1,2Gundula Schulze‐Tanzil, VMDResearch group “Bioreconstruction”

1Institute of Anatomy, Paracelsus Medical University, Salzburg and Nuremberg, Germany

2Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité- Universitätsmedizin Berlin, Campus Benjamin Franklin, Germany

1

•

•Education:1990‐1996199919992005

School of veterinary medicine, Freie Universität Berlin VMD Degree, Freie Universität BerlinPostdoctoral Fellow, Institute of Anatomy, Freie Universität Berlin Scientific assistant, Centrum of Anatomy, Charité

University of

•

•

•• Medicine, CCM, Berlin

Dep. of Trauma and Reconstructive Surgery, CBF/Centrum of Anatomy, CCM, Charité University of Medicine, BerlinLeader of the research laboratory of experimental surgery, AG BioreconstructionDep. of Trauma and Reconstructive Surgery, CBF, Charité University

2005‐2008

•2007

•

•

2008‐2014of Medicine, BerlinHabilitation: Anatomy and Cell BiologyProfessorship at the Institute of Anatomy, Paracelsus Medical University, Salzburg and Nuremberg, Germany

20092015•

• Teaching Activities:Dissection courses in gross anatomy, courses in histology, seminars and lessions in anatomy and cell biologyScholarships:

•

••

•1996‐19992005‐2008

Doctoral scholarship, Freie Universität Berlin

Rahel Hi‐ rsch habilitation Scholarship, Charité

Berlin

2

Ad hoc reviewer for

Acta Biomaterialia, Annals of Anatomy, Arthritis Rheumatism, Arthritis Research Therapy, Berliner Münchner Tierärztliche Wochenschrift, BMC Musculoskeletal Disorders, Biomaterials, Biotechnology Progress, Biotechnology Bioengineering, Bone, Cell Tissue Research, Cell Biology and Toxicology, Cells Tissues Organs, Clinical Medicine Insights: Arthritis Musculoskeletal Disorders, Clinical Experimental Immunology, Connective Tissue Research, Cytokine, Drug Dev reviews, European Cells Materials, European J Histochemistry, Experimental Biology and Medicine, Fibrogenesis and Tissue Repair, Folia Biologica, Gene Therapy, Histochemistry Cell Biology, Histology Histopathology, International J Molecular Science, J Biochips Tissue Chips, J Cellular Biochemistry, J Tissue Engineering Regenerative Medicine, J Biomedicine and Biotechnology, J Biomed Mater Res Part A, J Immunol Methods, J Orthop Surg Res, J Nanomedicine, Materials Letters, Medicinal Chemistry, Osteoarthritis Cartilage, Oxid Antioxid Med Sci, PLOSone, Review Material Letters, The Lancet, Tissue Engineering Part A.

3

Scientific interests

• cartilage and tendon/ligament biology• osteoarthritis (in vitro and in vivo models)• tendon injury and healing (in vitro and in vivo models)• interplay of anti‐ and proinflammatory cytokines in

tendon and cartilage, co‐culture models• cell‐ and biomaterial based cartilage and tendon

repair: cartilage and tendon tissue engineering• tendon/ligament ageing• comparative anatomy/cell biology of tendons and

cartilage subtypes

4

Cell biology●characterization of human and animal-derived primary cells: chondrocytes, tenocytes, ligament cells, synovial fibroblasts, PBMCs and neutrophils●three-dimensional cultures (pellet, organoid, spheroids, hydrogel and scaffold cultures, statical and dynamical seeding of biomaterials and self prepared cell-freed extracellular matrices● interaction of musculoskeletal cells in co-cultures● immunohistochemistry/-cytochemistry, immunofluorescence microscopy● confocal laser scanning microscopy● flow cytometry● histology/histopathology of musculoskeletal tissues● transmission-/scanning electron microscopy●ELISA, assays (cytokines, proliferation, DMMB-assay, cell vitality, cytotoxicity, caspase activity, TUNEL…)● SDS-PAGE and Western Blot Analyses● RT PCR and Real Time (RTD)-PCR● transfection of primary cells: adenoviral, lipofection, Electroporation● gene silencing using siRNA

Techniques

5

in the minipig and rabbiAnimal models●matrix assisted autologous chondrocytes transplantation (chondral vs. Osteochondral defects)Lohan A et al., 2014●partial Achilles tendon defect model in the rabbit (Stoll C et al., 2011, Biomaterials)● nude mice xenograft model: in vivo Chondrogenesis and

tendogenesisLohan A et al., 2011: Histochem Cell Biol● osteoarthritis model in the rat (surgically induced: MMT)

Techniques

t

gross anatomical preparation techniques of human and animal-derived samples

6

Some recent publications (2014)• Mrosewski I, Jork N, Gorte K, Conrad C, Wiegand E, Kohl B, Ertel W, John T, Oberholzer A, Kaps C, Schulze-

Ta nzil G (2014). Regulation of OA associated key mediators by TNFα and IL-10: Effects of IL-10 overexpression in human synovial fibroblasts and a synovial cell line. Cell Tissue Res 357(1):207-23.

• The Phantom 5 Consortium (2014). A promoter level mammalian expression atlas. Nature 27;507(7493):462- 70.

• Gröger D, Kerschnitzki M, Weinhart M, Schneider T, Kohl B, Wagermaier W, Schulze-Tanzil G, Fratzl P, Haag R (2014). Selectivity in Bone Targeting with Different Polyanionic Dendritic Dye Conjugates. Advanced Healthcare Materials 3(3):375-85.

• Lohan A, Marzahn U, El Sayed K, Haisch A, Müller RD, Kohl B, Stölzel K, Ertel W, John T, Schulze-Tanzil G (2014). Osteochondral cartilage defect repair using autologous orthotopic and heterotopic chondrocytes in the rabbit model. Ann Anat. 196(5):317-26.

• Girke G, Kohl B, Busch C, John T, Godkin O, Ertel W, Schulze-Tanzil G (2014). Tenocyte activation and regulation of complement factors in response to in vitro cell injury. Mol Immunol. 60(1):14-22.

• Stölzel K, Schulze-Tanzil G, Olze H, Schwarz S, Feldmann EM, Rotter N (2014). Immortalised human mesenchymal stem cells undergo chondrogenic differentiation in alginate and PGA/PLLA scaffolds. Cell Tissue Bank. 2014 May 16

• Hoyer M, Meier C, Breier A, Hahner J, Heinrich G, Drechsel N, Meyer M, Rentsch C, Garbe LA, Ertel W, Lohan A, Schulze-Tanzil G. In vitro characterization of self-assembled anterior cruciate ligament cell spheroids for ligament tissue engineering. Histochem Cell Biol. 2014 Sep 26.

• Jagielski M, Wolf J, Marzahn U, Völker A, Lemke M, Meier C, Ertel W, Godkin O, Arens S, Schulze-Tanzil G (2014). The influence of IL-10 and TNFα on chondrogenesis of human mesenchymal stromal cells in three- dimensional cultures. Int J Mol Sci. 15(9):15821-44.

• Hoyer M, Drechsel N, Meyer M, Meier C, Hinüber C, Breier A, Hahner J, Heinrich G, Rentsch C, Garbe LA, Ertel W, Schulze-Tanzil G, Lohan A. (2014). Embroidered polymer-collagen hybrid scaffold variants forligament tissue engineering. Mater Sci Eng C Mater Biol Appl. 1;43:290-9. 7

Cartilage tissue engineering

to characterize /compare the quality of tissue engineered cartilage produced by heterotopic chondrocytes seeded on

polyglycolic acid (PGA) scaffolds in vitro and in vivo

porcine auricular, nasoseptalarticular chondrocytes

dynamic culture3 weeks → analysis

Implanted ino nude mice 1, 6, 12 weeks

→ analysis

Lohan A et al., 2011Histochem Cell Biol. 136(1):57-69

Chondrogenesis by heterotopic chondrocytes in vivo1 week 6 weeks 12 weeks

arti

cula

r

explantation

auri

cula

rn

on

-see

ded

nas

ose

pta

l

resorbed

Lohan A et al., 2011Histochem Cell Biol. 136(1):57-69

articularChondrogenesis by heterotopic chondrocyte mono‐ and cocultures auricular articular / auricular nasoseptal articular / nasoseptal

lig

ht

mic

rosc

op

y

100 µm

liv

e / d

eath

as

say

100 µm

alci

an b

lue

stai

nin

g

20 µm

articular / nasoseptalarticular / auricular

cell

trac

kin

g

El Sayed K et al., 2013J Tissue EngRegen Med 7, 61-72.100 µm

Cartilage repair in vivo

intact articular cartilage

chondral full thickness defect

repair tissue after 6 month

gly

co

sa

min

og

lyca

n s

tain

ing

(re

d)

HE

Lohan A et al., 2013, 195(5):488-97.

influence of autologous leukocytes on tenocytes?

PBMCsinflammation tenocyte response

neutrophilstranswell system: indirect co-culture

● to cytokines?● to leukocytes?● to complement activation?● to cell injury?

Achilles tenocytes

10 ng/mL TNFα, 24 h 4x106 rabbit leukocytes105 rabbit tenocytes

MMP-110

8 *30

rela

tiv

e g

en

e e

xpre

ssio

n

IL-630 IL-1βTNFα 2.5

2.0

100 µm

6

4

2

0 0

10

20 *

0

10

20

**1.5

1.0

0.5

0.0

*

Al-Sadi O et al., 2012. Muscles Ligaments

coTNF

PBMCneutro

. coTNF

PBMC

neutro.co

TNFPBMC

neutro.

co

Tendons J. 2012 1(3):68-76.

TNFPBMC

neutro.

tenocyte response to cell injury

scratch assay

computer guided reproducible plotting system

„healing“ after injury

4 h 8 h 16 h 24 h

green: vital, red: dead cellshuman Hamstring tenocytes

Girke G et al., 2014 Mol Immunol 60(1):14-22.

tenocyte response to cell injury

10 tenocyte response

8

rela

tive

gen

e ex

pre

ssio

n *● to cytokines?● to leukocytes?● to complement activation?● to cell injury?

6

4

* *

*

0

2

* **

con

tro

l4

+ 2

4 h

4 h

24 h 4 h

24 h 4 h

24 h 4 h

24 h 4 h

24 h 4 h

24 h 4 h

24 h

C3a

R

C5a

R

CD

46

CD

55

TN

Fα

IL-1

β

MM

P-1

human Hamstring tenocytes

Girke G et al., 2014 Mol Immunol

decellularized tendon ECM scaffolds

human tenocytes

decellularization recellularization

porcine AS tendon

reseeded tendon ECM

tendon repair?

decellularizedECM

HE

Lohan et al., 2013, Schulze-Tanzil G et al., 2012Connect Tissue Res. 54(4-5):305-12. Cells 1:1010.

tenocyte implantation in partial tendon defects (rabbit)

rabbit AchillesAchilles tenocytes

tendon

expansion in

rabbit tenocytes

monolayerPGA-culture

PGA-culture

1 weekmedial M. gastrocnemius tendon

cell vitality

implantation intendon defect PGA-transplant

empty defect implanted PGA

6 weeks

healing?macroscopical and histological scoring

green = vital red = dead

Stoll C et al., 2011 Biomaterials 32:4806

tenocyte implantation in partial tendon defects (rabbit)

co empty defect tenocytes + PGAPGAH

E

macroscopical score

sulp

hat

ed

GA

Gs:

blu

e

*10

15

hea

lth

y =

17p

oin

ts

0

5empty PGA

PGA+cells

histological score, 6 weeks

hea

lth

y =

20

po

ints 15

elas

tic

fib

ers

Stoll C et al., 2011 Biomaterials 32:4806

5

10

empty PGAPGA+cells

0

Lapine anterior cruciate ligament (ACL) mean substance

posterior cruciate ligament enthesis

anteriorcruciate ligament

Dimensions:15 mm x 3 mm x 4 mm

Hoyer et al., 2014. Materials Science and Engineering 1;43:290-9.

embroidered scaffolds with collagen

●anterior cruciate ligament (ACL) tissue engineering using embroidered scaffolds supplemented with collagen

embroidery patternPLA-CL PLA + PLA-CL PLA

stitch length

stitch angle

loading axis

● Poly(lactic-co-ε-caprolacton) = PLA-CL, monofilament● Poly-L-lactic acid = PLA, multifilament● both materials combined

Dimensions of the lapine ACL (15 mm x 3 mm x 4 mm)

P(LA-CL)+ collagen foam

spheroid-based seeding

vita

lity

Hoyer et al., 2014. Materials Science and Engineering 1;43:290-9.

40

60M Cellularity

[nu

cle

i/µ

m2 ]ACL spheroid

characterizationHE Safranin O AzAn

20

4

0 d

ay

s

A B C

Total sGAG

***N

0 7 14 0 7

0days

[µg

/10

6 c

ells

pe

r s

ph

ero

id]

2

1

3200µm 200µm200µm

s yda

7 d

ay

s

sta

tic

al

D E F

0 7 14 0 7

0

7200µm 200µm 200µm

G H I

7 d

ays

d

yn

am

ical

Ø Ø

sy

Total collagenO ***

*

days

100

80

60

200µm200µm200µm

J K L

[µg

/10

6 c

ells

pe

r sp

her

oid

]43

40

20

da 14

14

da

ys

st

atic

al

200µm 200µm 200µm

0 7 14 0 7

210statical

Hoyer et al., 2014. Histochem Cell Biol. In press

dynamicaldays

Collagen scaffolds seeded with ligament cells

7 d

green: vital, red: dead cells

21Hoyer et al., 2014. Materials Science and Engineering 1;43:290-9.