irmi unige
TRANSCRIPT
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Bone structure
July 2013
Ranieri Cancedda, MD University of Genova &
IRCCS AOU San Martino – IST National Cancer Research Institute, Genova
Tissue Repair in Orthopedics: from Tissue Engineering to Regenerative Medicine
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Bone defects requiring repair/regeneration
§ Fractures § Traumatic bone loss § Bone tumor surgery § Congenital malformations
The “canonical” tissue engineering approach Crush injury on forearm in
a 30 year old man
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Bone Cell Therapy: ���a long way to the clinical application
ü in vitro studies (cell-biomaterial interactions)
ü nude mouse model
ü large animal model
ü clinical studies
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Tortelli et al. Tissue Engineering part A 2009
Bone ECM deposition in vitro in a 3D culture
Implant of MSC seeded scaffolds in nude mice
Mastrogiacomo et al. Tissue Engineering 2006 Giannoni et al. J. Tissue Eng. Regen. Med. 2008 Komlev et al. Tissue Eng. part C 2009
Tibia sheep model
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First bone repair by autologous MSC
Quarto R, et al: New Engl J Med 344: 385, 2001
10 months
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Number of novel cell therapy treatments for Bone Repair in Europe
Auto Allo Total HSC MSC HSC MSC HSC MSC
2008
Maxillo-facial 2
Orthopedics 10 12
12 12
2009
Maxillo-facial 1 1
Orthopedics 23 43 2
26 44
2010
Maxillo-facial 1 1
Orthopedics 26 19 10
28 29 Modified from Martin et al., Tissue Engineering: Part A; Volumes 16,17,18; 2010-12
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Reasons why bone tissue engineering is not making inroads
Regulatory issues (FDA, EMA, National Agencies …..)
Remaining unresolved scientific problems (identification of the “optimal” scaffold, large implant vascularisation ……)
Need of highly sofisticated cell culture facilities High cost of the procedure Difficult logistics Existence of therapeutic alternative
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• Haematoma occurs at the site of tissue injury • Platelets are activated and release bioactive substances • Innate immunity cells migrate (Granulocytes and Macrophages) • Endothelial progenitors move in and angiogenesis begins • Resident and recruited cells with a differentiation potential increase in number and new tissue formation initiates
Tissue Healing Overview (triggering of the process by platelet released factors)
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Bone structure
MSC can engraft in injured tissues and differentiate to replace damaged organs
However, MSC can also act as “factories” of bio-active molecules to limit tissue destruction or enhance repair by a variety of mechanisms
Mesenchymal Stem Cells as “medicinal” cells: induction of endogenous bone repair
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Can implanted MSC trigger a cascade of events leading to mobilization of macrophages, angiogenesis and endogenous bone formation?
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10.9!3.2!4.7!10.8!Day 7!
20.9!7.6!5.2!13.5!Day 3!
5.6!3.6!3.8!9.6!Day 1!
M2 (CD206+,CD51+)!
(% of total)!
M1 (CD86+,CD40+)!(% of total)!
M2 (CD206+,CD51+) !
(% of total)!
M1 (CD86+,CD40+)!(% of total)!
+ MSC !SKELITE!SKELITE! EMPTY!
0!0.5!
1!1.5!
2!2.5!
3!3.5!
4!
Empty Skelite!Skelite (+MSC)!
M2/
M1
ratio!
Day 1! Day 3! Day 7!
Innate immunity cells migration (Macrophages)
Tasso et al.; submitted
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Triggering of the process by platelet released factors
Activated Platelet
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Blood dona(on
Buffy coat isola(on
Buffy coat pool
Platelet concentrate (PRP)
PLATELET POOR PLASMA (PPP)
PLATELET LYSATE (PL)
Platelet adjustment to optimal concentration
High speed centrifugation
Freeze-thaw cycles Membrane removal
Validation test at the Transfusional Center
Platelet Derivative Production
RBC
PRP
PPP
PRP
Leukocytes & Platelets (<1% of total blood)
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BM-MSCs
16 hour stimulation with PL or IL-1α or PL + IL-1α
Gene activation analysis
NF-kB activation (binding to specific DNA sequences)
* *
* **
Fold
incr
ease
control"
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Increased synthesis of PGE2 induced by PL in an inflammatory
microenvironment
0
10000
20000
30000
40000
50000
60000
70000
80000
SF IL1 PL PL+IL1
PGE2
pg/m
l *
*
*
** *
control"
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Hypoxia and cell culture
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Research objectives within the IRMI project • Further investigate the influence of the microenvironment (clot formation, inflammation,
hypoxia) on the paracrine activity of stem cells: bone marrow Mesenchymal Stem Cells (MSCs), Amniotic Fluid Stem Cells (AFSCs)
• Perform a continuous “basic – translational research” feedback loop to remove identified bottlenecks to the use of allogenic (from donors) Platelet derived sub-fractions.
• Perform a continuous “basic – translational research” feedback loop to remove identified bottlenecks to the use of conditioned culture medium, and product thereof, from allogenic bone marrow MSCs and Amniotic Fluid Stem Cells (AFSCs)
• Anticipated bottlenecks include more detailed understanding of the recipient’s body immunoresponse.
• Develop and optimize products derived from platelets and/or stem cell conditioned media to be used as therapeutic agents either as an injectable solution or combined with biomaterials for the repair of: a) tendon and ligament lesion; b) cartilage and bone lesions and defects (bioactive scaffolds); c) skin lesions such as chronic ulcers, difficult wounds, bedsores, chinks (bioactive membrane).
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Biorigen Srl���(Spin off dell’Universita’ degli Studi di Genova)
Biorigen continuerà le ricerche in corso per mantenere ed aumentare il vantaggio competitivo derivante dai brevetti ottenuti e accrescere il potenziale mercato di
riferimento con la brevettazione di prodotti/servizi. Verra’ potenziata l’attivita’ di Ricerca e Sviluppo con particolare attenzione a tematiche relative a:
- Reagenti per colture cellulari basati su derivati piastrinici - Prodotti di medicina rigenerativa per terapia umana e veterinaria nel settori
dermatologico (prevalentemente ulcere e lesioni cutanee), ortopedico e odontoiatrico.
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