College 1 – “An introduction to Tissue Engineering” – 22nd of November 2012



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College 4 – “Bone” – 20th of December 2012



Chemical components of bone

  • The organic matrix is composed primarily of the protein collagen (type I) which provides ductility - 10% of adult bone mass.
     Collagen type I is causing the ductility of the bone.
     Collagen fibres are situated in between the ends of hydroxyapatite crystal plates and between the plates.


  • Mineral component is composed of hydroxyapatite, which is an insoluble salt of Ca and P - about 65% of adult bone mass
     This is why the bone is quite brittle.

  • Bone also contains small amounts of magnesium, sodium, and bicarbonate

  • Water comprises approximately 25% of adult bone mass

  • The ratio strain to failure is in bone relatively low (0.5-3%), this means it is a hard tissue.


Cells in the bone


  1. Osteoblasts are cuboidal and columnar in shape with a central nucleus found on the bone surface (occur in columns).

    1. Responsible from producing bone ECM

  2. Osteocytes live inside the bone and have long branches, which allow contact with each other as well as the lining cells on the bone surface.

    1. they sense any mechanical strain on the bone

    2. this directs bone remodeling to accomodate mechanical strain and repair fatigue damage
       mechanosensors

    3. they can secrete growth factors which activate the lining cells

  3. Osteoclasts are large cells with many nuclei, which share lineage with blood cells (especially macrophages).

    1. formed from fusion of the precursors, which circulate in the blood and bone marrow. RANK receptors on the osteoclast precursors are activated by the RANK-ligand which is secreted by osteoblasts (for communication). Osteoprotegerin (OPG) is a factor which also binds RANK-ligand, thus regulating osteoclast activation.

    2. they form sealed compartments next to the bone surface and secrete acids and enzymes which degrade the bone.

    3. after resorbing bone, they undergo apoptosis, a process regulated by proteins from other cells.

  • Osteoporosis (too much breakdown of bone)  common for women after the menopause. (There are also other illnesses that cause the breakdown of bone).



Other Bone Matrix Proteins
Need to be there for the remodelling of bone

  • Fibronectin - Relatively abundant, may help regulate osteoblast differentiation

  • Osteonectin - "Bone connector" may regulate mineralization

  • Thrombospondin - May inhibit bone cell precursors

  • Osteocalcin - Binds calcium onto phosphategroups

  • Matrix-gla-protein - Inhibits mineralization

  • Biglycan (a small proteoglycan)

Hormones (small proteins or organic molecules)

Parathyroid hormones, Calcitonin, Vitamin D, Gonaoidal Steroids, Growth Hormones, Glucocoticoids, Thyroid hormones


Microstructure of bone




  1. Osteon = centric lamella

  2. Cement line (cementum)
     often bone fractures are on this line

  3. Circumferential lamella (plexiform)

  4. Interstitial lamella

  • Bone is highly vascular (from the outside and from the bone marrow).


Macrostructure of bone



Growth in the growthplate
 growth in length

 cartilage calcified cartilage  bone



Bone types

  • Woven bone: generally immature

  • Cortical bone: compact bone

  • Cancellous bone (trabecular bone)
     The direction of the fibers are in the predominant loading direction

 So the weight of the bone is not too much


During growing the bone grows on the outside but is broken down at the inside.

Function of Skeletal Bone

  • Structural support for heart, lungs and bone marrow

  • Protection for brain, uterus, and other internal organs

  • Attachment sites for muscles allowing movement of limbs

  • Mineral reservoir for calcium and phosphorus

  • Defense against acidosis
     een ziekte waarbij de pH van je bloed te laag wordt.

  • Trap for some toxic minerals such as lead


Mechanical Properties of Bone

  • Testing modalities (Tension, compression, Bending, Torsion)

  • Strain gauges to measure bone deformations both in vitro and in vivo properties
     Strain gauges can only be used for bone and not for soft tissue. This is because the strain gauges need a rigid surface to work properly. On bone it is easy to measure the mechanical properties because of this.

  • Compressive Stiffness ranges 7 -30 GPa – directional effects

  • Changes with age – disease such as osteoporosis and osteopenia

Key Features

  • Morphogens are inductive signals that initiate and govern tissue morphogenesis, based on tissue interactions that are dynamic and reciprocal ( negative feedback)

  • Stem cells are primordial progenitors with immense replicative potential and multi-potential capacity for differentiation into multiple lineages

  • Biomaterial scaffolds to mimic ECM


BMP’s - Historical Perspective
Bone morphogenic growth (also in other tissues)
(recent work with knockout mice has revealed this  in these mice the ability of the gene is knocked out)

  • Huggins, over 60 years ago, found certain matrices were capable of new bone formation

  • Urist (orthopedic) (1965) established the key discovery, that de-mineralised lyophylized rabbitt bone can induce bone formation when implanted intra-muscularly
     ectopic – put tissue in the wrong place
     orthopic – put tissue in the right place

  • Reddi and Sampath showed that it acts in a sequential development cascade that mimics stages of osteochondral ossification similar to that in the limb bud, important in human development (in utero  how bone develops in the embryo).

  • Reddi and colleagues used dissociative extraction agents (guanidine, SDS (sodium dodecyl sulphate) and urea) to yield a soluble fraction (3%) and an insoluble type 1 collagen matrix. The two components need to be reconstituted for effectiveness. Bone induction markers ( what induces bone growth), such as alkaline phosphatase and RA calcium, are commonly used in bioassays

  • Wozney et al.(1988) were the first to clone BMP’s


BMP’s (“floating around in ECM”)

  • Decalcified bone implants have been used to treat patients with osteomyelitis

  • Bone contains a substance osteogenin (BMP-3) that initiates bone growth

  • Bone induction, as in morphogenesis through cartilage, involves a multi-stage process, each regulated by BMP’s involving

    • chemotaxis

    • mitosis

    • differentiation

  • BMP’s bind to extracellular matrix, such as heparin and collagen type IV. This converts a soluble morphogen into an insoluble matrix-bound morphogen that can act locally in the solid state and may protect it from proteolysis and prolong its half-life

  • There are 15 BMP’s
     It is not known why there are so many BMP’s, probably because they react in many tissues.

  • If you deliver the BMP’s in the right location and in the right way, they can work.


Pleiotropy of BMP’s
 The production by a single gene of at least two apparently unrelated effects
 Dependent on the concentration

  • Chemotaxis (optimal at fentomolar concentrations)
     or stimulate cell movement into a chemical gradient.

  • Mitosis (picomolar)

  • Differentiation (nanomolar)

    • cartilage in vitro

    • bone in vivo
       However, in vivo BMP’s are not “floating around” but are bound to ECM, so their local concentrations may be higher

  • Maintenance of phenotype (no differentiation)

    • cartilage

  • Stimulation of matrix production

  1. BMP’s bind to the ECM ( these are the key components for the development for bone)

  • ECM molecules play a key role in morphogenesis - explained by the binding of BMP’s to:

  • Collagen I - bulk matrix of bone

  • Collagen IV - part of invading capillaries. Vascular invasion is a pre-requisite for bone formation

  • Heparan sulphate - basement membranes

  • Heparin

  1. BMP Binding Proteins

  • NOGGIN
     role in head induction in amphibian embryos

  • CHORDIN

  • DAN

  • BMP’s have same affinity to these binding proteins in the ECM as to surface receptors on the cell membrane. (So there competition between BMP and the binding proteins).

  • This controls production and limits the possibility of hypertrophic bone (negative feedback control)


Signaling pathway for BMP’s




  • They are dimeric in form - types I and II receptors collaborate

  • Type I is a protein kinase. It phosphorylates intracellular substrates called SMAD’s (1&5), which enter into the nucleus to switch on gene expression

  • Once genes have been expressed, inhibitory SMAD’s within nucleus block protein kinases in the cytoplasm.

  • This regulates the activity of BMP’s, thus preventing them from “going into overdrive”




  • Also negative feedback in the cell (preventing from growing in the overdrive)


Activated BMP will go into the cell than there is the phosphorylation cascade, SMAD will express the genes and after that also “anti-SMAD” will be produced.

Biomimetic Biomaterials - Delivery Vehicle for BMP’s

  • Collagen

    • most effective vehicle for recombinant BMP. Commercial exploitation for use in cranial and facial applications

  • Hydroxyapatite

  • Fibronectin

  • Laminins

  • Geometry critical e.g. pore size (how easy is it for the cells to get in the pores?), beads/disc

  • The only use of BMP’s in tissue engineering:
    a fusion to help when there is pain in the back (vertebral column).

    The titanium cage is filled with bone chips from schrum, it will take about 6 months to grow. When BMP is used that will be faster.
     They had to prove with animals that it worked (monkeys because of the loading).
     Problem: by stopping the movement somewhere in the vertebral column, somewhere else is more movement and loading.

  • Most autografts are from the pelvis, since there is too much bone.



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