TISSUE ENGINEERING OF BONE

Presented ByS.Shashank Chetty2nd Year, M.tech NASTPondicherry University

OBJECTIVES

To understand the macroscopic aspect and functions of bone

To understand the mechanism behind Bone formation and repair

To indicate the different components of a bone tissue engineering

To summarize the different steps involved

Strategies for bone tissue engineering

In vitro models

Summary with Important terminology

INTRODUCTION: BONEBone is the main supporting system in the human body. It is a unique combination of minerals and tissue that provides excellent tensile and loading strength.

Bone serves a main biomechanical function with excellent tensile and loading strength, and is involved in calcium metabolism and hematopoiesis.

A scaffold for bone tissue engineering should ideally match the mechanical properties of the bone it will replace and participate in the natural remodeling process, which means that it should be resorbable.

Allograft: Tissue transplanted between genetically different individuals of the same species.

Autograft: Tissue transplanted within one individual.

Xenograft: Tissue transplanted between different species.

Calcification in tissues: Calcification is the deposition of insoluble calcium salts, which can be in extracellular bone matrix (a normal, physiological condition).

Ossification: Production of organic bone matrix and its subsequent mineralization or calcification.

Osteoconduction: Ability of a material/graft to allow ingrowth of vessels and osteoprogenitor cells from the recipient bed or spreading of bone over the surface proceeded by ordered migration of differentiating osteogenic cells.

Osteogenesis: Bone formation by determined osteoprogenitor cells.

Osteoinduction: The mechanism of cellular differentiation towards bone of one tissue due to the physicochemical effect or contact with another tissue

MACROSCOPIC ASPECT OF BONE Figure: A right femur (thigh bone, (A)) showing the mechanically strong cortical bone on the outside and the inner spongious bone in this metaphysic end of the bone;

(B) is a detail of the cortical bone and the inner spongious bone showing the trabeculae of which it is composed;

(C) is a detail of spongeous bone where a single trabecula is pictured with bone marrow cavities, lined with endosteum, on each side. The cavities make this type of bone rather porous as compared to the more dense cortical bone;

(D) shows a woven type of bone that is typically young and unorganised;

(E) is an example of lamellar bone that has been remodelled into a more mechanically strong type of bone.

On top of (D) an example of an osteoblast is portrayed that can typically be found in areas of active bone formation.

Similar to this, an osteoclast is found in the same areas for further remodelling of the bone (insert below (D)).

BONE FORMATION AND REPAIR Two distinct mechanisms can be observed separately or combined. These are intramembranous and endochondral bone formation.

Intramembranous bone formation Its is the highly efficient mechanism for the formation of flat bones like the cranial bones and the scapula.

Endochondral bone formation is the mechanism for long bone formation and lengthening. Characteristic of this mechanism is that bone is preceded by the formation of cartilage.

FRACTURE HEALING Figure: Schematic representation of the sequential steps in fracture healing:

(A) a fresh hematoma is formed,

(B) inflammatory and mesenchymal cells arrive after small vessels have invaded the area,

(C) callus is formed by both endosteal and periosteal reaction and

(D) the callus is remodeled.

BONE GRAFTING When the bone repair mechanism fails as a result of magnitude, infection or other causes, bone grafting has been shown to be a highly successful therapy.

Bone grafting means that bone from somewhere else is applied to stimulate bone formation. Bone of other humans (allogenic) or animals (xenogenic) is also applied

Figure Potential clinical orthopedic applications for the use of bone grafts:

(a) anterior spine fusion,

(b) posterolateral spine fusion,

(c) osteolytic defect (acetabular defect from a prosthetic implant),

(d) traumatic bone defect/pseudoarthrosis or tumor defect in femur diaphysis,

(e) cystic lesion femur condyle metaphysis and

(f) subtalar joint arthrodesis

STRATEGIES FOR BONE TISSUE ENGINEERING In general, tissue-engineered implants are constructs of a carrier (scaffold) and biologically active factors. These biological factors can be (a combination of) cells and proteins that stimulate host cells.

Constructs are designed to act for one or more of the following qualities as mentioned previously: osteoconduction, osteoinduction and osteogenesis.

Therefore, the ingredients for bone tissue engineering can basically be divided into scaffolds, growth factors and cells.

Scaffolds:

delivery vehicle for osteoinductive molecules and/or osteogenic cells.

fill the gap in a bone defect and facilitate healing

Exhibit biocompatibility, osteoconductivity, porosity, biodegradability, mechanical properties and intrinsic osteoinductivity

Growth factors:

signaling molecules that can influence

certain cellular functions through their binding

to specific cell membrane receptors

Stimulation of bone formation and fracture

Healing.

Figure Binding of growth factors to membrane-

bound receptors

(1) results in receptor oligomerization and

activation. Phosphorylation of the intracellular parts of the receptors

(2) activates intracellular signaling pathways

(3) and subsequently results in the transcription of target genes

(4). These transcription products can influence cellular behavior (such as cell proliferation and cell differentiation).

E.g. transforming growth factor (TGF)- , vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), fibroblastic growth factor (FGF) and platelet-derived growth factor (PDGF).

MATERIALS IN BONE TISSUE ENGINEERING Metals:

Examples-Stainless steel, Co-Cr-Mo, Ti-6Al-4V

Uses- Total joint replacement, Plates

Ceramics:

Examples: Hydroxyapatite, Bioactive glass

Uses: Bone filler, Scaffolds, Plates

Polymers:

Examples: Synthetic (Poly(lactide-co-galactide), Polycaprolactone),

Natural ( Chitosan, Gelatin)

Uses: Bone extenders, Scaffolds, Drug delivery

Characteristics

Biodegradable Interconnected porosity

Biocompatible Handleablity

Osteoconductive or osteoinductive Cheap

BIOMATERIAL SHORT CHARACTERISTIC

ADVANTAGES DISADVAMTAGES

CERAMICSBased mainly on hydroxyapatite, since this is the inorganic compound of bone

Able to form bone apatite-like material or carbonate hydroxyapatite on their surface, enhancing their osseointegration; Able to bind and concentrate cytokines, as in the case of natural bone

Brittleness and slow degradation rates

METALSMainly stainless steel and titanium alloys (i.e. Ti-6Al-4V)

Excellent mechanical properties, which makes them the most widely applied implant material used in bone surgical repairs

The lack of tissue adherence and the low rate of degradation results either in a second surgery to remove the implant or in permanent implantation in the body with the related risks of toxicity due to accumulation of metal ions due to corrosion

NATURAL POLYMETSCollagen and glycosaminoglycansSilk-based biomaterials

Biocompatibility and biodegradability, since they compose the structural materials of tissues

Low mechanical strength and high rates of degradation (they are used in composites or in chemical modification by cross-linking. These changes make cause cytotoxic effects and reduce compatibility).

Biocompatibility, excellent mechanical properties, long-standing use of silk as sature material.

SYNTHETIC POLYMERS The versatility of chemically synthesized polymers enables the fabrication of scaffolds with different features (forms, porosities and pore size, rates of degradation, mechanical properties) to match tissue specific applications

COMPOSITES Each individual material has advantages for osteogenic applications, each also has drawbacks associated in certain properties (i.e. brittleness of ceramics) that can be overcome by combining different materials.

IN VITRO MODELS In vitro models are the cornerstone of all developments in tissue engineering. Every idea should first be tested thoroughly in vitro before even thinking of in vivo tests. With respect to growth factors, this involves the purification, screening of release systems and effect on BMSCs or characterized cell lines like 3T3 fibroblasts.

Cell counting- a cytometer chamber

Colony-forming unit efficiency assay- To determine the quality of bone marrow aspirates via number of colonies per initially seeded number of nucleated cells.

Differentiation assays- specific markers

Cell type Origin Differentiation Function and phenotype

OSTEOBLASTS(Four categories)

pluripotential mesenchymalstem cells

Highly differentiated

!. Active osteoblasts - mononuclear cells with cuboidal shape; rich in alkaline phosphatase; synthesize and secrete collagen type I and glycoproteins (osteopontin, steocalcin), cytokines, and growth factors into a region of unmineralized matrix (osteoid) between the cell body and the mineralized matrix; produce calcium phosphate minerals extra- and intracellularly within vesicles; 2. Osteocytes – mature osteoblasts which have become trapped within the bone matrix and are responsible for its maintenance; 3. Bone-lining cells - found along the bone surfaces that are undergoing neither bone formation nor resorption, inactive cells that are believed to be precursors osteoblasts;4. Inactive osteoblasts - elongated cells, undistinguishable morphologically from the bone-lining cells.

OSTEOCLASTS hematopoietic stem cells (monocytes)

Highly differentiated

Polynuclear cells responsible for bone resorption (by acidification of bone mineral leading to its dissolution and by enzymatic degradation of demineralized extracellular bone matrix; important for growth and development

CHONDROCYTES mesenchymal stem cells

Highly differentiated

Cells found in cartilage that produce and maintain the cartilaginous matrix

MESENCHYMAL STEM CELLS AND OSTEOPROGENITOR CELLS

Adult mesenchimal stem cells can be isolated from bone marrow, adipose tissues, or amniotic membrane; non differentiated with self-renewal capacity; multipotent cells able to repopulate all the appropriate differentiation lineages (osteoblastic, myoblastic, adipocytic, chondrocytic, endothelial, and neurogenic). For the osteogenic lineage, mesenchimal stem cells sustain a cascade of differentiation steps as described by the following sequence: Mesenchimal stem cell → immature osteoprogenitor → mature osteoprogenitor → preosteoblast → mature osteoblast → osteocyte or lining cell or apoptosis. In bone marrow osteoprogenitor cells represent a very small percentage (e.g. < 0.005%) of nucleated cell types in healthy adult bone. Osteoprogenitor stem cell differentiation is controlled by the “osteogenic mastergene” Cbfa1/Osf2 that intervenes in skeleton and tooth mineralization.

THANK you for your KIND attention