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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