Support Provides structural support for the entire body

Функции скелетной системы • Support. Provides structural support for the entire body. A framework for attachment of soft tissues or organs. • Protection. Skull around brain and inner ear; ribs, sternum, vertebrae protect organs of thoracic cavity • Leverage. Act as levers for muscles that contract and produce movement by pulling on bones via tendons. • Storage. Acts as a reservoir for calcium and phosphorous. Fat stored in marrow cavities • Blood cell production (Hematopoiesis). Bone marrow gives rise to blood cells and platelets

Форма костей • Long bones – Upper and lower limbs • Short bones – Carpals and tarsals • Flat bones - Ribs, sternum, skull, scapulae • Irregular bones – Vertebrae, facial bones

Структура длинной кости • Diaphysis – Tubular shaft that forms the axis – Composed of compact bone that surrounds the medullary cavity – Yellow marrow (fat) stored in medullary cavity. – Shaft covered with periosteum – Medullary cavity lined with endosteum • Epiphysis – Expanded end of the bone – Interior is cancellous bone (spongy bone) – Joint surface covered with articular (hyaline) cartilage – Epiphyseal line: separates diaphysis from epiphysis • Composed of hyaline cartilage • Bone growth in lengths occurs here

Мембраны костей • Periosteum – double-layered protective membrane – Outer fibrous layer is dense regular connective tissue – Inner osteogenic (bone forming) layer is composed of osteoblasts and osteoclasts – Richly supplied with nerve fibers, blood, and lymphatic vessels, which enter the bone via nutrient foramina – Secured to underlying bone by Sharpey’s fibers • Endosteum – delicate membrane covering internal surfaces of bone

Структуры короткой кости, кости неправильной формы и плоской кости • Flat Bones – No diaphysis or epiphysis – Sandwich of cancellous between compact bone – Periosteum covers outer surface while endosteum lines interior • Some flat and irregular bones of skull have sinuses lined by mucous membranes (Frontal, Maxillary, Ethmoid, and Sphenoid bones)

Костный матрикс • Dry weight = 1/3 organic and 2/3 inorganic matter • Organic matter – collagen, glycosaminoglycans, proteoglycans and glycoproteins – Gives bone is resilience, flexibility • Inorganic matter – 85% hydroxyapatite (calcium phosphate) – 10% calcium carbonate – Other minerals (fluoride, potassium, magnesium) – Gives bone its hardness • Combination provides for strength and resilience – minerals resist compression; collagen resists tension – bone adapts by varying proportions

Гистология компактной кости • Osteon = basic structural unit – cylinders formed from layers (lamellae) of matrix around central canal (osteonic canal) • collagen fibers alternate between right- and lefthanded helices from lamella to lamella – osteocytes connected to each other and their blood supply by tiny cell processes in canaliculi • Perforating canals or Volkmann canals – vascular canals perpendicularly joining central canals

Костный матрикс • If mineral removed, bone is too bendable • If collagen removed, bone is too brittle

Основные типы костных клеток Mesenchymal lineage • Osteoblasts: bone surface, predominant bone-making cells, 90% collagen I • Osteocytes: terminal osteoblasts, embedded in bone matrix, cellular processess, gap junctions, mechanotransduction • Osteoclasts: bone resorption Hematopoietic lineage • Hepatopoietic progenitors

Клетки костной ткани (1) • Osteogenic cells in endosteum, periosteum or central canals give rise to new osteoblasts – arise from embryonic fibroblasts – multiply continuously • Osteoblasts mineralize organic matter of matrix – Synthesize and secrete collagen protein and other organic compounds of matrix – Produce new bone in a process known as osteogenesis • Osteocytes are osteoblasts trapped in the matrix they formed – cells in lacunae connected by gap junctions inside canaliculi

Клетки костной ткани (2) • Osteoclasts develop in bone marrow by fusion of 3 -50 stem cells • Remove and recycle bone matrix – Secrete acid and proteolytic enzymes that dissolve matrix and release the stored minerals; called osteolysis • Reside in pits that they ate into the bone

Структура губчатой кости • Spongelike appearance formed by plates of bone called trabeculae – spaces filled with red bone marrow • Trabeculae • Oriented along stress lines • Have few osteons or central canals –No osteocyte is far from blood of bone marrow • Provides strength with little weight –Trabeculae develop along bone’s lines of stress

Развитие кости (остеогенез и оссификация) • Osteogenesis and ossification – The process of bone tissue formation, which leads to: • Formation of the bony skeleton in embryos • Bone growth until early adulthood • Bone thickness, remodeling, and repair • Begins at week 8 of human embryo development • Two major methods used: – Intramembranous ossification • Takes place in connective tissue membrane – Endochondral ossification • Takes place in cartilage – Both methods of ossification – Produce woven bone that is then remodeled – After remodeling, formation cannot be distinguished as one or other

Два способа формирования костей (окостенения, или оссификации) • Intramembranous: cranial facial bones neural crest cells, mesoderm • Endochondral: axial and limb skeleton mesoderm (sclerotome, LPM)

Молекулярные различия двух способов окостенения • Molecular differences? Intramembranous bones: Msx 1/2, Dlx 5/6 Endochondral bones: Ihh • Why the differences? Intrinsic differences? Different extracellular signals? (vasculature? )

Рост кости в длину • Appositional growth - growth in length – Interstitial growth cannot occur because matrix is solid – Occurs on old bone and/or on cartilage surface • Growth in length occurs at the epiphyseal plate • Involves the formation of new cartilage by – Interstitial cartilage growth • Closure of epiphyseal plate: epiphyseal plate is ossified becoming the epiphyseal line. Between 12 and 25 years of age • Articular cartilage: does not ossify, and persists through life

Метафизис • Regions of the Epiphyseal Plate – Zone of reserve cartilage = hyaline cartilage – Zone of proliferation • chondrocytes multiply forming columns of flat lacunae – Zone of hypertrophy = cell enlargement – Zone of calcification • mineralization of matrix – Zone of bone deposition • chondrocytes die and columns fill with osteoblasts • osteons formed and spongy bone is created

Гиалиновый хрящ • If a thin slice is examined under the microscope, it will be found to consist of cells of a rounded or bluntly angular form, lying in groups of two or more in a granular or almost homogeneous matrix. • The cells, when arranged in groups of two or more, have generally straight outlines where they are in contact with each other, and in the rest of their circumference are rounded. • Hyaline cartilage also contains chondrocytes which are cartilage cells that produce the matrix. Hyaline cartilage matrix is mostly made up of type II collagenand chondroitin sulphate, both of which are also found in elastic cartilage. • Hyaline cartilage exists on the ventral ends of ribs; in the larynx, trachea, and bronchi; and on the articular surface of bones.

Гиалиновый хрящ • Hyaline cartilage (aka “Gristle") consists of a slimy mass of a firm consistency, but of considerable elasticity and pearly bluish color. It contains no nerves or blood vessels, and its structure is relatively simple. • Except where it coats and skin the articular ends of bones, it is covered externally by a fibrous membrane, the perichondrium. This membrane contains vessels that provide the cartilage with nutrition.

Внутримембранное окостенение • • • Takes place in connective tissue membrane formed from embryonic mesenchyme Forms many flat bones of the skull, part of mandible, diaphyses of clavicles When remodeled, indistinguishable from endochondral bone.

Внутримембранное окостенение • Produces flat bones of skull and clavicle.

Эндохондральное окостенение • Begins in the second month of development • Uses hyaline cartilage as model for bone construction • Bones of the base of the skull, part of the mandible, epiphyses of the clavicles, and most of remaining bones of skeletal system

Рост кости и ремоделирование • Bones increase in length – interstitial growth of epiphyseal plate – epiphyseal line is left behind when cartilage gone • Bones increase in width = appositional growth – osteoblasts lay down matrix in layers on outer surface and osteoclasts dissolve bone on inner surface • Bones remodeled throughout life – Wolff’s law of bone = architecture of bone determined by mechanical stresses • action of osteoblasts and osteoclasts – greater density and mass of bone in athletes or manual worker is an adaptation to stress

Факторы, влияющие на рост костей • Size and shape of a bone determined genetically but can be modified and influenced by nutrition and hormones • Nutrition – Lack of calcium, protein and other nutrients during growth and development can cause bones to be small – Vitamin D • Necessary for absorption of calcium from intestines • Can be eaten or manufactured in the body • Rickets: lack of vitamin D during childhood • Osteomalacia: lack of vitamin D during adulthood leading to softening of bones; pain when wt. put on affected bone – Vitamin C • Necessary for collagen synthesis by osteoblasts • Scurvy: deficiency of vitamin C • Lack of vitamin C also causes wounds not to heal, teeth to fall out

The PTH/PTHp transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis.

Развитие хряща и кости: Mice with homozygous inactivation of the PTHr. P gene die at birth, if not earlier. They manifest severe chondrodysplasia and premature epiphyseal closure, reflecting a developmental defect in proliferation and differentiation of cartilage. These and other types of studies indicate that PTHr. P stimulates the proliferation of chondrocytes and suppresses their terminal differentiation. These effects of PTHr. P appear due to interaction of the PTH-like peptide with the parathyroid hormone receptor

Паракринные факторы, участвующие в остеогенезе: • • Bone morphogenetic proteins (BMPs) Hedgehog (Hh) Wnt Notch Fibroblast growth factors (FGFs) Parathyroid hormone (PTH) Insulin-like growth factors (IGFs)

Indian hedgehog (Ihh) signaling critical for endochondral skeleton • • WT Ihhn/n Chondrocyte proliferation Chondrocyte maturation Osteoblast differentiation Cartilage vascularization (st. Jacques et al. , G&D, 1999)

Hh signaling Hh Smo Ptc . . . PKA Ci/Gli Ci. A/Gli. A Ci. R/Gli. R Ptc 1 Gli 1 OFF ON

Distinct roles of Gli proteins in mediating Ihh functions PTHr. P Prolif. Col 1 (I) Runx 2 Gli 3 Gli. A Ihh AP Osx Bsp inhibition Gli 3 Gli. A stimulation (Hilton et al. , Development, 2005; Joeng et al. , unpublished)

Dual mechanisms for Ihh to control bone formation • Direct requirement in perichondrial osteoprogenitors • Control of vascularization, which in turn induces osteogenesis (mechanisms unknown)

Hh signaling dual functions in postnatal bones? • Stimulates OB differentiation (Ohba et al. , 2008) • Signaling in mature OB inhibits osteoclast formation (Mak et al. , 2008) • Remaining issues: >discrepancy remains to be resolved, >genetic modifications not specific to adult stage

Canonical Wnt signaling Dkk 1 Lrp 5/6 Fz Wnt Lrp 5/6 Dvl xin GSK 3 Tcf Dvl G Rac 1 APC in GSK 3 Ax A Fz Jnk 2 Tcf 1 Dkk 1 Cyclin D 1 OFF ON (Wu et al. , Cell, 2008)

Lrp 5 and bone accrual • OPPG patients low bone mass (Lrp 5 loss-of-function) (Gong et al. , Cell, 2001) • High bone mass syndrome (Lrp 5 gain-of -function) (Boyden et al. , NEJM 2002; Little et al. , Am J Hum Genet 2002) • Lrp 5 knockout mice low bone mass(Kato et al, JCB, 2002) Lrp 5/6 compound knockout mice lower bone mass(Holmen et al. , JBMR, 2004)

-catenin and bone • Stimulates Opg in mature OB to inhibit OC formation (Glass et al. , 2005; Holmen et al. , 2005) • Indispensable in progenitors for OB formation, inhibits chondrocyte formation (Hu et al. , 2005; Day et al. , 2005; Hill et al. , 2005)

Remaining questions about canonical Wnt signaling in bone • Regarding bone accrual: Discrepancy between Lrp 5 KO and OBspecific -catenin KO >differentiation stage-specificity? >Lrp 5 function independent of -cat? >Lrp 5 function in other tissues? • Regarding osteoblast differentiation: Is canonical Wnt signaling indispensable?

Noncanonical Wnt signaling and bone • Wnt 3 a and Wnt 7 b can promote OB differentiation through G-protein–PIP 2 -PKC (Tu et al. , Dev Cell, 2007) Wnt DAG Plasma membrane PIP 2 PLC PKC Progenitor Runx 2 Osx q Dvl Fz Osteoblast • Wnt 4 promotes OB differentiation through p 38 (Chang et al. , JBC, 2007) • Wnt 5 a promotes OB differentiation from progenitors through inhibition of PPAR (Takada et al. , Nat Cell Biol. , 2007)

Notch signaling Signal Sending Cell Jagged 1, 2 Delta-like 1, 3, 4 Notch 1 -4 Signal Receiving Cell PS 1/PS 2 - secretase Proteolytic processing D NICCSL Hes / Hey

Deletion of Notch-RBPj increases bone mass WT PRBP A WT B PRBP C 1 D 1 C 2 D 2 (Xiaolin Tu)

Двойная функция Notch в кости • Inhibits OB differentiation from mesenchymal progenitors (Hilton et al. , Nat. Med. 2008) • Inhibits OC formation from macrophage precursors >indirect: Rankl and Opg expression in OB (Hilton et al. , 2008; Bai et al. , 2008; Engin et al. , 2008) > direct function in OC lineage (Bai et al. , 2008)

Механизмы действия Notch для угнетения дифференцировки остеобластов • Hes/Hey b. HLH transcription factors: Hes 1, Hey. L (Hilton et al. , 2008) • Physical interaction of Hes/Hey with and inhibition of Runx 2 (Hilton et al. , 2008; Garg et al. , 2005; Zamurovic et al. , 2004) • Physical interaction of NICD with and inhibition of Runx 2 (Engin et al. , 2008) • Other mechanisms: inhibition of -catenin or NFAT signaling (Canalis, Sci Signal. , 2008)

Участие BMP-индуцированных гомеодоменных транскрипционных факторов (Runx 2, Msx, Dlx, Hox) в дифференцировке остеобластов ? ? Col 1(I)low Runx 2 APlow Progenitor ? Col 1(I)low Col 1(I)high Runx 2 APlow APhigh Osx Bsp ATF 4 Bsp OC Osteoblast

Runx 2 is a mediator of molecular switches for Bone Development: (a) Wnt pathways and BMP signaling contribute to both chondrogenesis and osteogenesis. The Wnt/β-catenin pathway activate Runx 2 in mesenchymal stem cells (MSCs) through the transcriptional regulators TCF/LEF to support differentiation of osteoblast lineage cells. BMP 2 induction of Nkx 3. 2 recruits MSCs for chondrocyte differentiation with Sox factors and is the critical negative regulator of Runx 2 in MSCs for chondrogenesis to proceed. It is proposed that at the stage of hypertrophic chondrocyte maturation, the downregulation of Nkx 3. 2 permits Runx 2 reexpression to promote endochondral bone formation (EBF). Runx 2 upregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinases (e. g. , MMP 9), and matrix proteins (collagen type X) contribute to vascularization and calcification and of the growth plate for endochondral bone formation.

The program of BMP 2 mediated osteogenesis is through several parallel and intersecting pathways. BMP 2 induces homeodomain proteins that increase BMP 2 induced Runx 2 transcription. BMP 2 also induces Osterix (OSX) which is upregulated by Runx 2. Dlx 3, Dlx 5 and OSX can also directly contribute to expression of bone genes independent of Runx 2. Importantly, Runx 2 integrates the BMP 2 signal for osteogenesis by formation of Runx 2–Smad complexes that regulate target genes. (b) Runx 2 overexpression will repress other cell phenotypes; likewise, the adipogenic transcriptional regulator PPARγ can repress the bone cell phenotype by inhibiting Runx 2 DNA binding activity Rev Endocr Metab Disord (2006) 7: 1– 16 3

Направления будущих исследований роли паракринных сигналов в остеогенезе • Temporal control of gene manipulation embryo vs. adult drug-inducible Cre (TM, Dox) • Integration of diverse signals in the same cell signal-activatable Cre – Notch 1: : Cre, Kopan – Gli 1 -Cre. ERTM, Joyner