Physiology of wound healing

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Physiology of wound healing

• Wound healing is
• Complicated process that involves at least 4
  distinct cell types
• Commonly referred to as occurring in PHASES
• Affected by several factors

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Phases of wound healing process (WHP)
Maturation
Proliferation
Inflammation
Haemostasis
Where does a chronic wound get stuck?
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Platelet Activity
WOUND
Tissue factor
Exposed collagen
Extrinsic pathwayVII
Intrinsic pathwayXII
Messengers for Aggregation coagulation
Intrinsic pathway intermediates IX, VII
Platelet
Growth factors(PDGF)
Coagulation pathway intermediates V, X
Other enzymes (proteases)
Fibrinogen
Prothrombin thrombin
Xlila
Cross-linked fibrin clot (structural support for
wound healing)
FIBRIN
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Role of keratinocytes in wound healing
Keratinocyte
Migration/ Profileration
Protease release
ECM production
Angiogenesis

• Dissolves
• Nonviable tissue
• Fibrin barrier

Growth factor/ Cytokine production

• Epibloy
• Integrins

• Chemoattractants
• VEGF
• KGF (FGF-7)

• Matrix formation
• Basement membrane formation

• VEGF
• TGF-a
• PDGF
• PD-ECGF

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Selected growth factors important to wound healing

• EGF (epidermal growth factor). Stimulates wound
  re-epithelialization and stimulates blood vessels
  and fibroblasts.
• FGF (fibroblast growth factor). Stimulates new
  blood vessel and collagen formation.
• PDGF (platelet derived growth factor).
  Attracts/stimulates smooth muscle cells,
  fibroblasts, and other cells. Important in ECM
  formation.
• TGF-ß (transforming growth factor-beta). Slows
  buildup of epithelial cells, suppresses
  immunoglobulin secretion and is helpful in ECM
  formation.
• TNF-a (tumor necrosis factor-alpha). Activates
  neutrophils, causes fibroblasts to multiply,
  causes bone/cartilage resorption.
• IL-1 (interleukin-1). Attractant for epithelial
  cells, neutrophils, mono and lymphocytes also
  stimulates collagen synthesis.

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Chronic wounds characteristics

• Increased inflammatory cytokines
• Altered fibroblast phenotype
• Abnormalities growth factors
• Increased proteases
• Altered keratinocyte function
• Senescent cells (increased number)

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Wound bed preparation

• Debridement
• Bacterial balance
• Dressing therapies
• (f.i. silver dressings prevent of infections,
  help reduce healing time)

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Local wound care
debridement
moisture balance
Foams Calcium alginates Hydrogels Hydrocolloids Ad
hesive films Negative pressure therapy
Surgical Autolytic Enzymatic Biological
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Tissue engineering

• Biology, medicine and technology are today
  closely interleaved with each other

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• Tissue engineering combining cells and
  biomaterials into functional tissues
• Cells are seeded onto a biomaterial scaffold
  to be integrated into a specific tissue

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Tissue engineering

• Advances
• Biological wound dressings
• Material scaffolds and cell material interactions
• The use of stem cells for tissue engineering
• Combination of stem cells and material scaffolds
  into tissue engineered replacements of tissues
  and organs

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Tissue engineering implants

• Synthetic polymeric biomaterials
• Nonbiodegradable
• Is required to provide and maintain optimal
  cellular function -gt e.g. alginate, liposome,
• Biodegradable
• To restore the histological structure and
  replace the cellular function of recipients -gt
  e.g. poly L-lactic acid, poly glycolic acid,

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Wound healing promoting anti-adhesive matrix

• The collagen grafting is also applied to
  produce a healing / promoting antiadhesive
  membrane
• Particularly necessary in peritoneal surgery to
  prevent postoperative adhesion
• To produce skin wound dressing membranes

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Methods of tissue bioengineering

• Skin replacement
• Cultured epidermal graft
• Cultured human autologous and allogeneic
  keratinocytes
• Semi synthetic materials (composed of human
  neonatal dermal fibroblasts cultured onto a
  bioabsorbable mesh)

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Methods of tissue bioengineering

• Active dressings (f.i. with maggots excret,
  with honey)
• Photobiomodulation (modulate cellular activity in
  red to near infrared light)
• Hyperbaric oxygen therapy as therapeutic benefit
  in WT
• Growth factors (from blood)

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Allogeneic cultivated human skin keratinocytes

• Make rapid healing of the ulcers particularly
  those that are difficult to heal
• No clinical or laboratory evidence of rejection
• No evidence of preexisting cytotoxic antibodies
  specific fort the HLA class I antigens expressed
  on HSE cells
• A fibrin-based skin substitute produced in the
  defined keratinocyte medium could be safely used
  to threat a number of skin defect

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Preparation of autologous fibrin-based skin
substitutes
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Methods of tissue bioengineering

• Autologous platelet rich plasma product (platelet
  gel)
• Allogeneic platelet gel
• The effect is attributed to the growth factors

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Impaired healing
Delayed unionPseudoarthrosis - nonunion (bone
defect)
Fracture
Impaired healing
? Method of treatment?
Infection
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• Large bone defect
• Lack of osteogenic progenitor cells
• Diabetes, glucocorticoid treatment, chemotherapy,
  ...

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Accepted methods of treatment

• Autologous bone transplants
• Cancellous bone graft (contains all necessary
  characteristics of bone substitutes)
• Corticocancellous graft (possibly vascularized?
  limited amount)
• Homologous (allogeneic) graft
• Bone banks, treated (no rejection), contains
  only osteoconductive properties
• Ilizarow intercallary bone transport (traction
  method)

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Properties of bone grafts

• Osteogenesis (bone marrow, cancellous bone)
• Osteoinduction
• Demineralized bone matrix
• Growth factors (platelet rich plasma, bone
  morphogenic proteins BMPs)
• Osteoconduction
• Ceramics
• Collagen

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Alternatives

• Bone substitute (biomaterials for scaffold)
• Demineralized bone matrix
• Biocompatible ceramics
• Synthetic Calcium phosphate
• Mineral bone
• Collagen
• Composite grafts
• Osteoinductive collagen

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Alternatives

• Role of GROWTH FACTORS
• Role of STEM CELLS

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Collagen based matrices in tissue engineering

• Skin equivalent
• Cartilage repair
• Bone repair
• Matrices are also prepared from synthetic
  polymers

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Fracture healing promoting molecules

• Growth and different factors
• The transforming growth factor-ß (TGF-ß)
  superfamily
• Bone morphogenetic proteins(osteoprogenitors,
  mesenchymal cells, osteoblasts and chondrocytes
  within the extracellular matrix produce
  BMPs.)BMP-2, BMP-4BMP-5, BMP-6, BMP-7GDF-5
  (BMP-14), GDF-6 (BMP-13), GDF-7 (BMP-12)BMP-3
  (Osteogenin), GDF-10 (BMP-3b)
• Platelet-derived growth factor (PDGF)
• Fibroblast growth factor (FGFs)
• Insulin-like growth factor (IGFs)

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• Platelet rich plasma (contains high
  concentrations of growth factors) especially
  TGF-B and PDGF
• Autologous
• Allogeneic

PLATELETS
MONOCITE
NEUTROPHILS
PDGF TGF-ß
FIBROBLAST
SMOOTH MUSCLE
MACROPHAGE
ENDOTHELIUM
OSTEOBLASTS
PDGF TGF-ß
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• Mesenchymal stem cell the promise for treating
  skeletal disorders
• Adult stem cell are being isolated from various
  tissues

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Adult stem cells

• Bone marrow contains
• Hematopoietic stem cells (HSCs)
• All types of blood cells
• Bone marrow mesenchymal stem cells (MSCs)
• Generating bone, cartilage, fat, fibrous
  connective tissue

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Bone tissue formation

• Osteogenic progenitor cells
• Locations
• Periost
• Peritrabecular soft tissue
• Cancellous bone and bone marrow (in BM aspirate
  up to 40x less stem cells then in cancellous bone)

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Our method of tissue engineering

• Combined graft
• Autologous cancellous bone withstem cells
• Allogeneic platelet gel(source of GFs)

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• manually grounded autologous cancellous bone
  with stem cells
• corresponding amount of allogeneic platelet
  concentrate (app. 1,4x109 platelets per 1 ml)
• AND
• Added 0,06 ml human thrombin in 40 mM CaCl2
  for the activation of platelets
• in 1
  minute
• the resulting gelled graft can be shaped
  according to the bone defect and implanted

Mixed
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Our graft

• Autologous cancellous bone with stem cells and
  allogeneic platelet gel

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Conclusions

• The use of autologous cancellous bone with
  stem cells and allogeneic platelet gel is safety
  and effective method for the treatment of
  nonunion of long bones

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Future
Gene arrays for
gene discovery
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FUTURE
Cell and tissue engineering Detection of
numerous signal pathways activated during
physiological processes Self (re)restoration and
differentiation off mammalian embryonic, fetal
and stem cells of adult tissues
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• Thank you for attention !