STEFANO ZANASI VILLA ERBOSA HOSPITAL GRUPPO SAN DONATO ORTHOPAEDICS DEPARTMENT

1. RICOSTRUZIONE CON MSCs DELLE LESIONI CARTILAGINEE A STAMPO DEL GINOCCHIO E DELLA CAVIGLIA: • RISULTATI A 2 ANNI • SICOOP , MILANO 22 GIUGNO 2012 STEFANO ZANASI VILLA ERBOSA HOSPITAL GRUPPO SAN DONATO ORTHOPAEDICS DEPARTMENT IIIRD DIVISION – JOINT ARTHROPLASTY OPERATIVE CENTER CHIEF: STEFANO ZANASI M.D.

2. Cartilage has limited self-repair capabilitesarticular cartilage defects will ultimately result in chronic tissue losses To contrast this relentless outcome new reconstructive techniques have been developed such as 1. ACTlong-term results are encouragingbut present limitations2. MSCs abletodifferentiateintochondraland osseouslineages,thusabletofill the wholethicknessof a defect and secrete some trophicmolecules, whichcontributeofregenerationofdamagedtissue, the finalresultbeingcartilage on the top and bone on the bottom

3. MSCs Costituiscono una popolazione residente nel midollo osseo di cellule adulte non differenziate capace di autorigenerarsi e differenziarsi in cellule del tessuto cartilagineo, del tessuto osseo, del tessuto adiposo e nello stroma che supporta l’ematopoiesi

4. CELLULE STAMINALI DI MIDOLLO OSSEO AUTOLOGO CONCENTRATO BMAC Si ottengono in soli 15 minuti partendo da midollo osseo aspirato da cresta iliaca (60 o 120 ml) attraverso ciclo di centrifugazione operato da una centrifuga di piccole dimensioni, da usare in sala operatoria senza necessità di personale specializzato.

5. La procedura elimina i globuli rossi e il prodotto finale contiene • Cellule staminali emopoietiche • Cellule staminali mesenchimali • Progenitori vascolari • Cellule immunitarie e piastrine • Fattori di crescita (attivazione con trombina autologa) • in un volume finale di 10 o 20 ml • La procedura di concentrazione richiede l’utilizzo della centrifuga • e del kit BMAC composto di due confezioni B A (A) contiene il materiale utilizzato nel campo operatorio sterile per il prelievo del midollo da paziente (B) contiene il materiale per la procedura di concentrazione dell’aspirato midollare

6. Procedura • nella fase 1, si procede al prelievo del midollo da paziente, che viene raccolto • in una apposita sacca di sangue e infine trasferito in una siringa per essere • passato all’esterno del campo sterile nella fase 2, il campione di midollo viene immesso nella provetta, centrifugato, concentrato nel volume desiderato e di nuovo trasferito al campo operatorio per il definitivo utilizzo mediante connessione di 2 siringhe diverse

7. MSCs: 67 pts. from05/09 to05/10 forchondralkneedefectsOuterbridge stage III/IV accordingto Tom Minas’ classification simple30/6716sportmen coindefect (troclea, patellar, condyle/s, emi-tibialplate) complex14/679 sportmen shouldered massive unipolardefectof the lateral/medialcondyle plurifocalnotkissed and differentlycombined/sparedcoindefects (troclea, patellar, condyle/s, emi-tibialplate) salvage23/6714 sportmen shouldered, limitedkissinglesionsnotrequiringrealignment procedure unshoulderedkissinglesions and uni-compartmental OA concurrentlywithunloading/correctiveosteotomy 39/67 sportmen averageage25 ys (range19 - 50) - 47% F averagedefectsize 3.5 cm2 (range2.5 – 12.5cm)

8. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009 • Defect: bilateral patello-femoral kissing lesion • Location: massive involvement of the troclea and patella • Size: TROCLEA 2.5X1.5cm and PATELLA 3X1.5 cm /right • TROCLEA 1.5X1.5cm and PATELLA 2X1.5 cm / left • Patient: D.A. O.F., male, 44 years old. • History: grafted concurrently on 11/10/2009, • using fibrin glue as sealing (2 patches to fill the defect). • NMR at 1, 3, 6 and 12months post op • arthroscopic 2nd look on 30/07/2011

9. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009

10. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009

11. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009

12. 6 ms f.up

13. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009 Patient: Arthroscopy Time: 18 months Follow-up time: 18 months Subjective Evaluation Score: 93.5 (improvementfrombaseline: 49.0) Kneefunctionalgrade: Normal Cartilagerepairassessment: 12

14. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009 MOCART SCORING SYSTEM AT 12 MS F-UP A COMPLETE FILLING OF THE DEFECT A COMPLETE INTEGRATION OF THE BORDER ZONE TO THE ADJACENT CARTILAGE INTACT AND HOMOGENEOUS TISSUE REPAIR INTACT SUBCHONDRAL LAMINA AND SUBCHONDRAL BONE SCORE 95 12 ms f.up

15. Exemplificative case: salvage D.A.O.F., male, 44 years old - grafted on 11/10/2009 2nd look at 18 ms. follow-up STRONG COLLAGEN TYPE II DEPOSITION WELL-MATURED NEOCARTILAGE, WITH STRONG GLICOSAMINOGLYCANS DEPOSITION. COLUMNAR CHONDROCYTE REARRANGEMENT INSIDE THE GRAFTED TISSUE

16. Exemplificative case: salvage M.I., male, 23 years old - grafted on 11/1/2009 Defect: postraumatic ankle OA Location: massive involvement of the talar dome and tibia Size: 3x2/2.5 cm and 2.5x1.5 cm Patient: MalangaIvano , male, 23 years old. History: grafted on 11/1/2009, using fibrin glue as sealing (2 patches to fill the defect). NMR at 3, 6 and 9, 12, 18 months post op

20. MOCART SCORING SYSTEM AT 12 MS F-UP A COMPLETE FILLING OF THE DEFECT A COMPLETE INTEGRATION OF THE BORDER ZONE TO THE ADJACENT CARTILAGE INTACT AND HOMOGENEOUS TISSUE REPAIR SUBCHONDRAL LAMINA SUBCHONDRAL BONE ALMOST INTEGRATED SCORE 85

21. BaselineCharacteristics CRFsanalyzed: 56

22. IKDC: SubjectiveKneeEvaluation(n=67) (Score 0-100) Improved patients: 84.0 %

23. Subjective IKDC in relation tolesiontype SIMPLE, COMPLEX, SALVAGE Improved patients: 100.0% 66.7% 81.8% n= 30 n=14 n=23

24. Subjective IKDC in relation to lesionsize Improved patients: 71.4% 88.9%

25. EuroQol (EQ-5D) (N=67) Pain/discomfort Mobility Statisticallysignificantimprovement (painreduction) (Wilcoxonsignedrank test: p<0.0001) Statistically significant improvement in mobility (Wilcoxon signed rank test: p<0.0001) * Roset M et al. Sample size calculations in studies using EuroQol EQ5D. Quality of Life Research 8: 539-549, 1999

26. IKDC: KneeExamination Normal/Nearly Normal: 95.3 % Statistically significant improvement (Wilcoxon signed rank test: p<0.0001)

27. MOCART SCORING SYSTEM High-resolution 1.5T MRI was used to analyze the repair tissue with nine pertinent variables. A COMPLETE FILLING OF THE DEFECT was found in 92.5%, A COMPLETE INTEGRATION OF THE BORDER ZONE TO THE ADJACENT CARTILAGE in 94.1%. AN INTACT SUBCHONDRAL LAMINA was present in 84.6% AN INTACT SUBCHONDRAL BONE was present in 76.5%. Isointense signal intensities of the repair tissue compared to the adjacent native cartilage were seen in 92.3%. AVERAGE VALUE OF 78/100

28. Mean score: 11.4 100 % ArthroscopicEvaluation(N=4) Brittberg Score (1-12) Meanarthroscopictimefromgrafting: 17.3 months Significantlyimprovedappearanceof the tissue Total scaffoldbiodegradation Complete and uniformfibrocartilagineoustissueresurfacing discrete mechanicalresistenceto probe palpation Areasofunevencartilagestiffness

29. 2 1 1 2nd-Look BiopsyEvaluation 4 samplesanalyzed(meantime: 18 months) Based on criteriaofcellularity, celldistribution, matrixcomposition and collagentype I and II immunolocalization Hyaline-like: Mixedtissue: Fibrocartilage: Hyaline-like phenotype H&E Safranin-O Polarized Light

30. PRELIMINARY CONCLUSIONS: resurfacing by MSCs • Normal post-op withoutseriousadverseeventscorrelatedto the graft • 6/56 casesofincreased temperature (<39°) completelyceasedwithin 7 days • clinicalsympthoms(pain, effusion, catching, giving-way) significantlydecreasedwithin the 2nd month, and completelyceased, in allcases, within 3 monthsWITH GOOD/EXCELLENTJOINT FUNCTIONAL RECOVERY • Significative improvementof ROM (flex-ext >15%): • averagepre-op. active ROM 120° (range 80° - 140°) • average post-op active ROM 135° (range 110° - 140°) • SATISFACTORY CLINICAL RESULTS at 18 ms. average f. up

31. 2nd look arthroscopy at 12 ms f. up: biopsy DEMONSTRATES 2.5x 20x LIGHT STAINING FOR GAGS STRONG STAINING FOR GAGS LOW CONTENT AND NOT-UNIFORM DISTRIBUTION OF TYPE II COLLAGEN HIGH CONTENT AND UNIFORM DISTRIBUTION OF TYPE II COLLAGEN PRESENCE OF TYPE I COLLAGEN, NOT CELL CLUSTERING AND COLUMNAR ORGANIZATION ABSENCE OF TYPE I COLLAGEN, CELL CLUSTERING AND COLUMNAR ORGANIZATION THE MATURATION OF IMPLANTED TISSUE ENGINEERED CARTILAGE TO A CLEAR HYALINE-LIKE PHENOTYPE WITH PECULIAR CELL ORGANIZATION THE MATURATION OF IMPLANTED MSCS TO A CLEAR FIBRO-HYALINE-LIKE PHENOTYPE WITHOUT PECULIAR CELL ORGANIZATION

32. PRELIMINARY CONCLUSIONS: MSCs RECONSTRUCTION Need to verify the results at 3 and 5 years to appreciate the quality of the reconstructed tissue and the Maintainance/IMPROVEMENT of the (FIBRO)cartilage quality (no degenerative changes?)

33. In accordo con quanto scritto in • GianniniS., • “One-Step Bone Marrow-derived Cell Trasnsplantation in TalarOsteochondral Lesion”, • Clin. Orthop. Relat. Res. DOI 10.1007/s11999-009-0885-8 • (Associaton of Bone and Joint Surgeons 2009). • Questo studio riporta che, in seguito a inoculo del concentrato di midollo osseo su uno scaffold di acido ialuronico esterificato (HYAFF): • non si osserva alcuna complicanza locale nè sistemica • si ha la riformazione di tessuto cartilagineo • in modo del tutto sovrapponibile alla consolidata • tecnica del trapianto di condrocitiautologhi. • in un unico tempo operatorio, senza necessità di prelievo di cartilagine e clonazione della stessa in centro di coltura specializzato con reimpianto successivo dopo circa 30 gg • -Significativo minor costo della procedura