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Aspetti rigenerativi del disco lombare: è possibile?

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Presentazione sul tema: "Aspetti rigenerativi del disco lombare: è possibile?"— Transcript della presentazione:

1 Aspetti rigenerativi del disco lombare: è possibile?
Gabriela Ciapetti Fisiopatologia Ortopedica e Medicina Rigenerativa IOR

2 tessuto discale istologia fissazione sezionamento colorazione
Laboratorio di Fisiopatologia Ortopedica e Medicina Rigenerativa contenitore + terreno sterile intervento biologia molecolare °C x rtPCR coltura cellulare frammentazione semina espansione istologia fissazione sezionamento colorazione Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

3 IVD (#3387 – 45 anni, F- Pfirrmann 5)
Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

4 disco intervertebrale
scarsità cellule 5000/cm3 NP; 9000/cm3 AF avascolare basso pH ipossia (1-2% pO2) degenerazione precoce carichi meccanici complessi perdita di cellule notocordali (6 mesi nel coniglio;16 anni nell’uomo) immuno-privilegiato The IVD is confined by the two cartilage endplates and is composed of two distinct structures, the nucleus pulposus (NP), and the surrounding annulus fibrosus (AF). Although the two cartilage endplates offer anatomical limitation to the vertebral bodies, morphology along the plate is distinguished by a central articular-like cartilage under the NP and a peripheral fibrocartilage appropriately associated with the AF. During embryogenesis, the AF develops from the mesenchyme, whereas the NP is derived from the notochord. The AF consists of water (65–90%), collagen (50–70% dry weight), proteoglycans (10–20% dry weight) and noncollagenous proteins (e.g. elastin). The AF has a laminate structure consisting of a minimum of 15 (posterior) to a maximum of 25 (lateral) concentric layers. The layers are composed of type 1 collagen fibres that alternate in angles from 28 (peripheral AF) to 44 (central AF) with respect to the transverse plane of the disc. The spaces between the separate layers of the AF are called interlamellar septae, and they contain proteoglycan aggregates and a complex structure of linking elements creating interlamellar cohesion. At the periphery, some of the annulus fibres pass the endplates to penetrate into the bone of the vertebral body as ‘‘Sharpey’s fibres’’. Central fibres either insert into the cartilage of both endplates or bend with the NP. Da: Johannes Leendert Bron. Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges Eur Spine J (2009) 18:301–313. Il disco è praticamente avascolare perché ci sono solo pochi capillari che entrano per alcuni millimetri entro l’outer annulus. BMP2 Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

5 attività paracrina MSC
terapie ‘biologiche’ ingegneria tissutale medicina rigenerativa precursori scaffold fattori vascolarizzazione BMP2 attività paracrina MSC autoriparazione STRATEGIE RIPARATIVE tendono ad ‘accrescere’ o rimpiazzare il disco esistente introduzione cellule ‘attive’ su scaffold Regenerative strategies for the treatment of disc degeneration are focused on reviving or healing extant disc tissue. This can be done either by altering the phenotype of cells native to the ailing disc or by introducing new cell populations. Injection of growth factors such as bone morphogenetic protein 7 (BMP-7), transforming growth factor-β (TGFβ), growth/differentiation factor 5 (GDF-5) and others into the disc has been widely studied as a means to stimulate extracellular matrix production and cell proliferation (Masuda, 2008). In certain animal models of disc degeneration, treatments have successfully diminished or even reversed degeneration-like characteristics (Masuda, 2008). However, the translation of such treatments to human application and clinical use is hampered by the inability to accurately recreate the progressive, life-long degenerative transformation of the disc in an animal model (Alini et al., 2008). Moreover, the potential success of anabolic factors injected directly into the disc might be limited, both owing to the short biological half-life of the factors and their rapid diffusion away from the delivery site. Alternatively, cell populations within the disc can be manipulated through gene therapy approaches, which involve the delivery of genes into cells through viral-vector-mediated gene transfer (Sobajima et al., 2004). Finally, a more recent regenerative approach under investigation is cell therapy, whereby cells are delivered locally to the degenerated disc. The purpose of these cells is to either provide signaling cues that ameliorate the effects of disc degeneration, or adopt and/or maintain disc-like phenotypes themselves, producing extracellular matrix intended to re-establish healthy disc function (Leung et al., 2006; Sakai, 2008). Reparative strategies are focused on either augmenting or replacing degenerate disc tissue to re-establish healthy disc function. Although there are several non-biological reparative methods available, a recent focus in this area is on tissue engineering. The appeal of tissue engineering strategies is that, unlike non-biological materials that can wear with time, cell-generated tissues retain their capacity for remodeling and growth. The prevailing paradigm of tissue engineering is that cells on a biomaterial substrate or scaffold can be coaxed into forming new tissue when the appropriate stimuli (biological or physical) are provided. Hydrogels such as alginate-, collagen- and hyaluronan-based gels, among others, have been shown to support the survival of mature NP cells and to be conducive to matrix deposition (O’Halloran and Pandit, 2007). Although NP tissue engineering has been a particular focus over the years, interest has more recently turned to the AF and to whole disc composite tissues (Bowles et al., 2010; Mizuno et al., 2006; Nerurkar et al., 2010; Nesti et al., 2008). Most of these studies have used either disc cells or mesenchymal stem cells. Although experiments involving in vitro formation of disc-like structures have been used to make significant advances, important challenges remain to be addressed, including translation of these technologies to large animal models for pre-clinical trials and meeting in vivo nutritional requirements. STRATEGIE RIGENERATIVE tendono a rivitalizzare o ‘guarire’ il disco ancora esistente iniezione di fattori di crescita introduzione nuove popolazioni alterazione fenotipo cellule residenti Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

6 scaffold e cellule cellule indifferenziate cellule differenziate
STRATEGIE RIPARATIVE scaffold e cellule Nerurkar NL J Biomech 2010;43:1017 alginate/chitosan fibers carbossimetilcellulose gel atelocollagen honeycomb electrospun, nanofibrous nanofibrous+ agarose NP NP cells in alginate hyaluronic+ nanofibrous PCL multi.lamellar AF & DBM AF cells in PGA mesh Figure 3An array of strategies for disc tissue engineering. A) hybrid alginate/chitosan fibers synthesized for AF tissue engineering (Shao and Hunter, 2007). B) Carboxymethylcellulose gel seeded with NP cells (Reza and Nicoll, 2009a). C) Atelocollagen honeycomb scaffolds engineered from natural ECM (Sato et al., 2003a; Sato, et al., 2003b). D) Aligned, electrospun, nanofibrous scaffolds seeded with mesenchymal stem cells (Nerurkar, et al., 2007; Nerurkar, et al., 2008c; Nerurkar, et al., 2009a; Yang, et al., 2008). E) Engineered multi-lamellar AF constructed from poly(polycaprolactone-triol-malate) seeded with chondrocytes, and surrounded with a demineralized bone matrix (Wan, et al., 2008). Composte whole-discs constructed from an NP cell-encapsulated alginate hydrogel surrounded by an AF cell-seeded PGA mesh (Mizuno, et al., 2006). G) Disc formed from a composite hyaluronic acid/nanofibrous scaffold seeded with human mesenchymal stem cells (Nesti, et al., 2008). H) Polarized light microscopy of Picrosirius Red stained section from disc-like angle-ply structure formed from aligned nanofibrous scaffolds surrounding an agarose NP (Nerurkar et al., 2009b). Presa da : Nerurkar NL, Elliott DM, Mauck RL. Mechanical design criteria for intervertebral disc tissue engineering. J Biomech Apr 19;43(6): cellule indifferenziate coltura in 3D serum-free cellule differenziate coltura in 2D fattori di crescita ipossia co-cultura MSC-NP Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

7 fattori di crescita BMP-2, BMP-7 TGF-β1, IGF-1 GDF-5
STRATEGIE RIGENERATIVE fattori di crescita BMP-2, BMP-7 TGF-β1, IGF-1 GDF-5 incremento proliferazione cellulare aumento aggrecano, collagene tipo II riduzione delle cellule apoptotiche up-regolazione di Sox-9 ……. PRP BMP2 e 7 fattori anabolici secreti dal disco post invecchiamento e danno indotto… sia per addizione, che attraverso up-regolazione, trasfezione con AAV nelle cellule in vitro …aumentano coll II e aggrecano frase da: Than KD, Rahman SU, Vanaman MJ, Wang AC, Lin CY, Zhang H, Marca FL, Park P. Bone morphogenetic proteins and degenerative disc disease. Neurosurgery Sep 5. Spine J Jan;10(1): Epub 2009 Nov 18. Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture. Liang H, Ma SY, Feng G, Shen FH, Joshua Li X. GDF5 : membro della superfamiglia del TGF-b e della sub-famiglia delle BMP mancanza porta a degenerazione precoce dal disco post invecchiamento e danno indotto… in vitro e in vivo (in coniglio) aumenta coll II e aggrecano Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins. degeneration changes of discs punctured by different-size needles to develop a mice lumbar disc degeneration model and to evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene. Conclus:Disc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment. rhBMP-2/CRM/CD HORIZON® Spinal System Pivotal Study (phase III) March 2002 – February Medtronic Spinal and Biologics (463) Spine Fusion Instrumented With BMP-2 vs Uninstrumented With Infuse BMP-2 Alone Study Nov 2006 – Dec Capital District Health Authority, Canada (50) Intradiscal rhGDF-5 Phase I/II Clinical Trial June 2008 – January Advanced Technologies and Regenerative Medicine, LLC (48) Than KD, Neurosurgery 2011; Hayes AJ, Histochem Cell Biol 2011; Liang H, Spine J 2010 Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

8 terapia cellulare STRATEGIE RIGENERATIVE cellule NP condrociti MSC cellule autologhe : basso rischio infezione; no terapia immunosoppressiva cellule NP da disco sano o discectomia: reazioni avverse o resa scarsa attivazione delle cellule progenitrici endogene (cellule da disco degenerato: CD105, CD166, CD63, CD49a, CD90, CD73, LNGFr ) Svantaggi per ciascuna: NP o AF cells= sono poche, non è detto che siano ‘attive’, raccogliere cellule autologhe significa creare ‘danno’ (injury) al sito di prelievo Condrociti dalle superfici NON articolari del ginocchio, espanse e ri-iniettate= procedura piuttosto ‘pesante’ MSC= procedura POCO invasiva e midollo ‘ricco’ di MSC che possono differenziare in un fenotipo simil-condrocitico Il paper di KUH conclude che i condrociti sono quelli che producono più aggrecan e collageni rispetto a BM derived MSC e AF cells. Se stimolate con BMP2 tutti e tre i tipi di cellule producono … condrociti da cartil articolare : molto simili a cellule NP bassa resa e morbidità MSC da midollo osseo o tessuto adiposo : resa elevata e differenziamento in NP e condrociti ………. KUH SU Joint Bone Spine 2009;76:70 Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

9 MSC e IVD STRATEGIE RIGENERATIVE 48 settimane Sakai D, Biomaterials 2003; Yoshikawa T, Spine 2010 Fig. 3. Use of stem cells in a direct transplantation to degenerated IVD.da paper D Sakai 2011 stampato Il primo studio di trapianto di MSC in vivo è SAKAI nel Le MSC autogene erano marcate con GFP: The percentage of positive cells increased from 21% +-6% at 2 weeks to 55% +- 8% at 48 weeks; this increase proved that the MSCs survived and proliferated. Yoshikawa and colleagues nel 2010 transplanted autologous bone marrow MSCs into IVD showing vacuum phenomenon and instability in two patients going under decompression surgery for spinal stenosis. 2 anni post surgery radiography and CT showed improvements in the vacuum phenomenon in both patients. Yoshikawa T, Ueda Y, Miyazaki K, Koizumi M, Takakura Y. Disc regeneration therapy using marrow mesenchymal cell transplantation: a report of two case studies. Spine (Phila Pa 1976) May 15;35(11):E Qui sotto da: Sakai D, Mochida J, Yamamoto Y, Nomura T, Okuma M, Nishimura K, Nakai T, Ando K, Hotta T. Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials Sep;24(20): To evaluate the possible potential of MSCs in disc cell research and treatment of degenerative disc disease, autologous MSCs embedded in Atelocollagen gel were transplanted into the discs of rabbits which had undergone a procedure proven to induce degeneration. The results suggest that MSC transplantation is effective in decelerating disc degeneration in experimental models and provided new hopes for treatment of degenerative disc disease in humans. Atelocollagen gel served as an important carrier of MSCs in transplantation, permitting proliferation, matrix synthesis and differentiation of MSCs. Gli studi mostrano sì un rallentamento della degenerazione, maggior produzione di GAG, ma gli studi (su animali) sono condotti su difetti prodotti con tecnica ‘one shot’ ‘di colpo’, mediante iniezione di sostanze o insulto meccanico, e non dovuti alla situazione patologica che progredisce nel corso di mesi/anni…. Fig. 2. Use of stem cells for direct induction toward NP phenotype. Da SAKAI 2011 Testo: Using the multipotent differentiation capacity of stem cells, the author attempted to induce MSC differentiation in a mixed coculture system with NP or AF cells in alginate beads. Cell colorate con due differenti fluorocromi tipo PHK26.. MSC cocultured with NP cells expressed type II collagen and keratin sulfate, whereas the expression of type I collagen was more intense in cells cocultured with outer AF cells compared with the MSC before coculture. Gene expression analysis by reverse transcription–polymerase chain reaction (RTPCR) also confirmed that coculture with different IVD cells in the same 3-D environment led to differentiation of MSCs toward the direction of the cocultured opponent. These experiments showed that the mixed coculture system in alginate is an effective tool for inducing differentiation to MSCs. Safety and Preliminary Efficacy Study of Mesenchymal Precursor Cells (MPCs) in Subjects With Chronic Discogenic Lumbar Back Pain Aug 2011 – July Mesoblast, Ltd. (100)  injection of either 6 million (M) or 18M cells in a hyaluronic acid carrier into the degenerated lumbar disc nucleus pulposus Isolation and Authentication of Mesenchymal Stem Cell-like Progenitor Cells From the Degenerated Intervertebral Disc of Lumbar Spine Sept dec Taipei Medical University WanFang Hospital (20) Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

10 Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR
STRATEGIE RIGENERATIVE terapia genica Il fondamento della terapia genica per IVD è la trasduzione dei geni per citochine anaboliche …TGFb1, BMP-2, IGF-I, PDGF, GDF-5….. LIM mineralization protein (LMP), Sox-9… oppure per molecole anti-cataboliche, es. TIMP-1 cDNA per TGFb1 Il fondamento della terapia genica per IVD è l’identificazione di citochine anaboliche per il disco…TGFb1, BMP-2, IGF-I, PDGF, GDF-5…transforming growth factor b1, bone morphogenetic protein 2, insulin growth factor I, platelet derived growth factor The current concept of gene therapy has expanded to include the transfer of exogenous genes encoding therapeutic proteins into cells to treat disease. On transduction of this genetic material into the genome of the target cell, the host transcribes the transgene into mRNA, which is then translated by ribosome in the cytoplasm into the desired protein product. These protein products affect not only the metabolism of the host cell but also that of adjacent cells via a paracrine effect. AAV: adeno-associated virus trasduzione diretta o nelle cellula da trapiantare…. effetto paracrino: le proteine prodotte influenzano anche il metabolismo delle cellule adiacenti Woods B, Orthop Clin N Am 2011 Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

11 cellule esogene..trasfettate + fattori di crescita
limiti/rischi fonte non ben definita vita limitata delle cellule trapiantate microambiente ostile (bassa pO2, basso pH, no vasi..) effetti indesiderati (es. osteofiti) [Vadalà G. J Tissue Eng Regen Med 2011] terapia cellulare in assenza di cellule ‘responsive’ l’effetto è scarso BMP: reports discordanti uso off-label [Mroz TE .Spine 2010] subsidenza, infezione, radicolite, osso ectopico… Carragee EJ. Spine J 2011] fattori di crescita terapia genica Karin Wuertz, MSC response to pH levels found in degenerating intervertebral Discs Biochem Biophys Res Commun February 20; 379(4): 824–829.(da Nicola IVD) Results indicated that MSC functionality, phenotype, and viability were minimally restricted at pH 7.1, representing a fairly healthy disc, and severely compromised at pH 6.5, representing a severely degenerated disc. Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation. Vadalà G, Sowa G, Hubert M, Gilbertson LG, Denaro V, Kang JD. J Tissue Eng Regen Med Jun 13. Spine (Phila Pa 1976) May 1;36(10):E623-8.In vitro and in vivo testing of a novel regulatory system for gene therapy for intervertebral disc degeneration. Sowa G, Westrick E, Pacek C, Coelho P, Patel D, Vadala G, Georgescu H, Vo N, Studer R, Kang J. AAV……vettori non virali, i.e. liposomi, complessi DNA-ligando, micro-bolle (microbubbles) sistemi di ‘sicurezza’ per il vettore AAV [Sowa G. Spine 2011] cellule esogene..trasfettate + fattori di crescita Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

12 per ‘migliorare’ l’approccio rigenerativo all’IVD in degenerazione
conclusioni l’unione di due tessuti ‘disparati’ come l’AF fibroso e il NP gelatinoso rappresenta una sfida formidabile per l’ingegneria tissutale Nerurkar NL. J Biomech 2010 gli studi di ‘efficacia’ di cellule, fattori, scaffold sono condotti su animali, dove la degenerazione, che normalmente progredisce per anni nell’uomo, è simulato attraverso singolo insulto chimico o meccanico gli studi si concentrano su AF e/o NP: il ruolo della vascolarizzazione dal piatto vertebrale nel disco degenerato non è chiaro Moore RJ. Eurs Spine J 2006 Tissue Engineering of the Intervertebral Disc The union of two tissues as disparate as the fibrous AF and gelatinous NP poses formidable challenges in tissue engineering, and will likely require a combination of biomaterials, cell types, and chemical and mechanical factors.detto da: Nandan L. Nerurkar, J Biomech April 19; 43(6): 1017–1030. While MSC delivery by bolus injection has reduced degenerative changes such as GAG depletion and loss of disc-height, these studies are typically carried out in animal models where degeneration (which normally progresses pathologically over several years) is emulated rather abruptly by injury, injection of some agent, or mechanical stimulation (Boxberger et al., 2006; Elliott et al., 2008; Hoogendoorn et al., 2007; Hsieh et al., 2009; Imai et al., 2007; Wuertz et al., 2009) Eur Spine J Aug 24. [Epub ahead of print] Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells. Bertolo A, Mehr M, Aebli N, Baur M, Ferguson SJ, Stoyanov JV. Non usato per ‘migliorare’ l’approccio rigenerativo all’IVD in degenerazione la capacità del disco di autoriparazione = la presenza di ‘self progenitors’ e il processo di differenziamento Feng G. J Bone Joint Surg Am 2010 Fisiopatologia Ortopedica e Medicina Rigenerativa - IOR

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