Tissue engineering andtissue repair

Dragica Maja SMRKE, MD,Ph.D., Professor

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

Examples

• Artificial pancreas

• Artificial bladders

• Cartilage

• Artificial skin

• Artificial bone marrow

• Artificial bone

• Tissue engineered vessels, …

Types of cells• Autologous

– Harvested from the patient

• Allogeneic cells– Come from the body of a donor of the same species

• Xenogenic cells– Isolated from individuals of another species

• Isogenic cells– Isolated from genetically identical organisms such as twins, clones

• Stem cells– Undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells

Phases of wound healing process (WHP)

Haemostasis

Inflammation

Proliferation

Maturation

Where does a chronic wound get stuck?

Components of wound healing

CoagulationProcess

InflammatoryProcess

Migration/ProliferativeProcess

RemodelingProcess

Platelets

Cell Types Involved

PlateletsMacrophages

MacrophagesLymphocytesFibroblastsEpithelial cellsEndothelial cells

Fibroblasts

Injury Hours Days Weeks

Platelet ActivityWOUND

Exposed collagen

Intrinsic pathwayXII

Intrinsic pathwayintermediatesIX, VII

Extrinsic pathwayVII

Coagulation pathwayintermediatesV, 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• Basementmembraneformation

• VEGF• TGF-α• PDGF• PD-ECGF

• Chemoattractants− VEGF− KGF (FGF-7)

• Dissolves− Nonviabletissue

− Fibrinbarrier

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.

Local factors

• Growth factors

• Edema and ischemia

• Low oxygen tension

• Infection

Regional factors

• Arterial insufficiency

• Venous insufficiency

• neuropathy

Systemic factors

• Inadequate perfusion

• Metabolic disease (diabetes)

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

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, …

Biomaterials in surgery

• Collagen based biomaterials and tissue engineering

Extracellular matrix of connective tissue is composed of proteins:– Collagens (30% of all)

– Proteoglycans

– Glycoproteins

– Elastin

• Collagen provides the principal source of mechanical strength in tissues such as bone, cartilage, skin, tendon, ligaments

• Collagen contributes a structural framework to other tissues (blood vessels, most organs)

Local delivery systems contain gentamicin

• Bone cement (PMMA beads) -> PolyMethyl-Meth-Acrylate

• Collagen (fleeces)

as drug carrier

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

Methods of tissue bioengineering

•Photobiomodulation (modulate cellular activity in red to near infrared light)

•Hyperbaric oxygen therapy: as therapeutic benefit in WT

•Growth factors (from blood)

Methods of tissue bioengineering

Skin replacement

– Cultured epidermal graft

– Cultured human autologous and allogeneic keratinocytes

– Semi synthetic materials

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

FractureDelayed union

Pseudoarthrosis - nonunion

(bone defect)

Infection ?Method of treatment?

Impaired healing

Impaired healing

• Large bone defect

Lack of osteogenic progenitor cells

• Diabetes, glucocorticoid treatment, chemotherapy, ...

Reconstruction of bone defect

• Stability

• No infections

• Soft tissue defect reconstruction

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)

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

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

Growth factors (GF)

• Bone and cartilage GF are selected proteins

• Generally are produced locally in the skeleton microenvironment

• Regulate growth and repair

Fracture healing promoting molecules

Growth 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)

Current knowledge of clinical applications of bone

morphogenetic proteins• Marshal R. Wrist 40y ago discovered a substance in bone matrix that has inductive properties for the development of bone and cartilage

• Now are 20 individual human bone morphogenetic proteins (BMPs) with varying degrees of inductive activities

• BMP-2 and BMP-7 have the main role for the restoration and treatment of skeletal conditions

• Efficiency equivalent to autologous bone graft (clinical studies)

• Enhance bone healing

Reconstruction of bone defect

• BMPs

• Autologous cancellous bone with stem cells and allogeneic platelet gel graft

Methods of tissue bioengineering

• Autologous platelet rich plasma product (platelet gel)

• Allogeneic platelet gel

The effect is attributed to the growth factors

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

Autologous

AllogeneicPLATELETS

PDGFPDGF

TGFTGF--ββ

MONOCITE

MACROPHAGE

FIBROBLAST

ENDOTHELIUMOSTEOBLASTS

NEUTROPHILS

SMOOTH MUSCLE

PDGF

TGF-β

Autologous Platelet Separator (APS)

• Variable input of whole blood

• Two liquids

thrombin rich autologous serumAND

platelet rich plasma

Within a few seconds a gel will form

Advantages of APS

• Use intraoperatively

• Safe and rapid preparation of platelet poor plasma and platelet concentrate of whole blood

• Totally automated system for efficient time utilization

• Requires a small amount of blood

Stem cells

• May be useful for repair of diseased or damaged tissues

• May be used to grow new organs

•Embryonic stem cell (ESC) are derived from the inner cell mass of blastula - stage embryos

•The adult stem cell like populations are being isolated from various tissues

Stem cells

Some cells are totipotent in the earliest stages of embryo

Mesenchymal stem cell: the promise for treating skeletal disorders

Cancellous bone and BMwith stem cells

Tissue culture

The basic requirements of cells must be maintained in culture include:

– Oxygen

– pH

– Humidity

– Temperature

– Nutrients

– Osmotic pressure

In many cases also:– Growth factors, hormones, specific metabolites, chemical and physical stimuly

Scaffolds

• Support materials to growth cells into tissues

• Vary porosity, composition, biodegradability

• Tissue engineering of bone requires strategies to design 3D scaffolds that closely mimic the anatomical organization of bone and its tissue matrix

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 CaCl2for the activation of platelets

in 1 minute

the resulting gelled graft can be shaped according to the bone defect and implanted

Mixed

Tissue engineering

• Advances– In 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