Лекция 1 1

1. Лекция 11 Остеогенез

2. 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

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

4. 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

5. 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

6. Структура длинной кости Figure 6.3

7. Структуры короткой кости, кости неправильнойформы 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)

8. 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

9. 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 left-handed 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

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

11. 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 • Hepatopoietic progenitors Hematopoietic lineage

12. 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

13. 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

14. Cтруктура компактной кости Figure 6.6a, b

15. 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 orcentral canals • No osteocyte is far from blood of bone marrow • Provides strength with little weight • Trabeculaedevelop along bone’s lines of stress

16. 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

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

18. Молекулярные различия двух способов окостенения • Molecular differences? Intramembranous bones: Msx1/2, Dlx5/6 Endochondral bones: Ihh • Why the differences? Intrinsic differences? Different extracellular signals? (vasculature?)

19. Эндохондральное окостенение E10.5 E12.5 E14.5 E15.5 -18.5

20. Зрелая кость (строение) (Linheng Li)

21. Схематическое представление эндохондрального окостенения (Gilbert, 2003)

22. Стадии эндохондрального окостенения

23. по Gilbert, 2003 Локализация иРНК scleraxis (светлые области) в местах дифференци- ровки хондроцитов у 11.5-суточного зародыша мыши. Транскрипты scleraxis видны в конденсирующемся хряще носа и лица, а также в предшественниках конечностей и ребер. (по Cserjesi et al., 1995; фото- графия любезно предоставлена E.Olson.)

24. 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

25. Рост кости в длину

26. Рост кости и ремоделирование Figure 6.10

27. Активность остеокластов в костном матриксе (по Gilbert, 2003) Активность остеокластов в костном матриксе. (A) Электронная микро- фотография складчатой мембраны куриного остеокласта, культивиро- ванного на воссозданном костном матриксе. (Б) Срез складчатой мембраны, окрашенной на наличие АТФазы, способной транспорти- ровать ионы водорода из клетки. АТФаза связана с мембраной клеточ- ного отростка. (В) Солюбилизация неорганических и коллагеновых компонентов матрикса (на основе измерения выхода [45Ca] и [3H] пролина, соответственно) благодаря активности 10000 остеокластов, инкубированных на фрагментах меченой кости. (A иВиз Blair et al., 1986; Биз Baron et al.)

28. 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

29. Гиалиновый хрящ

30. 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.

31. 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.

32. 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.

33. Внутримембранное окостенение • Produces flat bones of skull and clavicle.

34. 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

35. 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

36. 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

37. The PTH/PTHp transgene promoted increased bone formation within prospectivemarrow 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.

38. Развитие хряща и кости: Mice with homozygous inactivation of the PTHrP 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 PTHrP stimulates the proliferation of chondrocytes and suppresses their terminal differentiation. These effects of PTHrP appear due to interaction of the PTH-like peptide with the parathyroid hormone receptor

39. 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)

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

41. Hh signaling Hh Smo Ptc . . . PKA Ci/Gli CiA/GliA CiR/GliR Ptc1 Gli1 OFF ON

42. Distinct roles of Gli proteins in mediating Ihh functions PTHrP Prolif. Col1(I) Runx2 Gli3 AP GliA Osx Bsp Ihh inhibition GliA Gli3 stimulation (Hilton et al., Development, 2005; Joeng et al., unpublished)

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

44. 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

45. Canonical Wnt signaling Dkk1 Wnt Lrp5/6 Fz Lrp5/6 Fz G Dvl Axin Dvl Rac1 Axin GSK3 APC GSK3 Jnk2 APC  (Wu et al., Cell, 2008)    Tcf Tcf  Tcf1 Dkk1 Cyclin D1 OFF ON

46. Lrp5 and bone accrual • OPPG patients low bone mass (Lrp5 loss-of-function) (Gong et al., Cell, 2001) • High bone mass syndrome (Lrp5 gain-of-function) (Boyden et al., NEJM 2002; Little et al., Am J Hum Genet 2002) • Lrp5 knockout mice low bone mass(Kato et al, JCB, 2002) Lrp5/6 compound knockout mice lower bone mass(Holmen et al., JBMR, 2004)

47. -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)

48. Существует мнение, что старение кости (остеопороз)связано с отвлечением бета-катенина канонического wnt-пути при оксидативном стрессе на активацию гена FOXO (Manolagas, Almeida 2007).

49. Remaining questions about canonical Wnt signaling in bone • Regarding bone accrual: Discrepancy between Lrp5 KO and OB- specific -catenin KO >differentiation stage-specificity? >Lrp5 function independent of -cat? >Lrp5 function in other tissues? • Regarding osteoblast differentiation: Is canonical Wnt signaling indispensable?

50. Wnt Fz PIP2 PLC DAG Plasma membrane q PKC Dvl Progenitor Runx2 Osx Osteoblast   Noncanonical Wnt signaling and bone • Wnt3a and Wnt7b can promote OB differentiation through G-protein–PIP2-PKC (Tu et al., Dev Cell, 2007) • Wnt4 promotes OB differentiation through p38 (Chang et al., JBC, 2007) • Wnt5a promotes OB differentiation from progenitors through inhibition of PPAR (Takada et al., Nat Cell Biol., 2007)