Diabetic wound healing in soft and hard oral

tissues

KANG I. KO, ANTON SCULEAN, and DANA T. GRAVES

PHILADELPHIA; AND BERN, SWITZERLAND

There is significant interest in understanding the cellular mechanisms responsible for

expedited healing response in various oral tissues and how they are impacted by
systemic diseases. Depending upon the types of oral tissue, wound healing may
occur by predominantly re-eptihelialization, by re-epithelialization with substantial
new connective tissue formation, or by a a combination of both plus new bone for-
mation. As a result, the cells involved differ and are impacted by systemic diaseses
in various ways. Diabetes mellitus is a prevalent metabolic disorder that impairs bar-
rier function and healing responses throughout the human body. In the oral cavity,
diabetes is a known risk factor for exacerbated periodontal disease and delayed
wound healing, which includes both soft and hard tissue components. Here, we
review the mechanisms of diabetic oral wound healing, particularly on impaired
keratinocyte proliferation and migration, altered level of inflammation, and
reduced formation of new connective tissue and bone. In particular, diabetes inhib-
its the expression of mitogenic growth factors whereas that of pro-inflammatory
cytokines is elevated through epigenetic mechanisms. Moreover, hyperglycemia
and oxidative stress induced by diabetes prevents the expansion of mesengenic
cells that are involved in both soft and hard tissue oral wounds. A better understand-
ing of how diabetes influences the healing processes is crucial for the prevention
and treatment of diabetes-associated oral complications. (Translational Research
2021; 236:72 86)

INTRODUCTION keratinized oral mucosa such as attached gingiva.1 The

The oral cavity contains a complex mixture of tissue
types and cells. Mucosal surfaces that cover muscle
and/or small amounts of loose connective tissue
include the soft palate, nasopharynx, and buccal
mucosa that extends through the floor of the mouth to
the tongue (Fig 1). These oral tissues are non-kerati-
nized, contain large amounts of elastin, and harbor epi-
thelium that expresses different keratins compared to
From the Department of Periodontics, School of Dental Medicine,
University of Pennsylvania, Philadelphia, 19104; Department of
Periodontology, School of Dental Medicine, University of Bern,
Freiburgstrasse 7, CH-3010, Bern, Switzerland.
Submitted for Publication March 29, 2021; received submitted May
6, 2021; accepted for publication May 6, 2021.
Reprint requests: Dana T. Graves, Department of Periodontics,
School of Dental Medicine, University of Pennsylvania, Philadel-
phia, 19104 e-mail: dtgraves@upenn.edu.
1931-5244/$ - see front matter
Ó 2021 Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.trsl.2021.05.001
attached gingiva contains dense connective tissue and
is found on the palate and masticatory gingiva that sur-
rounds dentition over alveolar bone. Healing of oral
mucosa covering loose connective tissue and muscle is
distinct from healing of attached gingiva that covers
alveolar bone. Epithelial coverage of excisional
wounds in the former is rapid and does not depend
upon stromal healing, while for the latter, epithelial
closure coincides with substantial new connective tis-
sue formation, which occurs more slowly. In addition,
wounds that cover muscle and loose connective tissue
heal by epithelial coverage plus contraction while those
that cover bone lack a significant contraction compo-
nent.2 The oral mucosal epithelium is thicker than the
epidermis in skin.3 Unlike skin wounds, oral mucosal
wounds lack dermal appendages such as hair follicles,
sweat glands and sebacious glands. This is significant
since the appendages contain stem cells that promote
the healing process, which may be particularly impor-
tant in healing of connective tissue as described below.

72
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Volume 236 KO et al 73

Fig 1. Re-epithelialization in diabetic oral wounds. Left, location of different types of oral mucosa. Right,
graphical diagram of diabetic impact on epithelialization in oral wounds. Diabetes causes hyperglycemia and
production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This in turn
increases neutrophil recruitment and redues microbial diversity. Moreover, salivary content for pro-healing fac-
tors (EGF and histatin) is reduced, whereas those that cause destruction (MMP2 and MMP9) are enhanced.
Another mechanism is FOXO1-mediated overexpression of MMP9 by keratinocytes. The overall outcome is
persistent inflammation and an environment that is conducive for keratinocyte apoptosis, coupled with reduced
keratinocyte proliferation and migration.

RE-EPITHELIALIZATION IN ORAL WOUNDS nonkeratinized oral mucosal compared to similar der-

Re-epithelialization of nonkeratinized tissue is faster
in oral wounds than in corresponding dermal wounds,
whereas re-epithelialization in keratinized attached
gingiva is not necessarily faster. Upon wounding, epi-
thelial keratinocytes at the wound edge proliferate and
migrate to cover the wound surface. It has been pro-
posed that non-keratinized oral mucosa has a higher
proliferation rate than skin epidermis, which may
account for the more rapid re-epithelialization.4 In
addition, wounds in nonkeratinized tissue appear to
have fewer leukocytes such as neutrophils and mast
cells that may lead to less inflammation and more rapid
healing.5 Growth factors that induce keratinocyte
migration are produced in the early phases of healing,
including epidermal growth factor (EGF), heparin-
binding EGF-like growth factor, transforming growth
factor-b (TGF-b), and fibroblast growth factor
(FGF).2,6 It has been proposed that saliva contains
growth factors that promote oral wound healing. In a
tongue mucosal wound healing model, removal of sub-
mandibular salivary glands reduced the salivary EGF
content and impaired healing.6,7 Saliva also contains
other biologically active growth factors including
nerve growth factor, TGF-b, insulin-like growth fac-
tor-1 and FGF.6 Furthermore, oral mucosal keratino-
cytes upregulate host response genes more quickly and
to a greater extent than dermal keratinocytes, which
may contribute to the more rapid healing of
mal wounds.1 In addition to growth factors, saliva con-
tains histatins, which are antimicrobial peptides and
have been proposed to promote keratinocyte migration
in oral wounds.8 There is some evidence that the bio-
logically active site of histatins that stimulates kerati-
nocyte migration is distinct from the antimicrobial
domain. It should be noted however, that saliva appears
to influence large mucosal wounds to a greater extent
than small mucosal wounds.9 Moreover, saliva alone is
unlikely to account for the differences in rates of heal-
ing between skin and nonkeratinized oral mucosal
wounds given that there are several intrinsic differen-
ces in the keratinocytes from oral mucosa and skin.10,11
Acute inflammation is necessary to initiate the
recruitment of cells to the wound site and to induce the
expression of factors that lead to repair. Although tran-
sient inflammation is needed for normal healing to
occur, prolonged inflammation limits the repair pro-
cess. When oral tongue wounds were compared to skin
wounds, the expression of pro-inflammatory MMPs,
peptidases, cytokines and chemokines was less in oral
wounds, which may reflect differences intrinsic to cells
in oral mucosa.12 However, the temporal sequence of
factors induced by wounding was similar in mucosal
and dermal wounds.12 Reduced cytokine and chemo-
kine expression may facilitate rapid healing in non-ker-
atinized mucosal wounds compared to dermal wounds.
An early event in wound healing is the development of
a hypoxic environment due to disruption of blood flow.

74 KO et al
Mucosal wounds produce less hypoxia inducible fac-
tor-alpha than similar skin wounds.13 These results
suggest that the level of hypoxia in mucosal wounds is
less than that in skin and that the response of oral
mucosal wounds of nonkeratinized tissue is milder.
DiPietro and colleagues have suggested that the differ-
ential responses to hypoxia may contribute to differen-
ces in rates of healing.13
THE IMPACT OF DIABETES ON RE-EPITHELIALIZATION
OF ORAL WOUNDS
Type 1 diabetes mellitus (T1DM) is caused by a loss of
beta cells in the pancreas, most frequently due to autoim-
mune etiology, and results in an insufficient amount of
insulin production. Type 2 diabetes mellitus (T2DM) is
due to insulin resistance, caused by an inadequate cellular
response to normal levels of insulin signaling, combined
with beta cell failure to provide a compensatory increase
in insulin.14 Oral mucosal wounds heal more slowly in
diabetic animals compared to normoglycemic controls in
both T1DM and T2DM models.15 Contributing factors
have consistently been shown reflect the direct effect of
high glucose levels, as well as indirect effects caused by
increased inflammation, high levels of advanced glyca-
tion endproducts (AGEs), and increased formation of
reactive oxygen species (ROS).16,15 These factors inhibit
migration of oral and dermal keratinocytes in vitro.17,30
Oral keratinocytes in diabetic wounds in vivo have
increased levels of inflammatory cytokines such as TNF
and IL-1b and reduced levels of growth factors such as
FGF-2 and TGF-b.15 The combination of reduced growth
factor expression by keratinocytes and increased expres-
sion of inflammatory mediators may negatively impact
keratinocyte migration.
Saliva has several constituents that are thought to
directly promote re-epithelialization, and diabetes has
been shown to alter saliva in a way that may be detri-
mental to re-epithelialization. Diabetes reduces EGF
levels in the saliva of humans and mice,18,19 which
may contribute to reduced oral wound healing. This
conclusion is supported by evidence that supplementa-
tion with EGF in the drinking water improves healing
of diabetic oral wounds but has little effect on healing
of non-diabetic oral wounds.6 Furthermore, diabetic
children have reduced levels of salivary antimicrobial
peptides including histatins.20 The reduced levels of
histatins may negatively impact keratinocyte migration
and lessen the effectiveness of the antimicrobial
defense in diabetic individuals. In the skin, reduced
migration is linked to high levels of MMP-9 in diabetic
humans and animal models.21 In vitro, keratinocyte
migration in high glucose is rescued by antibody to
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October 2021
MMP-9 and knockdown of the transcription factor,
FOXO122. Interestingly, MMP-2 and MMP-9 are ele-
vated in the saliva of diabetic patients,23 which may
impair re-epithelialization.
Diabetes delays both the early and late stages of oral
wound healing.24 Diabetic wounds have increased
inflammation as noted by greater numbers of neutro-
phils.24,25 Re-epithelialization of gingival wounds is
enhanced by TNF inhibitors in diabetic animals but not
in normal animals, indicating that the increased levels
of TNF in the diabetic oral wounds is problematic
whereas the normal level of TNF does not inhibit the
healing process.26 In skin wounds, diabetes reduces the
conversion from a M1 macrophage phenotype to a M2
macrophage phenotype, which may contribute to pro-
longed inflammation.27 Diabetic skin wounds also
have a greater senescence-associated secretory pheno-
type (SASP) with increased inflammatory cytokine
expression (eg IL-1a, IL-6, IL-8, and CXCL2) and
MMP expression.28 Moreover, aged mice have a simi-
lar increase in the SASP phenotype as diabetic mice,
suggesting that there may be similar mechanisms that
delay healing with both aging and diabetes.28
Mucosal wounds must deal with the influence of
bacteria since the oral cavity has a higher microbial
load than skin. Bacterial colonization impairs healing
by inducing prolonged inflammation, which intereferes
with transition to the next phase of healing, matura-
tion.29 Diabetic skin wounds have an impaired host
response to bacteria and an increased level of ROS that
reduces microbial diversity and increases bacterial col-
onization.30 Bacteria can also have direct effects on
keratinocytes by stimulating apoptosis, reducing
migration and decreasing proliferation.31 Bacteria such
as P. gingivalis and F. nucleatum can invade oral kera-
tinocytes to interfere with re-epithelialization by down
regulating genes that promote proliferation and integ-
rins needed for cell migration.31 The characteristics of
diabetic oral wound healing by re-epithelilization are
summarized in Fig 1.
RE-EPITHELIALIZATION, DIABETES AND FOXO1
As discussed above, diabetes negatively impacts kerati-
nocyte proliferation and migration to interfere with re-
epithelialization.32 Diabetic conditions impair keratino-
cyte function, which include high glucose levels,
increased formation of AGEs and ROS. Elevated ROS
levels have been shown to interfere with mucosal heal-
ing33 and increase inflammation in gingival epithelium.34
The epithelium of diabetic individuals has greater expres-
sion of RAGE, which increases inflammation and wor-
sens healing responses.35,36 A unifying explanation that
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links high glucose, AGEs and ROS formation to diabetes-
impaired re-epithelialization is the transcription factor
FOXO1. Under normal conditions, FOXO1 improves re-
epithelialization by protecting keratinocytes from oxida-
tive stress and by inducing expression of TGF-b, both of
which are needed for an appropriate healing response.37
In diabetic conditions, high glucose or AGEs cause an
alteration in FOXO1 binding to the promoter regions of
specific genes, most likely due to epigenetic modifica-
tions, that result in a diminished capacity to protect
against oxidative stress and induce TGF b1 transcription
is significantly reduced.32 These molecular changes are
observed in vivo and in vitro and in both skin and oral
mucosal keratinocytes. On a molecular level, diabetic
conditions interfere with FOXO1 induced transcription
by reducing FOXO1 binding to consensus response ele-
ments on gene promoters such as TGF b1 and tissue
inhibitor of matrix metalloproteinases 1. In contrast, high
glucose and AGEs increase FOXO1-promoter interac-
tions leading to overexpression of factors that reduce re-
epithelialization. More specifically, in a microenviron-
ment with high glucose/AGE levels, FOXO1 induces
expression of MMP9, serpin peptidase inhibitor clade B
member 2, chemokine CCL20 and IL-36g.17,22,32 Inter-
estingly, the negative effect of high glucose on keratino-
cyte migration is improved by addition of specific
inhibitors for MMP9, or CCL20 or IL-36g or by reducing
FOXO1 activity.22 The role of FOXO1 in inducing exces-
sive production of these factors is demonstrated in vivo
by lineage specific FOXO1 deletion in keratinocytes that
reduces high levels of MMP9, serpin peptidase inhibitor
clade B member 2, CCL20 and IL-36g expression.22 It
should be noted that while high levels of these factors
interfere with keratinocyte re-epithelialization, their total
absence is also problematic. For example, the total
absence of MMP9 is detrimental to keratinocyte migra-
tion,38 as is absence of IL-36 g.39 Thus, impaired re-epi-
thelialization in diabetes is due to overexpression of
factors that normally promote an appropriate healing
response at physiological concentrations. The expression
of FOXO1 provides a mechanistic explanation for how
diabetic conditions can lead to overexpression of these
factors as shown in Fig 1.
CONNECTIVE TISSUE HEALING IN GINGIVAL
WOUNDS
As discussed above, keratinized mucosa (e.g.
attached gingiva) covers the hard palate and alveolar
bone, and its healing differs from that of nonkerati-
nized mucosa by relying on substantial connective tis-
sue healing. Table I summarizes the findings from
studies that investigate specific mucosal sites. Attached
KO et al 75
gingiva has a denser connective tissue compartment,
which contains fibroblasts, leukocytes, endothelial and
other cells. Upon wounding, the coordinated healing
response often results in complete regeneration of
injured gingiva, which contrasts with skin wounds that
heal by fibrosis.40-42 Fibroblasts are the principal cell
type of connective tissues and are largely responsible
for stromal healing by producing extracellular matrix
proteins such as collagen and proteoglycans. It has
been proposed that the unique regenerative response of
gingival wounds is attributed to the intrinsic properties
of gingival fibroblasts that are distinct from skin fibro-
blasts. Cultured gingival fibroblasts are more prolifer-
ative and express reduced levels of TGF-b1, a pro-
fibrogenic cytokine, compared to skin fibroblasts.43
Importantly, gingival wounds have higher fibroblast
density and increased connective tissue fill compared
to skin wounds,42 which is associated with reduced lev-
els of TGF-b1 and elevated levels of anti-fibrogenic
TGF-b3 in oral wounds.44-47 In a similar vein, fetal
wounds that heal without scarring also exhibit low lev-
els of TGF-b1 and high TGF-b3 levels.47,48 Moreover,
it is proposed that fibroblasts may down regulate
immune responses in gingival wounds to facilitate res-
olution of inflammation and promote healing.11,42 Ani-
mal studies have shown that gingival fibroblasts are
derived from neural crest origin (Wnt1-lineage) and
can differentiate into tri-lineage mesenchymal cells in
vitro,49 which contrasts with mesoderm-derived skin
fibroblasts.49,50 Gingival fibroblasts cultured ex vivo
are known as gingival mesenchymal stromal cells
(GMSCs). GMSCs exhibit a pro-healing capacity by
their anti-inflammatory properties rather than by
directly differentiating into mesengenic cells.51 55 It is
speculated that gingival fibroblasts may exhibit similar
immunomodulatory function in vivo, which remains to
be proven. In vitro culture conditions drastically shift
the fibroblast transcriptome.56,57 Thus the phenotypic
and functional similarities of gingival fibroblasts and
GMSCs may not necessarily align in the context of
wound healing in vivo. It has been shown however
that gingival fibroblasts express toll-like receptors
and can secrete pro-inflammatory cytokines such as
IL-6 and IL-8 when exposed to bacterial products in
vitro and in vivo.58-61 Therefore, gingival fibro-
blasts, in addition to their traditional role in produc-
ing extracellular matrix, may exert polarizing
effects on inflammation depending on physiological
or pathologic condition.
Inflammation in gingival and mucosal wounds is
more transient than in skin wounds.11,42 Initial inflam-
mation is characterized by neutrophil infiltration which
is necessary to limit bacterial invasion into the wound
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76 KO et al October 2021

Table I. Wound healing characteristics in oral mucosa over loose connective tissue or bone
Mucosal healing over loose connective tissue
Mucosal type (model) Healing characteristics Reference
2
Buccal mucosa (mouse) Reduced collagen deposition and TGFb1 compared to skin healing
11
Buccal mucosa (human) Increased epithelial migration and proliferation, rapid resolution of
inflammation
63
Buccal mucosa (human) Increased NETosis and anti-microbial activity
5
Pharyngeal mucosa (human) Fewer numbers of immune cells in healthy and healed mucosa com-
pared to skin
7,10,13
Tongue (mouse) Enhanced mucosal healing by salivary EGF, and reduced apoptosis
and hypoxic response compared to skin wounds
45
Tongue (mouse) Reduced inflammation
74
Tongue (mouse) Reduced angiogenesis versus skin wounds
Mucosal healing over bone
Mucosal type (model) Healing characteristics Reference
42
Palatal gingiva (pig) Reduced inflammation, contraction and fibroblast density, improved
healing compared to skin wounds
54
Palatal gingiva (mouse) Reduced inflammation and improved healing by GMSC application
78
Palatal gingiva (mouse) Keratinocyte-derived VEGF controls angiogenesis in oral wounds
49,50
Palatal/marginal gingivae Presence of neural-crest-derived gingival MSCs
(mouse)
61
Marginal gingiva (human) Expression of IL-6 and IL-8 by gingival fibroblasts
66
Marginal gingiva (human, DEL-1 mediated resolution of inflammation in periodontitis
mouse)
69
Marginal gingiva (mouse) Homeostatic defense against masticatory stress by IL-17 driven
mechanism
70
Marginal gingiva (mouse) Protection by gamma-delta T cell-derived amphiregulin in periodon-
titis model

site via secretion of proinflammatory cytokines, phago-
cytosis and NETosis.62,63 Clearance of neutrophils
occurs via apoptosis,64 which may be targeted by bac-
teria to prolong their lifespan and inflammation.65
Another mechanism of neutrophil clearance is DEL-1-
mediated efferocytosis by macrophages, which in turn
reprograms macrophages toward a pro-healing pheno-
type to facilitate gingival regeneration.66 Furthermore,
lipid mediators such as resolvins and lipoxins have
shown to have a pro-resolving effect in gingival inflam-
mation from rabbit models.67,68 Lymphocytes have
also been shown to play an important role in shaping
immunity in gingival injury. In mice, induction of
mechanical damage to gingiva, either by mastication or
external abrasion, stimulates gingival epithelial cells to
secrete IL-6, which in turn leads to expansion of Th17
cells to produce defensins and neutrophil chemoattrac-
tants.69 Moreover, gingival gd T cells have been shown
to produce amphiregulin to enhance wound healing in
a periodontitis model.70 It is well established that unre-
solved inflammation results in impaired wound healing
and scarring.71,72 Therefore, transient inflammation
may partially contribute to rapid gingival wound heal-
ing.
Oral wounds have reduced angiogenic response
when compared to skin wounds,73,74 which may be due
to excessive angiogenesis of skin wounds that is
accompanied by inflammation. Inflammatory signals
such as IL-1b and TNF promote neovasculariza-
tion75,76 and are linked to aberrant angiogenesis in
rheumatoid arthritis.77 Moreover, oral keratinocytes
express reduced levels of VEGF compared to skin ker-
atinocytes,74 which may be advantageous. VEGF
expression by the oral keratinocytes is FOXO1-depen-
dent, and its deletion impairs angiogenesis and wound
healing parameters in gingiva.78
DIABETIC IMPACT ON CELLS INVOLVED IN
CONNECTIVE TISSUE HEALING
Diabetes negatively affects multiple cell types that
participate in connective tissue wound healing of
mucosal surfaces as summarized in Table II. In both
T1DM and T2DM mouse models, new connective tis-
sue fill in palatal gingival wounds is reduced compared
to normoglycemic control groups.79 This is attributed
to reduced fibroblast numbers in healing wounds and
downregulation of pro-collagen I and III expression.80
Mechanistically, high glucose levels promote apoptosis
and reduce proliferation of gingival fibroblasts,79,81
which is due to the impact of greater oxidative stress,
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Table II. Diabetes-induced alterations in wound healing

Cell or Tissue Effect of Diabetes Reference
33,34
Epithelium Elevated ROS
35
Epithelium Increased expression of receptor for AGE
22
Epithelium Alteration in downstream genes induced by FOXO1
17,78
Epithelium Reduced growth factor expression
15,17
Epithelium Increased cytokine expression
17,79
Epithelium Reduced re-epithelialization
6,20,23
Saliva Reduced growth factor and increased inflammatory proteins
79
Connective tissue Increased fibroblast apoptosis
15,79
Connective tissue Increased cytokine expression and inflammation
102
Connective tissue Reduce growth factor expression
79
Connective tissue Reduced fibroblast proliferation
79
Connective tissue Increased FOXO1 activity
35
Connective tissue Increased expression of receptor for AGE
78
Connective tissue Reduced angiogenesis
78
Connective tissue Reduced production of connective tissue matrix
81
Gingiva Increased inflammation and ROS
111,136
Alveolar bone Increased bone resorption and osteoclasts
111,132,134,144-146
Alveolar bone Reduced bone formation
129,147
Alveolar bone Increased inflammation
153,154,165
Alveolar bone Increased osteonecrosis
123,124
Mandibular bone Reduced bone formation
123
Mandibular bone Increased postoperative infection risk

excessive activation of FOXO1 and high levels of cas-
pase-3 expression.80,82 High glucose levels also cause
abnormal expression of MMP-1 from human gingival
fibroblasts in vitro.83,84 Moreover, AGEs upregulate
proinflammatory cytokine expression such as IL-6 and
IL-8 in human gingival fibroblasts.83,84 Human GMSCs
treatment improves wound healing in diabetic murine
models,85,86 but it has been shown that GMSCs isolated
from diabetic patients exhibit reduced proliferative
capacity compared to those from nondiabetic
patients.87 Similarly, bone marrow-derived MSCs iso-
lated from T2DM patients exhibit reduced immuno-
modulatory capacity in suppressing T cell
proliferation.88 Therefore, diabetes affects gingival
fibroblasts by reducing their numbers through
enhanced apoptosis, increases catabolic events in
wounds through upregulation of MMP-1 and down-
regulation of TIMP-1, and increases expression of
inflammatory cytokines, which altogether lead to sub-
optimal connective tissue fill in gingival wounds.
Diabetes impairs immune cell function and promotes
inflammation to delay gingival wound healing. It is
well established that diabetic wounds present with per-
sistent inflammation,81,89 particularly with high levels
of pro-inflammatory cytokine TNF. Inhibition of TNF
can reverse diabetes-induced apoptosis of fibroblasts
and mesenchymal stem cells in skin and osseous
wounds.26,90 A major source of TNF is macrophage/
monocyte population, and high glucose has been
shown to cause epigenetic changes in the promoter
region of TNF and MCP-1 to promote persistent
inflammation through over-activation of NF-kB.91,92
Moreover, diabetes and a high glucose environment
impair phagocytic activity of macrophages that is nec-
essary for clearance of apoptotic bodies and bacte-
ria.93,94 Diabetes has also shown to prime neutrophils
to undergo NETosis and cause aberrant inflammation
in mice and humans,95,96 which also may potentiate
gingival inflammation.97 Furthermore, metabolic
changes in obesity and T2DM have been linked to
reduced production of lipid mediators that are impor-
tant for resolving inflammation in diabetic wounds.98
Overall, the impact of diabetes on multiple immune
cell types is detrimental for wound healing by interfer-
ing with their function and promoting persistent inflam-
mation. This subsequently sets the stage for sub-
optimal connective tissue wound healing.
Diabetes can have an opposite effects on angiogene-
sis depending on various anatomic locations. In dia-
betic retinopathy and nephropathy, angiogenesis is
excessive and subsequently leads to vascular edema
that is linked to organ damage.99,100 In diabetic skin
wounds and chronic foot ulcers, angiogenesis is
reduced compared to normoglycemic animals or sub-
jects, which is associated with delayed healing and
reduced antimicrobial activity.99,101 In buccal mucosal
wounds of diabetic rats, new blood vessels are more
permeable, and the numbers are significantly reduced
compared to normoglycemic wounds.15,102 It has been
shown that TNF inhibition can reverse diminshed new

78 KO et al
Fig 2. Connective tissue healing in diabetic oral wounds. Gingiva is
composed of a thick layer of lamina propria overlying bone. Diabe-
tes-mediated hyperglycemia and ROS are the major driving force for
directly inducing fibroblast apoptosis and inhibiting proliferation.
Moreover, fibroblasts express enhanced levels of pro-inflammatory
cytokines IL-6, IL-8 and MMP1 in diabetic gingival wounds. Diabe-
tes also interferes with a phagocytic function of macrophages and
their polarization from pro-inflammatory (M1) to pro-resolving (M2)
type. Diabetes-induced epigenetic changes may prevent resolution of
inflammation. The promoter region of TGFb1 in keratinocytes are
methylated by high glucose whereas that of TNF in macrophages are
acetylated, resulting in reduced anti-inflammatory TGFb1 expression
and over-expression of pro-inflammatory TNF. The overall outcome
is persistent inflammation, which reduces stromal healing outcome.
blood vessel formation in diabetic osseous wounds by
restoring VEGF expression,103 providing a link between
persistent inflammation and reduced angiogenesis.
Because reduced angiogenesis in gingival wounds can
lead to delayed healing,78 diabetes-inhibited neovascu-
larization may represent an important pathogenic mech-
anism for altered gingival wound healing. Taken as a
whole, it is apparent that diabetes dysregulates the
expression of a number of different factors to impair
the wound healing process in various cell types as
shown in Fig 2.
KERATINOCYTES AND CONNECTIVE TISSUE HEALING
Crosstalk between keratinocytes and fibroblasts is
needed for normal connective tissue healing.104 Kerati-
nocytes produce growth factors such as TGF-b, CTGF
and VEGFA to stimulate connective tissue heal-
ing.17,105-107 TGF-b can directly and indirectly stimulate
connective tissue formation; the latter is mediated by
connective tissue growth factor (CTGF), also known as
cellular communication network factor-2.105 Applica-
tion of CTGF in vivo stimulates fibroblast proliferation
and deposition of collagen.108 Studies examining human
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October 2021
and animal dermal wounds demonstrate that VEGF is
produced by keratinocytes during the healing process.
The transcription factor FOXO1 activates wound heal-
ing responses in keratinocytes including the production
of TGF-b1 to promote connective tissue healing in oral
wounds.17 FOXO1 ablation in keratinocytes diminishes
TGF-b1 expression, which reduces CTGF expression by
cells in connective tissue.105 In type 1 diabetic oral
wounds, the production of TGF-b1 is reduced. This may
be explained by substantially reduced binding of
FOXO1 to the TGF-b1 promoter caused by high levels
of glucose or AGEs, which reduce TGF-b1 transcrip-
tional activity.17 Lineage specific ablation of FOXO1
reduces VEGF-A expression in keratinocytes in mucosal
wounds and delays wound closure and with reduced
angiogenesis.77,107
MOLECULAR MECHANISMS INVOLVED IN IMPAIRED
DIABETIC BONE HEALING
Dental and periodontal problems are exacerbated by
diabetes,109 often resulting in tooth extraction, bone aug-
mentation and restoration with dental implants. These
treatment modalities require an appropriate osseous
healing reponse that involves concerted activity of
osteoclasts, osteoblasts and mesenchymal stem cells
(MSCs). It is well established that diabetes has a detri-
mental impact on bone formation by affecting these cell
types. Diabetes uncouples bone remodeling by increas-
ing osteoclast numbers and inhibiting expansion of peri-
odontal fibroblasts and osteoblasts in an experimental
periodontitis model.110,111 This is linked to a negative
effect of high glucose on the proliferation and osteo-
genic differentiation of progenitor cells.112 The molecu-
lar mechanism by which diabetes affects osteoblast
progenitors and osteoblasts has been investigated exten-
sively in orthopedic injury models. The early phase of
diabetic bone healing is characterized by impaired oste-
oid matrix production and reduced cellularity, which
may be caused by defective MSC recruitment, prolifera-
tion and impaired osteogenic differentiation.113,114 Dia-
betes further suppresses mRNA expression of the Dlx5
and Runx-2 transcription factors, which are responsible
for the expression of osteoblastic phenotype.115 It also
suggests that the low rate of bone formation observed in
diabetic animals may be related to deficiencies in differ-
entiation of MSCs to osteoprogenitor cells and in the
osteoblastic cell maturation.114 Studies in fracture heal-
ing support this hypothesis as diabetes results in
impaired bone formation.116 Moreover, diabetes leads to
aberrant activation of NF-kB and enhances inflamma-
tion due in part to the loss of immunomodulatory
MSCs.117 In turn, persistent inflammation characterized
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Volume 236
by high levels of TNF reduces MSC expansion through
upregulation of FOXO190 and suppression of IHH118 to
ultimately impair healing in diabetic fractures. In the
oral cavity, animal studies have recently identified
genetically traceable MSC populations by a-SMA, Axin2
and Lepr expression,119-121 which reside in the periodon-
tal ligament space between the tooth surface and bone
and participate in bone healing after injury. These ani-
mal models may be crucial in understanding intraoral
MSC and osteoprogenitor behavior necessary for bone
healing and how diabetes alters it in vivo.
Guided bone regeneration (GBR) is a surgical proce-
dure that restores deficient bone volume with the use
bone graft. A key element is a barrier membrane that
separates the bone graft from the soft tissue to exclude
soft tissue infiltration that can interfere with bone for-
mation.122 The effect of experimental diabetes on GBR
has been investigated by means of histology and gene
expression analyses. GBR can sufficiently regenerate
bone even in diabetic rats, but the quantity of bone
formed is less than that formed in normoglycemic ani-
mals.123 Investigation of gene expression profiling has
shown that poorly controlled diabetes downregulates
pathways related to cell division, energy production
and osteogenesis during the proliferative phase of intra-
membranous bone healing after bone augmentation
treatment.124 Although detailed mechanisms responsi-
ble for the reduced cell proliferation rates are still not
completely elucidated, it has been suggested that diabe-
tes may suppress the expression of growth factors
needed for osteogenesis during the early phase of bone
healing.125,126 In support of this, diabetes reduces
expression of basic FGF, an important mitogenic factor
for MSCs during bone healing.127 Animal studies indi-
cate that experimentally induced type 1 diabetes dimin-
ishes the osteoprogenitor population in bone marrow,
possibly due to increased apoptosis via oxidative stress,
which in turn may impair osteoblastogenesis, bone for-
mation, and bone healing.128 Experimental studies
have also demonstrated reduced mRNA and protein
expression of platelet derived growth factor, TGF b1,
IGF-I and VEGF in femoral fractures of diabetic rats
during early osseous healing, which correlates with
reduced cell proliferation.125,126 Diabetes increases
TNF in the oral cavity that prolongs detrimental
inflammation. FGF-2, TGFb-1, bone morphogenetic
protein-2, and BMP-6 are suppressed during periodon-
tal bone formation, which is rescued when inflamma-
tion is reduced with a TNF inhibitor.129
The exogenous delivery of IGF-I or rhFGF-2
enhanced the healing of calvarial and periodontal
defects in mice and rats.130-132 Preclinical studies have
demonstrated that diabetic rats exhibit reduced peri-
odontal bone regeneration, which can be partly rescued
KO et al 79
with local application of enamel matrix derivative that
contains a milleu of growth factors.133,134 A recent pro-
spective study in human subjects showed that treatment
of periodontal bony defects with enamel matrix deriva-
tive, which contains a mixture of undefined growth fac-
tors, in diabetic patients, can achieve a regenerative
outcome that is comparable to non-diabetic patients
after 3 years.135
Diabetes has a significant impact on inflammatory
responses. Pathways linked to innate and adaptive
immune responses are generally altered in poorly con-
trolled diabetics compared to healthy controls. Pro-
longed inflammation is detrimental to bone formation,
in part, because NF-kB activation in response to inflam-
matory stimuli blocks the expression of bone matrix pro-
teins.136 Diabetes also interferes with proper immune
functions that are necessary for pathogen recognition
and removal.137-139 These findings may explain
increased risk for infectious complications following
GBR treatment in patients with uncontrolled diabetes.
Comparable findings were also reported by other authors
evaluating the healing of bone defects in animals140 and
humans.141 The regulation of osteoblasts and osteoblast
precursors is complex and involves timely expression of
genes encoding pro-inflammatory cytokines (Il1a, Il1b,
Il-6, TNF), chemokines (Ccl20, Cxcl1, Cxcl2, Cxcl10),
chemokine receptors (Ccr2, Ccr5, Ccr6) and cell adhe-
sion molecules (Vcam1 and Icam1) that are needed for
migration and activation of these cells.142,143 Prolonged
inflammation disrupts the level of expression and timing
of these factors.
Taken together, the available data suggest that sup-
pressed differentiation, proliferation and/or bone form-
ing capacity of osteoblastic cells during the critical
early healing period appear to play an important role in
the pathogenic mechanism underlying poor bone for-
mation in diabetes. In particular, poorly controlled dia-
betes appears to result in a higher and dysfunctional
inflammatory response during the early phase of bone
healing, which in turn may be responsible for the
increased risk of infection in subjects with uncontrolled
diabetes.
IMPACT OF DIABETES ON TOOTH EXTRACTION
The healing of bone in extraction sockets following
tooth removal in diabetes has been evaluated in a num-
ber of animal and human studies. Using a T1DM rat
model, alveolar bone remodeling following tooth
extraction revealed that the quantity and rate of alveo-
lar bone formation was significantly lower in the dia-
betic group compared to controls.144 After a healing
period of 10 days, diabetic extraction sockets contained

80 KO et al
thin and scanty collagen fibers whereas control groups
had thick collagen fibers that formed a pretrabecular
scaffold along the trabeculae.145 Interestingly, blood
vessel formation in the extraction sockets of diabetic
animals was comparable to that of healthy controls or
insulin-treated animals. In humans with T2DM, bone
healing of the extraction socket was delayed during the
early stages of healing, and in diabetic pigs, the delayed
healing was associated with decreased osteogenic differ-
entiation of MSCs.146 Although substantial bone forma-
tion still occurred in diabetic animals, there was
incomplete mineralization in the center of the socket
compared to that of control groups which were
completely filled with newly formed bone.146 The obser-
vations made in animals were in line with those made in
humans. Compared to nondiabetic control group, T2DM
patients had delayed bone fill in the extraction sockets at
multiple time points post surgery146 and 54.7% had
defective healing manifested by inadequate bone forma-
tion. In diabetic mice, the extraction sockets consistently
have a higher inflammatory profile, with more M1 mac-
rophages and TNF-a expression and less M2 macro-
phages and PPARg expression compared to non-
diabetic controls.147
While several studies point to insufficient bone for-
mation after dental extraction in diabetic patients, not
all reports reach this conclusion.148-151 For example it
has been reported that diabetics have increased neutro-
phil recruitment following tooth extraction, but bone
healing in diabetic and normal subjects were similar.149
Taken as a whole, the overall assessment of healing
following tooth extraction in diabetic patients is slower
compared to non-diabetic patients, especially in early
healing phases.151 The available evidence suggests that
a) in poorly controlled T1DM, bone formation is inhib-
ited, resulting in delayed healing and deficient bone
formation; and b) tooth extractions can be performed
in well controlled diabetic patients, without major post-
operative complications.
DIABETES AS A COMORBIDITY FOR MRONJ
In the oral cavity, osseous wounds such as extraction
socket or exogenous bone grafts eventually heal even
under diabetic condition, albeit at a delayed rate. How-
ever, major differences exist in bone healing for nondi-
abetic and diabetic patients that suffer from
medication-related osteonecrosis of the jaw (MRONJ).
MRONJ may be localized and easily treated or may
become widespread and develop into a serious clinical
condition characterized by progressive destruction of
the jawbones. It occurs in patients on anti-resorptive
medications that have undergone oral or periodontal
Translational Research
October 2021
surgery extraction.152 There is a strong association
between diabetes and the incidence of MRONJ;153-157
even though the exact pathogenetic mechanisms
remain unclear, several experimental studies have pro-
posed converging pathways. Much like diabetes-medi-
ated delay in extraction socket healing,158
bisphosphonate treatment delays bone formation in
extraction sockets in rats.159 There are a number of
potential mechanisms that may play a role including
diabetes-reduced coupled bone formation, and the
impact of diabetes on increased osteocyte and osteo-
blast apoptosis.160,161 Another possible converging
mechanism is altered immune system. Animal and
human studies have demonstrated that bisphosphonate
treatment impairs neutrophil and macrophage chemo-
taxis and function to promote immunosuppression.162-
164
Studies in mice have also demonstrated a pro-
inflammatory response by diabetes and zolendronate
treatment in macrophages, which was responsible for
MRONJ-like healing after tooth extraction.165,166 The
etiology for either disease is multifactorial and com-
plex, and further studies may identify pathways that
can be targeted to prevent MRONJ incidents in diabetic
patients. The impact of diabetes on intraoral bone heal-
ing as discussed above is summarized in Fig 3.
CONCLUSIONS AND FUTURE DIRECTIONS
Diabetes negatively impacts several aspects of oral
and dermal wound healing. When directly compared,
the effects of diabetes on both are relatively consistent.
Diabetes alters the rate of healing by reducing keratino-
cyte migration, the production of growth factors by a
number of different cell types including keratinocytes,
interferes with a number of cellular functions such as
cellular proliferation and differentiation and the
expression of genes that are essential for forming soft
connective tissue and bone matrix. Some of the cellular
dysregulation that occurs may be due to alterations in
the gene targets of transcription factors such as
FOXO1 or over activation of others such as NF-kB.
These changes lead to difficulty in down regulating
inflammation, increasing oxidative stress and enhanc-
ing apoptosis. In the skin these changes may lead to
biofilm formation in healing wounds that delays the
repair process and in the mouth, a biofilm may form on
surgically exposed bone that prevents healing. In oral
wounds that require substantial connective tissue heal-
ing, diabetes exerts its deterimental impact on multiple
cell types such as gingival fibroblasts, endothelial cells
and leukocytes through persistent inflammation and
aberrant cell apoptosis. Impaired healing by diabetes
can be rescued by controlling inflammation or by
Translational Research
Volume 236 KO et al 81

Fig 3. Diabetic impact on various cases of intraoral bone healing. In the oral cavity, bone healing takes place
under different scenarios such as, left to right, periodontal bone regeneration, guided bone regeneration, healing
after dental extraction, or pathological healing such as MRONJ. Diabetes significantly delays bone healing in
each of these processes. In periodontal bone healing, diabetes enhances osteoclastogenesis while reducing the
number of osteoblasts and periodontal fibroblasts. This is accompanied by persistent inflammation and reduced
growth factor expression. In guided bone regeneration, which restores the hard tissue volume necessary for den-
tal implant placement, diabetes has shown to inhibit genes associated with cell division and osteogenesis. In
exodontia, diabetes delays socket healing through elevation of pro-inflammatory cytokines such as TNF, which
may indirectly suppress proper expansion of osteogenic stem cells. Diabetes is a significant co-morbidity for
MRONJ, and although exact pathogenesis is unclear, experimental studies have shown enhanced apoptosis, per-
sistent inflammation and improper recruitment of leukocytes as a converging mechanism.

genetic inhibition of FOXO1 in experimental models,
thus targetting these processes and/or transcription fac-
tors with monoclonal antibody or small molecule
inhibitor may be a viable method to treat diabetic oral
wounds in humans. Similarly, diabetic intraoral bone
healing is characterized by reduced expression of
growth factors that are necessary for bone formation,
therefore the use of recombinant growth factors is an
attractive therapeutic option for diabetic patients.
Given the difficulty in resolving inflammation in dia-
betic patients, an optimal treatment may include a ther-
apeutic approach that facilitates resolution of
inflammation and delivers a growth factor to diabetic
wounds.
A mechanistic understanding of oral wound healing
is largely based on animal studies. These studies are
advantageous by targetting specific genes or factors
that may be implicated in different stages of the healing
process. However, they may not fully translate to
humans as diabetes itself is a multifactorial disease that
is often accompanied by other metabolic conditions.
Moreover, certain oral diseases in humans such as
MRONJ may not be fully replicated in mouse models.
Despite these limitations, animal studies have contrib-
uted significantly to understanding some detrimental
effects of diabetes on oral wound healing, and clinical
efforts to supplement growth factors to improve heal-
ing process are active underway. Future studies may
test a translational value of recombinant growth factor
therapy, anti-inflammatory drugs and/or small mole-
cule inhibitors of transcription factors that are impli-
cated in impaired diabetic healing.
ACKNOWLEDGMENTS
Conflict of interest: All authors have read the jour-
nal’s authorship policy on disclosure of potential con-
flict interest.
All authors have read the journal’s authorship agree-
ment and the manuscript has been reviewed by and
approved by all listed authors.
This study was supported by NIH grants to K.I.K.
(K08-DE027129, R01-DE030415) and D.T.G.
(R01DE019108).

82 KO et al
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