Mineralization Mechanisms 2 and 3 Flashcards Preview

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Flashcards in Mineralization Mechanisms 2 and 3 Deck (64):
1

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

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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

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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

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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

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• 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

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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

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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

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• 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

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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

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• 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

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• 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

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• 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

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• 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. •

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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

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• 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

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• 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)

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Important ECM proteins

• Signaling o____, ____

• SIBLINGs o _____ ____, _____

•_____

Important ECM proteins • Signaling o Osteocalcin o Fibronectin • SIBLINGs o Bone Silaoprotein o Osteopontin • Byglycan

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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)

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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

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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)

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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!

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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.

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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

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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

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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

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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

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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

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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

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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

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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.

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It is mineralizing at a later point (___ to enamel?) than the matrix was deposited and at a___  that is much later as well

Distance away and Later time

Something must be going on where its ____ that is not triggered by ___ ___

It is mineralizing at a later point (closer to enamel?) than the matrix was deposited and at a time that is much later as well Distance away and Later time Something must be going on where its mineralized that is not triggered by cell itself

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Dentinogenesis (hypothesis)

• ____ forms first, establishing the ____collagen scaffold.

o Still___ ____ MINERALIZATION→ Like Bone

• The ____ and ____ of the collagen fibrils is believed to be directed by the presence of ____.

o In bone we had our___ ___

• Mineralization occurs at a later time at a distance away from the ____

. o Presumably directed by ____ as well

Dentinogenesis (hypothesis) • Predentin forms first, establishing the Type I collagen scaffold. o Still COLLAGEN BASED MINERALIZATION→ Like Bone • The alignment and spacing of the collagen fibrils is believed to be directed by the presence of proteoglycans. o In bone we had our matrix vesicles • Mineralization occurs at a later time at a distance away from the odontoblasts. o Presumably directed by proteoglycans as well

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• Odontoblasts secrete ___ ____ (types I, V, no III) which have ___ ___ in register and the proteoglycans, ____.

• Odontoblasts ____, forming ___ ____

• Collagen fibrils ____ and grow in ____ with ____ guiding the creation of ____

• We are depositing matrix, collagen fibrils are going to grow and in bw the collegen fibrils is going to be a proteoglycan

 • Dentin proteins are secreted at the ___ ____ (where mineralization is going to occur)

o Two secretory portions of odontoblast • One close to __ ___→ secretes • One out twds the ____→ secretes proteins for ____ • Allows for temporal and spatial difference • Diff proteins secreted at each point

• Odontoblasts secrete collagen fibrils (types I, V, no III) which have gap regions in register and the proteoglycans, decorin. • Odontoblasts recede, forming tubular processes • Collagen fibrils mature and grow in diameter with decorin guiding the creation of spaces • We are depositing matrix, collagen fibrils are going to grow and in bw the collegen fibrils is going to be a proteoglycan • Dentin proteins are secreted at the mineralization front. (where mineralization is going to occur o Two secretory portions of odontoblast • One close to cell body→ secretes • One out twds the process→ secretes proteins for mineralization • Allows for temporal and spatial difference • Diff proteins secreted at each point

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Dentin biomolecules (secreted in process)

• Three matrix proteins found pre-dominantly in dentin:

___ ____ ____

. o D_P (dentin _____ or ____):

Involved in ______of Hap crystal formation.

o DSP (dentin sialoprotein): Function ____

o DGP (dentin ____ ): Function ____  

• All found on ___ ___(1 gene product) o Cleaved to make the 3 proteins o We call the precursor ____

Dentin biomolecules (secreted in process) • Three matrix proteins found pre-dominantly in dentin: DPP, DGP, and DSP. o DPP (dentin phosphoprotein or phosphophoryn): Involved in initiation of Hap crystal formation. o DSP (dentin sialoprotein): Function unknown o DGP (dentin glycoprotein): Function unknown • All found on same gene (1 gene product) o Cleaved to make the 3 proteins o We call the precursor DSPP

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Dentin Sialophosphoprotein (DSPP)

• Coding sequences for DSP, DGP, and DPP on same mRNA.

• D_P on 5’ End & D_P on 3’ End, with D_P in the middle

• Thus all are synthesized as a large precursor protein (D__P).

• Precursor must be proteolytically processed by ___ _____to release D_P (the active piece). • DPP is the most active • DSP is probably _____ DPP until you need it. o Some enzyme in the matrix will cleave it to be active so you can get mineralization

o We believe this is why we can get mineralization to occur so ___ ____/ and __ ___. Controlled by when this ___ ___

Dentin Sialophosphoprotein (DSPP) • Coding sequences for DSP, DGP, and DPP on same mRNA. • DSP on 5’ End & DPP on 3’ End, with DGP in the middle • Thus all are synthesized as a large precursor protein (DSPP). • Precursor must be proteolytically processed by unknown proteinases to release DPP (the active piece). • DPP is the most active • DSP is probably inhibiting DPP until you need it. o Some enzyme in the matrix will cleave it to be active so you can get mineralization o We believe this is why we can get mineralization to occur so far away/ and diff time. Controlled by when this cleaved off

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DSPP is transported to the Mineralization Front (the Predentin-Dentin Border).

DSPP is proteolytically processed to form DSP and DPP.

In this way, DPP is activated at the place where its activity is needed.

DSPP is transported to the Mineralization Front (the Predentin-Dentin Border). DSPP is proteolytically processed to form DSP and DPP. In this way, DPP is activated at the place where its activity is needed.

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DPP

• _____: Contains 36% ___ & 45% ____ : (PSE). o Lots of Asp and PSE o Lots of___ ___ o Attract ___ and ____ will follow

• Many ___ sequences: o (___ ____E)n and (___ ___ ___E)n

• DPP has a predicted structure of having repeated sequences, with the ___ and ___ groups on the ___

o They are always at ridge, not in the ___ o So its sticking___ into the environ so more able to get to Ca

• What makes it good for mineralization?

DPP • Polyanionic: Contains 36% Asp & 45% Phosphoserine : (PSE). o Lots of Asp and PSE o Lots of neg charge o Attract Ca • PO4 will follow • Many repeating sequences: o (ASP-PSE)n and (ASP-PSE-PSE)n • DPP has a predicted structure of having repeated sequences, with the carboxylate and phosphate groups on the ridges o They are always at ridge, not in the valleys o So its sticking out into the environ so more able to get to Ca • What makes it good for mineralization?

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DPP: Evidence of its importance in mineralization.

• Binds lots of ___ with ____affinity.

• Forms ____ in vitro when____ and incubated metastable Ca x P. o When you stabilize it, it can act as a nucleation site

• At higher concentrations in solution, DPP ___ ___ ___

• After synthesis, moves quickly to ___ ___

 • These data suggest that DPP has two roles: o ___ of ___ o___ of the ___ ___

DPP: Evidence of its importance in mineralization. • Binds lots of calcium with moderate affinity. • Forms HAP in vitro when immobilized and incubated metastable Ca x P. o When you stabilize it, it can act as a nucleation site • At higher concentrations in solution, DPP inhibits mineral formation. • After synthesis, moves quickly to mineralization front. • These data suggest that DPP has two roles: o Initiation of Hap o Control of crystal habit

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DPP is hypothesized to ____mineralization

• DPP binds to ____  in__ ___. o Its in the right place

• Here it ____formation of HAP mineral crystals.

• DPP would be a ___ ____ of HAP formation.

o If it initiates, a heterogenous nucleator means that it is a different molecule that does the initiation o Its not HAP

• As crystals grow, DPP binds to ___ ____ and ___ growth, leading to ____, ___ like HAP crystals that are ____ ___. o Negative feedback like Bone Sialoprotein

• Where does structure come from? o ___________ that are structured o ___ ___ o ____ slows down the process and makes sure its orderly

DPP is hypothesized to initiate mineralization • DPP binds to collagen in gap regions. o Its in the right place • Here it initiates formation of HAP mineral crystals. • DPP would be a heterogeneous nucleator of HAP formation. o If it initiates, a heterogenous nucleator means that it is a different molecule that does the initiation o Its not HAP • As crystals grow, DPP binds to external faces and slows growth, leading to oriented, plate-like HAP crystals that are biologically sound. o Negative feedback like Bone Sialoprotein • Where does structure come from? o Type I collagen that are structured o Odontoblast tubules o DPP slows down the process and makes sure its orderly

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DPP binds to collagen in gap regions. • Here it initiates formation of HAP mineral crystals. • DPP would be a heterogeneous nucleator of HAP formation.

 

• As crystals grow, ____is ___ and DPPs bind to external faces and slowly grow, leading to oriented, plate-like HAP crystals that are biologically sound.

• ____ is converted to____

DPP binds to collagen in gap regions. • Here it initiates formation of HAP mineral crystals. • DPP would be a heterogeneous nucleator of HAP formation. • As crystals grow, decorin is degraded and DPPs bind to external faces and slowly grow, leading to oriented, plate-like HAP crystals that are biologically sound. • Predentin is converted to dentin.

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A proteoglycan plays a significant role

• While the DSPP proteins are filling their role, a PG called ___ helps to ___ ___

• Secreted in among area of ___ __ ___

• Decorin: DON’T HAVE TO KNOW STRUCTURE

o Leucine-rich PG o 45 kDa with one CHS chain

o Bound to surface of ___ and ____ collagen & is ____ (degraded) as fibers in ___ and ____ grow in ____

o This ____ of collagen and ____of decorin may be an important event in mineralization.

o In dentin and bone, decorin is lost as collagen fibrils thicken. o Thus decorin may act as a spacer for mineralization o Spaces get filled in with____

A proteoglycan plays a significant role • While the DSPP proteins are filling their role, a PG called Decorin helps to create space. • Secreted in among area of Type I collagen • Decorin: DON’T HAVE TO KNOW STRUCTURE o Leucine-rich PG o 45 kDa with one CHS chain o Bound to surface of bone & dentin collagen & is removed (degraded) as fibers in osteoid and predentin grow in diameter. o This thickening of collagen and loss of decorin may be an important event in mineralization. o In dentin and bone, decorin is lost as collagen fibrils thicken. o Thus decorin may act as a spacer for mineralization o Spaces get filled in with HAP

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Dentinogenesis Imperfecta

Mutations in the ____ gene have been identified in people with type ___ and type ___ dentinogenesis imperfecta.

Type _ occurs as part of __ ___a.

Weaker dentin Poorly formed teeth, larger pulps

Can form enamel but bc of problems with dentin, the enamel doesn’t form equally as well

Sometimes seen as a result of osteogenesis imperfect. Problem with Type I collagen gene.

Dentinogenesis Imperfecta Mutations in the DSPP gene have been identified in people with type II and type III dentinogenesis imperfecta. Type I occurs as part of osteogenesis imperfecta. Weaker dentin Poorly formed teeth, larger pulps Can form enamel but bc of problems with dentin, the enamel doesn’t form equally as well Sometimes seen as a result of osteogenesis imperfect. Problem with Type I collagen gene.

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DSPP null mice show defects in mice teeth that mimic _____

DSPP null mice show defects in mice teeth that mimic DGI-III.

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DSPP knockout leads to retaining increased levels of ____and ____.

 Thus, supporting the role of PGs in converting ____ to ___. Problem with initiation of mineralization so may not have a signal for decorin to disappear Don’t have spaces for HAP Less mineralization in those areas

DSPP knockout leads to retaining increased levels of BGN and DCN. Thus, supporting the role of PGs in converting predentin to dentin Problem with initiation of mineralization so may not have a signal for decorin to disappear Don’t have spaces for HAP Less mineralization in those areas

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NO COLLAGEN BASED MINERALIZATION!!

Amelogenesis:

• Example of ___ ____based mineralization

• Apatite crystals are ___, ____ ____

• Mineralization process unusual: A set of___ ___ are used to create these huge, oriented crystals which must last a lifetime!

• Proteins are ___ to enamel

NO COLLAGEN BASED MINERALIZATION!! Amelogenesis: • Example of non-collagen based mineralization • Apatite crystals are large, highly oriented. • Mineralization process unusual: A set of extreme conditions are used to create these huge, oriented crystals which must last a lifetime! • Proteins are unique to enamel

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Compostitions

• Immature enamel contains a lot of ____r, 30%____ and has___,____-like apatite crystals.

Mature enamel contains ___% protein and ___, ____ crystals

Thus, the process of enamel maturation involves____ of proteins as the apatite grows (primarily in ____,).

We have a lot of proteins when we first start out and then we have almost no proteins when we end up. These proteins are like decorin and are ___ __ ____

 Its important for them to disappear but they need to disappear in an ____ fashion and they will help to direct what we are doing. 

Compostitions • Immature enamel contains a lot of water, 30% protein, and has thin, plate-like apatite crystals. 

Mature enamel contains <1% protein and large, oriented crystals

Thus, the process of enamel maturation involves removal of proteins as the apatite grows (primarily in width,).

We have a lot of proteins when we first start out and then we have almost no proteins when we end up. These proteins are like decorin and are holding some space.

 Its important for them to disappear but they need to disappear in an orderly fashion and they will help to direct what we are doing. 

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Enamel Proteins

• ______: 90-95% of proteins in ____ enamel. • 25-27 kDa, also smaller fragments—21, 15 kDa, etc

oProbably getting ____ o Doesn’t nec fcn as those smaller proteins but by the time we extract it, they have been cleaved

• ____ proteins.

• Form ____structures called _____.  About ___ amelogenins per sphere of 15-20 nM diameter. •

Belongs to the ____ ___ _____ ____ gene family • Another protein that ___ ___ • These nanospheres are going to be what is our structure. They take the role of ___ __ ___

Enamel Proteins • Amelogenins: 90-95% of proteins in immature enamel. • 25-27 kDa, also smaller fragments—21, 15 kDa, etc o Probably getting cleaved o Doesn’t nec fcn as those smaller proteins but by the time we extract it, they have been cleaved • Hydrophobic proteins. • Form spherical structures called Nanospheres: About 100 amelogenins per sphere of 15-20 nM diameter. • Belongs to the Secretory Calcium-binding phosphoprotein gene family • Another protein that binds Ca • These nanospheres are going to be what is our structure. They take the role of Type I collagen

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Atomic Force Microscopy shows spherical structures of nanospheres

We need ___ ___ ____ in order to be able to do this.

Ameloblasts starts ____ to ____and recedes

Atomic Force Microscopy shows spherical structures of nanospheres We need mineralized dentin surface in order to be able to do this. Ameloblasts starts close to dentin and recedes

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• Amelogenesis involves secretion of enamel specific proteins by ____

• Simultaneously they form ___ like oriented HAP crystals.

• Amelogenins form nanospheres,___ ___ ___of apatite crystals.

• Nanospheres___ ____ o Nothing orders them specifically • Then they form kind of like ____.

• They allow HAP to form in a ribbon like structure. Create a situation in which the only way HAP can grow is in one way. • Order comes from nanospheres • Has to grow in _____ direction.

• Enamel crystals are longer than others o This allows for it to be long crystal by forces it to grow only in ____ ____

• Bc these are tighly wound around it, it can’t grow ___ ___

• It will grow long first and then eventually laterally

• _____ will eventually get cleaved, then we can have ____ growth of those crystals o Length is most important in beginning

• Amelogenesis involves secretion of enamel specific proteins by ameloblasts • Simultaneously they form ribbon-like oriented HAP crystals. • Amelogenins form nanospheres, preventing lateral growth of apatite crystals. • Nanospheres self assemble o Nothing orders them specifically • Then they form kind of like helices. • They allow HAP to form in a ribbon like structure. Create a situation in which the only way HAP can grow is in one way. • Order comes from nanospheres • Has to grow in longitudinal direction. • Enamel crystals are longer than others o This allows for it to be long crystal by forces it to grow only in longitudinal direction • Bc these are tighly wound around it, it can’t grow laterally YET • It will grow long first and then eventually laterally • Amelogenins will eventually get cleaved, then we can have lateral growth of those crystals o Length is most important in beginning

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Ameloblasts secrete _____ allowing growth of apatite crystals.

Result is large, highly oriented apatite formed by ameloblasts.

Ameloblasts don’t ___ ____ . Once enamel is gone, its gone. We can’t make more

Ameloblasts secrete proteinases allowing growth of apatite crystals. Result is large, highly oriented apatite formed by ameloblasts. Ameloblasts don’t stay around. Once enamel is gone, its gone. We can’t make more

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Other, Minor Enamel Proteins

_____: Largest enamel protein: 120-140 kDa • Made &amp; secreted during ____ stage.  Believed to be unique to enamel. • Possibly important as a___ of crystal nucleation and growth. Appears to catalyze the _____ of enamel crystals along the c-axis (length) • Belongs to SCPP gene family

Other, Minor Enamel Proteins Enamelins: Largest enamel protein: 120-140 kDa • Made &amp; secreted during developing stage. Believed to be unique to enamel. • Possibly important as a regulator of crystal nucleation and growth. Appears to catalyze the extension of enamel crystals along the c-axis (length) • Belongs to SCPP gene family

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______:

Secreted only by ____

. • Hydrophobic, rich in Pro, Gly, Leu.

• Important in Mineralization: KO mice have enamel defects.

• Possible role is to maintain the ___ of ____ of the ameloblast

• Belongs to SCPP gene family

Recognize that these are enamel proteins Disruption in one of these would lead to defect in enamel DON’T NEED TO KNOW DETAILS ABOUT THESE

Ameloblastin: Secreted only by ameloblasts. • Hydrophobic, rich in Pro, Gly, Leu. • Important in Mineralization: KO mice have enamel defects. • Possible role is to maintain the state of differentiation of the ameloblast • Belongs to SCPP gene family Recognize that these are enamel proteins Disruption in one of these would lead to defect in enamel DON’T NEED TO KNOW DETAILS ABOUT THESE

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• ____: o May play a role in ____of HAp in enamel. o Uncertain. o Found in ___ ___.

• ____: role unknown

• Tuftelin: o May play a role in nucleation of HAp in enamel. o Uncertain. o Found in many tissues. • Amelotin: role unknown

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Enamel Proteinases

• ____: Matrix Metalloproteinase with broad specificity. o Secreted at the ___ ___of amelogenesis, with the SCPP proteins. Probably involved in the___ ___ o Important in start of the crystal and ____ growth of the crystal

• ____-__: secreted at the ____ stage. o Probably involved in degrading remaining enamel proteins to allow crystals to increase in ____. • Important for amelogenins to degrade • Need enzymes in the matrix for this to happen

Enamel Proteinases • MMP20: Matrix Metalloproteinase with broad specificity. o Secreted at the early stage of amelogenesis, with the SCPP proteins. Probably involved in the crystal formation. o Important in start of the crystal and longitudinal growth of the crystal • Kallikrein-4: secreted at the maturation stage. o Probably involved in degrading remaining enamel proteins to allow crystals to increase in width. • Important for amelogenins to degrade • Need enzymes in the matrix for this to happen

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Amelogenesis Imperfecta

• Mutations in the____ gene (AMELX) cause _-___ amelogenesis imperfect (most common), while mutations in the ____ gene (ENAM) cause ____ inherited forms of amelogenesis imperfecta.

• Recent reports involve ____, ____, and DLX3 genes in the etiologies of some cases.

• Common • Usually genetic • Patients die when you clean teeth. Exposed dentin • Often crown all the teeth or do implants • Yellow teeth because you see dentin

Amelogenesis Imperfecta • Mutations in the amelogenin gene (AMELX) cause X-linked amelogenesis imperfect (most common), while mutations in the enamelin gene (ENAM) cause autosomal inherited forms of amelogenesis imperfecta. • Recent reports involve kallikrein-4 (KLK4), MMP-20, and DLX3 genes in the etiologies of some cases. • Common • Usually genetic • Patients die when you clean teeth. Exposed dentin • Often crown all the teeth or do implants • Yellow teeth because you see dentin

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Substitutions- Fluoride

• Fluoride can substitute for ___-; it __ ___ in the ___of crystals;

• ____ soluble crystals of fluoroapatite result

o Bc its so electro(-) it ____ the Ca triangle so it’s a little ___ and less ___ by acids

• Fluoroapatite is more resistant to acid demineralization

• Fluoride on the tooth surface acts as a ____ for _________→ remineralization

Substitutions- Fluoride • Fluoride can substitute for OH-; it fits better in the center of crystals; • Less soluble crystals of fluoroapatite result o Bc its so electro(-) it collapses the Ca triangle so it’s a little closer and less permeable by acids • Fluoroapatite is more resistant to acid demineralization • Fluoride on the tooth surface acts as a catalyst for Ca and PO4 deposition→ remineralization

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____ ___ ____ (ACP)

• ____-ACP (casein phosphopeptide-ACP)

• Reacts with ____ ions, allowing release of ___ and ____, that helps to ___ teeth

• Its in everything now. Bleach, Toothpaste, gum

• It adds a lot of Ca and PO4 into an area

• Used to ___ ___ or ___

 • DON’T USE IF HAVE ___ ALLERGY

• We complex it with casein to act as a carrier

• Oral cavity is acidic so you will get H ion release anyway and by doing that you will release the Ca and PO4 ions into an area and then it will be there

Amorphous Calcium Phosphate (ACP) • CPP-ACP (casein phosphopeptide-ACP) • Reacts with hydrogen ions, allowing release of calcium and phosphate, that helps to remineralize teeth • Its in everything now. Bleach, Toothpaste, gum • It adds a lot of Ca and PO4 into an area • Used to decrease sensitivity or remineralize • DON’T USE IF HAVE MILK ALLERGY • We complex it with casein to act as a carrier • Oral cavity is acidic so you will get H ion release anyway and by doing that you will release the Ca and PO4 ions into an area and then it will be there

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Synthetic enamel of fluoroapatite They are working on creating synthetic enamels using fluoroapatites.

Synthetic enamel of fluoroapatite They are working on creating synthetic enamels using fluoroapatites.

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Emdogain

• A purified ___ ___ of developing ___ ____ derived from six-month old piglets

• Used to r_____ on teeth

• When we have separation and no longer have gingival tissue covering the teeth to help protect the cementum • You need something on the tooth surface to actually get tissue to ____and ___ __ tooth surface

• Its purpose is to act as a ___ ____ modulator that would mimic the events that occur during root development and to help stimulate___ ____

• Put emdogain and then do ___ ___ on top of that to cover back over again. • CT will adhere and set down on top of that • If you just put CT, it may not stick

Emdogain • A purified acidic extract of developing embryonal enamel derived from six-month old piglets • Used to resurface on teeth • When we have separation and no longer have gingival tissue covering the teeth to help protect the cementum • You need something on the tooth surface to actually get tissue to attract and stay on tooth surface • Its purpose is to act as a tissue-healing modulator that would mimic the events that occur during root development and to help stimulate periodontal regeneration • Put emdogain and then do CT graft on top of that to cover back over again. • CT will adhere and set down on top of that • If you just put CT, it may not stick

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Osteoconductive vs. Osteoinductive factors

• Osteoinduction o The process by which ____ is ____. It is a phenomenon regularly seen in any type of ___ ____ process.

o Osteoinduction implies the ____ of ____ cells and the _____of these cells to develop into _____.

o In a ___ ____ situation such as a fracture, the majority of bone healing is dependent on osteoinduction. o Induces bone formation (BMP2)

 

Osteoconduction o Bone grows ___ a ____ o This phenomenon is regularly seen in the case of___ ____

Osteoconductive vs. Osteoinductive factors • Osteoinduction o The process by which osteogenesis is induced. It is a phenomenon regularly seen in any type of bone healing process. o Osteoinduction implies the recruitment of immature cells and the stimulation of these cells to develop into preosteoblasts. o In a bonehealing situation such as a fracture, the majority of bone healing is dependent on osteoinduction. o Induces bone formation (BMP2) • Osteoconduction o Bone grows on a surface o This phenomenon is regularly seen in the case of bone implants.

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This is a sinus lift.

Sometimes we don’t have enough room bw maxillary sinus and teeth to put an implant. We pack ___ in here

You can use ___ to help jumpstart that You may be able to get it to work without BMP but if you need to create lots of___, use BMP mixed with graft material to jumpstart that

Take recombinant form of BMP2. Place it on a ___ bc it itself is dissolvable.

If you put it someplace it would just go away so have to put it on a scaffold to hold it in the place we want it P

ack that sponge with some bone in here and hope that we form bone.

This is a sinus lift. Sometimes we don’t have enough room bw maxillary sinus and teeth to put an implant. We pack bone in here You can use BMP to help jumpstart that You may be able to get it to work without BMP but if you need to create lots of bone, use BMP mixed with graft material to jumpstart that Take recombinant form of BMP2. Place it on a sponge bc it itself is dissolvable. If you put it someplace it would just go away so have to put it on a scaffold to hold it in the place we want it Pack that sponge with some bone in here and hope that we form bone.

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Sinus Augmentation Clinical Conclusions

• INFUSE Bone Graft o Induced de novo __formation o Proven clinical safety and effectiveness for generating bone in the __ ____ for dental resoration • __ used to jumpstart this process but you still have to ___ ___e in there

Sinus Augmentation Clinical Conclusions • INFUSE Bone Graft o Induced de novo bone formation o Proven clinical safety and effectiveness for generating bone in the maxillary sinus for dental resoration • BMP used to jumpstart this process but you still have to add bone in there

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____ bone as scaffolding

If you have infection of teeth, buccal plate can disappear.

Particulate bone as scaffolding If you have infection of teeth, buccal plate can disappear.

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Using natural bone processes in therapy

____Osteogenesis

• Distraction device creates a ____ ___ ____

• Repair of defect fills in with ___

• The amount of callus formed is inversely proportional to the amount of i_____n of the ____

• In fractures that are fixed with rigid compression plates there can be primary bone healing with little or no visible ___ ___

Using natural bone processes in therapy Distraction Osteogenesis • Distraction device creates a specified size fracture • Repair of defect fills in with bone • The amount of callus formed is inversely proportional to the amount of immobilisation of the fracture. • In fractures that are fixed with rigid compression plates there can be primary bone healing with little or no visible callus formation