Over the recent years, cellular senescence has emerged as a potential therapeutic target for combating age-related bone loss.^[86] While many of the previously discussed challenges to aging-complicated defect repair discussed in this review can be causal factors driving cellular senescence, many other types of in vivo stressors have been noted to or are hypothesized to contribute toward senescence. Thus, development of therapeutics targeting cellular senescence is becoming an attractive strategy to combat age-related bone disease and injury.

To date, several approaches aimed at combating senescence in bone have been described; from selective elimination of senescent cells (senolytics)^[87] to genetic/epigenetic^[88,89] approaches or inhibiting the senescence-associated secretory profile (SASP) of senescent cell populations.^[90] However, translation of many of these therapies into preclinical animal models is still in its infancy, as in vitro models are still lacking.

5.1. Stem Cell Protection

As mesenchymal stem cells (MSCs) are the main source of progenitor cells for driving bone regeneration, protecting the MSC population from senescence has shown demonstrable benefit to aiding new bone formation. Stem cell culturing, traditionally described as expansion of culture in 2D culture dishes, is seeing a move to 3D,^[91] and with it, benefits to protecting against markers of cell aging are becoming evident. Su et al. described a biomimetic scaffold culturing system to prevent MSC replicative senescence during long-term culture in conventional culture dish (Figure 5).^[92] A nanohydroxyapatite/chitosan/PLGA scaffold demonstrated improved the youthful nature of cells cultured after numerous passages and had comparable ectopic bone growth to much earlier cell passage numbers versus nonscaffold cultured cells. Human umbilical cord mesenchymal stem cells (hUCMSCs) cultured in tissue culture dish showed an increase in p53 mRNA and protein markers after numerous passages, whereas cells cultured on the 3D scaffolds saw little to no increase in these markers. Gene expression of BMP2, bFGF, EGF, and CXCL5 remained significantly higher after numerous passages on cells cultured in 3D scaffolds compared to tissue culture plate, yet further investigation into this particular mechanism remains to be seen. Zhou et al. cultured BMSCs isolated from aged mice with a cocktail of senolytic drugs (dasatinib and quercetin [Q+C]), and then demonstrated the improvements in new bone formation in mouse calvarial defect model when implanted in a collagen + HA scaffold.^[93] Q+C treated cells significantly enhanced proliferation rates of old BMSCs and displayed lower expression levels of senescence and inflammation markers p21, p16, Il6, among others, which eventually translated to more significant new bone formation in the mouse calvarial defect model. The study proposes that the clearance/elimination of senescent cells in vivo is the main mechanism of this improvement, by removing the SASP and associated inflammation from this relatively small population of cells due to the brief treatment window that provided relatively long-lasting results. Still, questions remain about other underlying mechanisms of impaired regenerative potential of aged BMSCs due to the experimental design utilizing donor BMSCs from aged mice in young host mice.

Figure 5.

a) Morphology of nHA/CS/PLGA scaffolds b) with cells cultured on scaffolds. c) Real-time PCR analysis of p53 mRNA levels in p3 and p27 of hUCMSCs cultured on dish or on scaffolds. d) Western blot of p53 protein levels. e) Real-time RT-PCR analysis of regenerative-expressing genes in p3 and p27 of hUCMSCs cultured on conventional dish or scaffolds. Adapted with permission.^[92] Copyright 2020, John Wiley and Sons.

5.2. Senolytic Therapeutics

Direct targeting and elimination of senescent cells in vivo is another significant challenge. Restoration of function of aged progenitor BMSCs has seen promise when moving from in vitro culture to in vivo models, but the complexity is high and more direct and facile therapeutic approaches are desired. Xing et al. recently developed a microenvironment-responsive hydrogel that could responsively release senolytic drug quercetin to locally eliminate senescent cells during aging bone repair in vivo.^[94] Quercetin, a natural flavanol, causes selective cell death in cancer cells and senescent cells via inhibition of PI3K, mTOR signaling, AKT pathway, and other various methods.^[95,96] Xing and co-workers utilized the high matrix metalloproteinase secretions of senescent cells as a trigger mechanism to degrade the hydrogel to release quercetin, helping limit systemic side effects and tightly localized the payload to the senescence site. Xing and co-workers also used another approach for targeted delivery of quercetin in vivo via use of a bone-targeting liposome delivery system.^[42] As compared to systemic intraperitoneal injection of drug, quercetin-loaded liposomes were significantly more effective at selectively eliminating senescent cells in mice, leading to improved osteogenic activity in a senile mouse osteoporosis model.

In classic fictional depictions of the “fountain of youth,” the vampires drinking the blood of the young is key to maintaining their youth appearance—which perhaps has partly inspired the bioengineering strategy of using young cells to rejuvenate the old. Lei et al. describe extracellular vesicles derived from neonatal umbilical cord MSCs that rejuvenate senescing adult bone-marrow-derived MSCs.^[97] Vesicles rejuvenated adult MSCs, increasing self-renewal capacity, and restoring telomere length, ultimately leading to improved regeneration in bone formation and angiogenesis. It was discovered that DNA repair-related gene PCNA was strongly induced in adult-derived stem cells after treatment with umbilical cord MSCs—mechanically through direct vesicle transfer into the adult stem cells, as identified via RNA-select stains. Lazzarini and co-workers co-cultured senescent human umbilical vein endothelial cells (HUVEC) to compromise bone marrow-derived MSCs, highlighting the role of miR 126a-3p in targeting SOX2 when investigating the mechanism of this phenomenon.^[98] BM-MSCs rapidly reduced proliferation rate when cultured with senescent HUVEC cells and not with young HUVEC cells, strongly indicating miR 126a-3p is an aging-associated miRNA. Downstream target SOX2, a stem-related gene, among others was significantly reduced with co-cultured MSCs, indicating a loss of stem cell potency from co-culture with aged HUVECs.

5.3. Author Insights

While elimination of local populations of senescent cellular populations has demonstrated potential to improve health outcomes, there is still a pressing need for determining universal markers to detect senescence, especially for complex multicellular tissue types like bone.^[99,100] Translation of cell culture models to clinical models, especially in terms of long-term culture and the effects of therapeutic interventions still require much innovation. Most current biomimetic approaches that regulate the microenvironment exclusively focus on protecting MSC populations, which may be insufficient in aged models that have an already limited number of MSCs to work with—this further raises the importance in identifying concrete markers for terminally differentiated cells in order to design effective therapeutics to combat senescence.

6. Concluding Remarks and Perspectives

Our increasing-age society is especially vulnerable to chronic and noncommunicable diseases that are commonly associated with aging. Reduced bone density is a unique challenge, as degeneration of bone is often undetectable and the risk of a potentially morbid fracture skyrockets with increasing age. As our world’s population age steadily rises, we can expect incidence of injury and fracture among the elderly to continue rising, making it imperative that we focus on effective therapeutic interventions to improve quality of life for the elderly moving forward.

Biomimetic strategies for bone repair are a rapidly developing field for therapeutics to face the challenge of aging bone repair in the 21st century. Many current and upcoming techniques are quickly evolving to address challenges in cost, biocompatibility, and reproducibility in biomimetic therapeutic design. A few examples include additive manufacturing technologies such as 3D bioprinting, which currently face challenges in nanoscale resolution and functional adaptations but are beginning to see progress in these areas; or RNA aptamers for precision medicine, which show great promise in precision cell targeting but still lack long-term safety data. One of the most significant challenges facing biomimetic therapeutic development is designing appropriate in vitro and in vivo models for aging bone environments—aged animal models while great tools are prohibitively more expensive than young models, making it essential to identify and develop high quality, reproducible aged models. Dissecting bone-specific diseases from comorbidities is a unique challenge to aged models that researchers should carefully approach, considering the wide range of age-driven diseases that can interact with each other, further complicating the challenge to designing effective models. To this end, effective and reproducible progressive-age models in vivo can help avoid premature death concerns and provide a more holistic lens to approach therapeutic interventions.

The direct cause and effect of chronic inflammation and ROS accumulation on cellular senescence, and other factors contributing to aging-induced reduced healing potential may not become clear for quite some time. In vitro and in vivo stressors undoubtedly have some level of crosstalk, thereby making it essential to identify distinct cellular markers to bone that can effectively mark senescence—and it is further essential that therapeutic interventions are specific and minimize off-target effects.

Biomimetic approaches hold great promise for aging-induced bone repair, with their high degree of safety and their cost-effectiveness as therapeutic interventions. As the mechanisms governing the changes in regenerative potential become clearer over time, they can be effectively addressed through biomimetic approaches to achieve great effect and improve public health outcomes worldwide.

Biographies

Jacob Miszuk is a research assistant professor at the Henry M. Goldman School of Dental Medicine at Boston University. He received his Ph.D. in biomedical engineering from the University of South Dakota (2019) and recently completed his postdoctoral work at the College of Dentistry at the University of Iowa, where his research interests focused on the development of novel biomimetic nanofibrous scaffolds for bone regeneration. At Boston University, his research in the Restorative Sciences and Biomaterials Department aims to push novel ceramic and tooth restoration techniques from the bench further toward the clinic to improve public dental health.

Hongli Sun is an associate professor atthe Iowa Institute for Oral Health Research and the Department of Oral and Maxillofacial Surgery, University of Iowa College of Dentistry. His research primarily centers on the development of bioinspired materials and advanced drug delivery techniques aimed at enhancing tissue regeneration, with a specific emphasis on addressing the unique challenges associated with repairing aged and inflamed dental and bone tissues.