The role of mechanical stimuli induced by prenatal movements in skeletal development

Transcription

1 The role of mechanical stimuli induced by prenatal movements in skeletal development Niamh Nowlan, PhD Department of Bioengineering, Imperial College London

2 Mechanobiology of Bone

3 Mechanoregulation of Skeletogenesis Rodriguez et al., JBJS, 1988

4 Developmental Mechanics (Entwicklungsmechanik) the goal of developmental mechanics (is) the ascertainment of formative forces or energies (Roux, 1894) Roux, W., Arch. Entwicklungsmech. Org. 1: Wilhelm Roux ( )

5 Developmental Mechanics (Entwicklungsmechanik) the goal of developmental mechanics (is) the ascertainment of formative forces or energies (Roux, 1894) To think that heredity will build organic beings without mechanical means is a piece of unscientific mysticism (His, 1888) Wilhelm His ( ) His, W., Proc. Roy. Soc. Edinburgh 15:

6 Developmental Mechanics (Entwicklungsmechanik) Wilhelm His ( ) His, W., Proc. Roy. Soc. Edinburgh 15:

7 Genetics Existence of genes; Gregor Mendel, 1866 Location of genes on chromosomes; Thomas Hunt Morgan, 1910 Structure of DNA; Watson & Crick, 1953 Watson, JD, Crick, FHC, Nature 171, pp

8 Research Approach Rot-Nicevic et al., 2006 Control Immobilised Animal Models of Abnormal Prenatal Movement 3-D Imaging of Embryonic Bones and Joints Finite Element Analysis

9 Relevance Treatment of congenital neuromuscular diseases alleviate or minimise effects on skeletal development Inform tissue engineering of cartilage and bone What biophysical stimuli needed for bone and/or articular cartilage? What cellular or genetic factors translate biophysical stimuli to developmental change?

10 Bone Development Mammalian Avian Fell, H.B., J. Morph. & Physiol., Hall, B.K., Clin. Orthop. Relat. Res., 1987

11 Part 1: Mechanoregulation of Bone Development in a Chick Immobilisation Model System

12 Part 1: Bone Development in Immobilised Chicks Hypothesis Mechanical forces induced by embryonic muscle contractions affect the development of skeletal tissues and structures through the action of mechanosensitive genes

13 Part 1: Bone Development in Immobilised Chicks Mechanosensitive Genes Genes which are up- or down- regulated as a result of a change in the mechanical environment Provot & Schipani, Biochem. Biophys. Res. Comm., 328(3), pp

14 Part 1: Bone Development in Immobilised Chicks Methods 1. Characterise biophysical stimuli in the embryonic limb using finite element (FE) analysis 2. Characterise expression patterns of candidate mechanosensitive genes involved in bone formation - Compare with patterns of biophysical stimuli 3. Examine effect of altered mechanical environment on mechanosensitive gene expression patterns

15 Part 1: Bone Development in Immobilised Chicks ¹Sharpe et al., 2002, Science 296, pp

16 Part 1: Bone Development in Immobilised Chicks Cartilage (Alcian Blue Staining) HH30 HH32 HH34 2.4mm 4.2mm 5.9mm

17 Part 1: Bone Development in Immobilised Chicks Muscle (Anti-myosin antibody staining) HH30 HH32 HH34 1 mm

18 Part 1: Bone Development in Immobilised Chicks Tendon (Scleraxis in situ)

19 Part 1: Bone Development in Immobilised Chicks Methods: Muscle load application HH32, extension contraction Muscle CSA Force Dorsal_ mm mn Dorsal_ mm mn Dorsal_ mm mn

20 Part 1: Bone Development in Immobilised Chicks Results: Shear Strain & Fluid Vel. Octahedral Shear Strain Fluid Velocity

21 Part 1: Bone Development in Immobilised Chicks HH30

22 Part 1: Bone Development in Immobilised Chicks HH30

23 Part 1: Bone Development in Immobilised Chicks HH32

24 Part 1: Bone Development in Immobilised Chicks HH34

25 Part 1: Bone Development in Immobilised Chicks Methods: Immobilisation Decamethonium Bromide (DMB) used to prevent muscle contractions in ovo

26 CONTROL (HH36) R J IMMOBILISED (HH36) S

27 Part 1: Bone Development in Immobilised Chicks Effect on Bone Collar Formation

28 Part 1: Bone Development in Immobilised Chicks

29 Part 1: Bone Development in Immobilised Chicks Effect on Indian Hedgehog Expression

30 Part 1: Bone Development in Immobilised Chicks Results on Collagen X Expression

31 Part 1: Bone Development in Immobilised Chicks Discussion: Chick Model Correlation between Ihh, ColX expression and patterns of biophysical stimuli No correlation between FGFr2 and PTHrP ColX and Ihh expression altered by immobilisation ColX and Ihh may be key mediators in translating information from the mechanical environment to the molecular regulation of bone formation in the embryo

32 Part 2: Skeletal Development in Muscleless Limb Mice

33 Part 2: Skeletal development in muscleless mice Hypothesis Embryonic muscle contractions are critical to normal development of skeletal tissues and structures in embryonic mice

34 Part 2: Skeletal development in muscleless mice Methods Altered mechanical environment induced in muscleless limb genetically modified mice Splotch(Pax3 Sp/Sp ) No limb muscle precursor cells and therefore no skeletal muscle[1] MyoD/Myf5 knockouts Muscleless: limb muscle precursor cells migrate but do not differentiate in doubleknockout(myf5 (nlacz/nlacz) :MyoD ( / ) )[2] Reduced muscle: if one copy of Myf5 present (Myf5 (nlacz/+) : MyoD ( / ) ) amountofmuscleisreducedby35-55%[2] TheilerStage(TS)23(e14.5)mainstageofinterest Initiation of major long bone ossification sites [1] Tajbakhsh et al., Cell, 1997; [2] Rudnicki et al., Cell, 1993.

35 Part 2: Skeletal development in muscleless mice Methods Stained for cartilage and mineralised tissue with Alcian Blue & Alizarin Red Measurements of 5 rudiments: length, length of bone, & mineralised proportion Optical Projection Tomography [1] used for 3-D imaging of control and mutant limbs Histological Analysis: Weigert's Iron Hematoxylin, Fast green and Safranin- O used to stain for cartilage and bone [1] Sharpe et al., Science 296: pp

36 Results: Gross Skeletal Morphology Abnormal ossification in scapula and humerus Delayed bone formation in scapular blade but not in scapular spine Irregular pattern of bone formation in humerus Apparently normal morphology in hindlimbs reduced muscle forelimbs & hindlimbs Part 2: Skeletal development in muscleless mice

37 Part 2: Skeletal development in muscleless mice Results: Abnormal Joint Formation

38 Part 2: Skeletal development in muscleless mice Results: Bone Initiation & Progression

39 Part 2: Skeletal development in muscleless mice Bone Proportion: Muscleless (Myf5 (nlacz/nlacz) : MyoD ( / ) ) and Reduced Muscle (Myf5 (nlacz/+) : MyoD ( / ) ) Littermates Scapula Humerus Ulna Femur Tibia

40 Part 2: Skeletal development in muscleless mice Conclusions: Part 1 Hypothesis partially corroborated: embryonic muscle contractions critical to normal development of some skeletal tissues& structures Skeletal rudiments differentially affected by lack of, or reduced, muscle Why the differential effects? Biochemical factors? someregionsmaybemoresensitivetothelackofdiffusiblefactors from the absent muscle bodies Biomechanical factors? different levels of mechanical forces could exist in different regions of skeleton in normal or mutant embryos

41 Part 3: Biomechanical role in the Differential Effects in Muscleless Limb Mice

42 Part 3: Biomechanical role in differential effects Hypothesis Differences in the local mechanical environment are responsible for differential effects in muscleless limb mice

43 Part 3: Biomechanical role in differential effects Methods Compare mechanosensitive gene expression patterns Compare patterns of biophysical stimuli

44 Part 3: Biomechanical role in differential effects Mechanosensitive gene analysis FGFr3 and ColX Provot & Schipani, Biochem. Biophys. Res. Comm.

45 Part 3: Biomechanical role in differential effects Methods in situ hybridisation performed on sections of femora and humeri of muscleless limb mice and normal littermate controls Myf5 nlacz/nlacz :Myod / and Pax3 Sp/Sp at (mostly) TS23 more extreme change in expression in humerus than in femur would indicate potential difference in mechanical environment between two regions

46 Part 3: Biomechanical role in differential effects Gene expression more severely affected in humerus than in femur

47 Part 3: Biomechanical role in differential effects Methods Compare mechanosensitive gene expression patterns Compare patterns of biophysical stimuli

48 Part 3: Biomechanical role in differential effects Patterns of Biophysical Stimuli Muscle Forces Limb Movements DeLaurier et al., 2008, BMC Dev Biol Normal mice only Compare humerus and femur Active movements in normal mice only Passive movements in muscleless mutants Compare forelimb and hindlimb

49 Part 3: Biomechanical role in differential effects Methods: Geometry Limbs stained for cartilage and bone using Alcian Blue and Alizarin Red 1 Optical Projection Tomography (OPT) 2 used to obtain 3-D geometries of limbs 1. Nowlan et al., 2005, J. Biomechanics, 2. Sharpe et al., 2002, Science

50 Part 3: Biomechanical role in differential effects Methods: Geometry TS22 TS23 TS24 Forelimb Hindlimb

51 Methods: Geometry Rhino and Cubit used to create meshes Part 3: Biomechanical role in differential effects

52 Part 3: Biomechanical role in differential effects Methods: Forces Muscle Forces Muscles visualised with OPT scans of myf-5 stained limbs Muscles and attachment sites identified using Mouse Limb atlas¹ Muscle forces estimated from cross sectional areas Limb Movements 10 µm displacement to distal end of limb 1µm, 50µm and 5mN also examined 1. DeLaurier et al., 2008, BMC Developmental Biology

53 Part 3: Biomechanical role in differential effects Methods: Muscle Forces TS22 TS23 TS24 Forelimb Hindlimb

54 Part 3: Biomechanical role in differential effects Methods: Boundary Conditions Muscle Forces Limb Movements

55 Part 2: Biomechanical role in differential effects Methods: Material Properties Muscle Forces Limb Movements Poroelastic material properties of murine embryonic cartilage and mineralised tissue: Tanck et al., 2004, Bone. Presumptive joint regions: Roddy et al., 2011, J. Biomechanics

56 Part 3: Biomechanical role in differential effects Results: Muscle Forces Similar patterns of biophysical stimuli between humerus and femur Low stimuli values induced by muscle forces

57 Part 3: Biomechanical role in differential effects Results: FE of Whole Limbs Higher levels of biophysical stimuli induced in hindlimb than in forelimb by 10µm displacement Stimuli magnitudes at least ten times greater than those directly induced by muscle contractions

58 Part 3: Biomechanical role in differential effects Conclusions: Part 3 Mechanosensitive gene expression patterns indicate a difference in the underlying mechanical environment between developing humerus and femur In mammalian embryos, limb movements rather than the direct application of muscle loads likely to drive mechanoregulation in normal mice In muscleless limb mice, passive movements compensate for lack of active movements, but to different degrees depending on region of the limb

59 Conclusion: Passive Movement may be Responsible for Differential Effects in Muscleless Mice Chick Mouse from Nowlan, Sharpe et al., 2010, Birth Defects Research Part C: Embryo Today

60 Future Work: The Role of Prenatal Movement in DDH Rot-Nicevic et al., 2006 Control Immobilised Animal Models of Abnormal Prenatal Movement Developmental Dysplasia of the Hip Therapeutic Passive Movement

61 Acknowledgements Collaborators James Sharpe(Centre for Genomic Regulation, Barcelona, Spain) Patrick J. Prendergast & Paula Murphy (Trinity College Dublin, Ireland) Shahragim Tajbakhsh(Pasteur Institute, Paris, France) Funding sources