Juan Pablo Rodríguez V.Doctor en Ciencias

Profesor TitularINTA - Universidad de Chile

- 2018 -

Proyecto FONDECYT # 1160214

Osteoporosis y el Microambiente de la Médula Ósea

Las células troncales residen en los tejidos y órganos adultos y son capaces de auto-renovación y de diferenciación multipotente.

Las células troncales adultas permanecen en un estado no proliferativo, (estado de quiescencia).

Célula madre

Auto-renovación Diferenciación

Célula madre

Células diferenciadas

Células Troncales

Kasper et al, Harrison. Principios de Medicina Interna

Watt and Driskell, Phil. Trans. R. Soc. B 365, 155–163, 2010

• Matriz Extracelular (ECM)• Células• Factores solubles:

Factores de crecimientoCitoquinas

• Factores físicos:Tensión de O2Rigidez

Nicho de Célula Troncal (Stem cell niche): Microambiente en el cual reside la célula troncal; promueve su mantención y regula su función.

Bone Marrow Microenvironment

Célula Progenitora Mesenquimática(MSCs)

Harada and Rodan, Nature 423: 349-355, 2003

Bone Marrow Microenvironment

El microambiente de las MSCs proporciona señales de otros fenotipos celulares, la matriz extracelular, factores locales o sistémicos, factores físicos (rigidez, tensión de O2).

MSCs

Osteoblastos

Adipocitos

En la osteoporosis, ¿El microambiente de la médula ósea, favorece el potencial adipogénico de las MSCs en desmedro del potencial osteogénico?

Demographic data Control Osteoporosis p

Age (years) 71.9 ± 6.4 (n=92) 73.0 ± 9.0 (n=99)

0.14

Height (cm) 153.8 ± 6.1 (n=91)

152.6 ± 6.4 (n=72)

0.12

Weight (kg) 69.8 ± 10.9 (n=90)

59.1 ± 10.5 (n=71)

0.0001

BMI 29.6 ± 4.4 (n=96)

25.7 ± 4.2 (n=81)

0.0001

Body Fat (%) 46.8 ± 6.6 (n=86)

41.8 ± 8.7 (n=53)

0.0001

BMD (g/cm2) 1.239 ± 0.131 (n=59)

0.812 ± 0.09 (n=36)

0.0001

t-score (L2-L4) 0.35 ± 1.15 (n=95)

-3.49 ± 0.826 (n=85)

0.0001

AMMI (kg/m2) 6.03 ± 1.11(n=88)

5.41 ± 0.99(n=69)

0.0002

Hip fracture number 2(3.4%)

15(26.3%)

Results are expressed as mean ± standard deviation. BMI: Body mass index.BMD: Bone mineral density.AMMI: Appendicular skeletal muscle mass index is defined as the sum of lean mass from legs and arms related to height.

Demographic Characteristics of Women

-6 -4 -2 2 4 60

2

4

6

8

0

10

t-score

AM

MI(k

g/m

2)

ControlOsteoporosis

Lo

wM

usc

leM

as

sC

on

tro

l

Fracture

800 x g5 min

FMO

Células

AspiradoMédula

Ósea

Obtención del Fluido de Médula Ósea(FMO)

Pino et al, J Bone Min Res, 25, 492-498, 2010

MSCs

Aspirado de Médula Osea(Cresta Ilíaca)

Gradiente de Densidad

(70% Percoll)

Cultivo Celular

Células Progenitoras Mesenquimáticas(MSCs)

DMEM - 10% FBS37°C - 5% CO2

Obtención de MSCs

Zhang et al, J Cell Physiol 233:3418–3428, 2018

Stiffness

Effect of ECM Stiffness on Differentiation

Stiff matrix(134 kPa)

(Bone Tissue)

Soft matrix(1.4 kPa)

(Adipose Tissue)

Rodríguez et al, J. Cell Biochem 79, 557-565, 2000

Synthesis and Depositionof Type I Collagen

Control Osteoporosis0

20

40

60

80

100

De

po

siti

on

of

Typ

eI C

olla

gen

(mg/

10

6ce

lls)

Control Osteoporosis

Typ

eI C

olla

gen

(mg/

10

6ce

lls)

0

2

4

6

8

10

12

Adipogénesis

Control Osteoporótica

Rodríguez et al. J. Cell. Biochem. 79, 557-565, 2000

Control Osteoporotic0

5

10

15

20

25

Ad

ipo

cyte

(%

)

30

*

Control Osteoporotic0

5

10

15

20

25

Ca

lciu

m D

ep

os

itio

n

(mg

/we

ll)

30

*

Non-osteoporotics

Osteoporotics P

Interleukin-6 (pg/ml) 4.8 ± 2.5(n=23)

6.2 ± 2.5(n=15)

0.044

Soluble interleukin-6 receptor (ng/ml)

33.7 ± 13.1(n=12)

47.0 ± 13.7(n=10)

0.02

TNF-a (pg/ml) 72.3 ± 55.0(n=15)

148.9 ± 82.0(n=12)

0.036

Adiponectin (mg/ml) 9.5 ± 2.4(n=15)

5.7 ± 2.7(n=14)

0.04

Soluble-RANKL (pmol/L)

0.27 ± 0.16(n = 8)

0.14 ± 0.05n = (8)

0.021n = 8

Osteoprotegerin (pmol/L)

2.9 ± 0.9(n = 8)

4.4 ± 1.8(n = 8)

0.035(n = 8)

Leptin (ng/ml) 14.5 ± 11.3(n=24)

7.0 ± 4.4(n=12)

0.004

Soluble leptin receptor (ng/ml)

44.6 ± 14.7(n=16)

48.9 ± 17.8(n=8)

0.282

Pino et al, J Bone Min Res 25, 492-498, 2010

Concentration of Cytokines and Soluble Receptors in the Bone Marrow Soluble Fluid

Signaling Regulator Levels in Bone Marrow Fluid of Control and Osteoporotic Donors

Control Osteoporosis p

Irisin (mg/ml) 1.53 ± 0.92 2.61 ± 1.11

Miranda et al, J. Cell Biochem 117: 2370-2376, 2016

Fatty Acids BMSF Blood Plasma

(mol%)

Saturated 43.2 ± 10.6 37.7 ± 5.6*

Monounsaturated 31.0 ± 5.6 28.8 ± 5.5*

Polyunsaturated 25.8 ± 12.7 33.4± 7.8**

Total Unsaturated 56.8 ± 10.6 62.3 ± 5.6*

Palmitic acid (C16:0) 32.3 ± 8.9 28.1 ± 4.9*

Palmitoleic acid (C16:1 n-7) 2.0 ± 0.9 2.3 ± 1.1

Stearic acid (C18:0) 10.9 ± 3.3 9.7 ± 2.7*

Oleic acid (C18:1 n-9 cis) 27.3 ± 5.3 25.1 ± 5.1*

Elaidic acid (C18:1 n-9 trans) 1.7 ± 1.7 1.5 ± 0.7

Linoleic acid (C18:2 n-6) 22.1 ± 10.3 28.9 ± 6.2**

Eicosatrienoic acid (C20:3 n-6) 0.6 ± 0.7 1.0 ± 0.8

Arachidonic acid (C20:4 n-6) 3.0 ± 3.0 3.5 ± 2.4

Fatty Acid Composition in BMSF and Blood Plasma from Postmenopausal Women

Results are expressed as mean ± standard deviation. * p

Control (n=10) Osteopenia (n=10) Osteoporosis (n=17)

Fatty Acids BMSF Plasma BMSF Plasma BMSF Plasma

(mol%) (mol%) (mol%)

Saturated 46.2 ± 12.0 38.1 ± 6.5 42.1 ± 12.3 35.4 ± 4.5 41.3 ± 8.5 38.8 ± 5.6

Monounsaturated 29.7 ± 5.1 32.2 ± 6.8 30.1 ± 4.2 26.1 ± 4.6* 32.3 ± 6.5 28.5 ± 4.5

Polyunsaturated 24.0 ± 13.4 29.7 ± 11.5 33.8 ± 9.2 38.5 ± 6.1 25.6 ± 11.6 32.7 ± 4.7*

Total unsaturated 53.8 ± 12.0 61.9 ± 6.5 57.9 ± 12.3 64.6 ± 4.5 57.9 ± 8.8 61.2 ± 5.6

Palmitic acid (C16:0) 34.8 ± 10.8 27.3 ± 5.1 31.3 ± 10.7 26.3 ± 5.0 31.5 ± 6.8 30.4 ± 3.7

Palmitoleic acid (C16:1 n-7) 2.0 ± 0.7 2.6 ± 1.4 1.9 ± 1.1 1.8 ± 0.7 2.1 ± 1.0 2.4 ± 1.0

Stearic acid (C18:0) 11.5 ± 2.0 10.9 ± 2.9 10.8 ± 2.5 9.1 ± 1.9 10.6 ± 4.3 9.3 ± 2.9

Oleic acid (C18:1 n-9 cis) 26.5 ± 4.8 28.1 ± 7.2 26.1 ± 4.3 23.1 ± 4.3 28.5 ± 6.1 24.5 ± 3.6*

Elaidic acid (C18:1 n-9 trans) 1.3 ± 0.8 1.5 ± 0.4 1.2 ± 0.8 1.2 ± 0.5 1.7 ± 0.8 1.6 ± 0.8

Linoleic acid (C18:2 n-6) 22.0 ± 9.2 25.3 ± 8.5 22.6 ± 11.2 31.9 ± 5.7* 23.1 ± 10.0 29.2 ± 4.1*

Eicosatrienoic acid (C20:3 n-6)

0.8 ± 0.7 0.9 ± 1.0 0.9 ± 0.6 1.3 ± 0.5 0.4 ± 0.6 0.9 ± 0.8

Arachidonic acid (C20:4 n-6) 3.3 ± 2.5 3.5 ± 3.0 4.3 ± 4.3 5.3 ± 1.4 2.0 ± 2.0 2.6 ± 1.9

Miranda et al, J. Cell Biochem 117: 2370-2376, 2016

Fatty Acid Composition in BMSF and Blood Plasma

Results are expressed as mean ± standard deviation. *p

Blood Plasma Bone Marrow Supernatant Fluid

Fatty AcidsWithout fracture

(n=8)With fracture

(n=9)Without fracture

(n=8)With fracture

(n=9)(mol%) (mol%)

Saturated 37.6 ± 4.6a,b 40.0 ± 6.5 45.6 ± 8.5 37.0 ± 6.3*Monounsaturated 29.5 ± 3.2 27.4 ± 5.5c 28.8 ± 5.3 34.1 ± 6.3*Polyunsaturated 32.8 ± 4.3 32.6 ± 5.2 24.1 ± 13.9 26.9 ± 9.8Total Unsaturated 62.4 ± 4.6a 60.0 ± 6.5 c 54.4 ± 8.5 63.0 ± 6.3

Palmitic acid (C16:0) 29.0 ± 3.7b 32.2 ± 3.2 32.4 ± 7.7 30.7 ± 6.2Palmitoleic acid (C16:1 n-7) 2.7 ± 0.9 2.0 ± 1.0 2.0 ± 1.0 2.3 ± 1.0Stearic acid (C18:0) 9.4 ± 1.3a 9.2 ± 4.0 13.2 ± 4.5 8.3 ± 2.7*Oleic acid (C18:1 n-9 cis) 25.1 ± 3.5 24.0 ± 3.7 c 24.9 ± 5.0 30.2 ± 5.3*Elaidic acid (C18:1 n-9 trans) 1.8 ± 0.5 1.4 ± 1.0 1.8 ± 0.8 1.6 ± 0.9Linoleic acid (C18:2 n-6) 28.5 ± 3.5a 30.0 ± 4.7 20.4 ± 10.9 25.6 ± 9.0Eicosatrienoic acid (C20:3 n-6) 1.1 ± 0.8 0.6 ± 0.7 c 0.8 ± 0.7 0.1 ± 0.1*Arachidonic acid (C20:4 n-6) 3.2 ± 2.3 2.0 ± 1.3 4.6 ± 1.0 1.5 ± 0.8***

Miranda et al, J. Cell Biochem 117: 2370-2376, 2016

Composition of Fatty Acid in Bone Marrow Fluid from Osteoporotic Donors with and without Hip Fracture.

Pino et al, Frontiers in Endocrinology 7:139, 2016

Relationship between Hip Fractures and Fatty Acid CompositionIn the Bone Marrow Fluid of Osteoporotic Women

Señales recibidas desde un microambiente alterado pueden tener efecto en las características funcionales de las MSCs:

Metabolismo Estado redox

Ito and Suda, Nature rev 15, 243-256, 2014

Does o-MSCs show an alteredmetabolic status, as compared

with c-MSCs?

Lactato Extracelular

mg

Lact

ato

/ 1

06

célu

las

0 3 140

0.5

1.0

1.5

2.0

Diferenciación Adipogénica(días)

A. Oxygen consumption rate (OCR). B. Extracellular acidification rate (ECAR), as a measured of glycolisis. Values were normalized at the protein content of each culture plate. Data shown correspond to the mean of at least three different assays by group.

c-MSCs o-MSCs

Oxidative Phosphorylation and Glycolisis in c- and o-MSCs

Rosen CJ. Personal communication

OXPHOS Glycolisis

Lact

ato

(m

g/1

06

célu

las)

Normal Osteopénica Osteoporótica

0.0

0.5

1.0

1.5

2.0

2.5

Lactato Extracelular(Basal)

OXPHOSTCA cycle

Pyruvate

Lactate

Glucose

G-6-P

Pyruvate

PPP

NADPH Glycolisis

IGF-1

PI3K

AKT

PDK

HIF1a

Acetyl CoAPDH

ROS

Catalase SOD

FOXO

Adipocytes

Osteoblasts

mTOR

NOX

TNX1 PRDX

Román et al, Reunión Anual Soc. Biol. Celular, 2016

Transcript levels quantified by qRT-PCR.

Normal OsteoporosisRe

lati

veEx

pre

ssio

n/

GA

DP

H

0

0.1

0.2

0.3

0.4Catalase ***

Normal Osteoporosis0

5

10

15

Re

lati

veEx

pre

ssio

n/

GA

DP

H SOD1

0

5

10

15

Re

lati

veEx

pre

ssio

n/

GA

DP

H

TNX1 ***

Normal Osteoporosis Normal Osteoporosis

**

Re

lati

veEx

pre

ssio

n/

GA

DP

H

0

0.001

0.002

0.003PRDX

Román et al, Reunión Anual Soc. Biol. Celular, 2016

0

0.005

0.010

0.015

0.020

0.025

Re

lati

veEx

pre

ssio

n/

GA

DP

H NOX4

Normal Osteoporosis0

0.01

0.02

0.03

0.04

0.05***

Re

lati

veEx

pre

ssio

n/

18

S PDK1

Normal Osteoporosis

0

0.05

0.10

0.15*

Re

lati

veEx

pre

ssio

n/

18

S

HIF-1a

Normal Osteoporosis

*

Foxo

Normal OsteoporosisRe

lati

veEx

pre

ssio

n/

GA

DP

H

0

0.02

0.04

0.06

0.08

0.10

Román et al, J. Cell Biochem 118: 585-593, 2017

Viability of MSCs after H2O2 Treatment

En resumen

El microambiente de la médula ósea normal es

diferente al de una médula ósea osteoporótica.

Diferente contenido de colágeno

Diferentes niveles de factores solubles (FMO)

Esto se puede traducir en diferente funcionalidad:

Metabolismo

Estado redox

Declaro no tener conflictos de interés.

Juan Pablo Rodríguez V.Doctor en Ciencias

Profesor TitularINTA - Universidad de Chile