Bnac 008

Fracture Healing in the Setting of Endocrine Diseases, Aging, and Cellular Senescence
Dominik Saul, MD1,2, Sundeep Khosla, MD1
Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

1
Kogod Center on Aging and Division of Endocrinology, Mayo Clinic, Rochester, MN 55905, USA.
t
ip
2
Department of Trauma, Orthopedics and Reconstructive Surgery, Georg-August-University of
Goettingen, Germany.
cr
us
an
Corresponding author
M
Sundeep Khosla, MD
d
Kogod Center on Aging and Division of Endocrinology

e
Mayo Clinic, Rochester, MN 55905, USA

pt
Tel: 507-255-6663
ce
khosla.sundeep@mayo.edu
Ac
ORCiD: 0000-0002-2936-4372
© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All
rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Supporting grants
Supported by NIH grants AG062413 and AG065868 (S.K.) and German Research Foundation
413501650 (Deutsche Forschungsgemeinschaft, DFG; D.S.).
Disclosures

The authors declare no conflict of interest.
t
ip
cr
us
an
M
e d
pt
ce
Ac
2
Abstract
Over 2.1 million age-related fractures occur in the United States annually, resulting in an immense
socioeconomic burden. Importantly, the age-related deterioration of bone structure is associated
with impaired bone healing. Fracture healing is a dynamic process which can be divided into four

stages. While the initial hematoma generates an inflammatory environment in which mesenchymal
stem cells and macrophages orchestrate the framework for repair, angiogenesis and cartilage
t
formation mark the second healing period. In the central region, endochondral ossification favors
ip
soft callus development while next to the fractured bony ends, intramembranous ossification
cr
directly forms woven bone. The third stage is characterized by removal and calcification of the
us
endochondral cartilage. Finally, the chronic remodeling phase concludes the healing process.
Impaired fracture healing due to aging is related to detrimental changes at the cellular level.
an
Macrophages, osteocytes and chondrocytes express markers of senescence, leading to reduced self-
M
renewal and proliferative capacity. A prolonged phase of “inflammaging” results in an extended
remodeling phase, characterized by a senescent microenvironment and deteriorating healing

d
capacity. Although there is evidence that in the setting of injury, at least in some tissues, senescent
e
cells may play a beneficial role in facilitating tissue repair, recent data demonstrate that clearing
pt
senescent cells enhances fracture repair. In this review, we summarize the physiological as well as
ce
pathological processes during fracture healing in endocrine disease and aging in order to establish a
broad understanding of the biomechanical as well as molecular mechanisms involved in bone repair.
Ac
Keywords
Aging; Bone; Bone healing; Fracture; Fracture healing; Osteoporosis; Senescence
3
Essential Points
 With the aging of the population and accumulation of co-morbidities, impaired fracture
healing is a significant clinical problem
 Four stages of fracture healing can be described, each of which may be impaired by aging and
various endocrine diseases
 Cellular senescence, which contributes to multiple co-morbidities of aging including bone

loss, may play a beneficial role in normal fracture healing but the accumulation of senescent
cells with aging may impair fracture healing
 A better understanding of the cellular and molecular impairments of fracture healing with
aging may lead to new therapeutic approaches to improve age-related deficits in fracture
repair
t
ip
cr
us
an
M
e d
pt
ce
Ac
4
t
ip
cr
us
an
Graphical_Abstract
5
d e
pt
ce
Ac
Introduction
In 2020, 53.5 million people were over the age of 65 years in the United States, representing 16 % of
the overall population (1). Due to frailty and/or neuromuscular impairment as well as osteoporosis
and comorbidities, the probability of falls and subsequent fractures in the elderly is high and the

consequences are dire. In fact, the one-year mortality after a hip fracture is estimated to be as high
as 30% in the elderly (2). The tremendous socioeconomic burden of fractures requires an orthopedic
t
awareness of the rise of patients with underlying pre-existing medical conditions (3). Since
ip
osteoporosis and aging represent fundamental aspects in this growing patient cohort, an in-depth
cr
understanding of mechanisms underlying fracture repair is crucial for improved treatment
us
approaches (4). Besides estrogen-deficiency, cellular senescence represents an independent
pathogenetic path accompanying aging which may lead to osteoporosis and represents a promising
an
future therapeutic approach (5).
M
Local cellular and molecular mediators of fracture healing

e d
Bone tissue is unique in that it can heal without a fibrous scar; however, the complex cellular and
pt
molecular interplay during the physiological and pathological bone healing process is not fully
understood (4). During the healing process, bones undergo endochondral ossification,
ce
intramembranous ossification and appositional bone formation. Various key molecular players
Ac
orchestrate the complex formation of new bone: the mechanical environment (i.e. stability) of the
fractured region, osteogenic and inflammatory cells, the vasculature providing mediators, and the
scaffold underlying the newly formed bone are all important in the process of bone healing (6).
Importantly, the processes of primary (direct) and secondary (indirect) fracture healing are clearly
distinct. The primary repair requires an anatomic reduction of the fracture site with high primary
stability to facilitate osteoclastic remodeling of the fracture and creation of “cutting cones”, in
6
humans forming new haversian channels (osteons). Direct intramembranous bone formation occurs
with immediate cortical remodeling and without greater callus formation. The inter-fragmentary
strain is reduced to a minimum and the fracture ends are anatomically restored (7). A primary
“tunnel” is established by osteoclastic cells to clear the way for blood vessels which are formed by
endothelial cells and perivascular mesenchymal cells. These mesenchymal cells serve as

osteoprogenitors and develop into osteoblastic cells, facilitated by the stable microenvironment (8).
t
The secondary (or indirect) fracture healing (via endochondral ossification) is characterized by micro
ip
motion between the fracture ends and subsequently the formation of a large callus, in which the de
cr
novo formation of embryonic bone is mimicked (9). Typically, this form of bone healing occurs after
us
intramedullary nailing and external fixation of fractures. Indirect fracture healing comprises
intramembranous (hard callus) and endochondral (soft callus) bone formation. In the former,
an
mesenchymal precursor cells of the inner periosteal layer proceed with direct bone formation via
differentiation into osteoblasts. Current data indicate that only newly formed osteoblasts contribute
M
to bone repair, whereas mature osteoblasts appear not to be involved in this step (10, 11). This
conclusion is based on genetic pulse-chase studies in C57BL/6 mice demonstrating a replicative

e d
population of stem cell/progenitors which dynamically replace osteolineage cells after injury. The
pt
mature osteoblasts present at the fracture site are almost completely (~90%) replaced after seven
days, and do not proliferate thereafter (10). No cartilaginous interim stage is built and the
ce
osteoprogenitor and undifferentiated mesenchymal cells directly form hard callus. In rabbit as well
Ac
as murine endochondral bone formation, a newly built cartilage matrix layer, established by
chondrocytes, is later converted into woven bone undergoing further remodeling into lamellar bone
(8, 10, 12).
7
Chronological progress of bone healing
Multiple attempts have been made to break down the complex temporal and spatial course of bone
healing (13–16). Since an exhaustive picture lacks accessibility and may result in oversimplification,
we tried to balance diverse paths for a comprehensive picture. For this purpose, we refer to a

temporal arrangement in four sections, accompanied by a spatial framework. In general, four
spatially divided regions can be distinguished: Cortical bone, bone marrow, periosteum, and external
t
(soft) tissue. However, these stages cannot be stringently distinguished and a simultaneous
ip
progression is more the rule than the exception. The main insights into the orchestrated process of
cr
bone formation were mostly obtained from mouse and rat fracture models. Regarding the time
us
course, we refer predominantly to murine data.
an
First healing period: Fracture hematoma and inflammatory response
M
Hematoma and Inflammation

d
Regardless of the fracture type, the first bone healing phase is invariably characterized by a robust
e
early inflammatory response and hematoma formation around the fracture site (Fig. 1). Around this
pt
cleft, the bone marrow architecture is lost and a cellular reorganization occurs (17). The initial
ce
function of the cells present at the fracture site is to debride the fracture site and prime a signaling
milieu in favor of the subsequent stages (4). Neutrophils migrate to the site of injury first and were
Ac
shown to be replaced by monocytes in mice after 24-48 hours (18, 19). These monocytes
differentiate into macrophages, secreting cytokines and growth factors (IL-1, IL-6, TNF-α, TGF-β,
iNOS, FGF2). Platelets and erythrocytes, as well as granulocytes and lymphocytes form an initial clot,
which partially reduces bleeding and serves as a provisional fibrin matrix for the upcoming
remodeling process. Periosteal cells were shown to proliferate and differentiate (mesenchymal stem
cells, MSCs) into osteogenic and chondrogenic progenitors at the fracture site in mice, developing
8
the subsequent reconstruction site (20). Transplantation experiments have demonstrated that
inflammatory cells in the callus area originate from the bone marrow, while some osteochondral
stem cells at the fracture site are derived from the local periosteum (21–23). The early hematoma
displays an enrichment in MSCs secreting bone morphogenic proteins (BMPs) promoting the
differentiation of progenitor cells to chondrogenic and osteogenic lineages (9). Next, an influx of

immunoregulatory cells occurs, including granulocytes, lymphocytes and macrophages, resulting in
increased expression of cytokines and growth factors, as demonstrated in mice and humans (6, 24,
t
ip
25). The overall role of IL-1, -6 and TNF-α, which peak early in the healing period, is critical as this
cr
leads to the recruitment of inflammatory cells and MSCs.
us
Macrophages act as coordinators of the healing process and their MCP1/CCR2 profile is essential for
MSC homing and migration (18, 26). Macrophages attract key cellular players in the initial healing
an
phase since they secrete multiple inflammatory factors and cytokines (IL-1, IL-6, TNF-α, iNOS, TGF-β,
PDGF, IGF, FGF2). Next to cytokine “homing”, the early coagulation leads to an “entrapment” of
M
immunoregulatory cells, primarily granulocytes, monocytes/macrophages and leukocytes (B cells, T
helper [Th] more than T cytotoxic [Tcyt] cells) from blood and bone marrow. MSCs differentiate into
e d
an osteoblastic phenotype at the periosteum and surrounding soft tissue, which is promoted by the
pt
Wnt/β-catenin pathway, as demonstrated in Lrp5-/- knockout mice (27). Upon activation of this
pathway, the number of osteoblasts is upregulated while the chondrocyte phenotype is decreased. T
ce
cells promote this osteoblastic differentiation as well. To limit the pro-inflammatory signaling, B cells
Ac
express IL10, reducing the early inflammatory response.
Osteocytes and Osteoclasts
As one of the most abundant cell types, osteocytes decrease in the acute phase by apoptosis,
mediated by caspase-3, and recruit osteoclasts to initiate bone resorption and release osteopontin
(OPN) to promote the recruitment of multiple cell types, as shown in stress fractures in rat ulnae
(28)and human femoral heads after fracture, where an age-related reduction in osteocyte viability
9
was detected (13, 29). During the early healing phase, sclerostin (SOST) expression of osteocytes is
downregulated directly after the fracture, demonstrated in fragility fracture patients with low-
energy hip fracture (30). In the later stages, high SOST-levels may facilitate endochondral ossification
by preserving cartilage, since sclerostin preserves chondrocyte metabolism, especially in mechanical
instability, as demonstrated in Sost-deficient mice (13, 31, 32). In the central hypoxic fracture area,

the osteoclasts promote the debridement of necrotic areas and formation of new soft callus. The
clearance of necrotic tissue and provisional matrix present after trauma is needed to further develop
t
ip
the provisional bone, which was demonstrated in rodents by Einhorn (17). As shown in
CX3CR1CreERT2/Ai14 tdTomato reporter mice, circulating CX3CR1+cells are recruited to the fractured
cr
bone by RANKL and macrophage colony-stimulating factor (M-CSF), lose CX3CR1 expression, and
us
become osteoclasts in order to partially clear and embed the de-sharpened fragments from the
an
forming callus area (33).
Matrix and Platelets

M
The structural backbone of newly formed tissue is formed by platelets interacting with extracellular
d
matrix collagen (type I [induced by IGF-1 and produced by osteoblasts] and type III) and fibronectin.
e
Platelets secrete, among other factors, PDGF, VEGF, TGF-β and IGF-1, resulting in both MSC
pt
differentiation and endothelial as well as fibroblastic cell proliferation. TGF-β (mainly TGF-β2 and 3)
ce
is initially produced by platelets and promotes the proliferation of MSCs as well as early osteoblastic
and chondrocyte cells. In addition, TGF-β also induces the expression of matrix components
Ac
(collagen I, fibronectin, osteopontin, osteonectin) and enhances bone formation, promotes
angiogenesis, and recruits immunoregulatory cells, as revealed in rat models and human long bones
(34, 35). Hence, chondrogenesis and endochondral bone formation are promoted. Thrombin and
soluble fibrinogen form a biological matrix in the hematoma, regulating the lysis rate. A number of
proteins (von Willebrand factor [vWF], tissue-type plasminogen activator [tPA], plasminogen
activator inhibitor [PAI] and fibroblast growth factor-2 [FGF-2]) regulate the early tissue formation.
10
IL-1, -6 and TNF-α are secreted by macrophages and other inflammatory cells, as well as
mesenchymal cells in the periosteum, driving the initial phase of fracture healing and reducing the
duration of cartilage formation (36). TNF-α is indispensable for the initiation of an intramembranous
stage on the periosteum, as shown in TNF-α receptor (p55 and p75) knockout mice (37). A bone
resident macrophage population referred to as “osteomacs” resides at the peri- and endosteum and

regulates osteoblast function, promoting fracture healing in mice (38). These CD68+ cells were also
detected in human bone samples. (39). The FGF level determines growth and differentiation of
t
ip
fibroblasts, osteoblasts and chondrocytes, cumulating in fibrin fibers sticking on early osteoblasts
cr
and subsequently leading to bone matrix accumulation (36).
us
Vascularization
Ruptured vessels from the periosteum, endosteum and surrounding tissue further facilitate the early
an
inflammatory response. As demonstrated in mice, a low oxygen tension at the site of the fracture
M
results in a hypoxic niche in which the early stages of fracture healing occur (27, 40). In this
environment, leukocytes and macrophages release vascular endothelial growth factor (VEGF) and
d
platelet-derived growth factor (PDGF). HIF and VEGF are central to regulating angiogenesis and
e
osteogenesis during the hypoxic stage. VEGF-dependent and angiopoietin-dependent pathways

pt
promote vascular growth into the developing callus.

ce
Ac
Second healing period: Angiogenesis, Cartilage formation/Soft callus
Since the remodeling process is neither strictly temporally nor spatially distinguishable, some distinct
fracture areas will undergo the healing process faster while others are still in early healing stages. In
the second healing period which is best described as an anabolic phase, the new formation of
vessels (angiogenesis) and formation of cartilage takes place in two distinct phases: endochondral
ossification (soft callus formation) and intramembranous ossification (hard callus formation) (Fig. 2).
11
Endochondral ossification (central parts, soft callus)
The endochondral ossification phase appears in the central parts of the fracture (as opposed to the
peripheral intramembranous ossification), where avascular cartilaginous tissue is built to deliver a
first “bridge” for mechanical microstability by chondrocytes (9, 41). This phase initiates the second

stage of mesenchymal cell activation, a process in which undifferentiated MSCs differentiate into
chondrocytes and osteoblasts from day three on. From day 3-7 onwards these cell types increase
substantially in number, with a maximum on day 6 after the fracture; these MSCs (now SMA9+)
t
ip
expand and develop characteristics of osteochondroprogenitor cells, as shown in vivo in
cr
αSMACreERT2 mice (OPCs) (42). In these cells, the Runx2 gene acts as regulator of
us
osteoblastogenesis, while downstream Sp7 (Osterix) suppresses RUNX2, leading to a higher
chondrogenic potential (43).

an
The overall cartilage building process in the soft callus starts with a collar of bone forming adjacent
M
to the fractured bone site at the periosteum that stays connected to the soft callus (17).
Undifferentiated MSCs proliferate here, giving rise to a subset of chondrocytes (Sox9+). These,
d
together with fibroblasts, form a type II collagen/aggrecan structure from day 3 on. This constitutes
e
the matrix for the avascular cartilaginous callus. The newly formed cartilage matrix mechanically
pt
bridges the fracture gap and provides stabilization; however, vertical (not transverse) cartilage is
ce
formed, as revealed in osteoclast-deficient mice (41, 44). The matrix is incrementally replaced by
cartilage. After the chondrocytes mineralize the cartilaginous matrix, they become hypertrophic and
Ac
produce collagen X, which appears to be dependent on TNF-α in mice (37). GDF-5 (BMP14) as well as
TGFβ2 and 3 reach their maximum on day 7, marking the maximum of cartilage formation (type II
collagen) and underlining the importance of chondrogenesis. The woven bone also covers the
external fibrocartilaginous callus surface and is thus the first step of mineralization of the
fibrocartilage scaffold. The induction of IL-6 is essential for mineralization and early remodeling of
12
the fracture callus, as shown in mice (6, 45, 46). This early remodeling requires the recruitment of
osteoclasts, osteoblasts and fibroblasts to the site of fracture.
In the inflammatory phase (Phase 1), the formation of classically activated macrophages (CAM) is
promoted. In the later soft callus phase (Phase 2), the macrophages (now polarized into M2 and

anti-inflammatory) are alternatively activated via IL-4 and IL-13 and participate in collagen
deposition, as was demonstrated in mice (47, 48). These cells promote healing and downregulate
t
the later phases of fracture healing (49). The high levels of IL-1, IL-6 and TNF-α that drove the initial
ip
repair phase is continuously reduced during cartilage formation (before rising again in late bone
cr
remodeling).
Vascularization
us
an
The vascularization of soft callus tissue is mainly driven by VEGF (primarily VEGF120 and VEGF164),
FGF-1 and TGF-β. VEGF levels of osteogenic cells are Runx2 dependent and driven by BMPs (from
M
osteoblasts and osteoblast-like cells (36)). VEGF represents a downstream target of HIF1-α and binds
to the cartilage matrix during endochondral ossification. The final phase of endochondral ossification
d
and remodeling is driven by bone matrix metalloproteinases, degrading cartilage and bone.
e
pt
Intramembranous ossification (hard callus)

ce
The intramembranous ossification (formation of hard callus) recapitulates embryonic
intramembranous ossification, where woven bone is formed (9). This bone formation route is mainly
Ac
driven by osteoblasts and takes place on the mechanically more stable periosteal region, where
MSCs differentiate into osteoblasts directly forming woven bone. Cytokines including CXCL12 (under
the control of HIF1α) from the periosteum lead to mobilization and adherence of these MSCs from
the periosteum close to the fracture site and the bone marrow in the fracture area. These MSCs
differentiate into osteoblastic cells and eventually begin to express an osteocyte phenotype, guided
by E11 and Cx43 as shown in vivo for knockout mice, rats and in vitro (13, 50–52).
13
Adjacent to the inner periosteal layer, the hard callus is formed out of type V and I collagen, mainly
driven by BMP-2, 4 and 7. However, in order to prevent excessive callus formation, the function of
BMPs needs to be antagonized, which mainly happens by inhibitory molecules such as Noggin (NOG)
(53). During this phase, the inflammatory cytokines are highly released by MSCs (not by
inflammatory cells) and their local descendants (osteoblasts and chondrocytes).

In summary, the chondrocytes produce cartilage spanning the fracture gap and connecting the bony
t
ends in order to mechanically stabilize the fractured area. This soft callus serves as scaffold for the
ip
endochondral bone formation, and a fibrocartilage tissue is formed in the central area. In contrast,
cr
osteoblasts form the cortical bone and osteoprogenitors form hard callus/woven bone on the
us
fracture ends.
an
Third healing period: Cartilage removal, Calcification/Hard callus
M
The third bone healing stage is characterized by the gradual enlargement of cartilage and beginning
d
calcification and it marks the initiation of a more catabolic process although osteoblastic and
e
osteoclastic processes alternate (Fig. 3). Intramembranous ossification and endochondral

pt
ossification are complementary processes required for the formation and remodeling of the callus
area.
ce
Intramembranous ossification
Ac
During intramembranous ossification, the bone is directly formed without a cartilage scaffold. It
takes place in mechanically more stable (peripheral) regions of the callus where it is restored to form
a collagenous network (hard callus). The osteoprogenitor cells from the local periosteum contribute
to the direct bone formation, while local endothelial cells transform into polymorphic cells with an
osteoblastic phenotype (36).
14
Endochondral ossification
After mechanical stability is reached, the previously formed cartilage becomes hypertrophic and
mineralizes (36). The hypertrophic chondrocytes are therein “buried” and participate in the
calcification of remaining extracellular matrix (ECM), and are subsequently removed by

chondroclasts (54). Although controversial, there is evidence of a change in the morphology of
hypertrophic chondrocytes, transitioning into an osteoblast/osteocyte phenotype with cellular
canalicular extensions both from developmental and fracture models. Thus, using Col10a1int2-
t
ip
Cre;ROSAEYFP mice, Yang et al. first demonstrated the specificity of Col10a1int2 for hypertrophic
cr
chondrocytes, before Col10a1int2-Cre;ROSAEYFP mice were used to detect the coexpression of Col1a1,
osteocalcin and bone sialoprotein within YFP+ cells, strongly indicating an osteoblastic transition. The
us
YFP+ cells accounted for 31% of all COL1A1+ osteoblasts in the metaphysis, and after two months,
an
YFP+ cells were entrapped in the bone, morphologically representing osteocytes (55). Additionally,
using Col10a1-Cre;Osxflox/+ mice (which express EGFP only in Osx-expressing cells following Cre-
M
mediated recombination), Zhou et al. demonstrated that Ocn+ and EGFP+ cells, representing mature
osteoblasts on the bone surface, originated from hypertrophic chondrocytes (56). In addition, within
e d
fractured Agc1-CreERT2; 2.3-GFP;ROSA-tdTomato mouse tibiae, many Tom+ cells were positive for
pt
GFP, indicating a transdifferentiation of chondrocytes to become osteoblasts. Similarly, Hu and
colleagues showed that within the transition zone, chondrocytes lose Sox9, Col2a1 and Col10a1
ce
expression, and express Col1a1 and Runx2. Later, osteocalcin and osteopontin were detected in
Ac
these cells as well. Performing lineage-tracing experiments (Ai9 to Col2CreERT and Agc1CreERT mice),
the authors showed that chondrocyte-derived cells line the bone surface of newly formed bone,
with the vascularization possibly triggering this transdifferentiation (54). Together, experimental
evidence suggests that, at least in part, these hypertrophic chondrocytes become true osteoblasts
(13, 47, 54). After vessel formation into formerly cartilaginous tissues has taken place, the entrapped
hypertrophic chondrocytes lose their Sox9 expression, leading to enhanced Runx2 and β-catenin
expression and subsequently expression of alkaline phosphatase, osterix, osteopontin and
15
osteocalcin. These transcriptomic changes were demonstrated to result in the calcification of the
cartilage matrix. Upon mineralization, bone matrix is formed on top of the calcified matrix. The
osteoblasts are marked by expression of the Wnt signaling components Dishevelled (Dsh) and β-
catenin and gradually replace chondrocytes (57). Osteoblasts initially form woven bone (58), and M-
CSF and RANKL recruit osteoclasts to resorb this bone which is followed by the formation of new

lamellar bone (41, 59).
t
One crucial molecule in the third healing period and endochondral ossification, binding to multiple
ip
BMP receptors, is Noggin, which opposes BMP effects on osteoblastic differentiation and impairs
cr
osteoblastogenesis. Noggin-overexpressing mice were more prone to fractures, osteopenia and
us
decreased bone formation, thus defining a role for Noggin in the reduction of callus formation (60,
61).
an
The important function of TNF-α includes the stimulation of osteoclastic function, which is essential
M
for the resorption of mineralized cartilage in the later phases of the third healing stage.
Synergistically with IL-1β, matrix mineralization by MSCs is also promoted by TNF-α as shown in vitro
d
(62, 63). As a consequence of these processes, the greatest amount of hard callus is reached on day
e
14 post fracture. At the same time, degradation of cartilage in endochondral ossification with
pt
matrix-metalloproteinases (especially MMP13 and MMP9) is initiated, degrading type II collagen to

ce
gelatin (36, 47). MEPE, similar to DMP-1 in the middle stage mineralization process, is present in the
osteocytes in later stages of newly formed bone and is subsequently externalized in osteocyte
Ac
lacunae (13, 64).
Fourth healing period: Chronic bone remodeling
The final remodeling phase in rodents begins approximately three weeks after fracture and can
continue for months or years and should, therefore, be referred to as a chronic stage. In this phase,
16
the woven bone is steadily replaced by superior lamellar bone while the overall callus size is reduced
and the normal hematopoietic and trabecular structure restored (14, 17) (Fig. 4).
Molecular pathways
In this stage, a resorptive cascade with remodeling of hard callus into lamellar bone with a central

medullary area ends the process of bone healing. The major drivers are IL-1, IL-6 and TNF-α (partially
BMP-2) (36, 47, 65, 66). Certain BMPs, such as 3, 4, 7 and 8, usually peak in expression levels in the
t
third week post fracture and drive cartilage resorption as well as osteoblastic recruitment (36, 67).
ip
Wnt is the crucial underlying pathway favoring osteogenesis and inhibiting chondrogenesis (13). In
cr
the late stages of fracture healing, sclerostin is upregulated and inhibits chondrogenic differentiation
us
of subperiosteal MSCs via competitive binding of BMP receptors (13). TNF-α promotes the apoptosis
of osteocytes (6), while some chondrocytes become apoptotic or necrotic in the remaining soft
an
callus. Multiple soluble factors are liberated in this process, including TGF-β, WNT10B, BMP6, SLIT3,
M
MMP13 and MMP9 which are crucial for subsequent osteogenesis (47).
Cellular compartment
e d
In the cellular compartment, the remodeling process is orchestrated by osteoclasts, which are the
pt
key responsive elements although a morphological difference between chondroclasts and
osteoclasts has not been formally demonstrated (6, 68). The hard callus is resorbed by osteoclasts,
ce
and lamellar bone is built by osteoblasts (47, 58).

Ac
In addition, the MSC/macrophage crosstalk plays a crucial role for the regulation of bone healing (6,
69). M1 polarized macrophages (pro-inflammatory) promote mineralization of MSCs, in part by
producing oncostatin M, while MSCs in turn promote the polarization shift from the M1 to M2
macrophage phenotype via TSG-6 and PGE2, decreasing pro-inflammatory cytokine secretion (6, 69).
Finally, the remaining osteoclasts become apoptotic via activation of their c-FMS receptor. In human
bone, this promotes the restoration of the Haversian system, a central vascular canal providing
17
supply to a branching canalicular network, with the osteoblasts and osteocytes as central elements,
and Mepe and osteocalcin as important regulators of that process (70–73). The ultimate stage of
stability is usually not reached until several months after the injury. In fact, the complex remodeling
process that re-establishes this Haversian system takes months or years to complete (6, 74, 75).

Regulation of fracture healing by systemic factors
t
ip
The bone healing process is influenced by systemic factors, endocrine diseases and medications, out
cr
of which the most important are discussed in detail below.
us
Parathyroid Hormone (PTH)
Mechanistically, anabolic therapy with PTH was suggested to facilitate fracture healing via the Wnt
an
signaling pathway (76). In preclinical murine femoral fracture models, daily administration of 1-34
M
PTH was found to facilitate fracture healing after 14 days. In contrast, a continuous infusion of PTH
1-34 (mimicking hyperparathyroidism) delayed fracture healing in very young mice, yet increasing
d
ultimate biomechanical properties on day 21, but not thereafter (77, 78). Treatment with 1-34 PTH
e
improved angiogenesis and recruitment of MSCs to the callus area in mouse models, whereas
pt
endogenous PTH deficiency in PTH knockout mice reduced RANKL expression in osteoblasts, thereby
ce
impairing osteoclast activity (77, 79).

Ac
While systematic prospective double-blind and controlled clinical trials for anabolic PTH therapy are
not available, enhanced fracture healing in osteoporotic pelvic fractures after daily treatment with
100 μg PTH 1-84 was observed (although in a small study population, n=21 treatment vs. n=44
control), whereas the improvements after treatment of osteoporotic distal radius fractures with 20
μg 1-34 PTH (teriparatide) were minor, but early cortical bridging was detectable (80, 81). In lumbar
fusion surgery, prospective studies have demonstrated improved union rates after daily
subcutaneous administration of 20 μg PTH 1-34 as compared to oral bisphosphonate therapy in
18
postmenopausal women (82–84). However, further randomized controlled trials in osteoporotic and
stress fractures are ongoing (NCT02972424, NCT04196855).
Growth hormone/Insulin-like growth factor 1 (GH/IGF1)
Increased GH and IGF-1 levels are hallmarks of acromegaly. These patients have secondary

osteoporosis with trabecular bone deficits and an increase in vertebral fracture rates, presumably
from concomitant hormonal deficiencies (e.g., hypogonadism) (85–87).
t
ip
Evidence from human studies is limited and although a role in consolidation of delayed union is
cr
possible, the clinical effect of an impaired GH/IGF-1 axis on fracture healing has not been sufficiently
characterized in human studies (88). Exposure of human MSCs to low doses of IGF-1 in vitro
us
increased the early osteogenic differentiation of MSCs. This finding suggests that IGF-1 treatment
an
could be a promising strategy for clinical trials, in cases of impaired fracture healing; however,
further in vivo evidence is missing (89, 90). Nonetheless, a clinical limitation is the expense and need
M
of i.v. or s.c. administration (91).

d
Besides its role in osteogenic differentiation, IGF-1 has been implicated in acromegaly. Apart from a
e
GH excess, the subsequent IGF-1 increase activates the Wnt/β-catenin pathway in acromegaly
pt
patients, leading to an increase of osteoblastic recruitment and, subsequently, enhanced callus
formation (evidence from dystrophin knockout mice (92)). Moreover, IGF-1 is crucial for the M1/M2
ce
macrophage transition in transwell experiments (93). Likewise, the important role of IGF1 becomes
Ac
evident in Igf1r knockout mice, where a reduced callus size and lower bone volume after tibial
fracture is accompanied by a mineralization defect and increased early osteoclast numbers –
although the effects in an inducible Igf1r knockout were inconsistent (94). In addition, local
administration of a combination of IGF-1 and TGF-β resulted in an increased bone defect healing
capacity in old rats (95).
19
Mineral metabolism
Ninety-nine percent of calcium present in the human body is deposited in bone and it is known to
play a crucial role in bone remodeling and mineralization (96). In rodent studies, a calcium deficit
resulted in reduced callus mineralization and biomechanical parameters (97, 98), while calcium

malabsorption could be counteracted by increased calcium release from the intact skeleton during
fracture healing, as shown in mice (99). Supplementation of calcium in combination with vitamin D
t
in osteoporotic C57BL/6 mice improved bone repair by reducing osteoclasts and increasing
ip
osteoblasts (100).
cr
Although one of the most frequent ions in bone, the effect of magnesium on fracture healing has not
us
been studied extensively. Interestingly, a link between systemic hypomagnesaemia and low BMD
(and partly fracture risk) in men has been drawn in large epidemiological studies, while the effects
an
on bone healing have not yet been experimentally defined (101, 102). In addition, according to a rat
M
study, locally administered magnesium is beneficial in promoting fracture healing. In this study, a
magnesium-containing nail facilitated femoral fracture repair by promoting CGRP-mediated

d
osteogenic differentiation (103).

e
Vitamin D
pt
Prospective clinical studies on the effect of vitamin D supplementation in healing bone are limited
ce
(104, 105). Most studies either pair vitamin D supplementation with calcium, or lack a control group.
Ac
Case series report vitamin D deficiency to be associated with non-union, but a causal connection has
not been made (106). However, a large inception cohort study of 309,330 human fractures reported
vitamin D deficiency to be associated with a slightly increased non-union rate (OR 1.14 [1.05-1.22]
(107)). In a randomized, placebo-controlled study with 30 postmenopausal women suffering from
proximal humerus fractures, supplementation with 800 IU vitamin D3 plus 1g calcium improved
BMD levels within the fracture after week 6 compared to the placebo group (108). In another RCT
20
with 32 postmenopausal women with distal radius fractures, however, low-dose (700 IU) vitamin D3
treatment did not lead to significant differences in pQCT parameters at the fractured radius site 12
weeks post fracture (cortical and trabecular bone density, stiffness) versus a high-dose (1800 IU)
vitamin D3 treatment group (109).

In a preclinical study, 24,25(OH)2D3 was injected subcutaneously (6.7 μg/kg daily) into wildtype
C57BL/6 mice and improved stiffness on day 10, 18 and 21 in a tibial shaft fracture model, while a
genetic lack of 24,25(OH)2D3 (Cyp24a1-/- mice) led to impaired endochondral callus formation,
t
ip
reduced callus volume and stiffness (110, 111).In another femoral shaft fracture model, C57BL/6 J
cr
mice, were fed a calcium/vitamin D deficient diet for eight weeks before being supplemented with
us
calcium/vitamin D for 23 days. While the deficient diet itself increased osteoclast activity and
reduced bone mass, supplementation after the fracture reduced bone resorption and improved
an
bone repair, and did not lead to reduced BMD in the fracture callus compared to control diet, as was
the case for permanent calcium/vitamin D deficiency (100).

M
In summary, the available data on vitamin D effects on human fracture healing remain inconclusive
d
with a lack of large RCTs. There may be some beneficial effect of vitamin D and/or the combination
e
with calcium, but this is mainly based on preclinical studies.

pt
ce
Impaired fracture repair in the setting of endocrine diseases

Ac
Diabetes mellitus (DM)
The Centers for Disease Control and Prevention (CDC) reported that 10.5% of the US population
(34.2 million) suffered from diabetes in 2020 (112). Importantly, diabetic patients are particularly
prone to fractures and impaired bone healing (113, 114).
21
According to multiple meta-analyses, an increased fracture risk and susceptibility towards fracture
malunion and reoperation in diabetic patients is not reflected by equally reduced BMD levels, at
least in patients with type 2 DM (113, 115–119). Possible underlying reasons for the impaired bone
healing in diabetic patients may be an increased concentration of TNF-α at the fracture site,
accompanied by a higher number of osteoclasts in the diabetic callus, as demonstrated in murine

diabetic tibia fractures (120, 121). Further studies of diabetic bone healing in rodents revealed
contradicting findings: Kayal et al. studied type 1 diabetic mice, which displayed increased
t
ip
chondrocyte apoptosis and osteoclastogenesis which accelerated cartilage loss and reduced
cr
endochondral bone formation; these deficits were reversed by insulin administration (122). By
contrast, reduced osteoclastogenesis and osteoclast activity were detected in a type 2 diabetic rat
model when glucose levels were high (123).

us
an
In human studies, Mangialardi et al. demonstrated dysfunction of CD146+ pericytic cells in patients
with type 2 diabetes, possibly reducing the cellular and vascular supply at the fracture site (124).
M
Along with the increased tendency of diabetic MSCs to differentiate into adipocytes instead of
osteoblasts, the reason for an increased fracture rate in diabetic patients may be both quantitative
e d
and qualitative (125).

pt
Interestingly, local administration of insulin using an intramedullary delivery system at the fracture
ce
site was shown to contribute to fracture healing in both non-diabetic rats and rats with type 1
diabetes (126, 127). In female type 1 diabetic mice, delayed fracture healing was reversed by
Ac
systemic insulin (and vitamin D3) treatment, possibly due to increased IGF-1 production in the
fracture callus (128).
Glucocorticoid (GC) excess
Glucocorticoids not only reduce overall bone mass (secondary osteoporosis), but also affect the
inflammatory bone healing process. The induction of inflammatory mediators in the initial healing
22
cascade is markedly reduced with GC treatment. GCs favor differentiation of specific anti-
inflammatory phenotypes of monocytes and macrophages (especially M2c) via activation of the
adenosine receptor A3 and inhibit the production of vasodilators such as VEGF, as demonstrated in
in vitro experiments (129–132). Prolonged GC treatment, similar to glucocorticoid-induced
osteoporosis, delays chondrocyte hypertrophy, attenuating endochondral bone healing via reduced

production of the chondrogenic differentiator TGF-β in murine experiments (133). Interestingly, the
early healing period (intramembranous ossification) appeared to be less altered after GC treatment
t
ip
compared to the late (endochondral ossification) healing period in this model (133).
cr
Correspondingly, the healing of murine shaft fractures was delayed after treatment with
dexamethasone, whereas the metaphyseal healing of femoral fractures was enhanced, confirming
us
an inhibitory effect of GC mainly in endochondral ossification (134). A similar impact on mainly
an
endochondral ossification was demonstrated after 3 months of prednisone treatment, delaying
endochondral ossification in mice (135)

M
Using a global glucocorticoid receptor knockout model (GRgtROSACreERT), Rapp et al. demonstrated
impaired fracture healing via an increase in the early inflammatory response and reduced late
e d
endochondral ossification, possibly due to negative regulation of osteoclast recruitment and

pt
osteoblast accumulation(131, 135–137). Interestingly, an impaired glucocorticoid receptor
dimerization ability protected from systemic trauma-induced compromised fracture healing (138).
ce
GC treatment impaired osteogenic differentiation via COX-2 and RUNX2 inhibition in a mandible
Ac
bone defect rat model (139). Although these animal studies provide insights into possible effects of
GCs on fracture healing, there is a paucity of clinical studies evaluating bone healing after GC
treatment (11).
23
Aging
Aging is associated with multiple disorders including diabetes, osteoporosis, atherosclerosis, cancer,
Alzheimer’s, and Parkinson’s disease. It has been challenging to separate the aging process from
comorbidities such as osteoporosis and their effect on fracture healing (140). Although a slower and

less reliable fracture healing process in elderly individuals appears likely, there is a significant lack of
prospective clinical trial data to verify this (4, 141). Summarized below is evidence from studies
t
examining fracture non-unions that provides some, albeit inconsistent, evidence for age as a risk
ip
factor for fracture non-union.
cr
Zura et al. found in a prospective cohort study using 2.5 million Medicare patients, 56,492 fractures
us
and 1,440 non-unions that increasing age is linked to a decreased risk of fracture non-union. Based
on their findings, the authors suggested that increased survival may reduce the impediments to
an
healing. The bones most prone to non-unions (highest odds ratio [OR]) were the scaphoid, femur,
M
humerus and tibia+fibula, followed by the clavicle. As acknowledged by the authors, however, a
weakness of this study is a potential lack of observing non-unions in the elderly due to inconsistent
d
follow-up (142). A likewise higher risk for younger patients for non-union was reported by Mills et al.
e
(143), who prospectively analyzed 4,715 non-unions over five years. The highest non-union rates
pt
appeared in the 30-44-year age group, and the most susceptible bones were the tibia+fibula,
ce
followed by the clavicle and humerus. A reason for a reduced non-union rate in femoral fractures in
older individuals may be the different treatment of choice (elderly more likely to receive
Ac
arthroplasty, while younger patients are internally fixated to a greater extent). A weakness of this
study is that just symptomatic non-unions were treated, and operatively treated non-unions were
included. In addition, the data were solely derived from the NHS Scotland database (excluding
patients within the private sector) (143). To overcome site-specific differences, a retrospective
review analyzed 1,003 reamed intramedullary nailed tibial fractures in a Scottish trauma center over
22 years, and found that age significantly influenced fracture non-union, with middle-aged patients
24
(30-49 years) having the highest risk of developing this complication. Selection bias and the
retrospective design as well as a biased cohort selection limit the applicability of that study (i.e.
patients 70-79 had a comparable non-union rate to patients in their 30s and 40s, around 15%) (144).
Overall, these studies do not support a higher rate of non-unions in the elderly population. However,
the study by Zura et al. reports distinct co-morbidities leading to higher non-union rates, including

smoking, alcoholism, obesity, cardiovascular disease, osteoarthritis, osteoporosis and type II
diabetes (142), introducing a possible confounding effect of a chronic inflammatory state. In this
t
ip
context, aging might be a modifying factor, and although female sex was not predictive of non-
cr
unions in this study, the non-union number in the Mills study peaked in women aged 65-74 years. It
may subsequently be advisable to evaluate these postmenopausal women separately in order to
us
detect the influence of menopause on fracture healing ability (142, 143).
an
The tibia, followed by the clavicle and the humerus are the primary bones prone to non-union (145).
In fact, perhaps the largest prospective study assessing non-unions in clavicular fractures highlighted
M
age consistently with the fracture displacement as risk factors for non-union (146). In osteoporotic
vertebral fractures, Inose et al. did not verify age as a risk factor for non-union in a unique
e d
prospective multicenter study (147). In retrospective studies on clavicular fractures, age remained a
pt
significant factor (odds ratio 1.07,), while in tibial diaphyseal fractures and scaphoid fractures, it was
not (148, 149). Intriguingly, in a meta-analysis of 41,429 tibial fractures, age > 60 was demonstrated
ce
to be predictive for fracture non-union (150). The importance of age in the appearance of non-
Ac
union, however, has been questioned since in retrospective studies of non-unions, age could not be
confirmed as significantly influencing the time to union in multivariate analyses (151). Thus, overall,
age cannot consistently be identified as a risk factor for non-union in generally every bone. However,
older age likely raises the risk of a pseudarthrosis in certain patients and sites (i.,e., clavicle, tibia).
Because co-morbidities such as smoking and diabetes mellitus have a detrimental effect on bone
healing as shown at the molecular level (113, 119, 152, 153), studies on the effect of aging alone
require a careful design (154).
25
Age-related changes in fracture repair
The single largest risk factor for various chronic diseases responsible for the majority of morbidity
and mortality is aging (155). Based on the “geroscience hypothesis”, the treatment of underlying

aging mechanisms may lead to new therapeutic approaches for our aging population (156, 157).
Since the identification of senescent cells in the bone microenvironment, numerous studies have
t
identified changes at the cellular and molecular level in the bone microenvironment. Drivers of these
ip
aging-related effects are accumulating senescent cells, which are characterized by high p16Ink4a and
cr
p21Cip1 levels, causing detrimental effects via secretion of a senescence-associated secretory
us
phenotype (SASP). However, the effects of these senescent cells and their secretome on the
dynamic bone healing process are largely unexplored (5, 158–160).

an
Generally, the overall molecular program of fracture healing appears to be unimpaired in aged mice,
M
while the callus volume, mineral content as well as rigidity and breaking load are reduced in elderly
mice and humans (154, 161). Interestingly, it was demonstrated that a “rejuvenation” of the bone
d
marrow affected bone healing: The experimental transplantation of bone marrow from young
e
(Rosa26) into old (irradiated C57BL/6J) mice resulted in larger calluses and more bone mass in the
pt
early healing period and rapid callus remodeling at later stages of healing in older mice, indicating
ce
that aging of bone marrow cells is affecting the later stages of bone healing as well (114–116). The
particular cellular components of fracture healing are each differently affected by aging.
Ac
Vascular supply
A reduced vascular capacity and angiogenesis delay fracture healing in the elderly that is not
necessarily associated with diabetes (4, 47). Vascular supply/perfusion is decreased in the aged
organism, subsequently impairing the number of total cells directed to the fracture area (162). One
proposed mechanism is reduced endothelium-dependent vasodilatation, as described in femoral
26
fractures in old rats through impairments in NOS signaling (162). Another possible underlying
mechanism might be reduced VEGF release: since the early inflammatory phase and chondrogenesis
are delayed in older animals due to a prolonged increase in TNF-α expression (mainly by MSCs and
macrophages), the overall VEGF release is reduced which is also a major contributor to delayed bone
healing in diabetic mice (163). However, it has also been demonstrated that low levels of TNF-α

promote the osteogenesis of muscle-derived stem cells (MDSCs) near the fracture site in the murine
model, expediting fracture repair (65). Interestingly, TNF-blockade via subcutaneous sTNFR1
t
ip
administration accelerated fracture healing in the aged mouse model and appeared to be mediated
cr
by p21 (Cdkn1a) (164). Overall, the precise role of TNF-α in ageing-related fracture healing is not
fully understood.
Macrophages
us
an
The importance of macrophages during bone healing is underlined by the fact that their deletion in
M
the inducible Mafia (macrophage Fas-induced apoptosis) transgenic mouse model in murine femoral
fractures resulted in a completely abolished callus formation (69, 165). As the macrophages, which
d
are polarized towards an M1 state throughout the early healing period, contribute substantially to
e
the fracture healing process, the impact of their age-related impairment is crucial. Aged
pt
macrophages are less responsive to granulocyte macrophage colony-stimulating factor (GM-CSF)

ce
stimulation, resulting in decreased proliferation, a chronic inflammatory state and impaired bone
healing. The GM-CSF-dependent stimulation decreases with age while the polarization toward an
Ac
M2 phenotype was reported to be favored, possibly due to increased levels of S-endoglin (166, 167).
The alternatively activated M2 macrophages show increased survival and reduced secretion of pro-
inflammatory factors with age, and appear more often in the late healing stages (18, 69). However,
Mahbub et al. demonstrated in vitro that an overall impaired polarization in elderly macrophages
might dysregulate the host response (168). Either way, the role of these aged macrophages on
fracture healing in the elderly appears to be more detrimental than helpful: in elderly mice, the
27
blockade of macrophage recruitment to the fracture site subsequentially enhanced fracture healing
(169). Interestingly, in ovariectomized mice, a reduction of both M1 and M2 macrophages in early
healing stages might contribute to impaired fracture healing (170).
The pivotal impact of macrophages for the aged versus young healing process has been

demonstrated for F4/80+ cells in heterochronic parabiosis studies by Vi et al. (171): Macrophages
which were isolated from old mice (20 months old) and transplanted into young (4 months) animals
t
led to reduced BV/TV and calcified callus volume after four weeks of tibia healing. By contrast,
ip
macrophages isolated from young mice and transplanted into old mice were able to rejuvenate the
cr
otherwise impaired healing capacity (171).
us
The close connections of macrophages and MSCs is likewise crucial for the healing process.
Macrophages are known to recruit MSCs via CCL2 and CXCL8 (69), and a reciprocal
an
immunomodulatory function via reduction of pro-inflammatory cytokines and induction of IL-10 in
M
macrophages by MSCs has been demonstrated in vitro (69, 172). Additionally, MSCs modulate
macrophage chemotaxis, and shift the monocyte polarization towards M2 macrophages in vitro
d
(173). The exact nature of the macrophage/MSC interaction is still not fully understood, however,
e
and direct cell-cell contact as well as soluble mediators are likely involved (69).
pt
Macrophages play a key role in eliminating senescent cells, and impairments in this process likely
ce
contribute to impaired tissue regeneration (174). Macrophages show critical regulatory activity in all
stages of tissue repair by clearing senescent cells (175). Senescent cells display certain hallmarks
Ac
associated with aging including increased expression of Cdkn2a (p16) and Cdkn1a (p21) (155, 156,
159). Interestingly, p16- and beta-galactosidase-positive macrophages are not necessarily senescent
but may be cleared by senolytic compounds (176); the impact of this on tissue repair, including
fracture healing, remains unclear.
MSCs
28
The bone marrow progenitor cells from the periosteal region and bone marrow derived stem cells,
out of which osteogenic and chondrogenic progenitors are recruited, are reduced in quantity and
quality, and a later initial periosteal reaction to the fracture can be detected in older mice (177).
However, the sole number of MSCs does not appear to be critical: while the total number of MSCs

from older mice was demonstrated to be higher, not as many osteoprogenitor cells develop out of
these as in younger mice (and are affected by sex) (178, 179). Additionally, their replicative lifespan
t
has been demonstrated to be substantially shortened, oxidative damage is enhanced and
ip
chondrogenic differentiation prolonged (16, 180, 181). As noted above, aged human MSCs have
cr
been described to overexpress TP53, CDKN2A (p16) and CDKN1A (p21), BAX and MYC (18, 182, 183).
us
Likewise, skeletal stem/progenitor cells (SSPC) secrete SASP factors with aging, resulting in an
activation of NF-κB in other SSPCs, inducing a state of senescence, leading to reduction of self-
an
renewal and proliferation, and decreasing the total cell number. The osteogenic ability of these aged
SSPCs, which express higher levels of Cdkn2a/p16 than their younger counterparts, has been shown
M
to be reduced in vivo and in vitro, and this was attributed to an increase in NF-κB mediated
inflammation (141).
e d
Recently, the osteoimmunological impact on fracture healing has gained attention (184, 185). Due to
pt
their low expression of MHC I and II molecules, MSCs do not induce an immunological response and
ce
can be used for transplantation experiments (186). Moreover, MSCs suppress T cell proliferation,
and induce Tregs to suppress the immune system, while T cells in general, particularly via IFN-γ,
Ac
reduce MSC-induced osteogenesis in murine allogeneic stem cell transplantation experiments (186,
187). These findings are corroborated by the detection of CD8+ effector memory T (TEMRA) cells in
humans in peripheral blood and the fracture hematoma which, by producing IFN-γ/TNF-α, inhibited
the osteogenic differentiation of MSCs(188).
In several meta-analyses, nonsteroidal anti-inflammatory drugs (NSAIDs) were reported to impair
fracture healing (increase delayed union or nonunion in humans (189) and decrease biomechanical
29
properties in animal models (190)). But it has likewise been shown that NSAIDS are able to inhibit
the SASP-associated activation of NF-κB, thereby potentially enhancing bone regeneration (141,
191). However, there is still no final conclusion regarding whether the beneficial or detrimental
effects of NSAIDs on bone healing are predominant due to the lack of appropriate prospective
randomized controlled trials (192).

Lymphocytes
t
As noted above, the role of the inflammatory system in an aging organism has been elucidated and
ip
summed up with the term ”inflammaging“ (193). A link to the inflammatory mediator
cr
cyclooxygenase (COX) and lipoxygenases (LOX) has been well-described: the inflammatory response
us
is more intense in aged female compared to young female mice, which delays bone healing, and can
be reduced by genetic deletion of 5-lipoxygenase (LOX) (194, 195) or pharmacologic inhibition via
an
NSAIDs or derivates (191, 196). Regarding the cellular components of this inflammatory phase, the
M
total number of T cells in general and regulatory T cells in particular is reduced in the old human
organism (197) T-cell migration is controlled by the chemokine receptor CCR2. One underlying
d
reason for the impaired immune function due to aging has been proposed as being a CCR2
e
(chemokine receptor for ligands including CCL2, CCL7 and CCL12) defect/deficiency, as
pt
demonstrated in mice (198). Apart from the osteochondral stem cells, some inflammatory cells
ce
originate in the bone marrow and their replacement in old mice via bone marrow transplantation
from young mice results in larger calluses and earlier bone formation in the early phases as well as
Ac
faster callus remodeling in the late stages (199). Proinflammatory cytokine expression from T cells as
well as B cells in the callus is mediated by IL-10, inducing proper healing, while a missing early
upregulation of IL-10 was associated with non-union in human tibia fractures(200). Affirming the
importance of IL-10 on fracture healing, B cells from patients with poor fracture healing had
decreased levels of IL-10 compared to regularly healing patients.
30
Chondrocytes
Chondrocytes and their precursors change their size and function throughout the healing period and
depending on age: in the early bone healing process, chondrocytes do not express collagen type II in
old mice but do so in young mice, indicating a faster adaptation to the acute fracture event (201).

Correspondingly, chondrocyte maturation is present earlier (at day 5) in young mice but not in older
and middle-aged mice (201). Older chondrocyte progenitors express significantly lower levels of
t
COX-2, a critical marker in the early inflammatory phase regulating chondrogenesis, bone formation
ip
and remodeling, as compared to young mice (202). This reduction of COX-2 expression leads to
cr
delayed remodeling in aged mice (202). In support of these data, pharmacological inhibition of COX-
us
2 led to an impairment in endochondral ossification in rabbits (203). Accordingly, a downstream
target of COX-2, i.e. EP4, has been targeted therapeutically to enhance bone healing (204).
an
Osteocytes
M
The osteocyte population translates mechanical signals to increase mineralization and accelerate
metaphyseal bone healing in the osteoporotic mouse fracture model (31). Osteocytes, although
d
long-lived and the most abundant cell population in bone, display increased rates of cell death
e
during aging, leaving behind empty lacunae (31, 205). The preservation of osteocytes is on the other
pt
hand influenced by mechanical stimulation, steroids and bisphosphonates as demonstrated in mice

ce
and partly in humans (206–209). Affecting the osteocyte secreted anti-anabolic factor, sclerostin,
sclerostin-antibodies were approved for clinical therapy of severe osteoporosis in 2019 and have
Ac
been demonstrated to improve bone healing in rat studies, but not in human tibia or femur bone
healing (210–213). A targeted deletion of SOST leads to an earlier removal of cartilage, affecting
especially the early remodeling phase (76, 214). This effect was consistent in delayed bone healing
due to osteoporosis in rats (215, 216).
31
Similar to the aging process in MSCs, aging osteocytes show telomere dysfunction-associated foci
(TAFs), a senescence-associated distension of satellites (SADS) and secrete dysfunctional factors
(SASP) linked to aging and chronic diseases (156, 158). One mechanism contributing to this
osteocyte aging is a lack of cellular autophagy (217, 218).

Osteoclasts
More than the number of osteoclasts itself, the osteoblast/osteoclast balance is of crucial
t
importance during the healing process to generate new bone and provide a proper re-formation of
ip
woven and lamellar bone (15). Increased osteoclast activity has been proposed to be causal for
cr
delayed fracture healing due to higher concentrations of tartrate-resistant acid phosphatase (TRAP)
us
and deoxy-pyridinoline (DPD) in a senescence-accelerated osteoporotic mouse model (219). The
molecular background of these mice indeed favors osteoclastogenesis: the increased IFN-γ
an
expression in aged mice increases RANKL expression, driving osteoclastogenesis and bone loss (220,
M
221). Additionally, a higher amount of TRAP-positive osteoclasts in the callus area was demonstrated
in old mice (194).

e d
pt
Beneficial versus adverse effects of cellular senescence

ce
While the initial descriptions of cellular senescence focused on its detrimental effects on aging, more
recent work has shed light upon its potential beneficial aspects, describing senescence as an
Ac
evolutionary advantageous, yet two-edged process (222, 223).
A senescent cell does not divide further, which is why suppressing cancer development has been a
potential explanation for cellular senescence (224). However, this non-dividing cell causes a
detrimental “bystander” effect on surrounding cells and whole tissues via secretion of reactive
oxygen species (ROS) and a miscellaneous cytokine cocktail, termed the SASP (225–228) (Fig. 5).
Along with the senescent cells, the SASP contributes to inflammaging, which may also be pro-
32
tumorigenic (193, 229). The senescent phenotype is not limited to mitotic cells and the elimination
of these SASP producing cells in mice has been demonstrated to be beneficial in ameliorating the
effects of aging in multiple tissues, including the heart and bone (159, 230, 230, 231). Overall, the
removal of accumulated senescent cells has beneficial effects on aging phenotypes (223). It is
therefore of importance to isolate, spatially restrict, or resolve an excessive accumulation of these

senescent cells, before they occupy cellular niches and lead to systemic disease and morbidity (232,
233). By mechanisms which are not fully understood, an imperfect clearance leads to the slow
t
ip
increase of the senescent cell burden with aging (234).
cr
Importantly, a beneficial influence of senescent cells on murine skin and liver regeneration (235,
us
236), in the murine placenta (237) and in chicken as well as murine embryonic development (238)
has been demonstrated (223). Moreover, a potentially important function of senescent cells in mice
an
and amphibians has been demonstrated to lie in wound healing and tissue repair (223, 236, 239). In
particular, the beneficial function of the SASP is mainly attributed to IL-6 in skeletal muscle, enabling
M
cellular reprogramming, and PDGF-AA in wound healing, as demonstrated in mice (235, 240). In the
injured skin, a transient induction of senescent cells is followed by a rapid clearance at the early
e d
reparative stage. The recruitment of immune cells to the area of damage by the SASP subsequently
pt
leads to the removal of senescent cells after completed healing, pointing to a primary role of these
cells in the damage repair process (223, 241, 242). In a deteriorating immune system, this clearance
ce
potential, which is attributed largely to local macrophages, may be impaired (175, 235, 243, 244).
Ac
In terms of skeletal repair, we recently established the appearance of senescent cells within murine
fracture healing in a time-dependent manner in young mice. In contrast to the above findings where
senescent cells had a beneficial role in tissue repair (235, 236), using genetic (Cdkn2aLUC) and
pharmacological (senolytic treatment with Dasatinib+Quercetin) models to clear senescent cells, we
demonstrated that a reduction of senescent cells and the SASP within the callus improved its
33
formation and biomechanical stability (245). Thus, the beneficial versus detrimental effects of
senescent cells following injury may vary across tissues.
Summarizing these contradictory influences of senescent cells, the local immune clearance of these
cells leads to a balance between beneficial and harmful effects, depending on the abundance and

duration of the senescent cell burden. Indeed, some senescent cells are not replaced and yet play
important local functional roles, whereas others accumulate and have a detrimental impact at a
t
systemic level, driving organ dysfunction and aging (246, 247). These contrarian roles of senescent
ip
cells are depicted in Figure 5 (223).
cr
Summary and conclusions
us
an
The cellular components of fracture healing that can be roughly subdivided into four temporal stages
are all affected by the aging process. A number of age-related impairments in fracture healing have
M
been identified, and further studies are needed to define the possible role of the accumulation of
d
senescent cells with aging in modulating fracture healing. In addition, studies are needed to evaluate
e
whether targeting senescent cells, or other impairments in fracture healing (e.g., inflammaging), will
pt
enhance fracture healing in older individuals.

ce
Ac
34
References
1. United States Census Bureau. National Population by Characteristics: 2010-2019.
https://www.census.gov/data/tables/time-series/demo/popest/2010s-national-detail.html
2. Rose S, Maffulli N. Hip fractures. An epidemiological review. Bull Hosp Jt Dis. 1999;58:197–201
3. Williams SA, Chastek B, Sundquist K, Barrera-Sierra S, Leader D, Weiss RJ, Wang Y,
Curtis JR. Economic burden of osteoporotic fractures in US managed care enrollees. Am J Manag
Care. 2020;26:e142-e149
4. Clark D, Nakamura M, Miclau T, Marcucio R. Effects of Aging on Fracture Healing. Curr
Osteoporos Rep. 2017;15:601–608

5. Farr JN, Rowsey JL, Eckhardt BA, Thicke BS, Fraser DG, Tchkonia T, Kirkland JL,
Monroe DG, Khosla S. Independent Roles of Estrogen Deficiency and Cellular Senescence in the
Pathogenesis of Osteoporosis: Evidence in Young Adult Mice and Older Humans. J Bone Miner
Res. 2019;34:1407–1418
t
6. Loi F, Córdova LA, Pajarinen J, Lin T, Yao Z, Goodman SB. Inflammation, Fracture and
ip
Bone Repair. Bone. 2016;86:119–130
7. Augat P, Simon U, Liedert A, Claes L. Mechanics and mechano-biology of fracture healing in
cr
normal and osteoporotic bone. Osteoporosis international a journal established as result of
cooperation between the European Foundation for Osteoporosis and the National Osteoporosis
Foundation of the USA. 2005;16 Suppl 2:S36-43
Environ Sci. 2015;28:57–71

us
8. Oryan A, Monazzah S, Bigham-Sadegh A. Bone injury and fracture healing biology. Biomed
9. Phillips AM. Overview of the fracture healing cascade. Injury. 2005;36 Suppl 3:S5-7
an
10. Park D, Spencer JA, Koh BI, Kobayashi T, Fujisaki J, Clemens TL, Lin CP, Kronenberg
HM, Scadden DT. Endogenous bone marrow MSCs are dynamic, fate-restricted participants in
bone maintenance and regeneration. Cell Stem Cell. 2012;10:259–272
M
11. Hachemi Y, Rapp AE, Picke A-K, Weidinger G, Ignatius A, Tuckermann J. Molecular
mechanisms of glucocorticoids on skeleton and bone regeneration after fracture. J Mol Endocrinol.
2018;61:R75-R90
12. Zhao L, Wang X, Pomlok K, Liao H, Yang G, Yang X, Chen Y-G. DDB1 promotes the
d
proliferation and hypertrophy of chondrocytes during mouse skeleton development. Dev Biol.
2020;465:100–107
e
13. Choy MHV, Wong RMY, Chow SKH, Li MC, Chim YN, Li TK, Ho WT, Cheng JCY,
pt
Cheung WH. How much do we know about the role of osteocytes in different phases of fracture
healing? A systematic review. J Orthop Translat. 2020;21:111–121
14. Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat Rev
ce
Rheumatol. 2015;11:45–54
15. Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions.
Nat Rev Rheumatol. 2012;8:133–143
Ac
16. Ferretti C, Lucarini G, Andreoni C, Salvolini E, Bianchi N, Vozzi G, Gigante A, Mattioli-

Belmonte M. Human Periosteal Derived Stem Cell Potential: The Impact of age. Stem Cell Rev
Rep. 2015;11:487–500
17. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res. 1998:S7-
21
18. Gibon E, Lu L, Goodman SB. Aging, inflammation, stem cells, and bone healing. Stem Cell Res
Ther. 2016;7:44
19. Kovtun A, Bergdolt S, Wiegner R, Radermacher P, Huber-Lang M, Ignatius A. The crucial
role of neutrophil granulocytes in bone fracture healing. Eur Cell Mater. 2016;32:152–162
20. Duchamp de Lageneste O, Julien A, Abou-Khalil R, Frangi G, Carvalho C, Cagnard N,
Cordier C, Conway SJ, Colnot C. Periosteum contains skeletal stem cells with high bone
regenerative potential controlled by Periostin. Nat Commun. 2018;9:773
35
21. Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a
post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell
Biochem. 2003;88:873–884
22. Nakahara H, Bruder SP, Goldberg VM, Caplan AI. In vivo osteochondrogenic potential of
cultured cells derived from the periosteum. Clin Orthop Relat Res. 1990:223–232
23. Colnot C, Huang S, Helms J. Analyzing the cellular contribution of bone marrow to fracture
healing using bone marrow transplantation in mice. Biochem Biophys Res Commun.
2006;350:557–561
24. Nam D, Mau E, Wang Y, Wright D, Silkstone D, Whetstone H, Whyne C, Alman B. T-

lymphocytes enable osteoblast maturation via IL-17F during the early phase of fracture repair.
PLoS ONE. 2012;7:e40044
25. Sun G, Wang Z, Ti Y, Wang Y, Wang J, Zhao J, Qian H. STAT3 promotes bone fracture
healing by enhancing the FOXP3 expression and the suppressive function of regulatory T cells.
t
APMIS. 2017;125:752–760
ip
26. Ito H. Chemokines in mesenchymal stem cell therapy for bone repair: a novel concept of
recruiting mesenchymal stem cells and the possible cell sources. Mod Rheumatol. 2011;21:113–
cr
121
27. Shiu HT, Leung PC, Ko CH. The roles of cellular and molecular components of a hematoma at
early stage of bone healing. J Tissue Eng Regen Med. 2018;12:e1911-e1925
us
28. Wu AC, Kidd LJ, Cowling NR, Kelly WL, Forwood MR. Osteocyte expression of caspase-3,
COX-2, IL-6 and sclerostin are spatially and temporally associated following stress fracture
initiation. Bonekey Rep. 2014;3:571
an
29. Dunstan CR, Evans RA, Hills E, Wong SY, Higgs RJ. Bone death in hip fracture in the elderly.
Calcif Tissue Int. 1990;47:270–275
30. Caetano-Lopes J, Lopes A, Rodrigues A, Fernandes D, Perpétuo IP, Monjardino T, Lucas R,
Monteiro J, Konttinen YT, Canhão H, Fonseca JE. Upregulation of inflammatory genes and
M
downregulation of sclerostin gene expression are key elements in the early phase of fragility
fracture healing. PLoS ONE. 2011;6:e16947
31. Cheung WH, Wong RMY, Choy VMH, Li MCM, Cheng KYK, Chow SKH. Enhancement of
d
osteoporotic fracture healing by vibration treatment: The role of osteocytes. Injury. 2020
32. Bouaziz W, Funck-Brentano T, Lin H, Marty C, Ea H-K, Hay E, Cohen-Solal M. Loss of
e
sclerostin promotes osteoarthritis in mice via β-catenin-dependent and -independent Wnt

pathways. Arthritis Res Ther. 2015;17:24
pt
33. Novak S, Roeder E, Kalinowski J, Jastrzebski S, Aguila HL, Lee S-K, Kalajzic I, Lorenzo
JA. Osteoclasts Derive Predominantly from Bone Marrow-Resident CX3CR1+ Precursor Cells in
ce
Homeostasis, whereas Circulating CX3CR1+ Cells Contribute to Osteoclast Development during

Fracture Repair. J Immunol. 2020;204:868–878
34. Wildemann B, Schmidmaier G, Ordel S, Stange R, Haas NP, Raschke M. Cell proliferation
Ac
and differentiation during fracture healing are influenced by locally applied IGF-I and TGF-beta1:
comparison of two proliferation markers, PCNA and BrdU. J Biomed Mater Res B Appl Biomater.
2003;65:150–156
35. Sarahrudi K, Thomas A, Mousavi M, Kaiser G, Köttstorfer J, Kecht M, Hajdu S,
Aharinejad S. Elevated transforming growth factor-beta 1 (TGF-β1) levels in human fracture
healing. Injury. 2011;42:833–837
36. Tsiridis E, Upadhyay N, Giannoudis P. Molecular aspects of fracture healing: which are the
important molecules? Injury. 2007;38 Suppl 1:S11-25
37. Gerstenfeld LC, Cho TJ, Kon T, Aizawa T, Cruceta J, Graves BD, Einhorn TA. Impaired
intramembranous bone formation during bone repair in the absence of tumor necrosis factor-alpha
signaling. Cells Tissues Organs (Print). 2001;169:285–294
38. Alexander KA, Chang MK, Maylin ER, Kohler T, Müller R, Wu AC, van Rooijen N, Sweet
MJ, Hume DA, Raggatt LJ, Pettit AR. Osteal macrophages promote in vivo intramembranous
bone healing in a mouse tibial injury model. J Bone Miner Res. 2011;26:1517–1532
36
39. Chang MK, Raggatt L-J, Alexander KA, Kuliwaba JS, Fazzalari NL, Schroder K, Maylin
ER, Ripoll VM, Hume DA, Pettit AR. Osteal tissue macrophages are intercalated throughout
human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo. J
Immunol. 2008;181:1232–1244
40. Lu C, Rollins M, Hou H, Swartz HM, Hopf H, Miclau T, Marcucio RS. Tibial fracture
decreases oxygen levels at the site of injury. Iowa Orthop J. 2008;28:14–21
41. Little DG, Ramachandran M, Schindeler A. The anabolic and catabolic responses in bone
repair. J Bone Joint Surg Br. 2007;89:425–433
42. Grcevic D, Pejda S, Matthews BG, Repic D, Wang L, Li H, Kronenberg MS, Jiang X, Maye

P, Adams DJ, Rowe DW, Aguila HL, Kalajzic I. In vivo fate mapping identifies mesenchymal
progenitor cells. Stem Cells. 2012;30:187–196
43. Yoshida CA, Komori H, Maruyama Z, Miyazaki T, Kawasaki K, Furuichi T, Fukuyama R,
Mori M, Yamana K, Nakamura K, Liu W, Toyosawa S, Moriishi T, Kawaguchi H, Takada
t
K, Komori T. SP7 inhibits osteoblast differentiation at a late stage in mice. PLoS ONE.
ip
2012;7:e32364
44. Deckers MML, van Beek ER, van der Pluijm G, Wetterwald A, van der Wee-Pals L,
cr
Cecchini MG, Papapoulos SE, Löwik CWGM. Dissociation of angiogenesis and
osteoclastogenesis during endochondral bone formation in neonatal mice. J Bone Miner Res.
2002;17:998–1007
us
45. Prystaz K, Kaiser K, Kovtun A, Haffner-Luntzer M, Fischer V, Rapp AE, Liedert A, Strauss
G, Waetzig GH, Rose-John S, Ignatius A. Distinct Effects of IL-6 Classic and Trans-Signaling
in Bone Fracture Healing. Am J Pathol. 2018;188:474–490
an
46. Wallace A, Cooney TE, Englund R, Lubahn JD. Effects of interleukin-6 ablation on fracture
healing in mice. J Orthop Res. 2011;29:1437–1442
47. Bahney CS, Zondervan RL, Allison P, Theologis A, Ashley JW, Ahn J, Miclau T, Marcucio
RS, Hankenson KD. Cellular biology of fracture healing. J Orthop Res. 2019;37:35–50
M
48. Schlundt C, El Khassawna T, Serra A, Dienelt A, Wendler S, Schell H, van Rooijen N,

Radbruch A, Lucius R, Hartmann S, Duda GN, Schmidt-Bleek K. Macrophages in bone
fracture healing: Their essential role in endochondral ossification. Bone. 2018;106:78–89
d
49. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and
disease. Nature. 2013;496:445–455
e
50. Loiselle AE, Paul EM, Lewis GS, Donahue HJ. Osteoblast and osteocyte-specific loss of
Connexin43 results in delayed bone formation and healing during murine fracture healing. J
pt
Orthop Res. 2013;31:147–154

51. Hadjiargyrou M, Rightmire EP, Ando T, Lombardo FT. The E11 osteoblastic lineage marker
ce
is differentially expressed during fracture healing. Bone. 2001;29:149–154

52. Staines KA, Prideaux M, Allen S, Buttle DJ, Pitsillides AA, Farquharson C. E11/Podoplanin
Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in
Ac
Murine in Vitro Models. J Cell Physiol. 2016;231:1392–1404

53. Yoshimura Y, Nomura S, Kawasaki S, Tsutsumimoto T, Shimizu T, Takaoka K.
Colocalization of noggin and bone morphogenetic protein-4 during fracture healing. J Bone Miner
Res. 2001;16:876–884
54. Hu DP, Ferro F, Yang F, Taylor AJ, Chang W, Miclau T, Marcucio RS, Bahney CS.
Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature
and induction of the core pluripotency genes. Development. 2017;144:221–234
55. Yang G, Zhu L, Hou N, Lan Y, Wu X-M, Zhou B, Teng Y, Yang X. Osteogenic fate of
hypertrophic chondrocytes. Cell Res. 2014;24:1266–1269
56. Zhou X, Mark K von der, Henry S, Norton W, Adams H, Crombrugghe B de. Chondrocytes
transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and
fracture healing in mice. PLoS Genet. 2014;10:e1004820
57. Duan P, Bonewald LF. The role of the wnt/β-catenin signaling pathway in formation and
maintenance of bone and teeth. Int J Biochem Cell Biol. 2016;77:23–29
37
58. Marsell R, Einhorn TA. THE BIOLOGY OF FRACTURE HEALING. Injury. 2011;42:551–555
59. Ikebuchi Y, Aoki S, Honma M, Hayashi M, Sugamori Y, Khan M, Kariya Y, Kato G, Tabata
Y, Penninger JM, Udagawa N, Aoki K, Suzuki H. Coupling of bone resorption and formation
by RANKL reverse signalling. Nature. 2018;561:195–200
60. Devlin RD, Du Z, Pereira RC, Kimble RB, Economides AN, Jorgetti V, Canalis E. Skeletal
overexpression of noggin results in osteopenia and reduced bone formation. Endocrinology.
2003;144:1972–1978
61. Aspenberg P, Jeppsson C, Economides AN. The bone morphogenetic proteins antagonist
Noggin inhibits membranous ossification. J Bone Miner Res. 2001;16:497–500

62. Vallés G, Bensiamar F, Maestro-Paramio L, García-Rey E, Vilaboa N, Saldaña L. Influence
of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem
cells. Stem Cell Res Ther. 2020;11:57
63. Osta B, Lavocat F, Eljaafari A, Miossec P. Effects of Interleukin-17A on Osteogenic
t
Differentiation of Isolated Human Mesenchymal Stem Cells. Front Immunol. 2014;5:425
ip
64. Lu C, Huang S, Miclau T, Helms JA, Colnot C. Mepe is expressed during skeletal development
and regeneration. Histochem Cell Biol. 2004;121:493–499
cr
65. Glass GE, Chan JK, Freidin A, Feldmann M, Horwood NJ, Nanchahal J. TNF-alpha
promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived
stromal cells. Proc Natl Acad Sci U S A. 2011;108:1585–1590
us
66. Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D, Gerstenfeld LC, Einhorn TA.
Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand)
and related proinflammatory cytokines during fracture healing. J Bone Miner Res. 2001;16:1004–
an
1014
67. Lissenberg-Thunnissen SN, Gorter DJJ de, Sier CFM, Schipper IB. Use and efficacy of bone
morphogenetic proteins in fracture healing. Int Orthop. 2011;35:1271–1280
68. Pazzaglia UE, Reguzzoni M, Casati L, Sibilia V, Zarattini G, Raspanti M. New morphological
M
evidence of the 'fate' of growth plate hypertrophic chondrocytes in the general context of
endochondral ossification. J Anat. 2020;236:305–316
69. Pajarinen J, Lin T, Gibon E, Kohno Y, Maruyama M, Nathan K, Lu L, Yao Z, Goodman
d
SB. Mesenchymal stem cell-macrophage crosstalk and bone healing. Biomaterials. 2019;196:80–
89
e
70. Yu B, Pacureanu A, Olivier C, Cloetens P, Peyrin F. Assessment of the human bone lacuno-
canalicular network at the nanoscale and impact of spatial resolution. Sci Rep. 2020;10:4567
pt
71. Toyosawa S, Kanatani N, Shintani S, Kobata M, Yuki M, Kishino M, Ijuhin N, Komori T.

Expression of dentin matrix protein 1 (DMP1) during fracture healing. Bone. 2004;35:553–561
ce
72. Lu C, Hansen E, Sapozhnikova A, Hu D, Miclau T, Marcucio RS. Effect of age on

vascularization during fracture repair. J Orthop Res. 2008;26:1384–1389
73. Ferron M, Wei J, Yoshizawa T, Del Fattore A, DePinho RA, Teti A, Ducy P, Karsenty G.
Ac
Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism. Cell.
2010;142:296–308
74. Oliver RA, Yu Y, Yee G, Low AK, Diwan AD, Walsh WR. Poor histological healing of a
femoral fracture following 12 months of oestrogen deficiency in rats. Osteoporosis international a
journal established as result of cooperation between the European Foundation for Osteoporosis
and the National Osteoporosis Foundation of the USA. 2013;24:2581–2589
75. Garcia P, Histing T, Holstein JH, Klein M, Laschke MW, Matthys R, Ignatius A,
Wildemann B, Lienau J, Peters A, Willie B, Duda G, Claes L, Pohlemann T, Menger MD.
Rodent animal models of delayed bone healing and non-union formation: a comprehensive review.
Eur Cell Mater. 2013;26:1-12; discussion 12-4
76. Schupbach D, Comeau-Gauthier M, Harvey E, Merle G. Wnt modulation in bone healing.
Bone. 2020;138:115491
38
77. Jiang X, Xu C, Shi H, Cheng Q. PTH1-34 improves bone healing by promoting angiogenesis and
facilitating MSCs migration and differentiation in a stabilized fracture mouse model. PLoS ONE.
2019;14:e0226163
78. Yukata K, Kanchiku T, Egawa H, Nakamura M, Nishida N, Hashimoto T, Ogasa H, Taguchi
T, Yasui N. Continuous infusion of PTH1-34 delayed fracture healing in mice. Sci Rep.
2018;8:13175
79. Sun P, Wang M, Yin G-Y. Endogenous parathyroid hormone (PTH) signals through osteoblasts
via RANKL during fracture healing to affect osteoclasts. Biochem Biophys Res Commun.
2020;525:850–856

80. Peichl P, Holzer LA, Maier R, Holzer G. Parathyroid hormone 1-84 accelerates fracture-healing
in pubic bones of elderly osteoporotic women. J Bone Joint Surg Am. 2011;93:1583–1587
81. Aspenberg P, Genant HK, Johansson T, Nino AJ, See K, Krohn K, García-Hernández PA,
Recknor CP, Einhorn TA, Dalsky GP, Mitlak BH, Fierlinger A, Lakshmanan MC.
t
Teriparatide for acceleration of fracture repair in humans: a prospective, randomized, double-blind
ip
study of 102 postmenopausal women with distal radial fractures. J Bone Miner Res. 2010;25:404–
414
cr
82. Ohtori S, Inoue G, Orita S, Yamauchi K, Eguchi Y, Ochiai N, Kishida S, Kuniyoshi K, Aoki
Y, Nakamura J, Ishikawa T, Miyagi M, Kamoda H, Suzuki M, Kubota G, Sakuma Y,
Oikawa Y, Inage K, Sainoh T, Takaso M, Ozawa T, Takahashi K, Toyone T. Teriparatide
us
accelerates lumbar posterolateral fusion in women with postmenopausal osteoporosis: prospective
study. Spine (Phila Pa 1976). 2012;37:E1464-8
83. Seki S, Hirano N, Kawaguchi Y, Nakano M, Yasuda T, Suzuki K, Watanabe K, Makino H,
an
Kanamori M, Kimura T. Teriparatide versus low-dose bisphosphonates before and after surgery
for adult spinal deformity in female Japanese patients with osteoporosis. Eur Spine J.
2017;26:2121–2127
84. Cho PG, Ji GY, Shin DA, Ha Y, Yoon DH, Kim KN. An effect comparison of teriparatide and
M
bisphosphonate on posterior lumbar interbody fusion in patients with osteoporosis: a prospective

cohort study and preliminary data. Eur Spine J. 2017;26:691–697
85. Padova G, Borzì G, Incorvaia L, Siciliano G, Migliorino V, Vetri M, Tita P. Prevalence of
d
osteoporosis and vertebral fractures in acromegalic patients. Clin Cases Miner Bone Metab.
2011;8:37–43
e
86. Mazziotti G, Bianchi A, Porcelli T, Mormando M, Maffezzoni F, Cristiano A, Giampietro A,

Marinis L de, Giustina A. Vertebral fractures in patients with acromegaly: a 3-year prospective
pt
study. J Clin Endocrinol Metab. 2013;98:3402–3410

87. Madeira M, Neto LV, Paula Paranhos Neto F de, Barbosa Lima IC, Carvalho de Mendonça
ce
LM, Gadelha MR, Fleiuss de Farias ML. Acromegaly has a negative influence on trabecular
bone, but not on cortical bone, as assessed by high-resolution peripheral quantitative computed
tomography. J Clin Endocrinol Metab. 2013;98:1734–1741
Ac
88. Weiss S, Henle P, Bidlingmaier M, Moghaddam A, Kasten P, Zimmermann G. Systemic

response of the GH/IGF-I axis in timely versus delayed fracture healing. Growth Horm IGF Res.
2008;18:205–212
89. Reible B, Schmidmaier G, Moghaddam A, Westhauser F. Insulin-Like Growth Factor-1 as a
Possible Alternative to Bone Morphogenetic Protein-7 to Induce Osteogenic Differentiation of
Human Mesenchymal Stem Cells in Vitro. Int J Mol Sci. 2018;19
90. Reible B, Schmidmaier G, Prokscha M, Moghaddam A, Westhauser F. Continuous
stimulation with differentiation factors is necessary to enhance osteogenic differentiation of human
mesenchymal stem cells in-vitro. Growth Factors. 2017;35:179–188
91. Kirby DJ, Buchalter DB, Anil U, Leucht P. DHEA in bone: the role in osteoporosis and fracture
healing. Arch Osteoporos. 2020;15:84
92. Deng Z, Gao X, Sun X, Cui Y, Amra S, Huard J. Gender differences in tibial fractures healing
in normal and muscular dystrophic mice. Am J Transl Res. 2020;12:2640–2651
39
93. Córdova LA, Loi F, Lin T, Gibon E, Pajarinen J, Nabeshima A, Lu L, Yao Z, Goodman SB.
CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2
transition in vitro. J Biomed Mater Res A. 2017;105:3069–3076
94. Wang T, Wang Y, Menendez A, Fong C, Babey M, Tahimic CGT, Cheng Z, Li A, Chang W,
Bikle DD. Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone
Formation During Fracture Healing. J Bone Miner Res. 2015;30:1572–1584
95. Blumenfeld I, Srouji S, Lanir Y, Laufer D, Livne E. Enhancement of bone defect healing in old
rats by TGF-beta and IGF-1. Exp Gerontol. 2002;37:553–565
96. Fischer V, Haffner-Luntzer M, Amling M, Ignatius A. Calcium and vitamin D in bone fracture

healing and post-traumatic bone turnover. Eur Cell Mater. 2018;35:365–385
97. Doepfner W. Consequences of calcium and-or phosphorus deficient diets on various parameters
of callus formation and on growth rate in young rats. Br J Pharmacol. 1970;39:188P-189P
98. Namkung-Matthai H, Appleyard R, Jansen J, Hao Lin J, Maastricht S, Swain M, Mason RS,
t
Murrell GA, Diwan AD, Diamond T. Osteoporosis influences the early period of fracture
ip
healing in a rat osteoporotic model. Bone. 2001;28:80–86
99. Haffner-Luntzer M, Heilmann A, Heidler V, Liedert A, Schinke T, Amling M, Yorgan TA,
cr
vom Scheidt A, Ignatius A. Hypochlorhydria-induced calcium malabsorption does not affect
fracture healing but increases post-traumatic bone loss in the intact skeleton. J Orthop Res.
2016;34:1914–1921
us
100. Fischer V, Haffner-Luntzer M, Prystaz K, vom Scheidt A, Busse B, Schinke T, Amling
M, Ignatius A. Calcium and vitamin-D deficiency marginally impairs fracture healing but
aggravates posttraumatic bone loss in osteoporotic mice. Sci Rep. 2017;7:7223
an
101. Kunutsor SK, Whitehouse MR, Blom AW, Laukkanen JA. Low serum magnesium levels
are associated with increased risk of fractures: a long-term prospective cohort study. Eur J
Epidemiol. 2017;32:593–603
102. Hayhoe RPG, Lentjes MAH, Luben RN, Khaw K-T, Welch AA. Dietary magnesium and
M
potassium intakes and circulating magnesium are associated with heel bone ultrasound attenuation
and osteoporotic fracture risk in the EPIC-Norfolk cohort study. Am J Clin Nutr. 2015;102:376–
384
d
103. Zhang Y, Xu J, Ruan YC, Yu MK, O'Laughlin M, Wise H, Di Chen, Tian L, Shi D,
Wang J, Chen S, Feng JQ, Chow DHK, Xie X, Zheng L, Le Huang, Huang S, Leung K, Lu
e
N, Zhao L, Li H, Zhao D, Guo X, Chan K, Witte F, Chan HC, Zheng Y, Qin L. Implant-
derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing
pt
in rats. Nat Med. 2016;22:1160–1169

104. Gorter EA, Hamdy NAT, Appelman-Dijkstra NM, Schipper IB. The role of vitamin D in
ce
human fracture healing: a systematic review of the literature. Bone. 2014;64:288–297

105. Chiavarini M, Naldini G, Fabiani R. The Role of Diet in Osteoporotic Fracture Healing: a
Systematic Review. Curr Osteoporos Rep. 2020;18:138–147
Ac
106. Brinker MR, O'Connor DP, Monla YT, Earthman TP. Metabolic and endocrine
abnormalities in patients with nonunions. J Orthop Trauma. 2007;21:557–570
107. Zura R, Xiong Z, Einhorn T, Watson JT, Ostrum RF, Prayson MJ, Della Rocca GJ,
Mehta S, McKinley T, Wang Z, Steen RG. Epidemiology of Fracture Nonunion in 18 Human
Bones. JAMA Surg. 2016;151:e162775
108. Doetsch AM, Faber J, Lynnerup N, Wätjen I, Bliddal H, Danneskiold-Samsøe B. The
effect of calcium and vitamin D3 supplementation on the healing of the proximal humerus
fracture: a randomized placebo-controlled study. Calcif Tissue Int. 2004;75:183–188
109. Heyer FL, Jong JJ de, Willems PC, Arts JJ, Bours SGP, van Kuijk SMJ, Bons JAP,
Poeze M, Geusens PP, van Rietbergen B, van den Bergh JP. The Effect of Bolus Vitamin D3
Supplementation on Distal Radius Fracture Healing: A Randomized Controlled Trial Using HR-
pQCT. J Bone Miner Res. 2021;36:1492–1501
40
110. Martineau C, Kaufmann M, Arabian A, Jones G, St-Arnaud R. Preclinical safety and
efficacy of 24R,25-dihydroxyvitamin D3 or lactosylceramide treatment to enhance fracture repair.
J Orthop Translat. 2020;23:77–88
111. Martineau C, Naja RP, Husseini A, Hamade B, Kaufmann M, Akhouayri O, Arabian A,
Jones G, St-Arnaud R. Optimal bone fracture repair requires 24R,25-dihydroxyvitamin D3 and
its effector molecule FAM57B2. J Clin Invest. 2018;128:3546–3557
112. Centers for Disease Control and Prevention. National Diabetes Statistics Report, 2020.:
Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of Health and Human
Services; 2020. https://www.cdc.gov/diabetes/data/statistics-report/index.html. Accessed 17

January 2021
113. Jiao H, Xiao E, Graves DT. Diabetes and Its Effect on Bone and Fracture Healing. Curr
114. Ferrari SL, Abrahamsen B, Napoli N, Akesson K, Chandran M, Eastell R, El-Hajj
t
Fuleihan G, Josse R, Kendler DL, Kraenzlin M, Suzuki A, Pierroz DD, Schwartz AV, Leslie
ip
WD. Diagnosis and management of bone fragility in diabetes: an emerging challenge.
Osteoporosis international a journal established as result of cooperation between the European
cr
Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA.
2018;29:2585–2596
115. Murray CE, Coleman CM. Impact of Diabetes Mellitus on Bone Health. Int J Mol Sci.
us
2019;20
116. Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic
reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009;6:e1000097
an
117. Janghorbani M, Feskanich D, Willett WC, Hu F. Prospective study of diabetes and risk of
hip fracture: the Nurses' Health Study. Diabetes Care. 2006;29:1573–1578
118. Vestergaard P. Discrepancies in bone mineral density and fracture risk in patients with type 1
and type 2 diabetes--a meta-analysis. Osteoporosis international a journal established as result of
M
cooperation between the European Foundation for Osteoporosis and the National Osteoporosis
Foundation of the USA. 2007;18:427–444
119. Henderson S, Ibe I, Cahill S, Chung Y-H, Lee FY. Bone Quality and Fracture-Healing in
d
Type-1 and Type-2 Diabetes Mellitus. J Bone Joint Surg Am. 2019;101:1399–1410
120. Kayal RA, Tsatsas D, Bauer MA, Allen B, Al-Sebaei MO, Kakar S, Leone CW, Morgan
e
EF, Gerstenfeld LC, Einhorn TA, Graves DT. Diminished bone formation during diabetic
fracture healing is related to the premature resorption of cartilage associated with increased
pt
osteoclast activity. J Bone Miner Res. 2007;22:560–568

121. Alblowi J, Kayal RA, Siqueira M, Siqueria M, McKenzie E, Krothapalli N, McLean J,
ce
Conn J, Nikolajczyk B, Einhorn TA, Gerstenfeld L, Graves DT. High levels of tumor necrosis
factor-alpha contribute to accelerated loss of cartilage in diabetic fracture healing. Am J Pathol.
2009;175:1574–1585
Ac
122. Kayal RA, Alblowi J, McKenzie E, Krothapalli N, Silkman L, Gerstenfeld L, Einhorn

TA, Graves DT. Diabetes causes the accelerated loss of cartilage during fracture repair which is
reversed by insulin treatment. Bone. 2009;44:357–363
123. Hu Z, Ma C, Liang Y, Zou S, Liu X. Osteoclasts in bone regeneration under type 2 diabetes
mellitus. Acta Biomater. 2019;84:402–413
124. Mangialardi G, Ferland-McCollough D, Maselli D, Santopaolo M, Cordaro A, Spinetti
G, Sambataro M, Sullivan N, Blom A, Madeddu P. Bone marrow pericyte dysfunction in
individuals with type 2 diabetes. Diabetologia. 2019;62:1275–1290
125. Vizoso FJ, Eiro N, Costa L, Esparza P, Landin M, Diaz-Rodriguez P, Schneider J, Perez-
Fernandez R. Mesenchymal Stem Cells in Homeostasis and Systemic Diseases: Hypothesis,
Evidences, and Therapeutic Opportunities. Int J Mol Sci. 2019;20
126. Dedania J, Borzio R, Paglia D, Breitbart EA, Mitchell A, Vaidya S, Wey A, Mehta S,
Benevenia J, O'Connor JP, Lin SS. Role of local insulin augmentation upon allograft
incorporation in a rat femoral defect model. J Orthop Res. 2011;29:92–99
41
127. Gandhi A, Beam HA, O'Connor JP, Parsons JR, Lin SS. The effects of local insulin
delivery on diabetic fracture healing. Bone. 2005;37:482–490
128. Cignachi NP, Ribeiro A, Machado GDB, Cignachi AP, Kist LW, Bogo MR, Silva RBM,
Campos MM. Bone regeneration in a mouse model of type 1 diabetes: Influence of sex, vitamin
D3, and insulin. Life Sci. 2020;263:118593
129. Ehrchen J, Steinmüller L, Barczyk K, Tenbrock K, Nacken W, Eisenacher M, Nordhues
U, Sorg C, Sunderkötter C, Roth J. Glucocorticoids induce differentiation of a specifically
activated, anti-inflammatory subtype of human monocytes. Blood. 2007;109:1265–1274
130. Barczyk K, Ehrchen J, Tenbrock K, Ahlmann M, Kneidl J, Viemann D, Roth J.

Glucocorticoids promote survival of anti-inflammatory macrophages via stimulation of adenosine
receptor A3. Blood. 2010;116:446–455
131. Ahmad M, Hachemi Y, Paxian K, Mengele F, Koenen M, Tuckermann J. A Jack of All
Trades: Impact of Glucocorticoids on Cellular Cross-Talk in Osteoimmunology. Front Immunol.
t
2019;10
ip
132. Huang X, Li Y, Fu M, Xin H-B. Polarizing Macrophages In Vitro. Methods Mol Biol.
2018;1784:119–126
cr
133. Zhang H, Shi X, Wang L, Li X, Zheng C, Gao B, Xu X, Lin X, Wang J, Lin Y, Shi J,
Huang Q, Luo Z, Yang L. Intramembranous ossification and endochondral ossification are
impaired differently between glucocorticoid-induced osteoporosis and estrogen deficiency-induced
us
osteoporosis. Sci Rep. 2018;8:3867
134. Sandberg OH, Aspenberg P. Glucocorticoids inhibit shaft fracture healing but not
metaphyseal bone regeneration under stable mechanical conditions. Bone Joint Res. 2015;4:170–
an
175
135. Liu Y-Z, Akhter MP, Gao X, Wang X-Y, Wang X-B, Zhao G, Wei X, Wu H-J, Chen H,
Wang D, Cui L. Glucocorticoid-induced delayed fracture healing and impaired bone
biomechanical properties in mice. Clin Interv Aging. 2018;13:1465–1474
M
136. Azetsu Y, Chatani M, Dodo Y, Karakawa A, Sakai N, Negishi-Koga T, Takami M.

Treatment with synthetic glucocorticoid impairs bone metabolism, as revealed by in vivo imaging
of osteoblasts and osteoclasts in medaka fish. Biomed Pharmacother. 2019;118:109101
d
137. Rapp AE, Hachemi Y, Kemmler J, Koenen M, Tuckermann J, Ignatius A. Induced global
deletion of glucocorticoid receptor impairs fracture healing. FASEB J. 2018;32:2235–2245
e
138. Hachemi Y, Rapp AE, Lee S, Dorn A-K, Krüger BT, Kaiser K, Ignatius A, Tuckermann
J. Intact Glucocorticoid Receptor Dimerization Is Deleterious in Trauma-Induced Impaired
pt
Fracture Healing. Front. Immunol. 2021

139. Li J, Wang X, Zhou C, Liu L, Wu Y, Wang D, Jiang H. Perioperative glucocorticosteroid
ce
treatment delays early healing of a mandible wound by inhibiting osteogenic differentiation.

Injury. 2012;43:1284–1289
140. Nikolaou VS, Efstathopoulos N, Kontakis G, Kanakaris NK, Giannoudis PV. The
Ac
influence of osteoporosis in femoral fracture healing time. Injury. 2009;40:663–668

141. Josephson AM, Bradaschia-Correa V, Lee S, Leclerc K, Patel KS, Muinos Lopez E,
Litwa HP, Neibart SS, Kadiyala M, Wong MZ, Mizrahi MM, Yim NL, Ramme AJ, Egol KA,
Leucht P. Age-related inflammation triggers skeletal stem/progenitor cell dysfunction. Proc Natl
Acad Sci U S A. 2019;116:6995–7004
142. Zura R, Braid-Forbes MJ, Jeray K, Mehta S, Einhorn TA, Watson JT, Della Rocca GJ,
Forbes K, Steen RG. Bone fracture nonunion rate decreases with increasing age: A prospective
inception cohort study. Bone. 2017;95:26–32
143. Mills LA, Aitken SA, Simpson AHRW. The risk of non-union per fracture: current myths
and revised figures from a population of over 4 million adults. Acta Orthop. 2017;88:434–439
144. Dailey HL, Wu KA, Wu P-S, McQueen MM, Court-Brown CM. Tibial Fracture Nonunion
and Time to Healing After Reamed Intramedullary Nailing: Risk Factors Based on a Single-Center
Review of 1003 Patients. J Orthop Trauma. 2018;32:e263-e269
42
145. Schmal H, Brix M, Bue M, Ekman A, Ferreira N, Gottlieb H, Kold S, Taylor A, Toft
Tengberg P, Ban I. Nonunion - consensus from the 4th annual meeting of the Danish Orthopaedic
Trauma Society. EFORT Open Rev. 2020;5:46–57
146. Robinson CM, Court-Brown CM, McQueen MM, Wakefield AE. Estimating the risk of
nonunion following nonoperative treatment of a clavicular fracture. J Bone Joint Surg Am.
2004;86:1359–1365
147. Inose H, Kato T, Ichimura S, Nakamura H, Hoshino M, Togawa D, Hirano T, Tokuhashi
Y, Ohba T, Haro H, Tsuji T, Sato K, Sasao Y, Takahata M, Otani K, Momoshima S, Yuasa
M, Hirai T, Yoshii T, Okawa A. Risk Factors of Nonunion After Acute Osteoporotic Vertebral

Fractures: A Prospective Multicenter Cohort Study. Spine (Phila Pa 1976). 2020;45:895–902
148. Makaram NS, Leow JM, Clement ND, Oliver WM, Ng ZH, Simpson C, Keating JF. Risk
factors associated with delayed and aseptic nonunion following tibial diaphyseal fractures
managed with intramedullary nailing. Bone Jt Open. 2021;2:227–235
t
149. Prabhakar P, Wessel L, Nguyen J, Stepan J, Carlson M, Fufa D. Factors Associated with
ip
Scaphoid Nonunion following Early Open Reduction and Internal Fixation. J Wrist Surg.
2020;9:141–149
cr
150. Tian R, Zheng F, Zhao W, Zhang Y, Yuan J, Zhang B, Li L. Prevalence and influencing
factors of nonunion in patients with tibial fracture: systematic review and meta-analysis. J Orthop
Surg Res. 2020;15:377
us
151. Taormina DP, Shulman BS, Karia R, Spitzer AB, Konda SR, Egol KA. Older age does
not affect healing time and functional outcomes after fracture nonunion surgery. Geriatr Orthop
Surg Rehabil. 2014;5:116–121
an
152. Hao Z, Li J, Li B, Alder KD, Cahill SV, Munger AM, Lee I, Kwon H-K, Back J, Xu S,
Lee FY. Smoking Alters Inflammation and Skeletal Stem and Progenitor Cell Activity During
Fracture Healing in Mice. J Bone Miner Res. 2020
153. Schmitz MA, Finnegan M, Natarajan R, Champine J. Effect of smoking on tibial shaft
M
fracture healing. Clin Orthop Relat Res. 1999:184–200

154. Lopas LA, Belkin NS, Mutyaba PL, Gray CF, Hankenson KD, Ahn J. Fractures in
geriatric mice show decreased callus expansion and bone volume. Clin Orthop Relat Res.
d
2014;472:3523–3532
155. Khosla S, Farr JN, Tchkonia T, Kirkland JL. The role of cellular senescence in ageing and
e
endocrine disease. Nature reviews. Endocrinology. 2020;16:263–275

156. Farr JN, Khosla S. Cellular senescence in bone. Bone. 2019;121:121–133
pt
157. Zhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding H, Giorgadze N, Palmer AK,
Ikeno Y, Hubbard GB, Lenburg M, O'Hara SP, LaRusso NF, Miller JD, Roos CM, Verzosa
ce
GC, LeBrasseur NK, Wren JD, Farr JN, Khosla S, Stout MB, McGowan SJ, Fuhrmann-
Stroissnigg H, Gurkar AU, Zhao J, Colangelo D, Dorronsoro A, Ling YY, Barghouthy AS,
Navarro DC, Sano T, Robbins PD, Niedernhofer LJ, Kirkland JL. The Achilles' heel of
Ac
senescent cells: from transcriptome to senolytic drugs. Aging Cell. 2015;14:644–658

158. Farr JN, Fraser DG, Wang H, Jaehn K, Ogrodnik MB, Weivoda MM, Drake MT,
Tchkonia T, LeBrasseur NK, Kirkland JL, Bonewald LF, Pignolo RJ, Monroe DG, Khosla S.
Identification of Senescent Cells in the Bone Microenvironment. J Bone Miner Res.
2016;31:1920–1929
159. Farr JN, Xu M, Weivoda MM, Monroe DG, Fraser DG, Onken JL, Negley BA, Sfeir JG,
Ogrodnik MB, Hachfeld CM, LeBrasseur NK, Drake MT, Pignolo RJ, Pirtskhalava T,
Tchkonia T, Oursler MJ, Kirkland JL, Khosla S. Targeting cellular senescence prevents age-
related bone loss in mice. Nat Med. 2017;23:1072–1079
160. Khosla S, Farr JN, Kirkland JL. Inhibiting Cellular Senescence: A New Therapeutic
Paradigm for Age-Related Osteoporosis. J Clin Endocrinol Metab. 2018;103:1282–1290
161. Meyer RA, Tsahakis PJ, Martin DF, Banks DM, Harrow ME, Kiebzak GM. Age and
ovariectomy impair both the normalization of mechanical properties and the accretion of mineral
by the fracture callus in rats. J Orthop Res. 2001;19:428–435
43
162. Prisby RD, Ramsey MW, Behnke BJ, Dominguez JM, Donato AJ, Allen MR, Delp MD.
Aging reduces skeletal blood flow, endothelium-dependent vasodilation, and NO bioavailability in
rats. J Bone Miner Res. 2007;22:1280–1288
163. Lim JC, Ko KI, Mattos M, Fang M, Zhang C, Feinberg D, Sindi H, Li S, Alblowi J,
Kayal RA, Einhorn TA, Gerstenfeld LC, Graves DT. TNFα contributes to diabetes impaired
angiogenesis in fracture healing. Bone. 2017;99:26–38
164. Wahl EC, Aronson J, Liu L, Fowlkes JL, Thrailkill KM, Bunn RC, Skinner RA, Miller
MJ, Cockrell GE, Clark LM, Ou Y, Isales CM, Badger TM, Ronis MJ, Sims J, Lumpkin
CK. Restoration of regenerative osteoblastogenesis in aged mice: modulation of TNF. J Bone

Miner Res. 2010;25:114–123
165. Raggatt LJ, Wullschleger ME, Alexander KA, Wu ACK, Millard SM, Kaur S,
Maugham ML, Gregory LS, Steck R, Pettit AR. Fracture healing via periosteal callus formation
requires macrophages for both initiation and progression of early endochondral ossification. Am J
t
Pathol. 2014;184:3192–3204
ip
166. Sebastián C, Herrero C, Serra M, Lloberas J, Blasco MA, Celada A. Telomere shortening
and oxidative stress in aged macrophages results in impaired STAT5a phosphorylation. J
cr
Immunol. 2009;183:2356–2364
167. Aristorena M, Blanco FJ, Las Casas-Engel M de, Ojeda-Fernandez L, Gallardo-Vara E,
Corbi A, Botella LM, Bernabeu C. Expression of endoglin isoforms in the myeloid lineage and
us
their role during aging and macrophage polarization. J Cell Sci. 2014;127:2723–2735
168. Mahbub S, Deburghgraeve CR, Kovacs EJ. Advanced age impairs macrophage
polarization. J Interferon Cytokine Res. 2012;32:18–26
an
169. Slade Shantz JA, Yu Y-Y, Andres W, Miclau T, Marcucio R. Modulation of macrophage
activity during fracture repair has differential effects in young adult and elderly mice. J Orthop
Trauma. 2014;28 Suppl 1:S10-4
170. Chen L, Cheng S, Sun K, Wang J, Liu X, Zhao Y, Yang J, Zhao D, Xue C, Tao Y, Zhao
M
S, Zhang H, Lu S, Shi Q, Wang Y, Shu B. Changes in macrophage and inflammatory cytokine

expressions during fracture healing in an ovariectomized mice model. BMC Musculoskelet Disord.
2021;22
d
171. Vi L, Baht GS, Soderblom EJ, Whetstone H, Wei Q, Furman B, Puviindran V, Nadesan
P, Foster M, Poon R, White JP, Yahara Y, Ng A, Barrientos T, Grynpas M, Mosely MA,
e
Alman BA. Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of
bone repair in mice. Nat Commun. 2018;9:5191
pt
172. Németh K, Leelahavanichkul A, Yuen PST, Mayer B, Parmelee A, Doi K, Robey PG,
Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star RA, Mezey E. Bone
ce
marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host
macrophages to increase their interleukin-10 production. Nat Med. 2009;15:42–49
173. François M, Romieu-Mourez R, Li M, Galipeau J. Human MSC suppression correlates
Ac
with cytokine induction of indoleamine 2,3-dioxygenase and bystander M2 macrophage

differentiation. Mol Ther. 2012;20:187–195
174. Franceschi C, Garagnani P, Vitale G, Capri M, Salvioli S. Inflammaging and 'Garb-aging'.
Trends Endocrinol Metab. 2017;28:199–212
175. Elder SS, Emmerson E. Senescent cells and macrophages: key players for regeneration?
Open Biol. 2020;10:200309
176. Hall BM, Balan V, Gleiberman AS, Strom E, Krasnov P, Virtuoso LP, Rydkina E,
Vujcic S, Balan K, Gitlin I, Leonova K, Polinsky A, Chernova OB, Gudkov AV. Aging of
mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that
can be induced in young mice by senescent cells. Aging (Albany NY). 2016;8:1294–1315
177. Quarto R, Thomas D, Liang CT. Bone progenitor cell deficits and the age-associated decline
in bone repair capacity. Calcif Tissue Int. 1995;56:123–129
178. Chen TL. Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow
stromal cell cultures by increased donor age and glucocorticoid treatment. Bone. 2004;35:83–95
44
179. Muschler GF, Nitto H, Boehm CA, Easley KA. Age- and gender-related changes in the
cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res.
2001;19:117–125
180. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of
telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion.
Stem Cells. 2004;22:675–682
181. O'Driscoll SW, Saris DB, Ito Y, Fitzimmons JS. The chondrogenic potential of periosteum
decreases with age. J Orthop Res. 2001;19:95–103
182. Shibata KR, Aoyama T, Shima Y, Fukiage K, Otsuka S, Furu M, Kohno Y, Ito K,

Fujibayashi S, Neo M, Nakayama T, Nakamura T, Toguchida J. Expression of the p16INK4A
gene is associated closely with senescence of human mesenchymal stem cells and is potentially
silenced by DNA methylation during in vitro expansion. Stem Cells. 2007;25:2371–2382
183. Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, LeBoff MS, Glowacki J. Age-
t
Related Intrinsic Changes in Human Bone Marrow-Derived Mesenchymal Stem Cells and Their
ip
Differentiation to Osteoblasts. Aging Cell. 2008;7:335–343
184. Ono T, Takayanagi H. Osteoimmunology in Bone Fracture Healing. Curr Osteoporos Rep.
cr
2017;15:367–375
185. Guder C, Gravius S, Burger C, Wirtz DC, Schildberg FA. Osteoimmunology: A Current
Update of the Interplay Between Bone and the Immune System. Front Immunol. 2020;11:58
us
186. Kovach TK, Dighe AS, Lobo PI, Cui Q. Interactions between MSCs and immune cells:
implications for bone healing. J Immunol Res. 2015;2015:752510
187. Dighe AS, Yang S, Madhu V, Balian G, Cui Q. Interferon gamma and T cells inhibit
an
osteogenesis induced by allogeneic mesenchymal stromal cells. J Orthop Res. 2013;31:227–234
188. Reinke S, Geissler S, Taylor WR, Schmidt-Bleek K, Juelke K, Schwachmeyer V, Dahne
M, Hartwig T, Akyüz L, Meisel C, Unterwalder N, Singh NB, Reinke P, Haas NP, Volk H-D,
Duda GN. Terminally differentiated CD8⁺ T cells negatively affect bone regeneration in humans.
M
Sci Transl Med. 2013;5:177ra36

189. Wheatley BM, Nappo KE, Christensen DL, Holman AM, Brooks DI, Potter BK. Effect of
NSAIDs on Bone Healing Rates: A Meta-analysis. J Am Acad Orthop Surg. 2019;27:e330-e336
d
190. Al-Waeli H, Reboucas AP, Mansour A, Morris M, Tamimi F, Nicolau B. Non-steroidal

anti-inflammatory drugs and bone healing in animal models-a systematic review and meta-
e
analysis. Syst Rev. 2021;10:201

191. Saul D, Weber M, Zimmermann MH, Kosinsky RL, Hoffmann DB, Menger B, Taudien
pt
S, Lehmann W, Komrakova M, Sehmisch S. Effect of the lipoxygenase inhibitor baicalein on

bone tissue and bone healing in ovariectomized rats. Nutr Metab (Lond). 2019;16:4
ce
192. Marquez-Lara A, Hutchinson ID, Nuñez F, Smith TL, Miller AN. Nonsteroidal Anti-
Inflammatory Drugs and Bone-Healing: A Systematic Review of Research Quality. JBJS Rev.
2016;4
Ac
193. Franceschi C, Bonafè M, Valensin S, Olivieri F, Luca M de, Ottaviani E, Benedictis G de.
Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci.
2000;908:244–254
194. Biguetti CC, Couto MCR, Silva ACR, Shindo JVTC, Rosa VM, Shinohara AL, Andreo
JC, Duarte MAH, Wang Z, Brotto M, Matsumoto MA. New Surgical Model for Bone-Muscle
Injury Reveals Age and Gender-Related Healing Patterns in the 5 Lipoxygenase (5LO) Knockout
Mouse. Front Endocrinol (Lausanne). 2020;11:484
195. Saul D, Ninkovic M, Komrakova M, Wolff L, Simka P, Gasimov T, Menger B,
Hoffmann DB, Rohde V, Sehmisch S. Effect of zileuton on osteoporotic bone and its healing,
expression of bone, and brain genes in rats. J Appl Physiol. 2018;124:118–130
196. Cottrell JA, O'Connor JP. Pharmacological inhibition of 5-lipoxygenase accelerates and
enhances fracture-healing. J Bone Joint Surg Am. 2009;91:2653–2665
197. Cossarizza A, Ortolani C, Paganelli R, Barbieri D, Monti D, Sansoni P, Fagiolo U,
Castellani G, Bersani F, Londei M, Franceschi C. CD45 isoforms expression on CD4+ and
45
CD8+ T cells throughout life, from newborns to centenarians: implications for T cell memory.
Mech Ageing Dev. 1996;86:173–195
198. Melton DW, Roberts AC, Wang H, Sarwar Z, Wetzel MD, Wells JT, Porter L, Berton
MT, McManus LM, Shireman PK. Absence of CCR2 results in an inflammaging environment in
young mice with age-independent impairments in muscle regeneration. J Leukoc Biol.
2016;100:1011–1025
199. Xing Z, Lu C, Hu D, Miclau T, Marcucio RS. Rejuvenation of the inflammatory system
stimulates fracture repair in aged mice. J Orthop Res. 2010;28:1000–1006
200. Sun G, Wang Y, Ti Y, Wang J, Zhao J, Qian H. Regulatory B cell is critical in bone union

process through suppressing proinflammatory cytokines and stimulating Foxp3 in Treg cells. Clin
Exp Pharmacol Physiol. 2017;44:455–462
201. Lu C, Miclau T, Hu D, Hansen E, Tsui K, Puttlitz C, Marcucio RS. Cellular basis for age-
related changes in fracture repair. J Orthop Res. 2005;23:1300–1307
t
202. Naik AA, Xie C, Zuscik MJ, Kingsley P, Schwarz EM, Awad H, Guldberg R, Drissi H,
ip
Puzas JE, Boyce B, Zhang X, O'Keefe RJ. Reduced COX-2 expression in aged mice is
associated with impaired fracture healing. J Bone Miner Res. 2009;24:251–264
cr
203. Janssen MP, Caron MM, van Rietbergen B, Surtel DA, van Rhijn LW, Welting TJ,
Emans PJ. Impairment of the chondrogenic phase of endochondral ossification in vivo by
inhibition of cyclooxygenase-2. Eur Cell Mater. 2017;34:202–216
us
204. Li M, Thompson DD, Paralkar VM. Prostaglandin E(2) receptors in bone formation. Int
Orthop. 2007;31:767–772
205. Hemmatian H, Bakker AD, Klein-Nulend J, van Lenthe GH. Aging, Osteocytes, and
an
Mechanotransduction. Curr Osteoporos Rep. 2017;15:401–411
206. Bellido T. Osteocyte-driven bone remodeling. Calcif Tissue Int. 2014;94:25–34
207. Kousteni S, Chen JR, Bellido T, Han L, Ali AA, O'Brien CA, Plotkin L, Fu Q, Mancino
AT, Wen Y, Vertino AM, Powers CC, Stewart SA, Ebert R, Parfitt AM, Weinstein RS, Jilka
M
RL, Manolagas SC. Reversal of bone loss in mice by nongenotropic signaling of sex steroids.
Science. 2002;298:843–846
208. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T.
d
Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest.
1999;104:1363–1374
e
209. Tomkinson A, Reeve J, Shaw RW, Noble BS. The death of osteocytes via apoptosis
accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab. 1997;82:3128–3135
pt
210. Kaplon H, Muralidharan M, Schneider Z, Reichert JM. Antibodies to watch in 2020.

MAbs. 2020;12:1703531
ce
211. Bhandari M, Schemitsch EH, Karachalios T, Sancheti P, Poolman RW, Caminis J,

Daizadeh N, Dent-Acosta RE, Egbuna O, Chines A, Miclau T. Romosozumab in Skeletally
Mature Adults with a Fresh Unilateral Tibial Diaphyseal Fracture: A Randomized Phase-2 Study.
Ac
J Bone Joint Surg Am. 2020;102:1416–1426

212. Schemitsch EH, Miclau T, Karachalios T, Nowak LL, Sancheti P, Poolman RW, Caminis
J, Daizadeh N, Dent-Acosta RE, Egbuna O, Chines A, Maddox J, Grauer A, Bhandari M. A
Randomized, Placebo-Controlled Study of Romosozumab for the Treatment of Hip Fractures. J
Bone Joint Surg Am. 2020;102:693–702
213. Suen PK, Zhu TY, Chow DHK, Le Huang, Zheng L-Z, Qin L. Sclerostin Antibody
Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult
Male Rats. Sci Rep. 2015;5:15632
214. Morse A, Yu NYC, Peacock L, Mikulec K, Kramer I, Kneissel M, McDonald MM, Little
DG. Endochondral fracture healing with external fixation in the Sost knockout mouse results in
earlier fibrocartilage callus removal and increased bone volume fraction and strength. Bone.
2015;71:155–163
46
215. Liu Y, Rui Y, Cheng TY, Huang S, Xu L, Meng F, Lee WYW, Zhang T, Li N, Li C, Ke
H, Li G. Effects of Sclerostin Antibody on the Healing of Femoral Fractures in Ovariectomised
Rats. Calcif Tissue Int. 2016;98:263–274
216. McDonald MM, Morse A, Mikulec K, Peacock L, Yu N, Baldock PA, Birke O, Liu M, Ke
HZ, Little DG. Inhibition of sclerostin by systemic treatment with sclerostin antibody enhances
healing of proximal tibial defects in ovariectomized rats. J Orthop Res. 2012;30:1541–1548
217. Piemontese M, Onal M, Xiong J, Han L, Thostenson JD, Almeida M, O'Brien CA. Low
bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast
lineage. Sci Rep. 2016;6:24262

218. Onal M, Piemontese M, Xiong J, Wang Y, Han L, Ye S, Komatsu M, Selig M, Weinstein
RS, Zhao H, Jilka RL, Almeida M, Manolagas SC, O'Brien CA. Suppression of autophagy in
osteocytes mimics skeletal aging. J Biol Chem. 2013;288:17432–17440
219. Histing T, Kuntz S, Stenger D, Scheuer C, Garcia P, Holstein JH, Klein M, Pohlemann
t
T, Menger MD. Delayed fracture healing in aged senescence-accelerated P6 mice. J Invest Surg.
ip
2013;26:30–35
220. Batoon L, Millard SM, Raggatt LJ, Pettit AR. Osteomacs and Bone Regeneration. Curr
cr
221. Cenci S, Toraldo G, Weitzmann MN, Roggia C, Gao Y, Qian WP, Sierra O, Pacifici R.
Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-
us
gamma-induced class II transactivator. Proc Natl Acad Sci U S A. 2003;100:10405–10410
222. Shay JW, Wright WE. Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol.
2000;1:72–76
an
223. Kowald A, Passos JF, Kirkwood TBL. On the evolution of cellular senescence. Aging Cell.
2020;19:e13270
224. Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol. 2013;75:685–705
225. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B,
M
Kirkland JL, van Deursen JM. Clearance of p16Ink4a-positive senescent cells delays ageing-
associated disorders. Nature. 2011;479:232–236
226. Bussian TJ, Aziz A, Meyer CF, Swenson BL, van Deursen JM, Baker DJ. Clearance of
d
senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature.
2018;562:578–582
e
227. Demaria M, O'Leary MN, Chang J, Shao L, Liu S, Alimirah F, Koenig K, Le C, Mitin N,
Deal AM, Alston S, Academia EC, Kilmarx S, Valdovinos A, Wang B, Bruin A de, Kennedy
pt
BK, Melov S, Zhou D, Sharpless NE, Muss H, Campisi J. Cellular Senescence Promotes
Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discov. 2017;7:165–176
ce
228. Nelson G, Wordsworth J, Wang C, Jurk D, Lawless C, Martin-Ruiz C, Zglinicki T von.

A senescent cell bystander effect: senescence-induced senescence. Aging Cell. 2012;11:345–349
229. Saul D, Kosinsky RL. Epigenetics of Aging and Aging-Associated Diseases. Int J Mol Sci.
Ac
2021;22
230. Anderson R, Lagnado A, Maggiorani D, Walaszczyk A, Dookun E, Chapman J, Birch J,
Salmonowicz H, Ogrodnik M, Jurk D, Proctor C, Correia-Melo C, Victorelli S, Fielder E,
Berlinguer-Palmini R, Owens A, Greaves LC, Kolsky KL, Parini A, Douin-Echinard V,
LeBrasseur NK, Arthur HM, Tual-Chalot S, Schafer MJ, Roos CM, Miller JD, Robertson N,
Mann J, Adams PD, Tchkonia T, Kirkland JL, Mialet-Perez J, Richardson GD, Passos JF.
Length-independent telomere damage drives post-mitotic cardiomyocyte senescence. EMBO J.
2019;38
231. Ogrodnik M, Zhu Y, Langhi LGP, Tchkonia T, Krüger P, Fielder E, Victorelli S,
Ruswhandi RA, Giorgadze N, Pirtskhalava T, Podgorni O, Enikolopov G, Johnson KO, Xu
M, Inman C, Palmer AK, Schafer M, Weigl M, Ikeno Y, Burns TC, Passos JF, Zglinicki T
von, Kirkland JL, Jurk D. Obesity-Induced Cellular Senescence Drives Anxiety and Impairs
Neurogenesis. Cell Metab. 2019;29:1061-1077.e8
232. López-Otín C, Kroemer G. Hallmarks of Health. Cell. 2021;184:33–63
47
233. He S, Sharpless NE. Senescence in Health and Disease. Cell. 2017;169:1000–1011
234. Karin O, Agrawal A, Porat Z, Krizhanovsky V, Alon U. Senescent cell turnover slows with
age providing an explanation for the Gompertz law. Nat Commun. 2019;10:5495
235. Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, Laberge R-M,
Vijg J, van Steeg H, Dollé MET, Hoeijmakers JHJ, Bruin A de, Hara E, Campisi J. An
essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev
Cell. 2014;31:722–733
236. Ritschka B, Storer M, Mas A, Heinzmann F, Ortells MC, Morton JP, Sansom OJ,
Zender L, Keyes WM. The senescence-associated secretory phenotype induces cellular plasticity

and tissue regeneration. Genes Dev. 2017;31:172–183
237. Gal H, Lysenko M, Stroganov S, Vadai E, Youssef SA, Tzadikevitch-Geffen K, Rotkopf
R, Biron-Shental T, Bruin A de, Neeman M, Krizhanovsky V. Molecular pathways of
senescence regulate placental structure and function. EMBO J. 2019;38:e100849
t
238. Gibaja A, Aburto MR, Pulido S, Collado M, Hurle JM, Varela-Nieto I, Magariños M.
ip
TGFβ2-induced senescence during early inner ear development. Sci Rep. 2019;9
239. Yun MH, Davaapil H, Brockes JP. Recurrent turnover of senescent cells during regeneration
cr
of a complex structure. Elife. 2015;4
240. Mosteiro L, Pantoja C, Martino A de, Serrano M. Senescence promotes in vivo
reprogramming through p16INK4a and IL-6. Aging Cell. 2018;17
us
241. Kale A, Sharma A, Stolzing A, Desprez P-Y, Campisi J. Role of immune cells in the
removal of deleterious senescent cells. Immun Ageing. 2020;17:16
242. Burton DGA, Stolzing A. Cellular senescence: Immunosurveillance and future
an
immunotherapy. Ageing Res Rev. 2018;43:17–25
243. Serrano M. Senescence helps regeneration. Dev Cell. 2014;31:671–672
244. Prata LGPL, Ovsyannikova IG, Tchkonia T, Kirkland JL. Senescent cell clearance by the
immune system: Emerging therapeutic opportunities. Semin Immunol. 2018;40:101275
M
245. Saul D, Monroe DG, Rowsey JL, Kosinsky RL, Vos SJ, Doolittle ML, Farr JN, Khosla S.
Modulation of fracture healing by the transient accumulation of senescent cells. Elife. 2021;10
246. Grosse L, Wagner N, Emelyanov A, Molina C, Lacas-Gervais S, Wagner K-D, Bulavin
d
DV. Defined p16High Senescent Cell Types Are Indispensable for Mouse Healthspan. Cell Metab.
2020;32:87-99.e6
e
247. Xu M, Pirtskhalava T, Farr JN, Weigand BM, Palmer AK, Weivoda MM, Inman CL,
pt
Ogrodnik MB, Hachfeld CM, Fraser DG, Onken JL, Johnson KO, Verzosa GC, Langhi
LGP, Weigl M, Giorgadze N, LeBrasseur NK, Miller JD, Jurk D, Singh RJ, Allison DB,
Ejima K, Hubbard GB, Ikeno Y, Cubro H, Garovic VD, Hou X, Weroha SJ, Robbins PD,
ce
Niedernhofer LJ, Khosla S, Tchkonia T, Kirkland JL. Senolytics improve physical function
and increase lifespan in old age. Nat Med. 2018;24:1246–1256
Ac
48
Figures
Figure 1: Initial steps after a traumatic fracture. Ruptured blood vessels lead to the formation of a
hematoma by platelets and erythrocytes. Adherent mesenchymal stem cells (MSCs) are recruited
and differentiate into osteoblastic and chondrogenic precursors. This results in a matrix of fibrinogen

and a microenvironment that has a central hypoxic state, where angiogenesis is promoted and the
key molecular players are IGF-1, TGF-β and IL-1/6, which lead to the promotion of the next healing
t
steps while maintaining an inflammatory state. A central cell in the first healing period is the
ip
classically activated macrophage (CAM=M1), which orchestrates the early inflammatory stage.
cr
us
Figure 2: Intramembranous ossification and endochondral ossification occur in parallel, but with
an
different underlying mechanisms. While periosteal MSCs adjacent to the fracture site form a hard
callus via intramembranous ossification, the central area with a hypoxic core forms a soft callus via
M
endochondral ossification. The macrophages are M2-polarized and alternatively activated (AAM) and
the role of chondrocytes in the soft callus development becomes more prominent.
e d
pt
Figure 3: Provisional cartilage is removed and the newly formed bone is calcified. In the third bone
ce
healing period, the central soft callus region undergoes a calcification process, while chondrocytes
become hypertrophic and are partially removed. Osteocytes are more and more incorporated, while
Ac
the previously built collagen II matrix is progressively degraded.
Figure 4: The fourth bone healing phase is characterized by bone remodeling. In a chronic process
which can last several months, the preceding osteoblast/osteoclast proportions are gradually
restored. Some osteocytes become apoptotic, some necrotic and the hypertrophic callus region is
reduced while favorable lamellar bone is built.
49
Figure 5: Senescent cell burden in the aging organism. Acute tissue damage causes a local, rapid
increase in senescent cell burden, briefly disturbing the healthy balance (green area), followed by a
rapid immune response aimed at clearing those cells. However, senescent cell burden increases with

age, due to the “bystander” effect and incomplete senescent cell clearance caused by a deteriorating
immune system. Further disturbance of this delicate senescent cell balance occurs due to
t
degenerative processes and cancer.
ip
cr
us
an
M
e d
pt
ce
Ac
50
t
ip
cr
us
an
Figure 1
51
d e
pt
ce
Ac
t
ip
cr
us
an
Figure 2
52
d e
pt
ce
Ac
t
ip
cr
us
an
Figure 3
53
d e
pt
ce
Ac
t
ip
cr
us
an
Figure 4
54
d e
pt
ce
Ac
t
ip
cr
us
an
Figure 5
55
d e
pt
ce
Ac

Bnac 008

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Bnac 008

Uploaded by

Copyright:

Available Formats

Fracture Healing in the Setting of Endocrine Diseases, Aging, and Cellular Senescence

Dominik Saul, MD1,2, Sundeep Khosla, MD1

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

Kogod Center on Aging and Division of Endocrinology

Mayo Clinic, Rochester, MN 55905, USA

413501650 (Deutsche Forschungsgemeinschaft, DFG; D.S.).

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

socioeconomic burden. Importantly, the age-related deterioration of bone structure is associated

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

renewal and proliferative capacity. A prolonged phase of “inflammaging” results in an extended

remodeling phase, characterized by a senescent microenvironment and deteriorating healing

Aging; Bone; Bone healing; Fracture; Fracture healing; Osteoporosis; Senescence

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

Local cellular and molecular mediators of fracture healing

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

conclusion is based on genetic pulse-chase studies in C57BL/6 mice demonstrating a replicative

(8, 10, 12).

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

Hematoma and Inflammation

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

immunoregulatory cells, primarily granulocytes, monocytes/macrophages and leukocytes (B cells, T

express IL10, reducing the early inflammatory response.

Osteocytes and Osteoclasts

by preserving cartilage, since sclerostin preserves chondrocyte metabolism, especially in mechanical

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

Matrix and Platelets

(collagen I, fibronectin, osteopontin, osteonectin) and enhances bone formation, promotes

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

osteogenesis during the hypoxic stage. VEGF-dependent and angiopoietin-dependent pathways

promote vascular growth into the developing callus.

Second healing period: Angiogenesis, Cartilage formation/Soft callus

peripheral intramembranous ossification), where avascular cartilaginous tissue is built to deliver a

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

chondrogenic potential (43).

osteoclasts, osteoblasts and fibroblasts to the site of fracture.

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

Intramembranous ossification (hard callus)

The intramembranous ossification (formation of hard callus) recapitulates embryonic

inflammatory cells) and their local descendants (osteoblasts and chondrocytes).

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

osteoclastic processes alternate (Fig. 3). Intramembranous ossification and endochondral

osteoblastic phenotype (36).

calcification of remaining extracellular matrix (ECM), and are subsequently removed by

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

hypertrophic chondrocytes, transitioning into an osteoblast/osteocyte phenotype with cellular

GFP, indicating a transdifferentiation of chondrocytes to become osteoblasts. Similarly, Hu and

expression and subsequently expression of alkaline phosphatase, osterix, osteopontin and

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

matrix-metalloproteinases (especially MMP13 and MMP9) is initiated, degrading type II collagen to

lacunae (13, 64).

Fourth healing period: Chronic bone remodeling

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

key responsive elements although a morphological difference between chondroclasts and

and lamellar bone is built by osteoblasts (47, 58).

69). M1 polarized macrophages (pro-inflammatory) promote mineralization of MSCs, in part by

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

impairing osteoclast activity (77, 79).

subcutaneous administration of 20 μg PTH 1-34 as compared to oral bisphosphonate therapy in

stress fractures are ongoing (NCT02972424, NCT04196855).

Growth hormone/Insulin-like growth factor 1 (GH/IGF1)

Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnac008/6532488 by guest on 25 March 2022

from concomitant hormonal deficiencies (e.g., hypogonadism) (85–87).