Bioceramics Lecture 5 Bones and Teeth: Composites of Ceramics and Proteins

Bioceramics Lecture 5 Bones and Teeth: Composites of Ceramics and Proteins Dr. rer. nat. Michael Maas Prof.Dr.-Ing. Kurosch Rezwan Keramische Werkstoffe und Bauteile - Advanced Ceramics Am Biologischen Garten 2, IW3 D - 28359 Bremen Tel: +49 421 218 64939 Fax: +49 421 218 64932 http:/// 1

Ø Course Outline Bioceramics 1. Introduction: Applications, Goals and Challenges 2. Biochemistry Basics 3. Ceramics at the Biology Interface: Fundamental Interactions 4. Characterization of Biomolecule Adsorption 5. Bones and Teeth: Composites of Ceramics and Proteins 6. Ceramics for Orthopedic and Dental Implants: Alumina and Zirconia 7. Scaffolds for Bone Tissue Engineering 8. Diamond and other Ceramic Biomaterials 9. Drug Delivery Using Ceramic Nanoparticles 10. Biomineralization 11. Biomimetic Materials 12. Student Talks on Selected Topics 13. Summary The summary lecture will be right before the exams. 2

Ø Sit in on a live orthopedic surgery at Klinikum Bremen Mitte! Dr. Thies,Chirung Klinikum Bremen Mitte Completely optional, but registration is binding. Basic German is required! 1.6. Hüft-TEP, 2.6.Hüft-TEP. 4.6. Hüft-TEP, 9.6, Knie-TEP 8:00 at Klinikum Mitte, registration desk (Pforte) 3

Ø Lecture Outline Bone Structure and Composition The Bone Remodeling Process Mechanical Properties of Bone Teeth 4

Ø Tropo-Collagen 3 protein subunits triple helical conformation high hydroxyproline content 25 35 % whole body protein content 200-1000 amino acids per monomer pi: 4.7 5

Ø Collagen Fibrils Kadler, K. E.; Holmes, D. F.; Trotter, J. A.; Chapman, J. A. Biochem J 1996, 316, 1 11. 6

Ø Example: Collagen Schematic view of some of the hierarchical features of collagen, ranging from the amino acid sequence level at nanoscale up to the scale of collagen fibers with lengths on the order of 10 µm. [ Bühler M. J., PNAS, 2006 ] 7

Ø Hydroxyapatite HA nanopowder HA nanoplatelets in bone geological HA crystal Hydroxy(l)apatite (HA): Formula: Ca 10 (PO 4 ) 6 (OH) 2 soluble at ph < 5.5 8

mineralized collagen. The fibril is sectioned through the xy plane, revealing plate-shaped apatite crystals (coloured in pink) embedded matrix. Scale bars: 100 nm. Ø Mineralization of Bone Tissue a b c Nudelman, F. et al, Nat Mater 2010 Figure 2 CryoTEM images of collagen at different stages of mineralization in the presence of 10 µg ml 1 of pasp. a, Mineralization f b, Mineralization for 48 h. c, Mineralization for 72 h. Scale bars: 100 nm. Artificial incorporation CaP into collagen fibrils the difficult banding but possible pattern along via PILP theprocess 67 nm repeat / control matched of with the Fig. S8). This site possesses the lowest electrostat positions pre-nucleation of theclusters. charged amino acids in the crystal structure of in the microfibril for interaction with the n collagen 5,24 (Fig. 3a,b, Supplementary S8, Fig. S7). Furthermore, the complex (Fig. 4b) and is therefore the most favo staining did not affect the mineral phase, because the same degree an attractive interaction with the negatively char of mineralization was observed as for unstained samples (Fig. 2). suggests that the attraction between these posit After 24 h of mineralization, ACP was observed surrounding and the negatively charged calcium phosphate pa and entering the fibril, associated with the a-bands (Fig. 1b, black a critical role in mediating the entry of the ACP int 9 circle and Fig. 3b e). These bands are 9 nm wide and span Uranyl acetate staining was also used to id both the overlap and gap zones at the C-terminal region of the nucleation sites within the collagen fibril. Apati Olszta et al, Materials Science and Engineering: R, 2007

Ø Bone Composition 1.5 % Non-collagenous proteins: osteocalcin osteonectin bone proteoglycan bone sialoprotein morphogenetic protein proteolipid 28.5 % Collagen Type I Bone Composition? 66.5 % Hydroxylapapite (mainly Calcium Phosphate) 3.5 % Chloride, Fluoride [ Felsenberg, D., Pharmazie in unserer Zeit, 2001 ] 10

Ø Other Matrix Proteins [ Young, M. F.(2003). Osteoporos Int 14 Suppl 3: S35-42 ] 11

Ø The role of Calciumphosphate: Mechanical Stiffness What happens if we remove Collagen? 12

Ø The role of Collagen: Elasticity What happens if we remove CaP? 13

Ø Hierarchical Structure of Bone Osteons (d ~ 100 µm) Collagen fibril (d ~ 500 nm) consisting of collagen molecules with embedded HAp-crystals (blue, d ~ 20 nm) Macroscopic bone Collagen fiber (d ~ 5 µm) consisting of Collagen fibrils Collagen triple helix (d ~ 1.5 nm) 1 cm 1 mm 1 µm 100 nm 1 nm Bones can be seen as nanocomposite materials of Calcium Phosphate and Collagen! 14

Ø The Skeleton Functions:? Stability of the Body Protection of Inner Organs Storage/Exchange of Minerals (mainly Calcium and Phosphate) Production of most Blood Cells Total Number of Bones: 208-214 Total Average Weight =? Total Average Weight 7 kg!!! 15

Ø Bone and Surrounding Tissues Adipose Tissue Blood Bone Cartilage Loose connective tissue Fibrous connective tissue 16

Ø Structure of Articular (Joint) Cartilage Chondrocytes 17 http://ajs.sagepub.com/content/26/6/853.full

Ø Bone Structure: Cortical and Spongy Bone ( Spongiosa ) Epiphysis Metaphysis Small vein Capillary Circumferential lamellae Osteons Periosteum Interstitial lamellae Diaphysis See Fig. 1.5 See Fig. 1.3 Metaphysis Epiphysis (a) Articular cartilage (b) Spongy bone 18

Ø Bone Structure: Spongiosa 3D Computertomograph of the spongiosa, cube size approx. 5x5x5 mm 3 Microtom of femur head showing spongiosa region. Environmental scanning electron micrograph of broken trabecula. 19

Ø Bones 20

Ø Classification of Bones Bones are classified as long bones ( Röhrenknochen ) short and sesamoid bones flat bones ( Plattenknochen ) irregular bones 21

Ø Long Bones ( Röhrenknochen, Ossa longa) Long bones are "long": their length is superior to their width. Long bones consist of a shaft (=diaphysis) and two expanded ends (=epiphysis, plural: epiphyses) that articulate with other bones. Epiphysis Metaphysis Diaphysis See Fig. Metaphysis Epiphysis (a) Articular cartilage (b) 22

Ø Short and Sesamoid Bones (Ossa brevia and ossa sesamoidea) Short bones are "short": cubelike. They do not have any cavity similar to the medullary cavity of the long bones. Short bones are made mostly of spongy bone tissue, but their outer parts are made of a thin crust of compact bone tissue. Sesamoid bones are a special type of short bones: they are embedded within a tendon and act to alter its direction of pull. They differ in size, shape and quantity from an individual to another. 23

Ø Flat Bones ( Platte Knochen, Ossa plana) Flat bones are "flat", platelike. They are thin and do not have any cavity similar to the medullary cavity of the long bones. The outer part of a flat bone is made of a layer of spongy bone tissue sandwiched between two layers of compact bone tissue. 24

Ø Irregular Bones ( Irreguläre Knochen, Ossa irregularia) Irregular bones are all the weird-shaped bones that do not belong in any other category. They do not have any cavity similar to the medullary cavity of the long bones. Irregular bones are made mostly of spongy bone tissue enclosed by a thin crust of compact bone tissue. 25

Ø Lecture Outline Bone Structure and Composition The Bone Remodeling Process Mechanical Properties of Bone Teeth 26

Ø Force Lines in the Femur Head ( Oberschenkelknochenkopf ) Bone Material is continuously remodeled in order to match different mechanical loads! 27

Ø Osteoblasts and Osteoclasts Osteoclast (Schematic) A multinucleated cell whose responsibility is to resorb bone. lysosome multiple nuclei 70 µm Osteoblast (Light Microscope) nuclei Cells responsible for laying down the protein matrix upon which calcium salts, particularly calcium phosphates, are deposited to form bone. tight seal to matrix bone matrix ruffled border of osteoclast 10 µm 28

Ø Bone Remodeling: Stimulation and Growth Factors http://depts.washington.edu/bonebio/asbmred/hormones.html 29

Ø Physiology of an Osteoclast and Bone Resorption carbonic anhydrase II ph drops down to 3-4 30

Ø Osteoclast Sealing Zone: Bone Resorption 31

Ø The Bone Remodeling Process (nonmineralized Collagen) The Osteoblasts and Osteoclasts at work are called Basic Multicellular Unit 32

Ø Basic Multicellular Unit at Work 1. Bone resorption by Osteoclasts (Black with red dots) 2. Osteoblasts (blue) deposit Osteoid (nonmineralized collagen, pink) 3. Mineralization of collagen with Calcium and Phosphate Ions (light green). http://depts.washington.edu/bonebio/asbmred/growth.html 33

Ø Many Basic Multicellular Units at Work At any time on average 20 % of the Spongiosa surface area undergoes remodeling! http://depts.washington.edu/bonebio/asbmred/growth.html 34

Ø Basic Multicellular Unit at Work in Real Life Osteoblasts Bone Marrow Cells Osteoclasts Osteoid Bone 35

Ø Basic Multicellular Unit at Work in Real Life Photomicrograph showing BMU (Basic Multicellular Unit ) along a trabecular surface. At left is osteoclastic resorption ( cutting edge ) and at the right are osteoblasts and osteoid. Osteoid = non mineralized collagen 36

Ø Remodeling of the Cortical Bone Osteclasts acting together in a small group excavate a tunnel through the old bone, advancing at a rate of about 50 μm per day. Osteoblasts enter the tunnel behind them, line its walls, and begin to form new bone depositing layers of matrix at a rate of 1-2 μm per day. At the same time, a blood capillary sprouts down the center of tunnel. 37

Ø Old and new Cortical Long Bone A transverse section through a compact outer portion of a long bone. The micrograph shows the outlines of tunnels formed by osteoclasts and then filled in by osteoblasts during successive rounds of bone remodeling. Note how older systems of concentric layers of bone have been partly cut through and replaced by newer systems. Small vein Capillary Circumferential lamellae Osteons Periosteum Interstitial lamellae See Fig. 1.5 Spongy bone 38

Ø Basic Multicellular Unit at Work: Animation http://courses.washington.edu/bonephys/gallery/bmuremodel.swf 39

Ø Lecture Outline Bone Structure and Composition The Bone Remodeling Process Mechanical Properties of Bone Teeth 40

Ø Effect of Different Mechanic Loads: Bone Densities in Athletes http://depts.washington.edu/bonebio/asbmred/exercise.html http://depts.washington.edu/bonebio/bonstrength/exercise/sports.html 41

Ø Bone Mechanics of Cortical Bone and Spongiosa Cortical Bone Spongiosa Fracture Toughness of Bone: 4 6 MPa. m 0.5 42

Ø Bone Ageing http://newscenter.lbl.gov/newsreleases/2011/08/29/thebrittleness-of-aging-bones- - more-than-a-loss-of-bone-mass/ bone mineral density Young adult Elderly adult with osteoporosis 43

Ø Lecture Outline Bone Structure and Composition The Bone Remodeling Process Mechanical Properties of Bone Teeth 44

Ø Teeth Schematics 45

Ø Teeth Schematics 46

Ø Enamel (Zahnschmelz) Enamel: Hardest material in human body Highly mineralized crystalline structure 95-98% inorganic matter by weight hydroxyapatite (HA) is largest mineral constituent (90-92% by volume) 1-2% by weight organic content 4% by weight water Enamel rods: about 4-5 µm thick and up to 3 mm in length. 47

Ø Dentin 48 (a) Scanning electron microscope (SEM) image of the pulp chamber dentinal wall of mouse molar. Pulp tissue, odontoblasts, and predentin have been mechanically removed, exposing the tubule openings. Magnification = 1,000 ; bar = 10 μm. (b) Number and radius of tubules with respect to the coronal dentin depth in human teeth. Tjäderhane, L.; Carrilho, M. R.; Breschi, L.; Tay, F. R.; Pashley, D. H. Endodontic Topics 2009, 20, 3 29.

Ø Dentin Composition similar to bone: 70 % HAp, 20 % organics (90 % collagen), 10 % water SEM images of resin-embedded, acid-etched dentin (a) and mandibular bone (b), demonstrating marked similarity between the odontoblast process and osteocyte lacunocanalicular networks. 49 Tjäderhane, L.; Carrilho, M. R.; Breschi, L.; Tay, F. R.; Pashley, D. H. Endodontic Topics 2009, 20, 3 29.

Ø Comparison of Teeth, Bone and Hydroxyapatite Properties Zahnschmelz Zahnbein Knochen Hydroxyl- (Enamel) (Dentin) apatit Calcium (g) 36,5 35,1 34,8 39,6 Phosphor (g) 17,7 16,9 15,2 18,5 Ca/P (molares Verhältnis) (g) 1,63 1,61 1,71 1,67 Natrium (g) 0,5 0,6 0,9 - Magnesium (g) 0,44 1,23 0,72 - Kalium (g) 0,08 0,05 0,03 - Carbonat (als CO 3 2- ) (n) 3,5 5,6 7,4 - Fluorid (g) 0,01 0,06 0,03 - Chlorid (g) 0,30 0,01 0,13 - Pyrophosphat (als P 2 O 7 4- ) (n) 0,022 0,10 0,07 - Gesamt anorganisch (n) 97 70 65 100 Gesamt organisch (n) 1,5 20 25 - Wasser (n) 1,5 10 10 - Gitterparameter (hexagonale Aufstellung) 50

Magnesium (g) 0,44 1,23 0,72 - Kalium (g) 0,08 0,05 0,03 - Ø Comparison of Teeth, Bone and Hydroxapatite Compositions Carbonat (als CO 3 2- ) (n) 3,5 5,6 7,4 - Fluorid (g) Zahnschmelz 0,01 Zahnbein 0,06 Knochen 0,03 Hydroxyl- - Chlorid (g) (Enamel) 0,30 (Dentin) 0,01 0,13 apatit - Calcium (g) 36,5 35,1 34,8 39,6 Pyrophosphat (als P 2 O 4-7 ) (n) Phosphor (g) 0,022 17,7 0,10 16,9 0,07 15,2-18,5 Gesamt anorganisch (n) Ca/P (molares Verhältnis) (g) 97 1,63 70 1,61 65 1,71 100 1,67 Gesamt organisch (n) Natrium (g) 1,5 0,5 20 0,6 25 0,9 - - Wasser (n) Magnesium (g) 1,5 0,44 10 1,23 10 0,72 - - Gitterparameter (hexagonale Aufstellung) Kalium (g) 0,08 0,05 0,03 - α-achse, Å Carbonat (als CO 2-3 ) (n) 9,441 3,5 9,421 5,6 9,41 7,4 9,430 - c-achse, Å Fluorid (g) 6,880 0,01 6,887 0,06 6,89 0,03 6,891 - Typische Kristallgröße / nm Chlorid (g) 100 µm 50 50 0,30 35 25 4 0,01 50 25 4 0,13 200-600 - Elastizitätsmodul (GPa) Pyrophosphat (als P 2 O 4-7 ) (n) 80 0,022 15 0,10 0,34-13,8 0,07 10 - Druckfestigkeit (MPa) Gesamt anorganisch (n) 10 97 100 70 150 65 100 100 Gesamt organisch (n) 1,5 20 25 - Wasser (n) 1,5 10 10 - Gitterparameter (hexagonale Aufstellung) 51

Ø Summary: Comparison Bone Mechanics with Bioactive Scaffolds Elastic Modulus [GPa] 1000 100 10 1 0,1 0,01 Challenge to all Materials Scientists: We need BETTER materials for Bone Tissue Engineering! Porous Biodegradable Composites Porous Biodegradable Polymers Dense Biodegradable Polymers Spongiosa Bone Dense Bioactive Ceramics Cortical Bone 0,001 Porous Bioactive Ceramics 0,0001 0,01 0,1 1 10 100 1000 10000 Compressive Strength [MPa] [Rezwan et al. Biomaterials. 2006] 52

Ø Course Outline Bioceramics 1. Introduction: Applications, Goals and Challenges 2. Biochemistry Basics 3. Ceramics at the Biology Interface: Fundamental Interactions 4. Characterization of Biomolecule Adsorption 5. Bones and Teeth: Composites of Ceramics and Proteins 6. Ceramics for Orthopedic and Dental Implants: Alumina and Zirconia 7. Scaffolds for Bone Tissue Engineering 8. Diamond and other Ceramic Biomaterials 9. Drug Delivery Using Ceramic Nanoparticles 10. Biomineralization 11. Biomimetic Materials 12. Student Talks on Selected Topics 13. Summary 53