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 Activity WOUND Exposed collagen Intrinsic pathway XII Intrinsic pathway intermediates IX, VII Extrinsic pathway VII Coagulation pathway intermediates V, X Tissue factor Messengers for Aggregation & coagulation Growth factors (PDGF) Other enzymes (proteases) Platelet Fibrinogen FIBRIN Prothrombin & thrombin Xlila Cross-linked fibrin clot (structural support for wound healing)

Role of keratinocytes in wound healing Migration/ Profileration Protease release Angiogenesis ECM production 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 debridementmoisture balance Surgical Autolytic Enzymatic Biological Foams Calcium alginates Hydrogels Hydrocolloids Adhesive films Negative 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 union Pseudoarthrosis - nonunion (bone defect) Infection ? Method of treatment? 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) graft Bone 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-4 BMP-5, BMP-6, BMP-7 GDF-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 PDGF TGF- β MONOCITE MACROPHAGE FIBROBLAST ENDOTHELIUM OSTEOBLASTS NEUTROPHILS SMOOTH MUSCLE PDGF TGF-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 formation Osteogenic progenitor cells Locations: Periost Periost Peritrabecular soft tissue Peritrabecular soft tissue Cancellous bone and bone marrow (in BM aspirate up to 40x less stem cells then in cancellous bone) Cancellous bone and bone marrow (in BM aspirate up to 40x less stem cells then in cancellous bone)

Our method of tissue engineering Combined graft Autologous cancellous bone with stem cells Allogeneic platelet gel (source of GFs)

manually grounded autologous cancellous bone with stem cells corresponding amount of allogeneic platelet concentrate (app. 1,4x10 9 platelets per 1 ml) AND Added 0,06 ml human thrombin in 40 mM CaCl 2 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 !