Transcript Hyaline cartilage

Лекция 11
Остеогенез
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
Структура длинной кости
Figure 6.3
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
Cтруктура компактной кости
Figure 6.6a, b
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: Msx1/2, Dlx5/6
Endochondral bones: Ihh
• Why the differences?
Intrinsic differences?
Different extracellular signals? (vasculature?)
Эндохондральное окостенение
E10.5
E12.5
E14.5
E15.5 -18.5
Зрелая кость (строение)
(Linheng Li)
Схематическое представление
эндохондрального окостенения (Gilbert, 2003)
Стадии эндохондрального окостенения
по Gilbert, 2003
Локализация иРНК scleraxis (светлые области) в местах дифференцировки 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; фотография любезно предоставлена E.Olson.)
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
Рост кости в длину
Рост кости и ремоделирование
Figure 6.10
Активность остеокластов в костном матриксе (по
Gilbert, 2003)
Активность остеокластов в костном матриксе. (A) Электронная микрофотография складчатой мембраны куриного остеокласта, культивированного на воссозданном костном матриксе. (Б) Срез складчатой
мембраны, окрашенной на наличие АТФазы, способной транспортировать ионы водорода из клетки. АТФаза связана с мембраной клеточного 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 и В из Blair et al.,
1986; Б из Baron et al.)
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
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 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
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
Chondrocyte proliferation
Chondrocyte maturation
Osteoblast differentiation
Cartilage vascularization
Ihhn/n
(st. Jacques et al., G&D, 1999)
Hh signaling
Hh
Smo
Ptc
...
PKA
CiA/GliA
Ci/Gli
CiR/GliR
Ptc1
Gli1
OFF
ON
Distinct roles of Gli proteins in
mediating Ihh functions
PTHrP
Prolif.
Col1(I)
Runx2
Gli3
GliA
Ihh
AP
Osx
Bsp
inhibition
Gli3
GliA
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
Dkk1
Lrp5/6
Fz
Wnt
Lrp5/6
Fz
Dvl
Dvl
G
Rac1

Jnk2



Tcf

Tcf
Tcf1
Dkk1
Cyclin D1
OFF
ON
(Wu et al.,
Cell, 2008)
Lrp5 and bone accrual
• OPPG patients low bone mass (Lrp5
loss-of-function)
(Gong et al., Cell, 2001)
• High bone mass syndrome (Lrp5 gainof-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)
-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)
Существует мнение, что старение кости (остеопороз) связано с
отвлечением бета-катенина канонического wnt-пути при
оксидативном стрессе на активацию гена FOXO (Manolagas,
Almeida 2007).
Remaining questions about
canonical Wnt signaling in bone
• Regarding bone accrual:
Discrepancy between Lrp5 KO and OBspecific -catenin KO
>differentiation stage-specificity?
>Lrp5 function independent of -cat?
>Lrp5 function in other tissues?
• Regarding osteoblast differentiation:
Is canonical Wnt signaling
indispensable?
Noncanonical Wnt signaling
and bone
• Wnt3a and Wnt7b can promote OB
differentiation through G-protein–PIP2-PKC
(Tu et al., Dev Cell, 2007)
Wnt
DAG
Plasma membrane
PIP2
PLC
PKC
Progenitor
Runx2
Osx
Osteoblast
q 
 Dvl
Fz
• 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)
Notch signaling
Signal
Sending Cell
Jagged 1, 2
Delta-like 1, 3, 4
Notch1-4
Signal
Receiving Cell
Proteolytic processing
PS1/PS2
 - secretase
CSL
Hes / Hey
Deletion of Notch-RBPj
increases bone mass
WT
PRBP
A
WT
B
PRBP
C1
D1
C2
D2
(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 bHLH transcription factors:
Hes1, Hey1, HeyL (Hilton et al., 2008)
• Physical interaction of Hes/Hey with and
inhibition of Runx2 (Hilton et al., 2008; Garg et al., 2005;
Zamurovic et al., 2004)
• Physical interaction of NICD with and
inhibition of Runx2 (Engin et al., 2008)
• Other mechanisms: inhibition of -catenin or
NFAT signaling (Canalis, Sci Signal., 2008)
Участие BMP-индуцированных гомеодоменных
транскрипционных факторов (Runx2, Msx, Dlx, Hox) в
дифференцировке остеобластов
?
?
Col1(I)low
Runx2
APlow
Progenitor
?
Col1(I)low Col1(I)high
Runx2
Runx2
APlow
APhigh
Osx
Osx
Bsp
ATF4
Bsp
OC
Osteoblast
Интеграционное действие множества
сигналов в ходе формирования кости
Notch
Notch
MSC
Runx2+
Hh
Wnt, Fgf
Osx+
Rankl
OB
Wnt, Bmp
OC
Notch, Wnt
Fgf, Bmp
Opg
MC
HSC
Факторы транскрипции, регулирующие
дифференцировку остеобластов
Факторы транскрипции, регулирующие
дифференцировку остеобластов
Множественные функции транскрипционного фактора
Runx2
Runx2 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 Runx2 in mesenchymal stem cells (MSCs) through the
transcriptional regulators TCF/LEF to support differentiation of
osteoblast lineage cells. BMP2 induction of Nkx3.2 recruits MSCs
for chondrocyte differentiation with Sox factors and is the critical
negative regulator of Runx2 in MSCs for chondrogenesis to proceed.
It is proposed that at the stage of hypertrophic chondrocyte
maturation, the downregulation of Nkx3.2 permits Runx2 reexpression to promote endochondral bone formation (EBF).
Runx2 upregulation of vascular endothelial growth factor (VEGF),matrix
metalloproteinases (e.g., MMP9), and matrix proteins (collagen type
X) contribute to vascularization and calcification and of the growth
plate for endochondral bone formation.
The program of BMP2 mediated osteogenesis is
through several parallel and intersecting pathways.
BMP2 induces homeodomain proteins that increase
BMP2 induced Runx2 transcription. BMP2 also
induces Osterix (OSX) which is upregulated by Runx2.
Dlx3, Dlx5 and OSX can also directly contribute to
expression of bone genes independent of Runx2.
Importantly, Runx2 integrates the BMP2 signal for
osteogenesis by formation of Runx2–Smad complexes
that regulate target genes.
(b) Runx2 overexpression will repress other cell
phenotypes; likewise,the adipogenic transcriptional
regulator PPARγ can repress the bone cell phenotype by
inhibiting Runx2 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
– Notch1::Cre, Kopan
– Gli1-CreERTM, Joyner
Мезенхимные стволовые
клетки костного мозга (MSC)
• Не гематопоэтические
• Молекулярная идентичность не установлена
• Субпопуляция клональных адгерентных
клеток (CFU-F) (~15%)
• In vitro дифференцируются в хондроциты,
остеобласты, адипоциты
• При трансплантации in vivo регенерируют в
клетки кости и костного мозга
Заключение и перспективы
• Два способа образования кости
Чем различаются?
• Образование остеобластов – сигналинг Hh,
Wnt и Notch.
Сигнал из кровеностных сосудов?
Интеграция сигналов? Роль таковых
у взрослого организма?
• MSC (стволовые клетки костного мозга)
Молекулярная идентичность?
Разнообразия и общность происхождения?