Fabio Valerio Sciarretta

Rome Italy

Accademia della BiorigenerazioneClinica Nostra Signora della Mercede

AMIC + ADIPOSE STEM CELLS FOR KNEE CARTILAGE REGENERATION

lunedì 27 gennaio 2014

GOAL• May adipose tissue represent the

alternative to bone marrow as mesenchymal stem cells source?

• Is it possible to use adipose tissue derived mesenchymal cells (ADSC) in

articular cartilage regeneration improving AMIC TECHNIQUE

ONE STEP SURGERY?

lunedì 27 gennaio 2014

....obesity > 30% of population

In last 40 yy.....

.... adipose tissue has been perceived as a real body unit

....significant increase in abdomenplasties & liposuctions

AdiposeAdipose

tissue tissue

Collagenase, centrifugationCollagenase, centrifugation

(floaters)(floaters)

StromalStromal--vascular cellsvascular cellsPrimary Primary adipocytesadipocytes

Selective cultureSelective culture

AdiposeAdipose Stem CellsStem Cells

11!!mm

Other Other

cellscells

11!!mm

Isolation of Adipose Stem CellsIsolation of Adipose Stem Cells

Zuk P et al. Tissue Engeneering 7, 2001Zuk P et al Mol Biol of Cell 13, 2002

Friedenstein identified .... .... then many other laboratories have confirmed that stromal cells similar to

those in the bone marrow could be isolated from adipose tissue resected or as intact tissue or aspirated

by liposuction

.... although the fat represented the surgical waste, plastic surgeons have found

it was a source of abundant and accessible multipotent stromal cells that might be used for

regenerative purposes

lunedì 27 gennaio 2014

Adipose tissue contains a great number of ADSC overlapping & mostly similar to BMSC but with distinct immunophenotypes & citokine profiles

Liu TM et al. Stem Cells 25:750, 2007 Gimble JM et al. Circ Res 2007

From 1% to 10% of SVF is constituted by progenitor cells & stem cells: about 0.5 × 104 a 2 × 105 stem cells

per gram of adipose tissue

(Zuk PA, De Ugarte, Mitchell JB, Guilak F 2006, Fraser 2008)

ADSC have with certainty the ability to transform in CHONDROCITES

ADSC’s

Lin G. et al. Stem Cells and Dev 17, 1053-64, 2008 - Guilak F et al. J Cell Phis 206, 2006 Fraser Jk Trends Biotechnol 24, 2006;

Winter A. Arth & Rheum, 2003 - Erickson GR Bioch and Bioph Res Com,2002 - de Girolamo L Cytothec, 2009

this means from 1,000 to 2,500-fold more than the frequency of stem cells in bone marrow

lunedì 27 gennaio 2014

Nathan et al, “Cell-based therapy in the repair of osteochondral defects: A novel use for adipose tissue” Tissue Engineering, 2003.

Rabbit Osteochondral Defect Repair Model

• 180 Day Study – Blinded/Placebo• Bilateral Hip OA – 18 dogs• Intra-articular stem cells – 1X• Vet/Owner assessments at pre, 30, 60, 90

180 Day Prospective, Non-randomized StudyUni or Bilateral Elbow OA - 14 dog studyIntra-articular stem cells 1xVet and Owner assessments at pre, 30, 60, 90, 180

ADSC - Animal Clinical Applications

J Pediatr Orthop. 2004 Jul-Aug;24(4):427-33.

Treatment of chondral lesions in advanced osteochondritis dissecans: a comparative study of the efficacy of chondrocytes, mesenchymal stem cells, periosteal graft, and mosaicplasty (osteochondral autograft) in animal models.Hui JH, Chen F, Thambyah A, Lee EH.Department of Orthopaedic Surgery, National University of Singapore, Singapore. doshuij@nus.edu.sg

lunedì 27 gennaio 2014

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ADSC - First Human Clinical Application

• 65 yr old male 28 months post hemimaxillectomy for keratocyst• Harvest autologous adipose tissue and expand ASC in autologous serum• Implant in rectus abdominis muscle above epigastric artery for 6-8 months• Transfer as free flap to repair palatal defect• Subsequent dental implants • Success out to 1.5 year follow up

lunedì 27 gennaio 2014

standard extraction process of stromal cells: enzymatic digestion at 37 º (collagenase, trypsin,

dispase), neutralization of the enzymes and centrifugation

whereby theStromal Vascular Fraction is separated from adipocytes

ADSC Isolation Process

Stromal Vascular Fraction consists ofan heterogeneous mesenchymal population:

Progenitor cellsStromal and hematopoietic stem cells

Endothelial cells, erythrocytes, fibroblasts, lymphocytes, macrophages, pericytes

further purification by washingeliminates hematopoietic cells and reveals:

Stem cells from adipose tissue (ADSC)

AdiposeAdipose

tissue tissue

Collagenase, centrifugationCollagenase, centrifugation

(floaters)(floaters)

StromalStromal--vascular cellsvascular cellsPrimary Primary adipocytesadipocytes

Selective cultureSelective culture

AdiposeAdipose Stem CellsStem Cells

11!!mm

Other Other

cellscells

11!!mm

Isolation of Adipose Stem CellsIsolation of Adipose Stem Cells

But requires 10 hoursNot applicable in clinical practice

lunedì 27 gennaio 2014

Francis MP, et al. Organogenesis 6, 1 2010

•about 100000 ADSCs per 100 ml of blood/saline with differentiation potential, characteristic cell surface markers and proliferative lifespan indistinguishable from BMSCs or conventionally processed ADSCs

Improved enhanced isolation of ADSC from adipose tissue

on the basis of this technique...

• Possibility of isolating MSCs on the basis of their characteristics of adhesion to culture surface, without resorting to collagenase tissue lysis, through direct culture of mechanically dissociated fragments of adipose tissue ( W. Lattanzi)

...In Line Tissue Engeneering

• Application of Tissue Engeneering tecniques in only one surgical step under control and authorization of the reference Blood Transfusion Centre

lunedì 27 gennaio 2014

ADSC My Stem withdrawal technique

Once the chondral defect has been arthroscopically identified and debrided, in local anesthesia, we extract, by a simple, minimally invasive method of liposuction aspirate, with a specifically created adipose tissue biopsy needle, the marrow tissue from adult adipose tissue of the abdomen. Every step is done in the OR: from the vacuum syringe the liposuction tissue is connected to the collection bag of pre-treatment, where it is mechanically filtered and then introduced in the centrifuge and centrifugated for 10 minutes in order to obtain the Stromal Vascular Fraction

lunedì 27 gennaio 2014

The stromal-vascular fraction of cells is separated from the mature lipid-laden adipocytes and the water cell-free component by centrifugation according to a specific centrifugation protocol for 10 minutes. This fraction, which represents a heterogeneous population of cells, contains the ADSCs in a large number, with yields of approximately 250,000 cells per gram of tissue. Then, adding fibrin glue to the concentrated ADSCc obtained by centrifugation, we obtain a sticky clot that is ready to be easily implanted in the osteochondral prepared defect, with or without the use of a collagen membrane scaffold.

ADSC Surgical Tecnique

Aust L et al. Cytotherapy 6;7-14, 2004

lunedì 27 gennaio 2014

• Non-unions• Bone loss• Femoral Head Avascular Necrosis • Chondral/Ostechondral Defects • Tendinopathies

• Few data with long term follow-up (Wakitani et al., TERM 2011)

• 45 joints with 6 yy. mean followup (range 1-11)

ADSC - One-step Technique Indications

Uysal AC, Mizuno H (2010) CurrStemCell Res Ther ObaidH, Connel D (2010) AmJ Sports Med BehfarM, Sarrafzadeh-Rezaei et al. (2012) Curr StemCell Res Ther

MSCs Safety

•No adverse event, infection or tumor

lunedì 27 gennaio 2014

ESTRAZIONE DELLE CELLULE

DA GRASSO

IN SALA OPERATORIA

GIURIDICAMENTE

LEGITTIMO CON MACCHINARI

MARCATI CE CHE EFFETTUANO

MINIMA MANIPOLAZIONE

TESSUTO ADIPOSO

FONTE DI STAMINALI

STROMALI DA TESSUTO ADIPOSO

COLLAGENASE

Legal Aspects of ADSCs one step techniqueAdipose Tissue

source of mesenchymal stem cells

ADSCs extraction from adipose tissue in OR is autologous & omofunctional

IT’S NOT transfusional activity & DOESN’T require passage through the

Cells Bank

legally legitimated with CE marked machineries that require minimal handling

ad hoc informed consent & with systems that DON’T use

enzyme debated in AIFA about it’s harmelessness on stem cells

EC DIRECTIVE 23/2004 CELLS & TISSUE BANK

1 di 18

MANUALE dell’ UTENTE

CENTRIFUGA da BANCO ( R-8D )

Leggere attentamente le istruzioni di sicurezza (a pagina 14 ) prima di utilizzare

la centrifuga

REMI MOTORS LTD.

VASAI , INDIA

SIOT Forensic Committee lunedì 27 gennaio 2014

..New My Stem Evo..New completely closed

system in which the introduction of a particular filtration system eliminates the need for centrifugation

Billig, 1983; Russel 2007

lunedì 27 gennaio 2014

MyStem Evo technique

lunedì 27 gennaio 2014

MyStem Evo technique

lunedì 27 gennaio 2014

Autologous matrix-induced chondrogenesisAMIC TECHNIQUE

A biological treatment option which repairs articular cartilage damage by microfracture surgery combine with the application

of a bi-layer collagen I/III membrane

lunedì 27 gennaio 2014

Hyalofast™ Scaffold•Hyalofast™ is a non-woven pad made o f H Y A F F® , a s e m i - s y n t h e t i c derivative of hyaluronic acid, major component of human cartilage.

•Acts as a scaffold which allows in situ residence of mesenchymal stem cells and other autologus elements after their mobilization due to microfracture or perforation procedures.  Upon degradation, the scaffold releases natural hyaluronic acid.

lunedì 27 gennaio 2014

•Chondro-Gide® is a resorbable collagen membrane, consisting of collagen types I and III. It’s particularity is due to it’s bilayer structure, with one compact and one porous side.

•The compact layer, which is cell occlusive, prevents cells from diffusion and also protects them from mechanical impact.

•The porous layer is made of collagen fibers in a loose, porous arrangement that favours cell invasion and attachment.

•This 3D collagen matrix stimulates the autologous cultured cells to differentiate into the chondrocyte phenotype and to produce collagen II and GAG.

Chondro-Gide® Membrane

lunedì 27 gennaio 2014

Our experience with AMIC + ADSCFirst 20 patients with III-IV degree chondral defects treated by ADSCs with scaffold membrane

Age (28-48 yy)

Arthroscopic assisted procedure/ mini open in patellar defects

Clinical: IKDC: initial improvement @ 6-12 months

No adverse reaction

Immediate ROM & muscle strenghtening

lunedì 27 gennaio 2014

AMIC + ADSC Surgical steps

Billig, 1983; Russel 2007

lunedì 27 gennaio 2014

• arthroscopic debridement & defect’s evaluation & sizing • defect’s preparation• template’s creation• microfractures• soaking double layer Chondrogide collagen membrane into SVF• fill the defect & the membrane with fibrin glue

• Check membrane adherence by repeated ROM movements

AMIC + ADSC TECHNIQUESurgical steps

lunedì 27 gennaio 2014

Surgical steps

AMIC + ADSC TECHNIQUE

lunedì 27 gennaio 2014

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lunedì 27 gennaio 2014

clinically valid

quick, perfect for one step surgery low cost

Discussion• The technique involves use of a fully closed device, ready for use, without centrifugation, in absolute sterility without risk of contamination or manipulation

Initial clinical, histological & MRI results encouraging

easy

lunedì 27 gennaio 2014

Discussion• With this system we have obtained ADSC with the defined caracteristichs •& in number as reported in the literature

• The cells are grouped in the stromal niches

•present the typical elongated splindle shape

Flow cytometry revealed CD45, CD34, CD31, CD105 & CD90 markers expression

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lunedì 27 gennaio 2014

Knee Surgery, Sports Traumatology, ArthroscopyJanuary 2013

Mesenchymal stem cells for the treatment of cartilage lesions: from preclinical findings to clinical application in orthopaedicsGiuseppe Filardo, Henning Madry, Mislav Jelic, Alice Roffi, Magali Cucchiarini, Elizaveta Kon

The systematic research showed an increasing number of published studies on this topic over time and identified 72 preclinical papers and 18 clinical trials. Among the 18 clinical trials identified focusing on cartilage regeneration, none were randomized, five were comparative, six were case series, and seven were case reports; five concerned the use of BMC, and 11 the use of bone marrow-derived MSCs, two the use of adipose-derived MSCs with preliminary interesting findings ranging from focal chondral defects to articular osteoarthritis degeneration.

lunedì 27 gennaio 2014

Adipose tissue is an ideal tissue to use as an autologous substitute with which to approach tissue deficiency. Clinically, the use of fat grafts and adipose-derived stem cells has dramatically increased worldwide for reconstructive and aesthetic purposes. Human adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. Adipose tissue is an abundant, expendable, and easily obtained tissue that may prove to be an ideal source of autologous stem cells for regenerating tissues. The recent identification and characterization of multilineage cells from human adipose tissue has been met with a great deal of excitement by the field of tissue engineering. The authors' laboratory has characterized a population of cells obtained from human adipose tissue that have the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, and neuron-like cells in vitro.

J Korean Med Assoc. 2012 Aug;55(8):757-769

Adipose-derived stem cells: characterization and clinical application

Jong Won Rhie

Chondrogenic differentiation from adipose derived stem cell. Alcian blue stain

lunedì 27 gennaio 2014

Stem Cells InternationalVolume 2012 (2012), Article ID 812693, 11 pages

Review Article

Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity

Patrick C. Baer and Helmut Geiger

Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs) yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells.

lunedì 27 gennaio 2014

lunedì 27 gennaio 2014

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Graziefvsciarretta@me.com

www.fabiosciarretta.com

lunedì 27 gennaio 2014