2nd Year, M.tech NAST
To understand the macroscopic aspect and functions of bone
To understand the mechanism behind Bone formation and
To indicate the different components of a bone tissue
To summarize the different steps involved
Strategies for bone tissue engineering
In vitro models
Summary with Important terminology
Bone 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
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
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 femur (thigh bone, (A)) showing the
Figure: A right
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
(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
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
BONE FORMATION AND REPAIR
Two distinct mechanisms can be observed separately or combined. These are
intramembranous and endochondral bone formation.
Its is the highly
of flat bones
like the cranial
bones and the
for long bone
that bone is
representation of the
sequential steps in
(A) a fresh hematoma is
(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
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
(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
(e) cystic lesion femur condyle metaphysis and
(f) subtalar joint arthrodesis
STRATEGIES FOR BONE
TISSUE ENGINEERING of a carrier (scaffold) and
In general, tissue-engineered implants are constructs
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.
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
signaling molecules that can influence
certain cellular functions through their binding
to specific cell membrane receptors
Stimulation of bone formation and fracture
Figure Binding of growth factors to membranebound 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
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
Examples-Stainless steel, Co-Cr-Mo, Ti-6Al-4V
Uses- Total joint replacement, Plates
Examples: Hydroxyapatite, Bioactive glass
Uses: Bone filler, Scaffolds, Plates
Examples: Synthetic (Poly(lactide-co-galactide), Polycaprolactone),
Natural ( Chitosan, Gelatin)
Uses: Bone extenders, Scaffolds, Drug delivery
Osteoconductive or osteoinductive
hydroxyapatite, since this is the
inorganic compound of bone
Able to form bone apatite-like material or carbonate Brittleness and slow degradation rates
hydroxyapatite on their surface, enhancing their
Able to bind and concentrate cytokines, as in the
case of natural bone
Excellent mechanical properties, which makes them The lack of tissue adherence and the low rate
Mainly stainless steel and the most widely applied implant material used in degradation results either in a second surgery
titanium alloys (i.e. Ti-6Al-4V) bone surgical repairs
remove the implant or in permanent implantation
the body with the related risks of toxicity due
accumulation of metal ions due to corrosion
Biocompatibility and biodegradability, since they Low mechanical strength and high rates of degradation
and compose the structural materials of tissues
(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.
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
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
Function and phenotype
!. 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
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 bonelining cells.
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
Cells found in cartilage that produce and maintain the cartilaginous matrix
STEM CELLS AND
Adult mesenchimal stem cells can be isolated from bone marrow, adipose tissues, or amniotic membrane; non differentiated with selfrenewal 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