Regenerative Medicine and Bone-Skin Tissue Engineering Assist. Prof. Dr. Murat Özmen Res. Assist. Dr. Emrah Şefik Abamor

Regenerative Medicine Regenerative medicine is the branch of medicine that develops methods to regrow, repair or replace damaged or diseased cells, organs or tissues. Regenerative medicine is a broad field that includes tissue engineering but also incorporates research on self-healing – where the body uses its own systems, sometimes with help foreign biological material to recreate cells and rebuild tissues and organs. 

Main Principles of Regenerative Medicine

Regenerative medicine includes the generation and use of stem cell therapy, tissue engineering, the production of artificial organs gene therapy

Artificial Organs The use of artificial organs has made remarkable progress in the last 10 years Biological compatibility is still not fully achieved and There are problems with preserving their functionalities after transplantation.

Organ Transplantation The problems in organ transplantation The number of donors is low immunologic rejection despite recent improvements in immunosuppressive therapy. .

Tissue Engineering Approaches The most accepted definition for tissue engineering is the application of biology, chemistry and engineering principles to repair or reconstruct living tissues using biomaterials, cells and biosynthetic molecules alone or in combination. According to this definition, there are 4 approaches for tissue engineering:                            1- Use of biomaterials only                             2- Use of cells only (cell transfer)                             3- Biomaterials + biosynthetic molecules                             4- Biomaterials + cell + biosynthetic                                 The most studied approach is the 4th one.

1. Cells According to obtained sources, cells that are used in tissue engineering are divided into 3 classes: Autologous cells; The patient's own cells Allogenic cells; Cells from outside the patient Xenogenic cells; Are cells of animal origin. Immunosuppressive therapy is required when new tissues are generated from allogenic and xenogeneic cells Autologous cells are the most suitable cell type for tissue engineering.

Cells can be also classified based on differentiation potential Cells can be also classified based on differentiation potential. The undifferentiated cells are embryonic stem cells and embryonic germ cells. These have differentiation and unlimited growth characteristics to all types of cells in the body.

Adult stem cells can differentiate into many different cells under appropriate conditions. Hematopoietic stem cells are found in the bone marrow and allow the formation of erythrocytes, megakaryocytes, osteoclasts, B and T cells. Bone marrow also contains mesenchymal stem cells. These cells are capable of differentiating many connective tissue cell types such as osteocytes, chondrocyte, adipocyte, myocyte, bone marrow stromal cells.

Many adult tissues contain progenitor cells that can differentiate to form organ-specific cell types. Keratinocytes and hepatocytes are examples of progenitor cells

2. Scaffolds Structures that are designed to mimic the extracellular matrix (ECM) It not only provides suitable adhesion surfaces for cells but also provides mechanical strength, helps to establish interaction with surrounding tissue to respond to physiological and biological changes, It also contributes to the regeneration of the true extracellular matrix

Features of Scaffolds Since scaffolding is not needed when cells reach the capacity to form a new extracellular matrix, the tissue scaffold must be produced from a material (biodegradable material) that can be broken down in the body environment. Do not lose the biocompatibility when the material is degraded and do not create toxic substances It must also be of porous structure to allow passage of cells and nutrients

3-Growth Factors Thanks to these molecules which trigger the division and multiplication of cells, the number of healthy cells increases and regeneration starts. Growth factors commonly referred to in tissue engineering are: Bone morphogenetic proteins (BMPs), Basic fibroblast growth factor (bFGF or FGF-2), Vascular epithelial growth factor Transforming growth factor-β (TGF-β)

Bone Tissue Engineering It is one of the roughest tissues of the body. It occurs when connective tissue turns into bone tissue. Bone is mechanically rigid and at the same time plastic, so it can renew itself in the face of mechanical interventions The substance between the cells in the bone consists of two parts, organic and inorganic molecules

Organic substances constitute 60-70% of bone tissue. Most of these materials contain Type 1 collagen fibrils The collagen fibers are arranged parallel to each other. Hydroxyapatite crystals are found between collagen fibers Hydroxyapatite is a substance that gives texture to the tissue

Bone can heal by itself

Some bone diseases, tumors or bad fractures can cause major bone damage. Bone tissue has the ability to renew itself, but it does not fill the large gaps that occur. At this situation, non-union bone formation is occured and the bone can not heal by itself

Autografts Histocompatible Non-immunogenic possess the essential components to achieve osteoinduction (i.e., bone morphogenetic proteins (BMPs) and other growth factors), osteogenesis (i.e., osteoprogenitor cells) and osteoconduction (i.e., three-dimensional and porous matrix)

Disadvantages of Autografts Autologous bone transplants are very expensive procedures, They may result in significant donor site injury and morbidity, deformity, scarring They are associated with surgical risks as well: bleeding, inflammation, infection, and chronic pain.

Disadvantages of Allografts In comparison to autografts, allografts are associated with risks of immunoreactions and transmission of infections. They have reduced osteoinductive properties and no cellular component, because donor grafts are devitalized via irradiation or freeze-drying processing.

Bone tissue engineering

Bone tissue engineering The classic BTE paradigm highlights several key players: (1) a biocompatible scaffold that closely mimics the natural bone extracellular matrix niche, (2) osteogenic cells to lay down the bone tissue matrix, (3) morphogenic signals that help to direct the cells to the phenotypically desirable type, and (4) sufficient vascularization to meet the growing tissue nutrient supply and clearance needs. 

There is roughness on the natural bone surface of about 100 nm in size. It is very important to include such nano details on the surface of bone implants commonly used in bone tissue damage

If the implant surface is smooth, the body will try to reject the implants. The smooth surface will trigger the production of threadlike tissue covering the implant surface, which reduces bone implant interaction The strength of the implant diminishes and the infection occurs. The nano-sized pieces reduce the risk of rejecting the body's implants and also promote osteoblast production.

The use of bioceramics such as hydroxyapatite (HA) and tricalcium phosphate in bone tissue engineering is the mostly applied technique. These structures are similar in chemical and structural terms to the minerals in the bone tissue. For this reason, bioceramics stimulate osteoblasts proliferation and osteogenic differentiation

Studies are underway to combine hydroxyapatite nanoparticles with polymers. The biodegradability of the polymer is combined with the osteoconductivity of hydroxyapatite. It was determined that the hydroxyapatite nanoparticles incorporated into the polymer scaffold changed the pore structure of the material and made protein absorption more suitable

Composites of hydroxyapatite and various polymers, including poly(lactic acid) (PLA), PLGA, gelatin, chitosan,  and collagen have been successfully fabricated and have demonstrated enhanced bone formation in vitro and/or in vivo.

Nanoparticles In recent years studies have used nanoparticles to increase the mechanical strength of these materials The use of nanoparticles allows for better imitation of the environment inside the body, as the organic and inorganic minerals present in the bone tissue are also nano-structured These structures, which provide a suitable surface geometry and mechanical strength, support the interaction between the material and the surrounding tissue

Growth Factors for Bone Tissue Engineering Growth factors that play a role in the restructuring of bone tissue These growth factors affect osteogenic differentiation by acting alone and together BMP (bone morphogenetic protein) TGF (transforming growth factor) PDFG (thrombocyte-derived growth factor) VEGF (vascular endothelial growth factor)

Skin Tissue Engineering The skin is the largest tissue of our body It forms one-tenth of your body weight. Therefore, the damage caused by traumas, illnesses and burns can cause serious consequences.

The skin consists of 3 layers; Dermis, epidermis, hypodermis Cell types in the epidermal layer: keratinocyte, melanocyte and sensory cells

Keratinocytes form the largest group of epithelial cells Keratinocytes produce keratin protein that protects the epidermis from chemical corrosion and mechanical injury, Melanocytes give color to the skin Sensory cells are differentiated to nerve cells.

Dermis is a connective tissue layer. Provides structural support and mechanical resistance to the skin Consisting of fibroblast, adipocyte and macrophages Fibroblast: Responsible for the formation of extracellular matrix components such as collagen, elastin and proteoglycan.

Collagen fibrils are formed from type I collagen These matrix components are responsible for Organization Integrity Stability Flexibility of dermis

Skin Wounds

First-degree injuries are limited to the epidermis and the injured area is characterized by pain, redness and local temperature increase. Second degree injuries surround the epidermis and dermis. Severe local pain is characterized by the formation of bubbles containing bloating, blood or other fluid Sweat glands and hair follicles may also be injured in second degree injuries

Third degree injuries surround the epidermis, dermis and subcutaneous tissue. It is characterized by the destruction of the skin layers of the skin. Third degree injuries may also involve subcutaneous tissue as well as other internal tissues such as skeletal muscle, tendon, ligament and bone.

In first and second degree injuries, the wound may heal itself, but if the wound has reached the deep subcutaneous layer (third degree injury), self-healing does not occur. For third degree injuries it is necessary to close the wound by changing the skin Regenerative approaches to the skin are based on the natural healing process of the wound.

Skin Tissue Engineering A number of cell types can be used to form skin tissue. These are; Embryonic stem cells Embryonic fetal cells Multipotent fetal stem cells Adult bone marrow stem cells Adult epidermal progenitor stem cells Adult dermal and epidermal cells

Embryonic and fetal stem cells are potential candidates for skin regeneration but may result in immunological rejection as they are derived from allogenic sources For this reason, the most ideal approach for skin renewal is; identify and use the autogenic epidermal stem cells. Hair follicles and sebaceous glands contain multipotent epidermal stem cells These stem cells are activated to produce epidermal cells, follicle cells and sebaceous gland cells in response to skin injury

The collagen matrix is ​​used as a scaffold for culturing epidermal cells and for tissue replacement to replace the skin. For this purpose, an artificially created collagen gel or a natural collagen matrix, which is allogeneically collected from the patient, is used. Other matrix components such as fibronectin, proteoglycan, and fibrin may be added to the collagen matrix to form the matrix tissue structure.

Growth Factors Stimulating the Proliferation of Epidermal Cells Keratinocyte Growth Factor (KGF) Epidermal Growth Factor (EGF) The Fibroblast Growth Factor (FGF) Platelet derived Growth Factor (PDGF) The Hepatocyte Growth Factor (HGF) Vascular Endothelial Growth Factor (VEGF) Insulin-like Growth Factor (IGF) Growth Factor (M-CSF) promoting macrophage colony formation Growth Factor (GM-CSF) promoting granulocyte- macrophage colony formation