Bone Research Flashcards
Angiogenesis during bone growth
When is it required
Bone development and growth
Fracture repair
Bone development
Bone remodelling cycle
Bone disease - pagets, osteaoperosis (ageing)
Paget’s disease
Men >70 yrs old
Highly vascularised bones
Rickets
Low vitamin D
Not enough calcium
Dysfunction of VGEF , dysfunction of vascularisation
Osteopetrosis
Too much bone and vascularisation
Osteoporosis
Not enough bone mass, increase fracture risk, decrease vascular use
Sexually dimorphism disease: female more likely due to loss of eatrogen during menopause as eastrogen is pro anabolic
Bone thinning
Decreased vascular growth factors
Bone growth and development
Mesenchymal cells directly differentiate into bone by intramembranous ossification - craniofacial skeleton (neural crest) NO CARTILAGE TEMPLATE -has to form quickly as protective layer
These cells can differentiate into cartilage which provided template for bone morphogenetic by endochondral ossification (long bones) CARTILAGE TEMPLATE
Endochondral ossification
Cartilage replaced by bone
Cartilage around joints remain
(Hypertrophic) Chondrocytes produce lots of proangiogenic factors eg VEGF and MMP13
Angiogenesis occurs due to blood vessel attraction to VEGF, (remove matrix via MMP9 & 14 to create space for blood vessels) provide bone with nutrients and oxygen
Osteoblasts create the bone
Growth of cartilage model
Hypertrophic chondrocytes attract blood vessels by producing VGEF
Development of primary ossification centre
Development of marrow cavity
Decelopment if secondary ossification centre
Formation of articulation cartilage
VEGF couples hypertrophic cartilage remodelling, ossification and angiogenesis during endochondral bone formation
Important - VEGF IMPORTANT IN PROCESS OF BONE OSSIFICATION AND MINERALISATION
Vascular endothelial growth factor (VEGF)
Angiogenesis - knock out single VEGF allele in mice causes lethal impairment of vascular development
Angiogenesis:formation of new blood vessels from pre-existing vascular use plays central role in adult tissue homeostasis
Pathological processes including wound healing, tissue remodelling, tumour development and growth of atherosclerosis plaques
VEGF-A gene exhibits splice variants isoforms
Studying VEGFs effects in vivo
Soluble VEGF receptor chimeric protein
mFlt(1-3)-IgG
VEGFR1/Flt1 is a negative regulator of angiogenesis
VEGFR1 - Decoy receptor little intracellular signaling capacity, bind VEGF at surface of cell and not induce any signalling cascades, takes VEGF and stops it binding from functional receptor
So too much VEGF = up regulation of VEGFR1 so stop endothelial proangogenic response
Make protein modelled on VEGFR1 to stop it binding to VEGFR2 to block the signalling
Angiogenesis
VEGFR2 - main receptor, VGEF floats around and binds to tyrosine kinase receptor, phosphorylation of tyrosine kinase molecules causing activation of signalling pathways and genes linked to angiogenesis like endothelial cell migration, proliferation and growth
What was found with injection of BEGFR1 proteins
Chimeric protein captures VEGF
Phenotypes seen: reduced CD31 (endothelial cell marker) staining
Increase hypertrophic chondrocyte zone so less bone formation (build up or cartilage cells)
Mineralisation is reduced
Where is VEGF coming from?
LacZ tagging of VEGF allele allowing precise analysis of patter of VEGF expression at cellular level
Antibodies not very good for this
LacZ reporter gene into untranslated region of endogenous VEGF locus by homologous recombination
BONE CELLS (OSTEOBLASTS) CAN COMMUNICATE WITH BLOOD VESSELS AND RELEASE VEGF
BONE (FORMING) SURFACES
Can bone cells influence and contribute to the vasculature?
Vasculature run through hersian canal
Endothelial cells <> osteoblasts
Osteoblasts can communicate with endothelial cells via angiogenesis signals such as VEGF and endothelial cells can communicate to osteoblasts via osteopenia signals eg BMP2
Hypothesis - cross talk and angiogenesis is bone is primarily driven by osteoblasts
Response to external factors eg low oxygen in a fracture or drop in eastrogen due to menopause
Influences VEGF which influence endothelial cells which influence vasculature
Parancrine relationship
Bone structure: cortical porosity
Low ct - slices of photos for 3D image (bone shape)
High res ct/SEM (bone cortex)
Whole bone > osteonal microstructures > lamellae > extra cellular matrix > fibril
29 yr old lots of hersian channels, age = increase in porosity
Expansion of hole, is there a blood vessel, is it functioning etc. can’t see in humans using imaging
Have to move to mice - 18 months, greater porosity
Examining blood vessel structure in adult murine bone by synchrotron and lab uCT analysis
Low resolution - miss loads of info
Syncatron light sources - higher resolution using radiation, can see the pores in bone (0.65um resolution)
Stack of images (1000) to created 3D stack, binary images, separate big and small pores, to quanitigy different structures
See differences in disease
VEGF DELETION Model
Osteocalcin VEGFKO mouse
Looked at vascular structures and bone matrix
Cre-Lox system - floxed allele, mouse with cre recombinase enzyme (driven by osteocalcin) expressed under control of transgenic promoter. Breed to create line that carries both - tissue specific deletion of floxed allele in tissue where cre recombinase is expressed (deleted in only osteoblast cells)
VEGF is released by osteoblasts due to hypoxia, mechanical strain and growth factors
VEGF bind to VEGF receptors on blood vessels, blood vessels produce factors that couple with bone formation to ensure we get angiogenesis and bone formation in coordinated way
Sexual dimorphism of cortic bone following OcnVEGF deletion
Adult female mouse vs knock out (not too bad)
Adult male mouse vs knock out (much more severe phenotype, increased bone porosity)
Clinically reduced VEGF in blood associated with osteoporosis
Sexual dimorphism in cortical porosity following deletion of VEGF (engineered)
Male much worse than females
Can bone matrix signatures influence sex specific vascular heterogeneity
Surrounding blood vessels in male knock out is unmineralised matrix (imagining)
VEGF KO in bone, dysfunctional blood vessel and then blood vessel cant mineralise the bone properly so mainly collagen based matrix
Sec effects of VEGF deletion on ECM occur at lvl of osteoblasts
ACP - immature matrix
CAP - mature matrix
Knock out male composition of matrix compared to femal knock out matrix- female have way more mature matrix present (CAP), male has more immature (ACP)
Sexually dysmorphia effect of VEGF deletion in endothelial cell function
Grew osteoblasts from male and female knock out mice
Condition media put on endothelial cells
Gene expression was sex specific eg increase in igf1 male had decrease of igf1
Effect of of VEGF DELETION IN BONE IN MALES AND FEMALES
Females conserved mature phenotypes so maintained skeletal integrity
Male had more immature phenotypes in matrix and had increase porosity and fractures
Translational benefits of targeting microcirculation
Sex specific mechanism controlling matrix production will impact cortical porosity and fracture risk with age
Stratified personalised approaches to fracture prevention and treatment (target vasculature therapies for males and females)
Sex bias towards women around development of OP drugs (because menopause) - health inequality
Need to improve understanding of male and female bone cell biology
Tibial macro and microstructure adaptations to prolonged arduous military training in women
High resolution scanning - basic training -14 weeks x 3
Look at bone vasculature to see differences over time and sex differences
Women more like to get fractures than men during training have to be removed from training. Why?
Army policy - same training
Women - more injuries
Men discharges due to overuse injury eg stress fractures - below 1.5%
Women - 4.6 to 11.1%
Same in athletic populations
Training = rapid adaptations of tibial density and geometry (14weeks)
Extract blood vessels - binary images
Men = higher cortical tissue volume
Significant difference between both cortical tissue volume and canal density between men and women
Fracture model - had higher cortical tissue volume, canal number debsity and volume density
Army study conclusion
Amend training to those more prone
Women more likely to fractures
Sex bias - health inequalities
