1,2Gundula Schulze‐Tanzil, VMDResearch Research groupgroup „„BioreconstructionBioreconstruction““

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

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

1

• Education:

• 1990‐1996  School of veterinary medicine, Freie Universität Berlin

• 1999 VMD Degree, Freie Universität Berlin

• 1999 Postdoctoral Fellow, Institute of Anatomy, Freie Universität Berlin

• 2005 Scientific assistant, Centrum of Anatomy, Charité University of , y, yMedicine, CCM, Berlin

• 2005‐2008 Dep. of Trauma and Reconstructive Surgery, CBF/Centrum of Anatomy, CCM, Charité University of Medicine, BerlinAnatomy, CCM, Charité University of Medicine, Berlin

• 2007 Leader of the research laboratory of experimental surgery, AG Bioreconstruction

• 2008 2014 Dep of Trauma and Reconstructive Surgery CBF Charité University• 2008‐2014 Dep. of Trauma and Reconstructive Surgery, CBF, Charité University of Medicine, Berlin

• 2009  Habilitation: Anatomy and Cell Biology

f h h f l d l• 2015 Professorship at the Institute of Anatomy, Paracelsus Medical University, Salzburg and Nuremberg, Germany

• Teaching Activities:

• Dissection courses in gross anatomy, courses in histology, seminars and lessions in anatomy and cell biology

• Scholarships:p

• 1996‐1999 Doctoral scholarship, Freie Universität Berlin

• 2005‐2008  Rahel‐Hirsch habilitation Scholarship, Charité Berlin 2

Ad hoc reviewer forAd hoc reviewer for

Acta Biomaterialia, Annals of Anatomy, Arthritis Rheumatism, Arthritis ResearchTherapy, Berliner Münchner Tierärztliche Wochenschrift, BMC MusculoskeletalDisorders, Biomaterials, Biotechnology Progress, Biotechnology Bioengineering,B C ll Ti R h C ll Bi l d T i l C ll Ti OBone, Cell Tissue Research, Cell Biology and Toxicology, Cells Tissues Organs,Clinical Medicine Insights: Arthritis Musculoskeletal Disorders, ClinicalExperimental Immunology, Connective Tissue Research, Cytokine, Drug Devreviews European Cells Materials European J Histochemistry Experimentalreviews, European Cells Materials, European J Histochemistry, ExperimentalBiology and Medicine, Fibrogenesis and Tissue Repair, Folia Biologica, GeneTherapy, Histochemistry Cell Biology, Histology Histopathology, International JMolecular Science J Biochips Tissue Chips J Cellular Biochemistry J TissueMolecular Science, J Biochips Tissue Chips, J Cellular Biochemistry, J TissueEngineering Regenerative Medicine, J Biomedicine and Biotechnology, J BiomedMater Res Part A, J Immunol Methods, J Orthop Surg Res, J Nanomedicine,Materials Letters Medicinal Chemistry Osteoarthritis Cartilage Oxid AntioxidMaterials Letters, Medicinal Chemistry, Osteoarthritis Cartilage, Oxid AntioxidMed Sci, PLOSone, Review Material Letters, The Lancet, Tissue EngineeringPart A.

3

Scientific interestsScientific interests

• cartilage and tendon/ligament biologyg / g gy

• osteoarthritis (in vitro and in vivo models)

• tendon injury and healing (in vitro and in vivo models)j y g ( )

• 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 cartilagesubtypes

4

TechniquesCell biology● characterization of human and animal-derived primary cells: chondrocytes,

Techniques

p y ytenocytes, ligament cells, synovial fibroblasts, PBMCs and neutrophils● three-dimensional cultures (pellet, organoid, spheroids, hydrogel and scaffoldcultures, statical and dynamical seeding of biomaterials and self prepared cell-freedextracellular matrices● interaction of musculoskeletal cells in co-cultures● immunohistochemistry/-cytochemistry, immunofluorescence microscopy● confocal laser scanning microscopy● flow cytometry● histology/histopathology of musculoskeletal tissues

t i i / i l t i● transmission-/scanning electron microscopy● ELISA, assays (cytokines, proliferation, DMMB-assay, cell vitality, cytotoxicity, caspase activity, TUNEL…)● SDS PAGE and Western Blot Analyses● SDS-PAGE and Western Blot Analyses● RT PCR and Real Time (RTD)-PCR● transfection of primary cells: adenoviral, lipofection, Electroporation● gene silencing using siRNA● gene silencing using siRNA

5

Techniques

Animal models

Techniques

● matrix assisted autologous chondrocytes transplantation in the minipig and rabbit(chondral vs. Osteochondral defects)Lohan A et al., 2014

ti l A hill t d d f t d l i th bbit (St ll C t l 2011● 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 BiolLohan A et al., 2011: Histochem Cell Biol● osteoarthritis model in the rat (surgically induced: MMT)

gross anatomical preparation techniques of human andgross anatomical preparation techniques of human andanimal-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-

Tanzil G (2014). Regulation of OA associated key mediators by TNFα and IL-10: Effects of IL-10 overexpressionin 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-• 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. AdvancedHealthcare Materials 3(3):375 85Healthcare 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 therabbit 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 andregulation 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 humanmesenchymal stem cells undergo chondrogenic differentiation in alginate and PGA/PLLA scaffolds. Cell Tissuey g g gBank. 2014 May 16

• Hoyer M, Meier C, Breier A, Hahner J, Heinrich G, Drechsel N, Meyer M, Rentsch C, Garbe LA, Ertel W, LohanA, Schulze-Tanzil G. In vitro characterization of self-assembled anterior cruciate ligament cell spheroids for ligament tissue engineering Histochem Cell Biol 2014 Sep 26ligament 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.H M D h l N M M M i C Hi üb C B i A H h J H i i h G R t h C G b LA• 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(PGA) scaffolds in vitro and in vivo

porcineauricular, nasoseptalarticular chondrocytes

dynamic culture3 weeks → analysis

Implanted ino nude mice1, 6, 12 weeks→ analysis→ analysis

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

6 weeks 12 weeks1 week

Chondrogenesis by heterotopic chondrocytes in vivo6 weeks 12 weeks1 week

cula

r

explantation

artic

arau

ricul

aos

epta

lde

dna

sono

n-se

ed

resorbed

n

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

articularChondrogenesis by heterotopic chondrocyte mono‐ and cocultures

articular / auricularauricular articular / nasoseptalnasoseptalt ro

scop

y

100 µmlight

mic

rth

100 µmlive

/ dea

tas

say

anbl

ueni

ng

20 µm20 µmalci

ast

ain

articular / nasoseptalarticular / auricular

ltra

ckin

g

100µmcell

100µm100 µm

El Sayed K et al., 2013J Tissue Eng Regen Med 7, 61-72.

Cartilage repair in vivo

intact articular cartilage

chondral full thickness defect

repair tissueafter 6 month

d)

after 6 month

HE

stai

ning

(red

min

ogly

can

glyc

osam

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?neutrophilsindirect co-culture ● to cell injury?

Achilles tenocytes

10 ng/mL TNFα, 24 h 4x106 rabbit leukocytes 105 bbit t t105 rabbit tenocytes

810

*30

*xpre

ssio

n

IL-630 IL-1βTNFα2 02.5

*MMP-1

100 µm

02468

0

10

20 *

ativ

e ge

ne e

x

0

10

20

**0 00.51.01.52.0 *

Al-Sadi O et al., 2012. Muscles Ligaments Tendons J. 2012 1(3):68-76.

co αTNF

PBMCneutro.

0co α

TNFPBMC

neutro.0

rela

co αTNF

PBMCneutro.

0co α

TNFPBMC

neutro.0.0

tenocyte response to cell injury 

scratch assayscratch assay

computer guided reproducible plotting system

4 h 8 h 16 h 24 h

„healing“ after injury

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

sion

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

6

ene

expr

ess ● to cell injury?

4

rela

tive

ge

* **

0

2* *

*

4 +

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 4 h

24 h

0

aR aR D46

D55 Fα -1β

ntro

l

P-1

human Hamstring tenocytesC3

C5

CD

CD

TN IL-

con

MM

Girke G et al., 2014 Mol Immunol

decellularized tendon ECM scaffolds

human tenocytes

decellularization recellularization

porcine AS tendon

reseeded tendon ECM

tendonrepair?

decellularized ECM

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 Achilles tendon

expansion in

rabbit tenocytes

monolayer PGA-culture

PGA-culture

1 week medial M. gastrocnemius tendoncell vitality

implantation in tendon defect PGA-transplant

1 weekempty defect implanted PGA

h li ?

6 weeks

healing?macroscopical and histological scoringgreen = vital

red = deadStoll C et al., 2011

Biomaterials 32:4806

f

tenocyte implantation in partial tendon defects (rabbit)co empty defect PGA tenocytes + PGA

HE

PGAPGA macroscopical score

d ue

10

15 macroscopical score

*

= 17

poin

ts

sulp

hate

dG

AG

s: b

lu

0

5empty PGA

PGA+cells

heal

thy

=

histological score, 6 weeks

oint

s 15

s

thy

= 20

po

5

10

empty PGAPGA+cellsas

ticfib

ers

heal

t

0

p y

ela

Stoll C et al., 2011Biomaterials 32:4806

Lapine anterior cruciate ligament (ACL)

mean substance

posterior cruciateligament enthesis

anterior cruciate ligament

Dimensions:15 3 4cruciate ligament 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 embroideredscaffolds supplemented with collagen

embroidery pattern

stitch length

PLA-CL PLA + PLA-CL PLAstitch length

stitch angle

loading axis

spheroid based

● Poly(lactic-co-ε-caprolacton) = PLA-CL,

P(LA-CL) + collagen foam

spheroid-based seeding

ty

monofilament● Poly-L-lactic acid = PLA, multifilament● both materials combined

vita

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

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

40

60M Cellularity

µm2 ]ACL spheroid characterization

HE Safranin O AzAn0

20[nuc

lei/µ

4

0 da

ys

A B C

Total sGAGN***

0 7 14 0 70

days

pher

oid]

2

3200µm 200µm200µm

7 days

days

atic

al

D E F

6ce

lls p

er s

p

0 7 14 0 7

0

17

7 d

sta

200µm 200µm 200µm

G H I

ays

mic

al

Ø Ø

Total collagen

[µg/

106

days

60

80

100200µm200µm200µm

7 da

dyna

mys

J K L

Total collagenO ***

*

er s

pher

oid]

34

20

40

60

14 day

14 d

ays

stat

ical

200µm 200µm 200µm

g/10

6ce

lls p

e

0 7 14 0 7

0123

statical dynamical

[µg

days

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

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.