Players in Osteogenesis
• Osteo___
• Osteo___
• Osteo___
• Osteoprogenitor cells come from the____ and are induced into becoming ____ and ____ cell.
• They will have all of the hallmarks of secretory cell (___ Golgi, __ ER, nucleus is___)
• Osteoblasts eventually get ___ into the ____ of bone and they become ____
• They become centralized and stuck in ___ ___ and form____l bone which is ___y and ___
• Initially you have ____ bone.
Woven bone is____
First bone that is laid down and its ___ ___ ___.
• Placing Implant: 1st 2 weeks you are dealing with woven bone so don’t go placing implant bc it will __ ___ __
o Have to go thru ____ to get more ___ bone called corticoid bone • This is very orderly
Players in Osteogenesis • Osteoblast • Osteoprogenitor • Osteoid • Osteoprogenitor cells come from the mesenchyme and are induced into becoming osteoblast and secretory cell. • They will have all of the hallmarks of secretory cell (big Golgi, big ER, nucleus is acentric) • Osteoblasts eventually get incorporated into the matrix of bone and they become osteoids • They become centralized and stuck in Haversion canals and form critical bone which is orderly and strong • Initially you have woven bone. Woven bone is irregular. First bone that is laid down and its not very strong. • Placing Implant: 1st 2 weeks you are dealing with woven bone so don’t go placing implant bc it will just come out o Have to go thru remodeling to get more orderly bone called corticoid bone • This is very orderly
Osteogenesis
• Two processes occur in bone:
o ___ ___ mineralization • Type I is major type
o ___ ___ ____ mineralization
Osteogenesis • Two processes occur in bone: o Collagen-based mineralization • Type I is major type o Matrix -vesicle mediated mineralization
Collagen-based Mineralization in Bone:
Build the ____
• Found in ____ bone and _____ dentin
Collagen of mineralized tissues is different than other collagen
• Little to no type ___
• Increased____-____ o This helps with the ____ g and makes it strong
• High amt. of___ ____ bc of OH-K
• Intermolecular space ____ o How the fibers pack together o Type I has a lot more space in bw o What goes in those spaces? • ____
Collagen-based Mineralization in Bone: Build the scaffold • Found in Lamellar bone and circumpulpal dentin Collagen of mineralized tissues is different than other collagen • Little to no type III • Increased OH-lysine o This helps with the crosslinking and makes it strong • High amt. of cross-linking bc of OH-K • Intermolecular space greater o How the fibers pack together o Type I has a lot more space in bw o What goes in those spaces? • HAP
Triple Helix→ Collagen
Collagen ___
Collagen____
From collagen molecules to higher order structures = bone
Collagen ____
Mineralized ___
___
___
___
____
Triple Helix→ Collagen Collagen Fibril Collagen Fiber From collagen molecules to higher order structures = bone Collagen Molecule Mineralized Fibril Fiber Bundle Lamella Osteon
• Mineralization initiated within __ ___ in the collagen ____
o Helix is very ___ and fibrils pack in orderly fashion
o If you packing something and everything around it is orderly, it also has to be ____
• Early stage mineralization (on left) o Begins in ___ (using ___ ___ ___)
• Later stage mineralization o Grows as ___ in ___ dimensions • Doesn’t have anywhere else to grow
•____ acts as a scaffold and shows it the only places it can go
• Will get orderly structure of HAP bc of collagen base
• Mineralization initiated within hole zones in the collagen fibrils o Helix is very orderly and fibrils pack in orderly fashion o If you packing something and everything around it is orderly, it also has to be orderly. • Early stage mineralization (on left) o Begins in gaps (using Matrix vesicle mediated) • Later stage mineralization o Grows as plates in all dimensions • Doesn’t have anywhere else to grow • Collagen acts as a scaffold and shows it the only places it can go • Will get orderly structure of HAP bc of collagen base
Gather the ingredients
• ____ solution of calcium and phosphate o Need high enough conc for mineralization to occur. o Don’t want so high of conc that mineralization with spontaneously occur (without nucleator) o We have metastable saturation of Ca and PO4
• ____ to seed the first crystal growth
•___ ___s to___ the components together
Gather the ingredients • Supersaturated solution of calcium and phosphate o Need high enough conc for mineralization to occur. o Don’t want so high of conc that mineralization with spontaneously occur (without nucleator) o We have metastable saturation of Ca and PO4 • Nucleator to seed the first crystal growth • Bridging molecules to connect the components together
Matrix vesicle-mediated mineralization
• Found in endochondral cartilage, ___ __, ___ __, and most types of pathologic mineralization •
This forms our “drop”→ ____
• This takes place in that little hole (gaps) where mineralization is initiated •
This type of mineralization occurs in areas that we have very ___y, very ___ mineralization.
• Not very____, just going to occur very ___.
o Orderliness occurs as bone continues to grow
Matrix vesicle-mediated mineralization • Found in endochondral cartilage, mantle dentin, woven bone, and most types of pathologic mineralization • This forms our “drop”→ nucleator • This takes place in that little hole (gaps) where mineralization is initiated • This type of mineralization occurs in areas that we have very early, very rapid mineralization. • Not very orderly, just going to occur very quickly. o Orderliness occurs as bone continues to grow
• Matrix vesicle mediated mineralization
o ____ cells __ ___ to form a ___ bound ____ ____structure
o Contain ___ ____ and ___ ____
o Function as ____ for __ ____
o Eventually____and released into the ___, in between the ___ ___, promoting further ____.
• Further biomineralization is regulated by other___ ____
• Matrix vesicle mediated mineralization o Blastic cells bleb off to form a membrane bound extracellular vesicular structure o Contain acidic phospholipids and alkaline phosphatase o Function as catalysts for apatite formation o Eventually degraded and released into the ECM, in between the collagen fibers, promoting further mineralization • Further biomineralization is regulated by other bone proteins
Blebbing off of matrix vesicles from cell
• We have matrix vesicles • These are not really cells, they are vesicles __ ___ our ____ • Like a cytoplasmic inclusion • It will take these off from the cell
Blebbing off of matrix vesicles from cell • We have matrix vesicles • These are not really cells, they are vesicles blebbed off our osteoblasts • Like a cytoplasmic inclusion • It will take these off from the cell
• Inside of them, there is __ ___
. • It is just a little ____
• Mineralization can occur easier if we have a contained unit • Contain Ca and PO4 in ___ ___ to increase levels of Ca and PO4 and isolate it into a small area.
• This is our defined space • We are going to increase our Ca and PO4 levels to ___ ___ metastable. It is quite supersaturated in this area so that when we initiate the mineralization, it will work
• On the outside we have____ ____(____→ __ + ___) • Phosphates are ___to come ___ the matrix
• Inside of them, there is no nucleus. • It is just a little container. • Mineralization can occur easier if we have a contained unit • Contain Ca and PO4 in one area to increase levels of Ca and PO4 and isolate it into a small area. • This is our defined space • We are going to increase our Ca and PO4 levels to higher than metastable. It is quite supersaturated in this area so that when we initiate the mineralization, it will work • On the outside we have Alkaline Phosphatase. (R-PO4→ ROH + PO4) • Phosphates are free to come into the matrix
• We have shut off ___ ___ so no ___is pumped ___
• Ca allowed to come in but not allowed to leave
•___ [] of Ca and by ____alone, all the available ___ will com in as well
o Attraction to + Ca inside • So now we have a bunch of Ca and PO4 inside
• We have shut off ATP pump so no Ca is pumped out • Ca allowed to come in but not allowed to leave • Increase [] of Ca and by diffusion alone, all the available PO4 will com in as well o Attraction to + Ca inside • So now we have a bunch of Ca and PO4 inside
• Across here we have a nucleation site
o It is created by ___ and___ ___sites o This is in the membrane
o Acts as our _____
• Now we have Ca, PO4 and nucleator so it should ____ly start to mineralize within this vesicle o ____ mineralization within the vesicle, but its controlled by the fact that its in a ___
• Across here we have a nucleation site o It is created by annexin and phosphatidyl seine sites o This is in the membrane o Acts as our grain of sand • Now we have Ca, PO4 and nucleator so it should spontaneously start to mineralize within this vesicle o Uncontrolled mineralization within the vesicle, but its controlled by the fact that its in a membrane
• Eventually we have to get it ____ of the membrane
• The crystals are going to ____ as we go thru threr.
• Then membrane starts_____
• Once the membrane degrades, this is a crystal that acts to ____ other areas of mineralization.
• If this is sitting in my little gap region, this will act as a nucleator for more mineralization to occur in that gap region
• Spindles of hydroxyapatite can be seen in degrading matrix vesicles. •
• Eventually we have to get it out of the membrane • The crystals are going to proliferate as we go thru threre. • Then membrane starts degrading • Once the membrane degrades, this is a crystal that acts to nucleate other areas of mineralization. • If this is sitting in my little gap region, this will act as a nucleator for more mineralization to occur in that gap region • Spindles of hydroxyapatite can be seen in degrading matrix vesicles. •
Degradation of the vesicle allows ____ of the crystal to a physiological _____ solution in the body.
Crystals are able to ____ to grow.
They will grow in ___ fashion bc they are trapped in ___ ____
Degradation of the vesicle allows exposure of the crystal to a physiological metastable solution in the body. Crystals are able to continue to grow. They will grow in orderly fashion bc they are trapped in collagen matrix
• You get these clumps • We call them _____
• As they grow they make ___ of mineralization • Eventually what happens is these will ____ together and form bigger ___ ____
• You get these clumps • We call them calcospherules • As they grow they make globules of mineralization • Eventually what happens is these will coalesce together and form bigger mineralization plates
• Later stage mineralization
o More ___ of ___
o It will be ___
o Grows as plates in __ ___s
o ___, ___ and ____ of crystal growth is determined by ___ ___ ___ found in the ___
• ECM proteins ____ the ___ and ____ components
• We also have other molecules (not just collagen) that regulate growth)
• Later stage mineralization o More deposition of mineral o It will be orderly o Grows as plates in all dimensions o Direction, size and speed of crystal growth is determined by non-collagenous proteins found in the ECM • ECM proteins interconnect the mineral and organic components • We also have other molecules (not just collagen) that regulate growth)
Important ECM proteins
• Signaling o____, ____
• SIBLINGs o _____ ____, _____
•_____
Important ECM proteins • Signaling o Osteocalcin o Fibronectin • SIBLINGs o Bone Silaoprotein o Osteopontin • Byglycan
Additional Proteins of Interest
• ___ ___ __(DMP)
• ___ __ ___ (osteocalcin, BGP)
• ___ __ ___ (MGP)
• _____OPN)
• ___ __ ___ (BMP)
Additional Proteins of Interest • Dentin matrix protein (DMP) • Bone gla protein (osteocalcin, BGP) • Matrix gla protein (MGP) • Osteopontin (OPN) • Bone morphogenetic proteins (BMP)
SIBLINGs S___ i___-b___ l___ N-___ g___
• Found in ___ and ___ (___ and ___ found in higher amounts in ___)
- o Dentin Sialoprotein, Dentin Matrix Protein
• ____: ___ ____ o ___ ___ ____ o They all have this sequence o Integrin binding o May be a signal for ___ to ___ ___
• Osteoclast
o Signals “Be here”
• Gene _____ are similar
• Genes are clustered on ____ ___q21-23 o All found in same area of the csome
• Similar ___ __ ___
SIBLINGs Small integrin-binding ligand N-linked glycoprotein • Found in bone and dentin (DSPP and DMP-1 found in higher amounts in dentin) o Dentin Sialoprotein, Dentin Matrix Protein • RGD cell attachment o Arginine Glycine Aspartic Acid o They all have this sequence o Integrin binding o May be a signal for cell to attach here • Osteoclast o Signals “Be here” • Gene organizations are similar • Genes are clustered on chromosome 4q21-23 o All found in same area of the csome • Similar post-translational modifications
What is the nucleator? • We don’t know but its believed to be ___ ____
• ___ ____ of • 75 kDa.
• Contains ___ sequence, attaches to ____ on bone cell similar to OPN.
• _____ mineral formation, but inhibits it at ___ ___
o You need something to tell it to stop o Feedback mechanism
• Found in high amounts in ___, ___, ____. (Mineralized tissue)
What is the nucleator? • We don’t know but its believed to be Bone Sialoprotein • Phosphorylated glycoprotein of • 75 kDa. • Contains RGD sequence, attaches to integrins on bone cell similar to OPN. • Initiates mineral formation, but inhibits it at higher concentrations. o You need something to tell it to stop o Feedback mechanism • Found in high amounts in bone, dentin, cementum. (Mineralized tissue)
Dentin Matrix Protein-1 (DMP-1)
• ___ ___ has never been isolated.
___ ___ have been isolated (37Kd and 57Kd) o Protein is degraded!
• Overexpression of Dmp-1 induces ___ of ___ ___ to ___t-like cells and ___ ____
• Ectopic addition of DMP-1 seems to have __ ___ on mineralization o So its ___ a ____
. Not osteo___
• Majority of information comes from in vivo studies
• Initially found in___ but found in ___ too!
Dentin Matrix Protein-1 (DMP-1) • Intact protein has never been isolated. Proteolytic fragments have been isolated (37Kd and 57Kd) o Protein is degraded! • Overexpression of Dmp-1 induces differentiation of mesenchymal cells to odontoblast-like cells and enhances mineralization • Ectopic addition of DMP-1 seems to have no effect on mineralization o So its not a nucleator. Not osteoinductive. • Majority of information comes from in vivo studies • Initially found in dentin but found in bone too!
DMP-1 null mice
•__ ____ molar teeth
• Decreased layer of____
• ___ pulp
• Must be important for deposition.
May have to do with ___ cell differentiation.
DMP-1 null mice • Poorly forming molar teeth • Decreased layer of dentin • Larger pulp • Must be important for deposition. May have to do with less cell differentiation.
Osteopontin
• Reduction of OPN allows ___ ___ healing and ____ the ___ ___
• Matrix molecule found in ___
• Seems to have to do with inflammatory response
• If you injure a cell, you should see ___level of OPN being produced
• If you reduce OPN, response becomes more ___. You will have ability to allow that to heal more ____
___-___ healing if you ___ amt of OPN
• OPN indicates that you need ___ formation and repair • You need lower levels of OPN if you want orderly tissue
Osteopontin • Reduction of OPN allows more organized healing and decreases the inflammatory response • Matrix molecule found in bone • Seems to have to do with inflammatory response • If you injure a cell, you should see higher level of OPN being produced • If you reduce OPN, response becomes more orderly. You will have ability to allow that to heal more smoothly • Scar-free healing if you decrease amt of OPN • OPN indicates that you need rapid formation and repair • You need lower levels of OPN if you want orderly tissue
Osteocalcin
• ___ ___ dependent ____ of three ___ ___ residues (___ carbon)
o Vit K dependent→ Think ____ o In coagulation Vit K triggers carboxylation. Its involved in gamma carboxylation
o Normally those carboxyl groups are not 100% carboxylated but with Vit K dependent ones they will carboxylate the gamma
• Gla residues are on __ ___ ___
• Carboxylation→ more ___charged o ___ ___. Gets Ca into an area • Attract calcium ions o By charge alone, PO4 will follow • Around this molecule we have ____ [ ] of Ca and PO4 in that area o Getting molecules in right spot
___ to ____ o Create ___ ____
• A form that is not gamma carboxylated and found in ___ may have some ___ ____ functions
Osteocalcin • Vitamin K dependent carboxylation of three glutamic acid residues (gamma carbon) o Vit K dependent→ Think coagulation o In coagulation Vit K triggers carboxylation. Its involved in gamma carboxylation o Normally those carboxyl groups are not 100% carboxylated but with Vit K dependent ones they will carboxylate the gamma • Gla residues are on one planar surface • Carboxylation→ more negatively charged o Chelates Ca. Gets Ca into an area • Attract calcium ions o By charge alone, PO4 will follow • Around this molecule we have supersaturated [ ] of Ca and PO4 in that area o Getting molecules in right spot • Attaches to hydroxyapatite o Create intramolecular bridges • A form that is not gamma carboxylated and found in serum may have some hormonal regulatory functions
MGP:
Matrix Gla Protein
• Gla→ indicates that its ___ ____
• MGP Null Mice: See ____
o It ___ ____ In aorta and blood cells
MGP: Matrix Gla Protein • Gla→ indicates that its gamma carboxylated • MGP Null Mice: See mineralization o It inhibits mineralization In aorta and blood cells
Bone Morphogenic Protein-2
• ___ occurring protein
o Produced by ____
o Stored in __ ___
• ____ o Induce new bone formation without autogenous bone or bone marrow • Stick it in muscle→ will get bone • Can force mesenchymal cells to differentiate and make bone
• Produced in ___ ___o Same ____ as native protein o ____ feasible product • Used in dentistry
Bone Morphogenic Protein-2 • Nataurally occurring protein o Produced by Osteoblasts o Stored in bone mineral • Osteoinductive o Induce new bone formation without autogenous bone or bone marrow • Stick it in muscle→ will get bone • Can force mesenchymal cells to differentiate and make bone • Produced in recombinant form o Same activity as native protein o Commercially feasible product • Used in dentistry
Osteogenesis Imperfecta
• Mutations in the COL1A1, COL1A2, CRTAP, and LEPRE1 genes cause osteogenesis imperfecta. o ____ genes o Genes important for__ ___ of collagen
• Mutations in the COL1A1 and COL1A2 genes are responsible for more than ___ percent of all cases of osteogenesis imperfecta.
• Characterized by ___ ___
• “__ ___ disease” o Their bones break a lot o Think its abuse at first
Osteogenesis Imperfecta • Mutations in the COL1A1, COL1A2, CRTAP, and LEPRE1 genes cause osteogenesis imperfecta. o Collagen genes o Genes important for cross linking of collagen • Mutations in the COL1A1 and COL1A2 genes are responsible for more than 90 percent of all cases of osteogenesis imperfecta. • Characterized by blue sclera • “Brittle Bone disease” o Their bones break a lot o Think its abuse at first
Summary
• Biomineralization of bone occurs in___ ___
• Calcium and phosphates are brought into the ___ and____within the space
• ___ ___ act as scaffolds, nucleators, and regulators of the mineralization process
• ___ ____ is deposited in and among the collagen fibers
• Crystal ___ and ___ is regulated by macromolecules in the ___
Summary • Biomineralization of bone occurs in defined spaces • Calcium and phosphates are brought into the space and held within the space • Organic macromolecules act as scaffolds, nucleators, and regulators of the mineralization process • Mineralized hydroxyapatite is deposited in and among the collagen fibers • Crystal size and shape is regulated by macromolecules in the matrix
Denting
Composition
Dentin similar to bone
A lot of ___ proteins and____water
Comparison of % contribution of each component with respect to weight vs. volume.
• Wet weight : higher %inorganic, higher % water and organic compared to volume
• Volume: ____% inorganic, ____% water and organic
Differences suggest that there is ___ ___ of ____ elements between the ____ components
• Don’t have that much ____ matter but you are packing tons of stuff very tighly in bw inorganic matter
DentingComposition Dentin similar to bone A lot of organic proteins and some water Comparison of % contribution of each component with respect to weight vs. volume.
• Wet weight : higher %inorganic, higher % water and organic compared to volume
• Volume: lower % inorganic, higher % water and organic Differences suggest that there is tight packing of inorganic elements between the organic components • Don’t have that much inorganic matter but you are packing tons of stuff very tighly in bw organic matter
Dentinogenesis
• Odontoblasts: ___ __ ___
o Forms __ ___
o Does this in ____ to enamel
• They form in opposite directions
• Osteoblasts have to move ____ from enamel to deposit the dentin
• It will put down a predentin,____ matrix, like bone • At some time later, this will ____
• Very orderly because the __ ___ from odontoblast stays ____
o Dentin is still ____ bc you have process going to the surface o Have to numb patient to work on dentin o Enamel you don’t bc its not a live tissue.
Dentinogenesis • Odontoblasts: Dentin forming cell o Forms Pre-Dentin o Does this in opposition to enamel • They form in opposite directions • Osteoblasts have to move away from enamel to deposit the dentin • It will put down a predentin, unmineralized matrix, like bone • At some time later, this will mineralize • Very orderly because the dentin process from odontoblast stays in tact o Dentin is still alive bc you have process going to the surface o Have to numb patient to work on dentin o Enamel you don’t bc its not a live tissue.