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

Phases of wound healing process (WHP)

Haemostasis

Inflammation

Proliferation

Maturation

Where does a chronic wound get stuck?

Platelet ActivityWOUND

Exposed collagen

Intrinsic pathwayXII

Intrinsic pathwayintermediates IX, VII

Extrinsic pathwayVII

Coagulation pathwayintermediates V, X

Tissue factor

Messengers forAggregation &coagulation

Growth factors(PDGF)

Other enzymes(proteases)

Platelet

Fibrinogen

FIBRIN

Prothrombin & thrombin

XlilaCross-linked fibrin clot(structural support for wound healing)

Role of keratinocytes in wound healing

Migration/Profileration

Protease release

AngiogenesisECMproduction Growth factor/

Cytokine production• Epibloy• Integrins

• Matrix formation• Basement membrane formation

• VEGF• TGF-α• PDGF• PD-ECGF

• Chemoattractants− VEGF− KGF (FGF-7)

• Dissolves− Nonviable tissue− Fibrin barrier

Keratinocyte

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-β (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.

Chronic wounds – characteristics

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

Wound bed preparation

• Debridement• Bacterial balance• Dressing therapies

(f.i. silver dressings prevent of infections, help reduce healing time)

Local wound care

debridement moisture balance

SurgicalAutolyticEnzymaticBiological

FoamsCalcium alginatesHydrogelsHydrocolloidsAdhesive filmsNegative pressure therapy

Tissue engineering

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

Tissue engineering – combining cells and biomaterials into functional tissues

Cells are seeded onto a biomaterial scaffold to be integrated into a specific tissue

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

Tissue engineering implants

Synthetic polymeric biomaterials• Nonbiodegradable

Is required to provide and maintain optimal cellular function -> e.g. alginate, liposome,…

• Biodegradable To restore the histological structure and

replace the cellular function of recipients -> e.g. poly L-lactic acid, poly glycolic acid, …

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

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)

Methods of tissue bioengineering

• Active dressings (f.i.: with maggot’s 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)

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

Preparation of autologous fibrin-based skin substitutes

Methods of tissue bioengineering

• Autologous platelet rich plasma product (platelet gel)

• Allogeneic platelet gel

The effect is attributed to the growth factors

Fracture

Delayed unionPseudoarthrosis - nonunion (bone defect)

Infection

?Method of treatment?

Impaired healing

Impaired healing

• Large bone defect

Lack of osteogenic progenitor cells

• Diabetes, glucocorticoid treatment, chemotherapy, ...

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) graftBone banks, treated (no rejection), contains only osteoconductive properties

• Ilizarow intercallary bone transport (traction method)

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

Alternatives

Bone substitute (biomaterials for scaffold):

– Demineralized bone matrix– Biocompatible ceramics– Synthetic Calcium phosphate– Mineral bone– Collagen– Composite grafts – Osteoinductive collagen

Alternatives

Role of GROWTH FACTORS

Role of STEM CELLS

Collagen based matrices in tissue engineering

• Skin equivalent• Cartilage repair• Bone repair

Matrices are also prepared from synthetic polymers

Fracture healing promoting molecules

Growth and different factors– The transforming growth factor-β (TGF-β) 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)

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

Autologous Allogeneic

PLATELETS

PDGFPDGF

TGF-TGF-ββ

MONOCITE

MACROPHAGE

FIBROBLAST

ENDOTHELIUM OSTEOBLASTS

NEUTROPHILS

SMOOTH MUSCLE

PDGFTGF-β

• Mesenchymal stem cell: the promise for treating skeletal disorders

• Adult stem cell are being isolated from various tissues

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

Bone tissue formationBone tissue formation

OOsteogenic progenitor cellssteogenic progenitor cells

Locations:Locations:• PeriostPeriost• Peritrabecular soft tissuePeritrabecular soft tissue• Cancellous bone and bone marrow Cancellous bone and bone marrow

(in BM aspirate up to 40x less (in BM aspirate up to 40x less stem cells stem cells then in cancellous bone)then in cancellous bone)

Our method of tissue engineering

Combined graft

Autologous cancellous bone withstem cells

Allogeneic platelet gel(source of GFs)

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

Our graft

Autologous cancellous bone with stem cells and allogeneic platelet gel

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

Future

Gene arrays for gene

discovery

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

Thank you for attention !