BMP Seminar

BONE MORPHOGENETIC PROTEINS The bone morphogenetic proteins are the only morphogens so Far found in mammalian species
and are detected not only in the embryonic but also post fetal bone development. They are critically important during embryogenesis, not only in regards to skeletal system but quite possibly in the morphogenesis and pattern formation of other tissues as well. It has been observed that extra skeletal tissues under certain pathologic conditions may ossify. This extra skeletal bone is called heterotrophic or ectopic bone containing all the morphologic and metabolic features of orthotopic bone, including bone marrow. Causes are: repeated trauma, burns, tumors-osteosarcoma, osteoid osteoma,osteoblastoma; neurologic injury resulting in periarticular ossification.
Downloaded from Perio Craze (drprem.blogspot.com) Prepared by Dr. Jai Ganesh ; Copyright free material
Urists triphasic theory of calcification: In the early 1960s several researchers Were investing the process of calcification. Urist and co workers Discovered that control samples of untreated decalcified bone Implanted into muscle pouches of rabbits and rats resulted in new Cartilage and bone formation. This led to the hypothesis of bone Formation by auto induction, in which an inductor cell(wandering Histiocyte) acts upon an induced cell young connective tissue cell, Causing it to differentiate into either an osteoprogenitor or chondro Progenitor cell. In 1938, Levander implanted living Bone fragments 1-1.5cm in length either subcutaneously or intra Muscularly. Upon obtaining regenerated bone, levander was able to Show that it was neither the periosteum nor the cells on the surface Or within the graft that were responsible for new bone growth.
He concluded that there is an extractable substance from bone which Is able to activate mesenchymal cells to form bone tissue.
Isolation of protein components with bone inductive property: Even though urist was not the first to speculate on the existence of a bone inducing substance, he must be credited With much of early work in isolation of BMP. In late 1960s Evidence was established that the substance which was responsible For bone induction is bound intimately with collagen. It was still Uncertain whether the inducing property was due to specific Chemical or physical structure of collagen itself was in some way Directing the mech. For mesenchymal cell differentiation.
Transmembrane diffussion:(urist 1968) An experiment deseigned to test the morphogenetic activity of various sizes of bone particles used Preparations of bone matrix pulverised in a freezer mill at 109 deg. In order to minimize the possible protein damage resulting from Heat. Diffusion chambers made up of cellulose acetate membranes having a thickness of 150 micrometer were also used. In the controls, particle sizes of 400-1000mic resulted in new bone Formation while 75-149mic,44-74 mic particles produced no new Bone. Larger particle size of 400-1000 mic the amount of new bone Formation on the outside of the membrane to the farthest. Therefore The amount of new bone formation was shown to be inversely Proportional to the distance which the bone morp.property must Travel. This Proved the evidence of diff.bone morphogen property When previously none was believed to be exist. Downloaded from Perio Craze
(drprem.blogspot.com) Prepared by Dr. Jai Ganesh ; Copyright free material
Structure of BMP: BMP proteins have similar structural charac: A hydrophobic secretory leader sequence, a large propeptide region and a mature domain. The mature contains the active component Of the molecule. Within this domain reside seven cysteine residues Positioned. Domain resembles TGF beta.
Examining the relationships between the BMP proteins has Fascilitated to organize them into three groups based on their amino Acid sequence homologies. * BMP 2,4 are closely related proteins, nearly identical in their Seven cysteine domains and showing more variability in the amino Terminal regions of mature proteins. *similarly BMP5 through BMP 8 share a great deal of sequence Homology and form another sub group of BMP.
*BMP3 (osteogenin) forms third group. Till now BMP1-16 have been isolated. Of all the bmps known BMP 2,7 are reported to be most important with regards to periodontal Regeneration.
Purification: isolation and purification of BMP has been difficult due To the fact that so little of it is present in bone and because of its Relative insoluability. 1.frozen cortical bone was ground to a particle size of 1mm cube Demineralized in 0.6N hcl at 4 deg for48 hrs, then defatted in1:1 Chloroform methanol. 2.using different conc.of cacl2 and urea the non collagenous proteins Were extracted from the insoluable bone matrix gelatin and separated Into different fractions. 3.sub fractionation was then accomplished using ultra filtration,sds Gel electrophoresis and various types of chromatography in order to Determine which molecule weight components were responsible for BMP activity.
4.in order to determine which subfractions were capable of Inducing bone formation, a reconstitution assay was utilized,this Most commonly accomplished in the rat. The protein to be tested was reconstituted with collagenous bone Matrix which was devoid of endogenous bmp and then implanted Subcutaneously. The formation of bone indicated a positive resp. To the implanted protein. Wang et al through sds polyacryl.gel electroph, identified a group Of proteins from bovine bone of 30kd and then proceeded to isolate recombinant clones for each. Amino acid seq. of 16,18, 30kd components were used to develop probes to screen bovine C dna libraries. Thus recombinant human proteins bmp 1,2,3 were Obtained. Upto bmp9, recombinant BMP proteins were isolated.
Bmp family: BMP proteins can be divided into sub groups based on the primary Amino acid sequence in the mature regions of the molecule. BMP Family Bmp1 Bmp2/ 4
Bmp3
Genetic name
Bmp1 Bmp2A Bmp2B osteogenin
BMP designation
BMP1 Bmp2 Bmp4 BMP3
chrom osome 20 20 14
4
Bmp7/ Op1
Bmp5 osteogenic protein1
Bmp5 Bmp7
6 20
Bmp Family
Genetic name vegetal related protein1 osteogenic protein2 osteogenic protein3
Bmp desig Bmp6 Bmp8 Bmp8A Bmp 9 Bmp10 Bmp11 Bmp14 Bmp13 Bmp12 Bmp15 Bmp16
chrom 6 -
Others
Bmp 9 Bmp10 growth and diff.factor11 cartilage derived morp.prot cartilage der morp.prot2 cartilage der morp.prot3 Bmp15 Bmp16
20
CDMP
Mechanism of action of BMP: The biological actions of Bmps are mediated in spec. Bmp receptors. They are of two types: 1 and 2- serine and threonine Protein kinases. These kinases are enzymes that phosphorylate proteins called Smad Small mothers against decapentaplegia and activate them. These activated SMADS are then translocated to the nucleus, where They participate in the transriptional regulation of the expression of Genes involved in cartilage and bone formation. There are eight different smads.smad 1,5,8 are substrates for BMP Receptors. Smad2 and 3 are substrates for TGF beta and activin Receptors. These smads are classified into 3 groups:R smad(receptor Regulated smads), co smads(common partner smads),Ismad (inhibitory smads).
Phosphorylation of smad1,5,8 activates it to interact with common Functional partner smad4 and this heteromeic complex enters the Nucleus to active turn on BMP responsive genes. There are two inhibitory smads6 and 7 that normally reside in the Nucleus and act as a relay to inhibit or turn off BMP type I receptor Kinase mediated phosporylation of smads1,5,8. So there is intricate homeostatic regulation of the BMP receptor Activated turning on of genes and their turning off by smads6and7 Through the inhibition of typeI BMP receptor kinase phosphorylation.
BMP specific antagonists: Dr.Hom and zimmermann identified BMP specific Antagonists and member of DAN family. The member of DAN Family mcr1, gremlin, PRDC and several genes are antagonistic To Bmp. These antagonists bind to the BMPs with the same affinity as BMP receptors do. Local irradiation also inhibits BMP induced bone formation. An over expression or dysregulation of BMP pathway may lead to heterotrophic bone formation or fibroplasia Ossificans progressiva.
BONE INDUCTION CASCADE Time after implantation: one min Cellular events: blood clot formation and platelet release. Molecular processes: fibrin network formation, release of platelets derived Growth factors. Binding of plasma fibronectin to implanted matrix.
After one hour Cellular ev: arrival of PMN by chemotaxis Molec: release of collagenase and elastase. After 18 hrs Cellular ev: accumulation of PMN. Adhesion of cells Molec: limited proteolysis and release of chemotactic factors for Fibroblasts.
Day I Cellular ev: chemotaxis of fibroblasts and cell attached to the Implanted extra cellular matrix. Molecular pr: release of peptide of fibronectin. Role of microtubules And micro filaments. Day 2 Cellular ev: continuation of chemotaxis for fibroblasts Signal transduction from matrix to cell surface. Molecular ev: initiation of protein and nucleic acid synthesis and Release of growth factors. Day3 Cellular ev: cell proliferation Molec ev: thymidine incorporation into DNA and increase in Ornithine decarboxylase activity.
Day 5 Cell ev: differentiation of chodroblasts Mol.ev: increase in so4 incorporation inside proteoglycans. Day7 Cell ev: chondrocytes synthesis and form of matrix Mol ev: type II collagen synthesis, cartilage specific proteoglycans Synthesis. Day 9 Cell ev: hypertrophy of chondrocytes and calcification of cartilage Matrix Mol ev: increase in ca incorp and alkalin phosphatase activity Type IV collagen synthesis and laminin , factor VIII in blood vessels.
Day 10-12 Cell ev: osteoblasts aggregation. Bone formation and mineralization. Mol ev: type I collagen syn, bone proteoglycan syn, increase in ca And alkaline phosphatase activity. Day 12-18 Cell ev: osteoclasts bone remodelling Mol ev: increase in acid phosphatase , aryl sulphatase. Release of Collagenases. Day 21: Cell ev: bone marrow differentiation. Mol ev: type III collagen synthesis.
FUNCTIONS OF BMP Osteogenesis, chondrogenesis. BMP 3 play a role in embryonic skeletogenesis. BMP7 expressed in kidney raising the possibility of its effects in Endocrine function. BMP7 m rna expressed in brain suggesting a possible role in cns Development. BMP 3 expressed in lungs. OP2 transcript were found in 8 day Mouse embryos suggesting a developmental role.
BMP 4 m rna expressed in developing tooth bud, odontoblast layer Downloaded from Perio Craze and palatal shelves. (drprem.blogspot.com) Prepared by Dr. Jai
Ganesh ; Copyright free material
Carrier systems: although BMPs possess activities beyond Osteoinduction, the primary interest in the proteins arises from Their potential use in clinical bone regeneration. collagen based carriers are being used in clinical Sitting. Advantage is it is a natural component of bone whose Degradation and degradation products can be mediated by Physiological means. Dis adv: weak mechanical performance, immunogenic response.
While the molecular biology of BMPs and related members in the Formulation and of inorganic carrier substrate with defined Geometrices capable of delivering BMPs in the absence of collag Matrix is the crucial goal for periodontists and skeletal Reconstructionists.
Ripamonti et al- composites of porous hydroxyapatite and mature BMP fractions, absorbed into the hydroxyapatite induced rapid Bone differentiation in calvarial defects of adult baboon The porous substratum of hydroxyapatite allows a spatially Controlled osteogenesis, restricting bone differentiation locally To surgical sites.
BMP AND PERIODONTAL REGENERATION The healing process initiated by a single molecular species of bone Morphogenetic protein such as BMP2,7 sets in motion a cascade of cellular events resulting in differentiation of progenitor cells into Phenotypes involved in periodontal regeneration. The optimal effects of BMPs are modulated by a Range of factors that need careful evaluation in clinical studies. 1.influence of root conditioning, occlusal loading, BMP dose, release Characters of carrier. 2.although BMP2 initiates stem cells along an osteogenic pathway The dose may have to be of sufficient concentration to ensure other Growth and differntiation factors do not redirect or retard the Osteogenic potential of the cell.
3.understanding when to manipulate the cells differentiation Pathway with the application of single or multiple doses of BMPs At the appropriate concentration is required to optimize the effect Of BMPs in periodontal wound healing. 4.the effect of BMPs could be further modulated by cross talk of The BMP smad pathway with other signal transduction pathways Including the TGF beta pathway as well as MAPK pathway. BMPs are involved with type I and II receptors and their Effects are modulated by a multitude of ligands many with similar Activities and capacity which form heterodimers.
BMP activities many be modulated by inhibitory binding protein, Certain nuclear co factors that co operate with smads in Regulatory specific target genes. Thus it needs to kept in mind That at the molecular level the additions of BMPs alone in Certain situations may not yield the desired clinical response.
King et al- cells responsible for the formation of cellular cememtum Are derived from osteo progenitor cells. These cells are target for BMPs. Cellular cementum may represent reparative formation that Is similar to bone. Partial root demineralization by acid conditioning may Augment the therapeutic effect of BMP2. benefits may include the Ability to remove smear layer after root preparation. Conditioned root surface provides a substrate that promotes Chemotaxis, migration and attachment of peridontal cells Encouraging connective tissue attachment to denuded root surface.
Saito et al used polymer coated gelatin sponge (pgs) as a spacer In PGS coated rh BMP2 and root surface in horizontal defects In beagle dogs. On histometric analysis after three months, bone regeneration, Cementum regeneration was evident. Root resorption occurred In all cases.
Lee et al reported enhanced bone augmentation by controlled Release of rh BMP2 from bio absorbable membrane.
Pang et al evaluated rh BMP4 effects on bone formation in Rat calvarial defect model. After eight weeks dense fibrous Connective tissue, limited bone formation. No difference in healing and degree of remodelling with Respect to collagen sponge and tricalcium phosphate carrier. King, hughes reported BMP2 stimulates cell recruitment And cementogenesis during early wound healing in rats.
Takakis, wozney et al evaluated the effect of rh BMP2 on Alveolar ridge augmentation and dental implant Osseo integration In beagle dogs. Peri implant defects were created in dogs. Rh BMP2 (0.05,0.1,0.2mg/ml) in ACS molded around openings Of the titanium fixtures and implanted into the peri implant Defects. After 8 weeks there was significant decrease in defect Height and increase in induced bone area.
Huang et al effect of BMP6 on periodontal wound healing in a Fenestration defect of rats. Surgically created bone window in 24 male rats followed by application of BMP6(1,3,10 micro gm) After eight weeks complete healing for all test sites, new cementum Formation seen. Resorption of root and bone for BMP dose of 10micro gm.
Release kinetics: Although a matrix carrier is not essential to promote bone formation There are a number of advantages to an appropriate carrier, including Localization and retention of BMP to the site of application thus Reducing the loading dose, providing a matrix mesenchymal cell Infiltration, provision of a substrate for cell growth and differentiation A shape that help define the resulting new bone and a degradation Rate that does not inhibit bone growth and resulting in fibrous tissue Formation or fiber encapsulation of the carrier. As BMP has an affinity for type I collagen, it would Seem reasonable to use a collagen carrier. It has been reported that The half life of BMP in a collagen sponge was less than 12hrs. This May not provide a sufficient exposure for the cells involved in Wound healing. So to alter the release kinetics of a carrier has advantages.
One way would be to change the degradation rate of the carrier. Additionally the drug release rate could be controlled chemically by Altering the cross linking pattern of the carrier. The emission of photons at certain wave lengths or the use of Magnetic fields or ultra sound could be used to alter the binding Characteristics of the growth factor from its carrier. Ex: the release profile of BMP from gelatin can be modified by cross Linking the gelatin with glutaraldehyde. Ultra sound can also alter the release kinetics of carrier systems by Promoting carrier degradation. The adv of this approach is that the Agent can be released from the carrier and titrated during the early Healing period as directed by the clinician.
The PEM polymer system can be manipulated to control both Amount and the pattern of release by varying the preparation method The copolymer ratios and using different monomers to control Hydrophilicity. This suggest that the inert carrier system may have potential Therapeutic applications in regenerative periodontics as a carrier for BMP as well as providing the space maintainence by retaining its Resilient structure.
Dis advantage of using a non resorbable material is that it requires Surgical procedure to remove the membrane.
Limitations: So far studies have been done in animals only, in which surgically The defect is created and BMP placed. This dont replace the Disease conditions. Half life of BMP is 12 hrs. need for sustained release from the Carrier material. Large quantities of bone is required for micro gm of BMP
Regenerated cementum is cellular cementum which is less conductive For fiber attachment
Human studies are needed.
Future directions: Towards Maintaining the periodontal ligament space. Effect of BMP under physiological and pathological conditions. Differences between BMPs also need careful evaluation as many Feedback loops can modulate the expression levels and activity Of essential components of the BMP signaling pathway both at The extracellular and intra cellular levels.
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BMP Seminar

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BONE MORPHOGENETIC PROTEINS The bone morphogenetic proteins are the only morphogens so Far found in mammalian species

Bmp5 osteogenic protein1

Genetic name vegetal related protein1 osteogenic protein2 osteogenic protein3

Dose and delivery systems.

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