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.