Diabetic wound m Pathophysiology.........

Diabetic wound

2. Normal anatomy

3. Abnormal pathology

4. Normal Skin anatomy
Epidermis - contains sebacseous glands, hair follicles, sweat glands

Dermis -ECM, vasculature, mechanoreceptors

5. Normal wound healing process: Acute phase
4 overlapping
phases:
Hemostasis
Inflammation
Proliferation
Remodeling

7. Hemostasis
Primary hemostasis: Aggregation of platelets with arterial vasoconstriction to limit
blood loss
Vasoconstriction- caused by endothelin released from damaged epithelium,
others include norepinephrine, prostaglandins, epinephrine, Plts release platelet
derived growth factor activates mesenchymal cells
ADP, integrin(alphaIbbeta3) initiates Platelet clumping,
Secondary hemostasis- activation of coagulation cascade fibrinogen is
converted to insoluble strands that form a fibrin mesh→thrombus formation.

9. Inflammatory phase 48-96h
Vasodilation to get WBCs and thrombocytes release cytokines
Platelet derived growth factor + transforming growth factor- attracts fibroblasts
and enhance division and multiplication of fibroblasts which produce collagen.
Factors promote: collagen degradation, transformation of fibroblasts,
angiogenesis and re-epithelialization
Plts- release histamine and serotonin- increase cellular permeability, fibrin scaffold

10. Inflammatory phase
Inflammatory cells are recruited by Damage associated molecular patterns,
hydrogen peroxide, lipid mediators, chemokines
Chemokines ELR + attract neutrophils preferentially. ELR- chemokines attract
lymphocytes
TNF alpha, pro-inflammatory factors, cleaved fibrin and bacteria stimulate
chemokine release

Neutrophils, monocytes and macrophages- phagocytosis of bacteria

11. Role of neutrophils in wound healing. A: neutrophils are
recruited to the wound in response to calcium waves,
damage-associated molecular patterns (DAMPs), hydrogen
peroxide, lipid mediators, and chemokines that are released
by resident cells immediately after wounding. B: neutrophils
combat pathogens through release of proteases from their
intracellular granules. They also produce neutrophil
extracellular traps (NETs) that capture pathogens through a
process called NETosis. In this process, neutrophils extend
chromatin filaments coated with proteases outside of the cell
to aid in the elimination of pathogens. C: neutrophils also
perform phagocytosis in the wound. They probe antigens
using surface receptors and integrins and form a phagocytic
cup that engulfs antigens. Internalized antigens are
degraded by proteases within the neutrophil granules. D:
timely clearance of neutrophils is critical for the resolution of
inflammation. They are either engulfed by macrophages
through efferocytosis or they can re-enter the circulation and
leave the wound through a process called reverse migration.

12. Inflammatory phase
Macrophages CD45+, CD11b+, CD66B- surface markers accumulate in 24-48h
Monocytes are recruited via plt, mast cell degranulation, increased hypoxia
inducible factors, chemokines(stromal derived factor 1)--> differentiate into
macrophages
Studies show that decreased monocytes lead to impaired wound healing
Early stages: M1 macrophages release TNF alpha, IL-6 and IL 1beta,
phagocytose bacteria.
Synthesize MMPs allows them to digest ECM fragments

13. Inflammatory phase
M2 macrophages anti-inflammatory cell type= express Tie2 participate in blood
vessel anastomosis by fusing endothelial vessels and connecting to systemic
vasculature.
Release VEGF-angiogenesis
Signal dermal fibroblasts to transition ot myofibroblast which increases
collagen and alpha smooth muscle actin deposition
M2aTransition into fibrotic cells- deposit collagen and ECM components

14. Granulation phase
Granulation tissue comprised of activated
fibroblasts- synthesize ECMand contract wound.
Scaffolding for other cells and components
Angiogenesis during wound healing. New blood vessel formation
is one of the most important stages of wound healing. Endothelial
cells at the leading edge or tip branch out or “sprout” to form new
capillaries in response to vascular endothelial growth factor
(VEGF) and other growth factor signals from epidermal cells,
macrophages, and the subcutaneous adipose tissue. The
endothelial cells during angiogenesis are leaky to allow for
immune cells and other circulating cells to extravasate from the
blood vessel lumen into the wound. Pro-angiogenic macrophages
release growth factors for endothelial cell growth and fuse newly
forming capillaries. Activated endothelial cells upregulate surface
markers intercellular adhesion molecule (ICAM)-1, vascular cell
adhesion molecule (VCAM)-1, E-selectin, and P-selectin that help
with cell-cell interactions with leukocytes. Deletion of these
surface markers during wound healing impairs wound repair.

15. Proliferative or granulation phase
Fibroblasts- lay down collagen and glycosaminoglycans form the core of the
wound.
Re-epithelialization- occurs with cell migration form wound periphery and adjacent
edges with a thin superficial layering of epithelial cells but eventually replaced by
thicker layer

Macrophages
M2c or Mreg-like release proteases and phagocytize excessive cells and
matrix

16. Granulation phase
Neovascularization- angiogenesis and vasculogenesis from endothelial progenitor
cells
Angiogenesis- Endothelial cells respond to hypoxia responsive growth factors
(VEGF and PDGF)

17. Maturation phase
Wound contraction, excess collagen degradation

18. Abnormal skin
Diabetics- delayed chemokine
expression→ delayed monocyte and
macrophage activation→ delayed
efferocytosis of neutrophils, ECM and
wounded ebris→ delayed onset of
proliferation phase results in chronic
remodeling stage and inflammation.

19. Wound healing overview: Hyperglycemia
Atherosclerosis→ prevents nutrients from reaching wounds
Endothelial cell dysfunction
Disruption of re-epithelialization(keratinocytes, fibroblasts)
Free radical damage
Reactive oxygen species

20. Disruption of re-epithelialization by disrupting protein
expression
Cytoskeletal keratin proteins (K2,K6,k10 keratinocyte differentiation) and almini-5
alpha3 chain precursor protein (LM-3A32)- binds epithelial cells to the basement
membrane. Reduction also leads to decreased keratinocyte survival and
differentiation.

21. Free radicals
Free radical damage results in reduced activity of antioxidant enzymes glutathione
peroxidase and superoxide dimutase
Advanced glycation end produces (AGEs) and receptors)

22. ROS
Damage blood supply, metabolism and structure of peripheral nerves.

24. Wound healing impairment: Neuropathy
Decreased sweat gland activity→ dry cracked skin
Diabetic neuropathy associated with pruritus
Motor neuropathy→ increased pressure on plantar surface of foot, leading to
ischemia and death
Reduced neuron density

25. Other contributing factors
Peripheral artery disease- ischemia, reduced nutrient supply to affected area
Hypoxia-
Anemia
Transepidermal water loss

26. Hypoxia
Leads to change in gene expression

27. TEWL
Uppermost skin layer (Stratum corneum) denucleated keratinocytes with keratin
fibers and crosslinked envelope proteins(corneocytes)= supprpunded by a
hydrophobic lipid layer to prevent water loss through the skin
Diabetic skin- less lipid content, decreased stratum corneum hydration and
increased AGEs

28. Antimicrobial peptides
Healthy skin- produces antimicrobial peptides- regulate natural skin microbiome.
In response to infection (S.Aureus) dermal fibroblasts differentiate into adipocytes
and produce cathelicidin which stimulates keratinocyte migration and
angiogenesis
Decreased in DFUs

29. Bacterial diversity
Changed in DM- higher colonization of S. aureus, S. epidermidis, Pseudomonas,
Enterobacteriaceae,
Staph and Strep- disrupt barrier by expressing proteolytic factors
SepB cleaves desmoglein 1 and 3.

30. pH and Microbiome

DM: have higher skin pH which can increase biofilm formation (Pseudomonas
species)

31. Inflammation and Immune system dysfunction