molecular aspects of a fracture healing
TRANSCRIPT
SAI PRASANTH G TMODERATOR : DR N RAJ KUMAR
MOLECULAR ASPECTS & IMPLANT INFLUENCE ON
BONE HEALING
Rockwood and Green’s treatment of fractures 8th ed
Campbell’s Orthopedics 12th ed
Molecular mechanisms regulating skeletal tissue formation in utero are involved in fracture healing.
The four components at # site : cortex, periosteum, bone marrow & soft tissues – contribute to healing.
Direct (primary) cortical fracture healing
Indirect (secondary) fracture healing
Molecules involved in Bone Healing
Fracture healing molecules Proinflammatory cytokines TGF – beta superfamily Growth factors Metalloproteinases and angiogenic factors
Inhibitory molecules Inhibitors of BMP signalling
Proinflammatory cytokines
IL - 1, TNF – alpha, IL-6Produced by macrophages, mesenchymal cells of
the periosteumFunctions :
Chemotactic effect on other inflammatory molecules
Enhancement of extracellular matrix synthesis Angiogenesis Recruitment of endogenous fibrogenic cells
Levels peak within 24 hrs, reduce in the period of cartilage formation
Increase again in the period of bone remodelling
TNF - Alpha
Important in endochondral ossification and remodelling
Promotes the recruitment of Mesenchymal stem cells (MSC’s)
Induces apoptosis of hypertrophic chondrocytes Induces osteoclastic activation and function
The loss of TNF – alpha persistence of cartilage
delayed bone healing
Molecules involved in Bone Healing
Fracture healing molecules Proinflammatory cytokines TGF – beta superfamily Growth factors Metalloproteinases and angiogenic factors
Inhibitory molecules Inhibitors of BMP signalling
TGF – beta Superfamily Bone morphogenetic proteins (BMP’s) Transforming Growth Factor beta (B1, B2) Growth Differentiation factors (GDF’s) Activin Inhibin Mullerian inhibiting substance
Bone Morphogenetic Proteins
Osteoprogenitor cells, osteoblasts, mesenchymal cells, chondrocytes produce BMP’s
Four subgroups of BMP family : Group 1 : BMP 2 & BMP 4 Group 2 : BMP 5, BMP 6 & BMP 7 Group 3 : BMP 14, BMP 13 & BMP 12 Group 4 : BMP 3 (osteogenin) & GDF 10
Induction of cascade of events for chondro-osteogenesis, mesenchymal & osteoprogenitor cell proliferation, production of ECM
Pathway of signal transduction
BMP Binding to Type II receptor Activation of Type I r receptor
Initiation of Smad
signalling cascade
Formation of heteromeric complexes
Translocation into nucleus and transcription of target genes
Formation of ECM proteins, proliferation of cells
Functions of various BMP’s
Transforming growth factor - Beta
Produced by platelets, osteoblasts & chondrocytesInitiates callus formationEffect exerted by Type I/ Type II receptors which
activate the Smad pathwayEnhances the proliferation of MSC’s, osteoblasts
and chondrocytesProduction of extracellular proteins – collagen,
proteoglycans, osteopontin, osteonectin & ALP.
Main role : Chondrogenesis and endochondral ossification
Molecules involved in Bone Healing
Fracture healing molecules Proinflammatory cytokines TGF – beta superfamily Growth factors Metalloproteinases and angiogenic factors
Inhibitory molecules Inhibitors of BMP signalling
Growth factors Platelet Derived Growth Factor (PDGF) Insulin like Growth Factor (IGF’ s) Fibroblast Growth Factor (FGF’ s)
Platelet Derived Growth Factor (PDGF)
Synthesized by platelets, monocytes, macrophages, endothelial cells, osteoblasts.
Acts via the tyrosinase kinase pathwayActions :
Potent chemotactic stimulator for inflammatory cells
Proliferation of MSC’s & osteoblasts Increases the callus density
Fibroblast growth factor
Synthesized by monocytes, macrophages, mesenchymal cells, osteoblasts & chondrocytes.
Actions : Differentiation of fibroblasts, myocytes,
osteoblasts & chondrocytes Needed for angiogenesis & MSC’s proliferation Alpha – FGF : regulation of chondrocyte
prolifertion and maturation Beta – FGF : expressed by osteoblasts
Insulin like growth factors (IGF’s)
IGF-I : somatomedin – C, IGF- II – skeletal growth factor
Produced by : Bone matrix, endothelial cells, osteoblasts, chondrocytes.
Actions : Promotes formation of bone matrix (Ty I collagen) IGF II – acts on stage of endochondral ossification IGF II – cartilage matrix synthesis, cellular
proliferation
Molecules involved in Bone Healing
Fracture healing molecules Proinflammatory cytokines TGF – beta superfamily Growth factors Metalloproteinases and angiogenic factors
Inhibitory molecules Inhibitors of BMP signalling
Metalloproteinases and angiogenic factors
Terminal stage of endochondral ossification involves degradation of cartilage cells and infiltration of blood vessels
Metalloproteinases degrade cartilage & bone allowing vascular channels to grow
Angiogenesis : VEGF pathway
Neoangiogenesis & endothelial cell proliferation Angiopoetin – dependent pathway
Formation of larger vessels & development of collateral branches from existing blood vessels
Molecular events during fracture repair
Fracture of long bone and hematoma formation
Activation of immune system
Initial inflammatory stage
Release of IL-I, IL-6, TNF- alpha, BMP’s
Chemotactic effect on other inflammatory cells and recruitment of MSC’s
Release of PDGF & TGF – beta from the platelets
MSC’s proliferation and formation of a chondrogenic/ osteogenic matrix
Production of VEGF/ angiopoetin
Vascular ingrowth into the developing callus
Intramembranous ossification endochondral ossification
Intramembranous ossification
Formation of bone with hard callus formation
Mechanically stable constructs
Periosteal cambial layer is the source of cells
Osteoblasts & osteoprogenitor cells form woven bone by day 3
Increased levels of BMP – 2, 4 & 7 are found.
Endochondral ossificationOccurs at # sites that
are less mechanically stable
Enhanced by soft tissues surrounding the #
Differentiation of MSC’s into chondrocytes
Formation of soft callus in 21 days
Chondrocytes synthesize proteoglycans, type II collagen & cartilage specific matrix
Once mechanical stability is attained – chondrocyte hypertrophy & calcification
Vascular invasion – removal of hypertrophic chondrocytes by chondroclasts
Formation of woven boneRemodelling of woven
bone.
Molecules involved in Bone Healing
Fracture healing molecules Proinflammatory cytokines TGF – beta superfamily Growth factors Metalloproteinases and angiogenic factors
Inhibitory molecules Inhibitors of BMP signalling
Inhibitory molecules in fracture healing
Involvement of negative feedback loops
Decrease cellular exposure to signalling molecules
Inhibition occurs at various levels : Extracellular compartment level Intracellular compartment level Receptor level Intranuclear level
Inhibitors of BMP signalling
Modulation of BMP signalling occurs at numerous levels
Presence of inhibitors control the level of BMP’sInhibition at extracellular levelInhibition at the receptor levelInhibition at intracellular level
Inhibition at extracellular level
Antagonistic molecules specifically bind to BMP’s inactivating them
The balance b/w the antagonists and BMP’s govern the fracture repair
Synthesized by : Osteoblasts BMP’s : expression of antagonists
Noggin
Binds to BMP - 2, 4, 5, 6, 7 & GDF – 5 & 6Noggin binds to BMP’s extracellularly and prevents
BMP binding with the Type II receptorsPrevent tissue overexposure to BMP’s BMP Noggin production BMP
antagonismNoggin/ BMP 4 balance an important factor that
regulates the callus formationDiseases with an antibody to Noggin :
osteoblastogenesis, skeletal abnormalities, multiple joint lesions.
d/s with null noggin gene: fibrodysplasia ossificans progressiva, multiple synostosis syndrome .
Chordin
Binds to BMP-2, 4, 7 & prevents their binding to the receptors
Chd levels are high in skeletogenesis – in condensing cartilage elements
Predominantly expressed in epiphyseal regionsChd antagonizes BMP-induced chondrocyte
differentiation
A negative regulator of endochondral ossificationChd + TSG (Twisted gastrulation) : inhibition of
osteoblast differentiation and mineralization
Gremlin, Sclerostin & Follistatin
Gremlin acts complementary to NogginBlocks BMP – 2, 4, 7 activity and inhibits osteoblastic
differentiationSclerostin expressed exclusively by osteocytes &
competes with BMP-2, 4, 6, 7 in binding to their receptors
Decreases osteoblast activity, MSC’s proliferation, reduction of osteoprogenitor differentiation
Sclerostin also promotes apoptosis of bone cells.Critical role in bone remodelling, bone repairAlso may have an antagonistic action over Noggin :
thereby increasing BMP activity
SOST mutation : Sclerosteosis – massive, progressive overgrowth of bones
Follistatin binds to BMP- 2, 4, 15 and mainly to BMP – 7
Formation of Trimeric complexes : BMP, Follistatin, Type II receptors
Inhibits all aspects of BMP activity in embryogenesis (further research needed)
Role of Gremlin and Follistatin dysregulation being studied in Osteoarthritis
Receptor level inhibition
Negative regulation by BAMBI (BMP & activin membrane bound inhibitor)
BAMBI is a pseudoreceptor It stably associates with Type II receptor as it
shares similar structure to Type I BMP receptorBut the complex doesn't travel intracellularly : no
signallingRole in embryonic development
Intracellular level inhibition
I- Smads ( SMAD-6 & 7)Interaction of I- Smads with activated Type I BMP
receptors thereby preventing activation of R SmadsCompetition with coSmad-4 & formation of inactive
Smad-1/Smad-6 complexSmad 6 : supression of Smad-1 induced
transcriptional activity
Other inhibitory molecules : Cytokines
Osteoprotegerin (OPG) : inhibitor of bone resorption – inhibits terminal differentiation of osteoclasts maturation
IL – 1 Ra : prevents the interaction between IL-1 and its receptor
TGF – Beta FGF IGFBP’s :
Insulin like growth factor binding protein : IGFBP-2 and 4 bind to IGF 1 and IGF 2 and prevent osteogenesis
Clinical application of these molecules
Osteoinductive properties of BMP’s have been used to enhance # healing in : Healing of bone defects Treatments of # non unions Spinal fusion
MDSC’s used to produce BMP-4 and retroviral vector to produce Noggin in a critical skull defect
Inhibitory molecule Ahsg : controls the osteogenic potential of ectopically added BMP.
Involvement of follistatin & gremlin in osteoarthritis
IMPLANT INFLUENCE ON FRACTURE HEALING
Fracture healing following intramedullary nailing
Healing pattern depends on type of fracture & degree of stabilisation
Reaming produces thermal necrosis of inner 2/3 rd of medullary canal
Periosteal vessels and the medullary vessels growing longitudinally enter the fracture site.
Formation of woven bone if construct stable and fibrous cartilage if construct not stable
Application of a plate to an intact bone leads to circulatory deficiencies.
Extent of circulatory deficiencies – degree of contact
Complete immobilization (plate) Direct bone healing - No callus formation, direct
deposition of lamellar bone.
Incomplete immobilization ( IM nail ) : Indirect healing – formation of fibrous connective
tissue – fibrous cartilage – woven bone – lamellar bone.
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