1. Musculoskeletal Integrity Program Research Opportunities Adele L Boskey, PhD Director, Musculoskeletal Integrity Program (HSS) Starr Chair in Mineralized Tissue Research (HSS) Professor, Program in Physiology, Biophysics, and Systems Biology (Weill) Professor, Biochemistry (Weill) Professor, Bioengineering Field (Cornell, Ithaca)

2. Laboratories in the Program Mineralized tissue laboratory Adele L Boskey Spectroscopy and imaging laboratory Nancy Pleshko Camacho Molecular Imaging laboratory Phillip Mayer-Kuckuk Biomechanics and biomaterials laboratory Timothy M. Wright

3. Overall Goal Understand mechanism of vertebrate biological calcification in health and disease : www.farnes.com/ bone_density.htmwww.farnes.com/ bone_density.htm

4. Diseases of Interest Osteoporosis Osteogenesis Imperfecta Osteoarthritis Juvenile dermatomyositis Skeletal dysplasias

5. Research Questions What causes mineral to form at specific sites in mineralizing tissues? How do extracellular matrix proteins affect the formation and proliferation of mineral crystals? Which genes regulate initial mineral deposition? Mineral crystal growth? How do mineral and matrix properties change in osteoporosis (with and without treatment)? What molecular changes occur in bone and cartilage during aging and in disease? How can we apply the concepts learned from in vitro analyses to tissue engineering of bones, teeth and cartilage?

6. Biomineralization: The cell mediated process by which insoluble inorganic materials are deposited by a living organism. Physiologic Dystrophic / Aberrant Apatite in vertebrate bone, calcified cartilage, dentin, cementum, tendon-ligament insertions, enamel, Calcite & Aragonite in Shells Silicates in Diatoms Iron oxides in magneto- bacteria, etc. Apatite in atherosclerotic plaque, prosthetic heart valves, tumoral calcinosis, kidney and salivary stones, JDM deposits, and other ectopic calcification. Calcite in pancreatic stones Iron oxides in treated thalessemia patients.

7. Common Features of Biomineralization The mineral may be amorphous or crystalline Mineral deposits in an oriented fashion on an oriented matrix (template) Cells and matrix regulate the mineralization process Matrix proteins associated with biomineralization are anionic

8. Bone and Dentin: Mineralization Facts The mineral is a poorly crystalline analog of hydroxyapatite Mineral crystals deposit in an oriented fashion on an oriented collagen matrix Mineralization is regulated by osteoblasts and Noncollagenous matrix proteins. The functions of the matrix proteins are redundant. Physicochemical effects (Ca*P, P/PP) important Debate- ACP? OCP? other Debate – do phosphoproteins simply regulate Pi levels or do they have other function?

9. Ca 10 (PO 4 ) 6 (OH) 2 Ca P O H

10. Why some collagenous tissues mineralize while others do not? Exposure and/or modification of nucleators Removal or modification of inhibitors

11. Bone and Dentin ECM protein Families associated with Mineralization SIBLINGs SLRPS Gla-Proteins Phospholipids (Enzymes)

12. To prove a Macromolecule is Important for Mineralization Solution Cell Culture Animal Models Human Diseases Structural Present or modified at calcification front

13. Hypothesis Post-translational modifications of extracellular matrix proteins enables them to regulate the mineralization process in bones, teeth, and other tissues.  Fragmentation  Phosphorylation  Dephosphorylation  (Interaction with collagen)

14. SIBLINGS (Small Integrin Binding Ligand N-linked Glycoproteins) OPN, DMP-1, DSP/DPP (dspp), BSP, MEPE (matrix extracellular phosphorylated glycoprotein). found on human chromosome 4. sialic acid rich, low pI, one or more integrin binding domains, and phosphorylation sites. have in vitro and in vivo effects on mineralization.

15. ASARM peptides The SIBLINGs all contain domains with an acidic serine-aspartate-rich motif (ASARM). These domains are thought to interact with HA crystals in regulation of the mineralization process.

16. Bone, Dentin, and Cartilage Matrix Protein Regulators of Mineralization phosphophoryn DSP Pre-biglycan Cleaved proteins with significant effects nucleators inhibitors OPN phosphophoryn Intact MEPEASARM peptide MEPE dspp DMP157K + 37K DMP1DMP1 N term peptides biglycan Pepsin/MMP4&7 BMP1

17. Bone, Dentin, and Cartilage Matrix Protein Regulators of Mineralization phosphophoryn DSP Pre-biglycan Cleaved proteins with significant effects nucleatorsinhibitors OPN phosphophoryn Intact MEPEASARM peptide MEPE dspp DMP157K + 37K DMP1DMP1 N term peptides biglycan Pepsin/MMP4&7 BMP1

19. Small Leucine Rich Proteoglycans Associated with Mineralization Biglycan nucleator Decorin inhibitor Osteoadherin ? Osteoglycin ?? (culture inhibitor) Fibromodulin ? Lumican ?

20. Cell Culture Investigations Chick limb-bud mesenchymal cells differentiate in micro- mass culture forming chondrocyte nodules and a matrix resembling the chick growth plate. The matrix forms “physiologic mineral” in the presence of 4 mM inorganic phosphate (4P). Chondrocyte nodules d12 mineralizing culture- type X collagen antibody. (whole mount )

21. Selected Genes associated with Mineralization in Chick limb-bud cultures UP REGULATED Calbindin (20x) Osteoglycin (24x) SPP2 (30X) NaPi Co-transporter (42x) Matrilin (10x) DOWN REGULATED Aggrecanase (12x) Thrombospondin 4 COMP

22. 2000 1800 1600 1400 1200 1000 800 Phosphate Amide I Absorbance Wavenumber (cm -1 ) FTIR Imaging

23. IR Spectrum of Bone 200018001600140012001000800 Phosphate Amide I Absorbance Amide II C O N H Stretching Bending symmetric stretch ( 1 ) antisymmetric stretch ( 3 ) Carbonate 200018001600140012001000 Amide I Amide II

24. Cortical Bone Spectra -60 -40 -20 0 20 40 60 0 1 2 3 4 800 1000 1200 1400 1600 1800 2000 CO 3 2- Amide IAmide II PMMA PO 4 3- OSTEONAL CENTER WAVENUMBERS (cm -1 ) DISTANCE (  m) -60 Paschalis et al Bone, 1997

25. Methods for Deconvoluting Phosphate Spectra Second derivatives and curve-fitting 2D spectroscopy Principal Component Analysis Factor Analysis Cluster Analysis

26. Parameters for HA containing Materials Mineral:Matrix Ratio Carbonate:Phosphat e Ratio Type of Carbonate Substitution Crystallinity Collagen Maturity Parameters validated by independent assays D21 chick limb- bud mineralizing culture

27. Osteoporosis Studies Material properties (determined by FTIRI) predict why individuals with the same BMD do or do not suffer fragility fractures. Human studies – Baboon studies – age changes Sheep studies - therapies

29. Baboon data Age (yrs) % distance from osteon center Samuel Gourion

30. 10 % 50 % 100 %

31. – Mineral & Matrix Changes in Osteoporosis – Dan Faibish XLR 9/104/51/109/104/5# fractures

32. 0.5 1 1.5 2 2.5 3 Placebo 6week Treated 6week 0.1 FT-IRIS(PG) Alcian Blue Infrared Imaging Spectroscopy & Fiber Optics of Cartilage Repair and Degeneration Musculoskeletal Imaging & Spectroscopy Lab, P.I. Nancy Pleshko Camacho

33. IR fiber optic probe and spectrometer Normal cartilage Degraded cartilage Musculoskeletal Imaging & Spectroscopy Lab, P.I. Nancy Pleshko Camacho PLS Chemometric Analysis Mankin Grade

34. Techniques Routinely Used Analytical chemistry X-ray diffraction Mechanical testing Micro-computed tomography Cell culture – including siRNA Infrared Imaging, Raman Imaging, Fiber-optic Probe In vivo molecular imaging

35. NIH Support DE04141 AR037661 AR043125 AR046121