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GAMBA Report Summary Proje ct re fe re nce : 245993 Funde d unde r: FP7-NMP
Final Repo rt Summary - GAMBA (Gene Activated Matrices fo r Bo ne and Cartilage Regeneratio n in Arthritis)
Exe cutive Summary: The GAMBA cons ortium has aime d at cre ating a nove l ge ne -activate d matrix platform for bone and cartilage re pair with a focus on os te oarthritis -re late d tis s ue damage . The S&T obje ctive s of this proje ct have be e n comple me nte d with an innovative program of public outre ach, active ly linking patie nts and s ocie ty to the e volve me nt of this proje ct. The GAMBA platform has be e n conce ive d to imple me nt a conce pt of s patiote mporal control of re ge ne rative bioactivity on command and de mand. The platform compris e s module s that can be inde pe nde ntly addre s s e d with e ndoge nous biological and e xoge nous phys ical or pharmacological s timuli, re s ulting in a te mporally and s patially coordinate d growth factor ge ne e xpre s s ion patte rn. This has be e n inte nde d to re produce ke y e le me nts of natural tis s ue formation. The module s are growth factor-e ncoding ge ne ve ctors , me s e nchymal s te m ce lls , magne tic nanoparticle s , a ce ramic matrix and a biomime tic hyaluronan ge l. Anatomical adaptivity was to be achie ve d with e ngine e re d the rmal prope rtie s of the hyaluronan matrix, which e mbe ds the othe r module s , s e le cte d according to functional re quire me nts . Me chanical s upport was to be provide d by the Micro Macroporous Biphas ic Calcium Phos phate (MBCP™), a re s orbable mate rial approve d for clinical us e . Spatiote mporal control of bioactivity and re s pons ive ne s s to phys iological conditions was to be re pre s e nte d, firs tly, in the s patial dis tribution and re le as e profile s of ge ne ve ctors within the compos ite matrix and, s e condly, by le tting local and e xte rnal biological or phys ical s timuli activate the promote rs driving the e xpre s s ion of ve ctor-e ncode d trans ge ne s . The proje ct had four major obje ctive s : 1) Es tablis hing GAMBA, cre ating the cons truction kit – matching mate rials s cie nce and ge ne ve ctor de ve lopme nt to achie ve hie rarchie s of s patiote mporal control of bioactivity on command and de mand; 2) Validating GAMBA – matching biophys ics and ce ll biology in orde r provide proof of conce pt in ce ll culture mode ls and e ve ntually in animal mode ls ; 3) Linking GAMBA to s ocie ty through innovative outre ach me thods including te aching s tude nts , dis cus s ing chance s and ris ks in patie nt and citiz e n pane ls and rais ing aware ne s s for ge nde r e quality within and outs ide the cons ortium; 4) Dis s e minating and e xploiting proje ct re s ults through publications , public re lations e fforts and e ve ntually through comme rcial product de ve lopme nts . To achie ve the s e obje ctive s , the proje ct was s tructure d in 7 s cie ntific workpackage s comple me nte d by the outre ach workpackage . Two furthe r workpackage s we re de dicate d to dis s e mination / e xploitation and proje ct manage me nt. The e s s e ntial goals of the proje ct have be e n re ache d. In a firs t s te p, all re quire d ve ctor cons tructs we re e s tablis he d and dis tribute d among the partne rs . Inducible ge ne e xpre s s ion was de mons trate d in ce ll line s . Howe ve r it turne d out that s ome of the inducible s ys te ms did not work e qually we ll in me s e nchymal s te m ce lls which are an e s s e ntial e le me nt of the GAMBA platform. He nce , only thos e cons tructs could be us e d that did work in this ce ll type . While s ome of the GAMBA partne rs continue d the ir bas ic re s e arch on the biology of bone and cartilage re ge ne ration, notably with patie nt ce lls , in orde r to de fine the pre conditions for the GAMBA platform to work, the ve ctor and biomate rial e ngine e rs within the proje ct s tarte d to as s e mble the module s to de mons trate s patial and te mporal control of re porte r ge ne e xpre s s ion in 3-dime ns ional s e tups of the GAMBA module s . The individual e ngine e ring tas ks proce e de d quite s ucce s s fully with re porte r ge ne s . It turne d out much more challe nging to de mons trate cartilage (re )ge ne ration and anti-inflammatory action with the individual ge ne s that had be e n chos e n be fore hand bas e d on the s cie ntific lite rature while the induction of bone formation was de mons trate d s ucce s s fully in vitro and in vivo. Achie ving s patio-te mporal control with thre e diffe re nt the rape utically re le vant ge ne s re mains to be accomplis he d. The GAMBA proje ct has s ucce s s fully cre ate d a toolbox of individual e le me nts that will be us e d in follow-up proje cts . Pos s ibly the mos t s ucce s s ful and e xciting part of the proje ct was public outre ach in patie nt and citiz e n pane ls which was gre atly appre ciate d by the participating lay pe rs ons and s cie ntis ts alike . As part of the GAMBA proje ct, 20 pape rs we re publis he d and 9 mas te r and PhD the s e s we re comple te d. Nume rous pre s s article s and broadcas ts made the wide r public aware of the proje ct. Thre e comme rcial ge ne de live ry products for tis s ue e ngine e ring we re launche d and one pate nt application is unde r way. Summariz ing, this proje ct is a s ucce s s upon which future ge ne rations of follow-up proje cts can build on.
P a ge 1 o f 23 Research and Innovation
Proje ct Conte xt and Obje ctive s : Conte xt and obje ctive s Due to de mographic and life s tyle change s , de ge ne rative dis e as e s are an e normous me dical and s ocio-e conomic challe nge in indus trialis e d nations . Among the m, the mus culos ke le tal dis e as e s os te oarthritis (OA), rhe umatoid arthritis and os te oporos is are the mos t pre vale nt. The re is curre ntly no gold s tandard for the re pair or pre ve ntion of ons e t of OA. The re are thre e major challe nge s : re ducing inflammation, cartilage re pair and s ubchondral bone re pair. Biomime tic approache s for tis s ue re pair in os te oarthritis re quire tight s patiote mporal control of bioactivity in orde r to addre s s the s e challe nge s in a coordinate d fas hion. No s ingle e s tablis he d tre atme nt of tis s ue de fe cts – including OA - imple me nts the fe ature s of natural tis s ue formation, name ly ce ll diffe re ntiation in re s pons e to s patiote mporally controlle d growth factor ge ne e xpre s s ion patte rns on command and de mand. GAMBA was conce ive d to imple me nt the s e fe ature s and has focus s e d on innovative mate rials that induce and s us tain bone and cartilage he aling agains t the background of os te oarthritis . Os te oarthritis (OA) is a de ge ne rative dis e as e of the joints affe cting, above the age of 45, more wome n than me n, with an incide nce incre as ing with age . It is e s timate d that 80 % of the population will have radiographic e vide nce of OA by age 65. The cartilage of the affe cte d joint be come s rough and de ge ne rate s . With dis e as e progre s s ion, the cartilage dis appe ars and bone rubs on bone . The population burde n of OA will incre as e ove r the ne xt ye ars due to the age ing population and the ris ing pre vale nce of obe s ity, be ing the principal non-ge ne tic ris k factor for OA. This le ads to tre me ndous e conomic burde n. The e tiology of OA is s till unknown. The curre nt manage me nt of os te oarthritis is not re ge ne rative but me re ly s ymptomatic, aime d at re duction of pain, controlling inflammation with non-s te roidal anti-inflammatory drugs with an ultimate option of total joint re place me nt. He althy tis s ue fe ature s unique plas ticity characte ris e d by continuous re mode lling in re s pons e to phys iological and e xte rnal s timuli, re s ulting in a controlle d balance of anabolic and catabolic proce s s e s . Tis s ue damage is characte ris e d by imbalance , los s of control and in the cas e of os te oarthritis a dominance of catabolic proce s s e s . Innovative the rape utic conce pts are re ge ne rative in nature , thriving on the re ge ne rative pote ntial imprinte d in our ge ne tic background. The challe nge is s e iz ing this pote ntial. This can be achie ve d, in principle , with growth factors and s te m ce lls . While the latte r are inhe re ntly multipote nt, the forme r can be us e d to re awake n s ile nce d e ndoge nous programs of tis s ue formation, in a s imilar way to the s patiote mporal conce ntration gradie nt of morphoge ne s is obs e rve d during e mbryoge ne s is . The s e programs cons is t initially of te mporally and s patially coordinate d ge ne e xpre s s ion patte rns , le ading ultimate ly to a s patially and te mporally conce rte d action of growth factors . In the conte xt of tis s ue re ge ne ration with biomime tic implants , s uch conce rte d action is e xce e dingly challe nging to re produce with re combinant growth factors as the y have no inhe re nt e le me nts for re s ponding to phys iological conditions or e xte rnal s timuli. In contras t, ge ne ve ctors can be e ngine e re d to have the s e e le me nts and thus can re produce s patiote mporally controlle d ge ne e xpre s s ion patte rns in s itu. He nce , the main obje ctive of the GAMBA proje ct was de ve loping a biomime tic implant s ys te m us ing a nanobiote chnological approach that de live rs re ge ne rative bioactivity in a te mporally and s patially controlle d fas hion in re s pons e to e ndoge nous and e xte rnal s timuli. In this manne r, the s ys te m was de s igne d to re s pond to and control inflammation and induce cartilage and s ubchondral bone re pair in OA-re late d tis s ue de fe cts . The implant s ys te m was de s igne d alte r the phe notype of ce lls in a phys iologically me aningful way to e licit the de s ire d the rape utic outcome . For this purpos e , the GAMBA cons ortium has aime d at cre ating a nove l ge ne -activate d matrix platform for bone and cartilage re pair with a focus on os te oarthritis -re late d tis s ue damage . Thre e the rape utically re le vant ge ne s unde r the control of inde pe nde ntly inducible promote rs we re to be ve ctoriz e d and co-incorporate d with me s e nchymal s te m ce lls in a 3-dime ns ional compartme ntaliz e d matrix. The compartme nts we re to mimic the s ituation in an os te ochondral bone de fe ct, name ly damage d bone and cartilage to be re ge ne rate d while the cartilage facing an inflammatory de s tructive e nvironme nt. He nce , the re was to be a ve ctoriz e d ge ne e ncoding an os te oinductive factor in the firs t compartme nt, anothe r one e ncoding a cartilage -inducing s e cond compartme nt and a third one e ncoding an anti-inflammatory factor in the third compartme nt. The compartme ntaliz ation was to be re aliz e d with bone re place me nt mate rial (MBCP™, a re s orbable mate rial approve d for clinical us e ) impre gnate d with the bone -inducing ve ctor and co-incorporate d with the s te m ce lls in a hydroge l matrix that in the uppe r laye r would compris e the cartilage inducing and inflammatory ve ctors . While re s iding in the compartme ntaliz e d matrix, the s te m ce lls would be trans fe cte d, s tart to e xpre s s the the rape utic ge ne s and in cons e que nce diffe re ntiate to bone and cartilage ce lls while als o fighting inflammation by producing the anti-inflammatory factor. To make the s ys te m controllable in a te mporal fas hion, the thre e the rape utic ge ne s we re to be s witchable by inde pe nde ntly inducible promote rs . This s cie ntific conce pt was to be comple me nte d with an innovative program of public outre ach, active ly linking patie nts and s ocie ty to the e volve me nt of this proje ct. Conce que ntly, the proje ct had the following obje ctive s : Obje ctive 1: Es tablis hing GAMBA, cre ating the cons truction kit – matching mate rials s cie nce and ge ne ve ctor de ve lopme nt to achie ve hie rarchie s of s patiote mporal control of bioactivity on command and de mand 1. Cre ate s patiote mporal control e le me nts bas e d on biological/pharmacological principle s with nonviral and ade noviral ve ctors with fe e dback re s pons e e le me nts driving trans ge ne e xpre s s ion. Ve ctors we re to be cons tructe d s uch that inducible promote rs drive trans ge ne e xpre s s ion: Ve ctors re s ponding to inflammatory s ignals with the e xpre s s ion of P a ge 2 o f 23 Research and Innovation
anti-inflammatory factors (Cox-2 promote r). Ve ctors re s ponding to te mpe rature (HSP70 promote r driving growth factor ge ne e xpre s s ion; induction by AC magne tic fie ld hype rthe rmia). Ve ctors re s ponding to doxycycline (te t-on s ys te m driving the growth factor ge ne e xpre s s ion). 2. Cre ate s patiote mporal control e le me nts bas e d on phys ical principle s . A the rmo-re s pons ive hyaluronan-bas e d biomime tic polyme r and e mbe dde d the re in MBCP™, a calcium phos phate ce ramic, we re to be us e d as carrie rs for ge ne ve ctors . Magne tic nanoparticle s we re to be co-e mbe dde d. Spatial control was to be achie ve d by diffe re ntial ve ctor pos itioning within the compos ite . Spatiote mporal control was to be de mons trate d bas e d on ve ctor re le as e patte rns including the rmally induce d re le as e ge ne rate d by AC magne tic fie ld hype rthe rmia and magne tic nanoparticle s . Obje ctive 2: Validating GAMBA – matching biophys ics and ce ll biology 1. De mons trate s patiote mporal control of trans ge ne e xpre s s ion with s e le cte d ve ctor/biomate rial combinations in ce ll and tis s ue culture mode ls , by e valuating trans duction/trans fe ction e fficie ncie s , trans ge ne e xpre s s ion duration and e ffe ctive ne s s of growth factor s e cre tion with time . As control e le me nts we will us e diffe re ntial ve ctor re le as e , mole cular biological / pharmacological me ans (doxycycline ), phys ical (he at) and mole cular-biological (HSP70) me ans . 2. Obtain anti-inflammatory re s pons e in vitro with ge ne activate d compound mate rials e ncoding IL-10 unde r the control of Cox-2 promote r. The Cox-2 promote r re s ponds to inflammatory cytokine s and the re by provide s a fe e dback control e le me nt for the e xpre s s ion of the anti-inflammatory cytokine IL-10. This proce s s was to be characte ris e d in vitro, e x vivo and finally in a s mall animal mode l. 3. Us ing the GAMBA platform to obtain s patiote mporally controlle d chondroge ne s is or os te oge ne s is with MSCs e mbe dde d in ge ne activate d compound mate rials . For induction of chondroge nic diffe re ntiation TGF-ß e xpre s s ion and for s timulation of os te oge ne s is BMP-2 e xpre s s ion was to be induce d by promote rs that re s pond to doxycycline or he at to be able to re gulate location, time and duration of ge ne e xpre s s ion. As s e s s me nt was to be pe rforme d in vitro, e x vivo and in s uitable s mall animal mode ls of os te ochondral de fe cts . 4. Obtain proof of conce pt for GAMBA. To finally as ce rtain the s ucce s s of GAMBA, a large animal mode l (goat) was to be us e d. The choice of the mode l will de pe nd on the re lative s ucce s s e s of the pre vious obje ctive s with an os te ochondral de fe ct be ing the mode l of choice as s uming the s ucce s s of all pre vious obje ctive s . Obje ctive 3: Linking GAMBA to s ocie ty through innovative outre ach me thods 1. Educating s tude nts in nanome dicine . Each Partne r curre ntly participate s in local e ducation programs for unde rgraduate s tude nts . We will incorporate the GAMBA conce pt in the s e e xis ting cours e s as we ll as attract unde rgraduate and graduate s tude nts to participate in the proje ct. 2. Inte grate patie nts (e s p. fe male /e lde rly) into the de ve lopme nt of innovative tre atme nts . This was to be accomplis he d with s o-calle d Patie nt Pane ls . OA patie nts we re to be invite d to participate in works hops toge the r with the s cie ntis ts carrying out this proje ct and we re to be as ke d to communicate the ir ne e ds and vie ws in re lation to this proje ct. The patie nts will compile the ir e xpe ctations and re comme ndations on re ge ne rative me dicine in writing. Proce e dings from the patie nt pane ls we re to be made available to s cie ntis ts , the inte re s te d public, re gulators and politicians via an inte rne t dis cus s ion forum and othe r PR me as ure s (s e e chapte r 3.2) toge the r with re s ults from (3) be low. 3. Dis cus s chance s , ris ks and e thical as pe cts of GAMBA with the ge ne ral public. In a format s imilar to the Patie nt Pane ls , Citiz e n Pane ls we re to be he ld in orde r to e nhance aware ne s s of nanome dicine in the public and take into account public e xpe ctations and re s e rvations . Randomly s e le cte d citiz e ns we re to be invite d to dis cus s the chance s , ris ks and e thical as pe cts of GAMBA toge the r with e xpe rts from diffe re nt s cie ntific dis cipline s facilitate d by ne utral communication e xpe rts . The participants we re to compile a Citiz e n Re port with re comme ndations on re ge ne rative me dicine that we re to be publis he d and dis s e minate d to inte re s te d s cie ntis ts , me dical pe rs onne l, re gulators , the me dia and the inte re s te d ge ne ral public. 4. Rais e aware ne s s for and dis cus s ge nde r e quality. A Partne r works hop was to be organis e d to rais e aware ne s s for ge nde r is s ue s within the Partne r organis ations and to dis cus s e xpe ctations and ne e ds of fe male e mploye e s re garding ge nde r e quality. The goal of this works hop is to e s tablis h the profe s s ional ne tworks for fe male proje ct me mbe rs that are e s s e ntial for a s us taine d profe s s ional care e r. Achie ving this goal is facilitate d by the fact that 4 out of 8 of the Principal Inve s tigators in this proje ct (including the s ubcontractor PIs ), among the m notably the coordinator, are fe male . Obje ctive 4: Dis s e minating and e xploiting GAMBA 1. Trans fe r of proje ct re s ults into re s e arch and clinics through the participating companie s . Thre e of the Partne rs are locate d in acade mic hos pitals and are in clos e contact with patie nts . Me dical doctors we re to be active ly involve d in the proje ct from an e arly s tage . The companie s involve d will guarante e prote ction of IP rights . We will organis e s pe cific me e tings with me dical s pe cialis t and bus ine s s pe ople to dis cus s the ne e ds and pos s ibilitie s to ge t our ide a into a clinical application. The re was to be at le as t one s uch me e ting at the s tart, one at midpoint and one at the e nd of the proje ct. Stake holde rs will als o be invite d to participate in Patie nt Pane ls and Citiz e n Pane ls of Obje ctive 3. 2. Public re lations campaign. The pre s e nce of me mbe rs of inte rnational/national as s ociations in re ge ne rative me dicine within the cons ortium will s ignificantly e nhance the pos s ibility to dis s e minate ne w data to as wide an audie nce as pos s ible . Afte r the IP pote ntial has be e n e valuate d and s e cure d, data and re s ults we re to be made public to the s cie ntific and wide r community. As the proje ct is progre s s ing the ne w information was to be dis s e minate d. Proje ct Re s ults :
P a ge 3 o f 23 Research and Innovation
WP 1: Ve ctor de ve lopme nt for s patiote mporally controlle d ge ne e xpre s s ion Le ad be ne ficiary: TUM WP le ade r: Chris tian Plank, Martina Anton The aim of this WP was to cons truct and characte riz e ge ne ve ctors s uitable to inde pe nde ntly re gulate ge ne e xpre s s ion of re porte r ge ne s or the rape utically active ge ne s that is to s ay ge ne s that are known to be involve d in re ge ne ration of cartilage or bone or in re ducing inflammation in joints . The aim was to produce non-viral and viral ve ctors , optimiz e the m for trans duction e fficie ncy, s how the ir functionality in ce ll culture e xpe rime nts and pre pare and analyz e as s e mblie s of ge ne ve ctors and s caffolds with ce lls . TUM s ucce s s fully cons tructe d a s e rie s of inducible plas mid and ade noviral ge ne ve ctors combining as re gulatory e le me nts e ithe r the TET-on s ys te m, a he at-inducible Hs p70B bas e d promote r or an inflammation inducible Cox-2 promote r. The s e we re us e d to drive e xpre s s ion of bone morphoge ne tic prote in 2 (BMP-2), trans forming growth factor be ta1 (TGFb1 or viral Inte rle ukin 10 (vIL10), which was chos e n, s ince vIL10 was re porte d to e xhibit s ole ly antiinflammatory activity. Additionally ge ne ve ctors carrying the re s pe ctive cDNAs unde r control of cons titutive ly active promote rs we re produce d as controls . The s e ve ctors we re analyz e d for the ir functionality afte r infe cting or trans fe cting tumor ce lls , me s e nchymal s te m ce lls or chondrocyte s and by applying the re s pe ctive s timulus . He at induction was by s imple incubation at e le vate d te mpe rature s and turne d out to be optimal at 43°C; inflammation was mimicke d by e ithe r applying LPS and TPS or IFNgamma and TNFalpha and the choice of induce rs was de pe nde nt on ce ll type ; pharmacological s timulation was by addition of doxycycline (DOX) to tis s ue culture me dium. Induction was de mons trate d by re porte r ge ne as s ays , fluore s ce nce micros copy and flow cytome try and by ELISA for s e cre tion of the growth factors and the cytokine . All re gulation s ys te ms s howe d inducibility by the re s pe ctive s timulus in tumor ce ll line s . Howe ve r induction le ve ls of the he at-inducible ve ctors was low as we ll as for the inflammation inducible Cox-2 promote r. Additionally no inducibility of both promote rs was s e e n in MSC e ithe r due to toxicity of non-viral ve ctors with he at or background activity of the Cox-2 ve ctors in hMSC (NUI Galway, EMC). Production of the rape utic prote ins was e xpe cte d not to re ach critical le ve ls that might drive diffe re ntiation of MSC. In contras t, the TET-on s ys te m re s ulte d in high induction le ve ls for re porte r ge ne s and BMP-2 in diffe re nt ce lls , mos t importantly in MSC, with only background e xpre s s ion in the abs e nce of induce r. The s e highly favourable characte ris tics le ad to cons truction of ne w ve ctors for TET-on vIL10 e xpre s s ion, which re s ulte d in tightly re gulate d and high vIL10 e xpre s s ion afte r infe ction of rMSC. Since re s ults of WP5 had s hown that TGFb ne e ds to be e xpre s s e d for e xte nde d pe riods of time , which cannot be achie ve d by he at s hock the continuous ly active CMV promote r was chos e n. This way the ability of the ve ctor s ys te ms for te mporal ge ne e xpre s s ion was cle arly de mons trate d for all induction s ys te ms te s te d, but the choice of promote rs ne e de d to be adapte d to the e xpre s s ion time s and le ve ls ne e de d for pote ntial bias ing MSC diffe re ntiation. Due to the ir highe r e xpre s s ion le ve ls , ade noviral ve ctors we re chos e n for furthe r analys is for functionality of produce d the rape utic prote ins . The influe nce of s witche d “on” (and “off”) ge ne e xpre s s ion of the growth factors BMP-2 and TGFb and the cytokine vIL10 on os te oge nic and chondroge nic diffe re ntiation of MSC was analyz e d. Rat MSC infe cte d with diffe re nt Dox-inducible and cons titutive ly e xpre s s ing ade noviral ve ctors we re s ubje cte d to s tandard in vitro diffe re ntiation as s ay for os te oge ne s is (Ca2+ quantification and aliz arin re d s taining) and pe lle t mas s as s ays for os te oge nic diffe re ntiation (glycos aminoglycan quantification and alcian blue s taining). At the s ame time the le ve l of the rape utic ge ne e xpre s s ion was monitore d ove r time and in the cas e of cons titutive ge ne e xpre s s ion turne d out to be de cre as e d as e xpe cte d, e ithe r due to los s of ve ctor (non-inte grating s ys te ms ) or due to e pige ne tic s ile ncing. Unde r diffe re ntiation conditions DOX-inducibility was re taine d. At e arly time points high induction le ve ls we re achie ve d, but DOX-induce d le ve l of the rape utic prote ins de cre as e d ove r time although induction was s till pos s ible at the e nd of diffe re ntiation as s ays . Expe rime nts re ve ale d that the Te t-induce d BMP-2 e xpre s s ion e nhance s os te oge nic and to a le s s e r e xte nt chondroge nic diffe re ntiation of MSC. Non-re gulate d TGFb1 e nhance s chondroge nic diffe re ntiation, although be ing produce d at a low le ve l, thus re placing re combinant TGFb prote in. vIL10 ove rproduction had a ne gative impact on os te oge nic diffe re ntiation, but did not influe nce chondroge nic diffe re ntiation ne gative ly. Thus with the Te t-induce d s ys te m we could s ucce s s fully de mons trate te mporal control of growth factor e xpre s s ion and s howe d the functionality of all the rape utic ge ne ve ctors in a 2D mode l of chondroge ne s is and os te oge ne s is . Spatial control of ge ne e xpre s s ion was s hown by as s e mbly of module s (OZB) with re porte r ge ne s us ing diffe re nt s caffolds for ge ne ve ctors and ce lls provide d from partne rs (ARI, BIM, INSERM) or comme rcially available ge ls . Spatially diffe re ntial e xpre s s ion of re porte r ge ne s was s e e n us ing re d and gre e n fluore s ce nce prote in re porte rs (TUM), but we re difficult to quantify and de mons trate une quivocally us ing the the rmos e ns itive hyaluronan ge l (ARI). With 3D-as s e mbly of diffe re nt s caffolds , ve ctors and ce lls TUM was able to confirm te mporally re gulate d BMP-2 e xpre s s ion combine d with non-re gulate d TGFb e xpre s s ion. So far us ing qRT-PCR as s ays of MSC in/on the diffe re nt s caffolds did not de mons trate any influe nce on diffe re ntiation by the the rape utic prote ins produce d due to low ce ll numbe rs . The s e e xpe rime nts ne e d optimiz ation and are s till ongoing. Howe ve r e vading ce lls hinte d at incre as e d Ca2+de pos ition unde r Doxycycline addition and Te t-re gulate d BMP-2 e xpre s s ion was incre as e d. It was de mons trate d by ELISA, that e ve n in 3D- culture the Te t-on s ys te m is functional for BMP-2 e xpre s s ion and that Dox has no ne gative influe nce on the cons titutive TGFb e xpre s s ion in the s ame as s e mblie s . P a ge 4 o f 23 Research and Innovation
As an alte rnative to de mons trate s patial control of ge ne e xpre s s ion, a 2D biolumine s ce nce imaging s ys te m was chos e n by TUM. He re one of the induce rs of the Cox-2 promote r was locally produce d, s e cre te d into the me dium and afte r diffus ion re s ulte d in induction of Cox-2 promote r-drive n lucife ras e biolumine s ce nce of ce lls s e e de d in a diffe re nt locus of the s ame dis h. Thus within the GAMBA proje ct the conce pt of s patial and te mporal control of ge ne e xpre s s ion was s ucce s s fully de mons trate d. Additional tas ks of WP1 de alt with optimiz ing ge ne ve ctors by s hie lding and e mbe dding of ge ne ve ctors into/onto s caffolds and magne tic ge ne ve ctors and magne tofe ction. On the nonviral s ide , a high throughput s cre e ning of a lipid library by OZB le d to the s e le ction of two DNA trans fe ction candidate s for both 3D-hydroge ls and 3D-Scaffolds . The s e two compounds we re optimiz e d on a varie ty of matrice s and we re late r comme rcially launche d (3D-Fe ct™ & 3D-Fe ctIN™). The re afte r, ne w lipids and polyme rs we re furthe r s ynthe s iz e d to s olve the trans fe ction proble ms as s ociate d with the combination of the rmo-re s pons ive HA-PNIPAM ge l with the hydrophobic lipids . Ne w lipids and polyme rs candidate s with re duce d hydrophobic prope rtie s we re s ynthe s iz e d, s e le cte d and s e nt to partne rs 1, 2 and 7 (MRI, ARI and IRCCS). Phys ical and che mical characte riz ation and biological e valuation we re pe rforme d for the s e compounds (s e e WP2 ). OZB has the n s e le cte d the be s t formulation to trans fe ct chondrocyte s and hMSC in clas s ical ce ll culture conditions (be fore implantation in the biomate rials ) (1) with high e fficie ncy, (2) low toxicity and (3) without inducing any vis ible phe notypic change s . The lipids DOGTOR and NL-37 induce d highe s t trans fe ction e fficie ncy in te rms of numbe r of ce lls trans fe cte d and trans ge ne e xpre s s ion. More ove r, CombiMag (Magne tofe ction) allowe d rais ing the trans fe ction e fficie ncy of both re age nts . Importantly OZB als o de fine d critical parame te rs that influe nce the trans fe ction e fficie ncy including ce ll de ns ity, culture conditions , pas s age numbe r, days of trans fe ction, me dium change e tc. OZB has the n produce d a de taile d protocol to s tandardiz e the trans fe ction proce dure of hMSC that was dis s e minate d with the re age nts to all partne rs . DOGTOR was finally adopte d by all partne rs to be the re age nt of choice for trans fe cting s te m ce lls in 2D. With re s pe ct to ve ctor s hie lding, the partne rs had pre -e xis ting knowle dge and compounds re ady. Polyme rs and/or magne tic nanoparticle s (MNP) and we re analys e d for the ir phys ical prope rtie s , including re le as e from s caffolds and ge ne e xpre s s ion le ve ls . Surpris ingly, magne tic nanoparticle s alone e xe rte d an e xce lle nt s urface s hie lding e ffe ct on the ade noviral ve ctor which was at the s ame time trans fe ction e nhancing whe n the ve ctor was co-incorporate d into 3D culture format toge the r with ce lls . The ge ne ration of magne tic particle -as s ociate d ve ctors was an important tas k in the proje ct als o for anothe r re as on. One modality of the induction of trans ge ne e xpre s s ion was via he at ge ne ration by AC magne tic fie ld action on magne tic nanoparticle s . This re quire d a high conce ntration of the particle s in the 3D phas e . And luckily this turne d out to e nhance ade noviral trans duction, as me ntione d. From a colle ction of MNP, two type s we re ide ntifie d by TUM that re s ulte d in a s atis fying incre as e of te mpe rature unde r AC magne tic fie ld. Furthe rmore , the s e particle s we re compatible with the ge lation prope rtie s of the hyaluronan-bas e d hydroge l (ARI, TUM). The s e particle s we re finally us e d to induce lucife ras e e xpre s s ion by AC magne tic fie ld induction in a ce ll line that carrie s the ge ne unde r control of a he at-inducible promote r. The s ys te m s till ne e ds improve me nt, but proof of principle was achie ve d. As AC-MF ins trume nts are not wide ly available , a s wift te chnology trans fe r to othe r partne rs was not pos s ible . As a s ide -product of re s e arch in nanomagne tic ve ctors , OZB de ve lope d and publis he d the i-MICST me thod for inte grate d ce ll s orting and trans duction of MSC with ade noviral ve ctors . This me thod is highly e fficie nt in ge ne rating ge ne tically modifie d me s e nchymal s te m ce lls and can be us e d advantage ous ly for ce rtain obje ctive s of the GAMBA proje ct. Conclus ion: In WP1 we s ucce s s fully cons tructe d re gulate d non-viral and ade noviral ge ne ve ctors and de mons trate d functionality with re s pe ct to re gulation (TUM). The s e ve ctors we re made available to all partne rs . Non-viral and viral magne tic ge ne ve ctors have be e n s ucce s s fully produce d. Two compounds we re optimiz e d by OZB on a varie ty of matrice s and we re late r comme rcially launche d (3D-Fe ct™ & 3D-Fe ctIN™). A de taile d protocol to s tandardiz e the non-viral trans fe ction proce dure of hMSC was dis s e minate d with the re age nts to all partne rs . DOGTOR was finally adopte d by all partne rs to be the re age nt of choice for trans fe cting s te m ce lls in 2D. Like wis e the AC/MF he at induction had be e n inve s tigate d and de mons trate d, but ne e ds improve me nt. As AC-MF ins trume nts are not wide ly available a s wift trans lation to othe r partne rs was not pos s ible . Some of the re gulatory e le me nts (Cox-2- and Hs p70B promote rs ) turne d out to be functional, but too we ak to be e xpe cte d to work in driving diffe re ntiation of MSC. The re fore the following alte rnative s we re chos e n, that allowe d for s tronge r or cons titutive e xpre s s ion: Te t-s ys te m for the re gulate d e xpre s s ion if vIL10 and the cons titutive CMV promote r for TGFb e xpre s s ion. With the Te t-induce d BMP-2 s ys te m we could s ucce s s fully de mons trate te mporal control of growth factor e xpre s s ion and s howe d the functionality of all the rape utic ge ne ve ctors in a 2D mode l of chondroge ne s is and os te oge ne s is . TGFb1 P a ge 5 o f 23 Research and Innovation
e nhance d chondroge nic diffe re ntiation The 3D-as s e mbly of diffe re nt s caffolds , ve ctors and ce lls was s ucce s s ful. Although we we re not able to de mons trate bias of diffe re ntiation by ge ne ve ctors within the 3D as s e mblie s due to low ce ll numbe rs , we we re able to de mons trate that s witching on of BMP-2 e xpre s s ion was s ucce s s ful in 3D as we ll as 2D culture . Thus the te mporal control of ge ne e xpre s s ion by the Te t-on s ys te m was s ucce s s fully imple me nte d in 3D culture as we ll. Spatial control of ge ne e xpre s s ion was de mons trate d by re porte r ge ne imaging and une quivocally in an inde pe nde nt s ys te m of lucife ras e imaging that re lie s on the local production of TNFalpha, which le ads to Cox-2 promote r me diate d lucife ras e e xpre s s ion in a s patially s e parate locus . WP2: De ve lopme nt of a biomime tic polyme r as carrie r of ge ne ve ctors Le ad be ne ficiary: ARI WP le ade r: Mauro Alini Thus , the major obje ctive of the workpackage 2 was to cre ate a biomime tic polyme r bas e d carrie r platform that allowe d s patiote mporal control of ce ll be haviour us ing a combination of te chnological bre akthroughs achie ve d by Partne rs of the cons ortium. The s pe cific te chnical module de ve lope d in the workpackage 2 was a te mpe rature -s e ns itive hyaluronan (HA) hydroge l and its combination with MBCP™ tailor-made granule s (Micro macroporous Biphas ic Calcium Phos phate ), nanos iz e d nonviral or viral ge ne ve ctors e ncoding de s ire d growth factors unde r the control of inducible promote rs , s upe rparamagne tic nanoparticle s and me s e nchymal s te m ce lls . All the partne rs we re involve d in this major workpackage . The biomime tic hydroge l carrie r de ve lope d in this proje ct was bas e d on hyaluronan which has important role s in organ de ve lopme nt and ce ll s ignaling (Le ach e t al., 2004). Commonly, hyaluronan hydroge ls are obtaine d by cros s -linking re actions involving che micals and/or UV light (Lapcik e t al., 1998), which are pote ntially de trime ntal to biologics to be e mbe dde d in the ge ls . In the workpackage 2, the partne r 2 (AOF) pe rforme d the grafting of the rmo-re s pons ive line ar polyme rs (poly(N-is opropylacrylamide )) onto the hyaluronan e ithe r via Cu(I) catalyz e d alkyne -az ide cycloaddition (CuAAC) re action or via dire ct amidation. The mate rials we re characte riz e d with notably; 1H nucle ar magne tic re s onance , the rmal analys is , rhe ology, and s we lling-s hrinkage me as ure me nts , as we ll as s tability te s t. The mate rials we re s ucce s s fully s ynthe s iz e d and s howe d a ge l point in aque ous s olution at 30-32•C. The grafting via amidation re action did not le ad to de gradation of hayluronan as obs e rve d in the CuAAC re action pe rforme d in the pre s e nce of the catalys t; CuSO4, 5H2O with s odium as corbic acid s alt. This le ade d to improve re producibility and pote ntial s caling-up. More ove r, the abs e nce of pote ntially toxic coppe r trace s in the amidation re action product incre as e d the like line s s for us e in vivo. The the rmore s pons ive ne s s of the hydroge ls and the rhe ological fe ature s of the ge ls we re modulate d from room te mpe rature to above body te mpe rature de pe nding on compos ition and conce ntration, he nce providing te mpe rature -de pe nde nt s patiote mporal control of the mate rial prope rtie s . In the me antime , a biomime tic hyaluronan hydroge l platform bas e d on a thiol-e ne re action cros s linking was de ve lope d in orde r to compare with the pure ly phys ical cros s linke d hyaluronan carrie r. Biomime tic functionalitie s , s uch as RGD binding e pitope we re e as ily adde d in this 2nd hyaluronan platform, e nhancing hMSCs s pre ading in 3D hydroge l matrix. Pre liminary ce ll s tudy was pe rforme d in orde r to as s e s s the viability of ce lls dis pe rs e d in a the rmore s pons ive hyaluronan compos ition afte r inje ction through a ne e dle . Ce ll viability at 24 h afte r inje ction was 90 ± 2% for the the rmore s pons ive hydroge l compare d to 71 ± 9% for alginate hydroge l control, confirming that the the rmore s pons ive hydroge l can be us e d as an inje ctable ce ll carrie r. Furthe r s tudy on the e ncaps ulation of human me s e nchymal s te m ce lls (hMSCs ) has be e n pe rforme d by s e ve ral partne rs s howing that hMSCs viability at 7 days was highe r or ide ntical in the the rmore s pons ive hydroge l compare d to alginate control hydroge l. Furthe r, ce ll culture de mons trate d the chondroge nic pote ntial of the the rmore s pons ive hydroge l in vitro. Afte r, the optimiz ation of the biomime tic hydroge ls platform, combination with micro and macroporous biphas ic calcium phos phate (MBCPTM and MBCP+TM) de ve lope d and produce d by Biomatlante (BIM) was pe rforme d in orde r to cre ate the os te ochondral GAM. The MBCPTM was firs t cons ide re d to be the ge ne ve ctor carrie r e ncoding a firs t factor which will the n be e mbe dde d in the hydroge l which acte d as a cohe s ion e nhance r for the MBCPTM particle s , ce lls carrie r or 2nd ge ne ve ctor carrie d. In a whole , the compos ite GAM conce pt provide d an e le me nt of s patiote mporal control in this proje ct. The combination of MBCPTM granule s of diffe re nt compos ition and varying conce ntration with the the rmore s pons ive hydroge l s olutions was pe rforme d in collaboration be twe e n AOF, BIM and INSERM partne rs . The pre paration of compos ite mate rials was s ucce s s ful. It was found that the loading of the hydroge l s olution with a quantity of particle s up to e ight time highe r in we ight of the polyme r in s olution, 1) incre as e d the vis cos ity of the compos ition at te mpe rature be low the lowe r critical s olution te mpe rature (LCST), 2) de cre as e d the LCST with the amount of granule s adde d, 3) cons e rve d the ge lling prope rty of the hydroge l and 4) incre as e d the final s torage modulus with the granule conte nt. Full characte riz ation of the compos ite s was pe rforme d ranging from IR s pe ctros copy, rhe ology, X-ray diffraction, e le ctron micros copy, e tc. The s tability of the formulations was e valuate d obs e rving the s e ttling of the particle s . Focus ing on the handling of the compos ite matrice s pre pare d e xtrus ion force was me as ure d on an Ins tron 4302 e le ctrome chanical te s ting machine e quippe d with a cus tomiz e d load ce ll. The diffe re nt formulations we re characte riz e d by diffe re nt s tability de pe nding on the particle s iz e . It was s e e n that particle of s iz e above 200µm te nd to s e ttle . Such compos itions ne e de d to be formulate d at highe r vis cos ity (for ins tance , addition of unde rivatiz e d hyaluronan) at room te mpe rature . All the compos itions we re e xtrudible at room te mpe rature through a s yringe . The y had the re fore s uitable handling for us e in a s urgical the ate r. P a ge 6 o f 23 Research and Innovation
Additionally, s upe rparamagne tic nanoparticle s have be e n combine d with the biomime tic hydroge l platform: Magne tic nanoparticle s are wide ly us e d in biology, bioe ngine e ring, bios e paration, me dical diagnos tics as we ll as in pre clinical and e arly clinical s tudie s with innovative the rapie s . The hydrodynamic diame te rs of the particle s us e d we re of fe w nanome te rs (about 10%) was re quire d to maintain the comple xe s into the ge l. Howe ve r unde r this condition, the hydroge l was difficult to handle . Cons e que ntly, OZB produce d ne w lipids and polyme rs with minimiz e d hydrophobic prope rtie s . The ne w compounds we re e ffe ctive in trans fe cting ce lls in 3D hydroge l made of ate locollage n but much le s s e ffe ctive with hyaluronan ge l. In orde r to gain a be tte r ins ight on the phys icoche mical characte riz ation of the comple xe s , OZB produce d fluore s ce ntly labe le d trans fe ction re age nts for s tudying the capacity of fluore s ce nt comple xe s to be maintaine d ins ide the matrice s according to the s caffold compos ition. The s tudy allowe d to gaining ins ights on s e ve ral points : s uch as the diffe re nt nonviral ve ctor re te ntion capacity of matrice s . A cle ar outcome was that the e mbe dding MBCP and MBCP+ granule s , into hydroge l was ne ce s s ary to re tain the comple xe s ins ide the biomate rials and e fficie ntly trans fe ct coloniz ing ce lls . The s e re s ults we re in clos e re lations hip to the obs e rve d ce ll trans fe ction data. The s e obs e rvations we re of high importance for in vivo e xpe rime nts as GAM be aring comple xe s mus t re main active s e ve ral days to allow coloniz ation and trans fe ction of ce lls . OZB and ISTGE collaborate d on in vivo mous e mode l and lipids non-viral ve ctor ne wly s ynthe s iz e d and ne w GAM protocol. Pre liminary re s ults s howe d that fluore s ce nce e xpre s s ion of trans fe ct ce lls can be obs e rve d in the implante d s caffold. In addition to the above re porte d in vivo s tudy, ARI has de mons trate d that viral trans fe ction of hMSCs in the rmore s pons ive hydroge l was highly e fficie nt and that s e le cte d biomime tic hydroge l compos ition was biocompatibility (s e e WP7). For this las t tas k, s te riliz ation me thods we re s cre e ne d, mate rial batch s ynthe s is was s cale d up and as s e s s e d, and in vivo s tudy pe rforme d. To conclude , although high e fficacy of non-viral ve ctor into the the rmore s pons ive hydroge l matrix could not be re ache d, the GAM de ve lope d by the GAMBA te am, as a combination of biomate rials and a biomime tic polyme r, has re ache d a le ve l whe re it can be us e d as ge ne ve ctors and ce lls carrie rs for pre -clinical s tudie s . WP3: Biomime tic Calcium Phos phate Bioce ramics granule s as carrie rs for ge ne ve ctors Le ad be ne ficiary: BIM WP le ade r: Pas cal Borge t/Guy Daculs i The conce pt of s caffold for the invas ion of ne wly forme d tis s ue s at the e xpe ns e of the latte r is the corne rs tone of bone tis s ue e ngine e ring. The challe nge of the Europe an proje ct Gamba to combine ge ne the rapy and bone tis s ue e ngine e ring involve s optimiz ing bioactive mate rials s upporting the re cons truction thanks to adhe s ion of hos t ce lls and ve ctors for ge ne trans fe ction. Biomatlante company, s ynthe tic bone s ubs titute s manufacture r, and LIOAD Ins e rm laboratory of Nante s Unive rs ity, de ve lope d ne w formulations of bioactive ce ramics to achie ve obje ctive s . Re s e arch and De ve lopme nt tas ks ne ce s s ary for the comple tion of this proje ct are the s cie ntific and te chnical he art. Biocompatibility te s ts we re the n pe rforme d in vitro and s pe cific in vivo animal mode ls have be e n s e t up to be tte r me e t the s pe cifications and accurate ly as s e s s the cons is te ncy of e s tablis he d biologics s olutions . At maturity of 3 ye ars of Gamba proje ct, Biomatlante company and Ins e rm LIOAD laboratory we re able to de mons trate une quivocally the e ffe ctive ne s s of ce ramic s caffolds (MBCP+®) in te rms of ce lls or trans fe ction ve ctors adhe s ion, diffe re ntiation and bone ingrowth P a ge 7 o f 23 Research and Innovation
e ve n in the mos t adve rs e s ituations (os te one cros is ). I. Scaffolds optimiz ation and ne w gale nic formulations de ve lopme nt The de mand place d on biomate rials for bone re ge ne ration involve s a total control of crys tallographic, che mical and me chanical prope rtie s . Biological re s pons e s to the s e alloplas tic grafts de pe nd on the s e multiple factors that cannot be le ft to chance . That is why Biomatlante company in collaboration with the laboratory Ins e rm LIOAD de e ply characte riz e d its ne wly de ve lope d me dical de vice s for Gamba proje ct in orde r to anticipate the ir pos itive bioactivity. 1) MBCP® te chnology (granule s or blocks ) [Tas k T3.1] Bone knowle dge is the bas e of biomime tic approach which le ads to improve ce ramic biomate rial s ynthe s is approaching native bone in te rms of che mical e le me nts through the crys talline phas e name d hydroxyapatite . Biological apatite is form as nanocrys tals afte r pre cipitation proce s s . The mate rials e ngine e ring joining biomime tic and che mical s trate gie s was us e d to de ve lop me thods to combine intimate ly crys talline phas e of hydroxyapatite with calle d Be ta-Tricalcium Phos phate (TCP), the latte r be ing abs e nt from the body. Re s orption prope rtie s of TCP give the biomate rial, biphas ic (HA / TCP), a "s mart de s ign". Inde e d, the s ynthe s is proce s s control at high te mpe rature s and the addition of poroge ns , allow offe ring dual-poros ity, micros copic and macros copic to the s e biphas ic bioce ramics with crys tallographic grains s iz e monitoring. Crys tal s iz e will influe nce microporos ity, re s orption and me chanical re s is tance . From this e xpe rtis e was the n cre ate d the MBCP conce pt, “M” for Macro-Microporos ity, and “BCP” for Biphas ic Calcium Phos phate . 2) Ne w granule s formulations with lowe r de ns ity and inte rnal large concavity [Tas k T3.1] As ge nic the rapy and bone tis s ue e ngine e ring s tate me nts of Gamba proje ct focus e d on hos t ce lls adhe s ion on os te oconductive s caffolds , Biomatlante and Ins e rm laboratory have inve nte d a ne w gale nic formulation of granule s . The s e granule s have be e n s pe cifically de s ign for s e ve ral re as ons : - To prote ct ce lls from tribological force s be twe e n granule s s urface s , which could damage ce lls on conve xical e xte rnal s urface s . - To improve ce ll adhe s ion in a large inte rnal concavity acting as a favorable os te opromotive calcium phos phate re le as ing “niche ”. - To de cre as e de ns ity of s caffolds which incre as e s the re s orption rate thanks to thin microporous walls and e nhance s the fluids pe ne trations . In othe r te rms , with a lowe r mas s of granule s , you could obtain at le as t as much ne w bone volume than with high de ns ity granule s . The s e ne w granule s are curre ntly be ing pate nte d by Biomatlante and Ins e rm LIOAD laboratory toge the r. 3) Compos ite Ce ramic/Hydroge l [Tas k T3.2] Cohe s ion be twe e n hydroge l made of e ithe r hyaluronic acid (AO foundation, Davos , Switz e rland) and ce ramic granule s (Biomatlante ) have be e n te s te d, as we ll as che mical e ve nt monitoring afte r as s ociation with trans fe ction ve ctors . Two formulations of granule s we re us e d for as s ociation, the firs t was cons titute d with 60% HA and 40% TCP, and the s e cond with 20% HA and 80% TCP. Re s ults de mons trate d a highe r s tability of s caffolds with ge ne ve ctors for 60% HA/40% TCP, than for 20% HA/80%TCP formulation, be caus e of dis s olution/pre cipitation phe nome non of TCP more re active phas e during as s ociation proce s s . 4) Compos ite matrice s Ce ramic/Polyme r Fibe rs [Tas k T3.3] As bone is a biological compos ite mate rial made of collage n and apatite , the biomimicry s take for Biomatlante company and Ins e rm laboratory in the conte xt of Gamba proje ct was to de ve lop a fibe r-bas e d compos ite matrix able : - To improve ce ll adhe s ion in 3D, along bioine rt polyme r fibe rs and on/in os te oconductive ce ramic granule s . - To e mbe d ce ramic granule s in fibe r me s h, which provide controlle d s pacing and avoid frictions be twe e n granule s . - To incre as e me chanical s tre ngth and allow be tte r handle ability. To obtain s uch compos ite fibe r-bas e d s caffolds , a one -s te p, room te mpe rature proce s s was us e d: e le ctros pinning. This me thod is available to s ynthe s is nano or microfibe rs from a polyme r s olution thanks to high voltage pote ntial be twe e n s yringe containing s olution and a me tallic colle ctor. The chos e n polyme r was polylactic acid (PLA), s ince its hydrolytic de gradation rate in vivo and bionie rty cons titute as s e ts to be e ligible in s uch me dical de vice s . Macros copic granule s e mbe dde d in PLA fibe r matrix we re the ne wly formulate d granule s de s cribe d above . The ne xt s te p would have be e n the as s ociation of the compos ite with the ve ctors to te s t the inte grity of the trans fe ction mate rials . P a ge 8 o f 23 Research and Innovation
5) Phys ico-che mical as s e s s me nt [Tas k T3.1; T3.2; T3.3; T3.4; T3.5] Characte riz ation me thods us e d for quality as s e s s me nt and control monitoring of our s caffolds we re : • X-Ray diffraction (XRD) to ide ntify and quantify crys talline phas e s (HA/TCP). • Infra-re d s pe ctros copy (FTIR) to ide ntify che mical groups and highlight unde s irable mole cule s . • Me rcury intrus ion poros ime try (MIP) to e valuate pore dis tribution and bulk de ns ity. • He lium pycnome try to me as ure the s ke le tal “true ” de ns ity. • Phys is orption te s t (BET) to me as ure the s pe cific s urface are a (i.e . are a available to prote ins ads orption from body fluids invas ion). • Ele me ntal analys is (EDX) to de te ct pos s ible impuritie s . • Ze ta pote ntial to e valuate e le ctric s urface charge s in s olutions • Scanning Ele ctron Micros copy (SEM) to vis ualiz e macro- micros tructure and grain boundarie s . A s e rie s of ads orption-re le as e te s t of prote ins have als o be e n s ubcontracte d to e xpe rt in the s e fie lds : CIC-IT ins titute (Borde aux, France ) and Atlantic Bone Scre e n (ABS) company (St-He rblain, France ). 6) Manufacturing and s te riliz ation proce s s [tas k T3.4, T3.5] Equipme nt qualification, proce s s validation for up-s caling from laboratory, s te riliz ation e fficie ncy according inte rnational quality s tandard and s taff training was s che dule d in orde r to e ns ure s afe ty, e fficie ncy and re producibility of the de vice which has to be combine d with the ve ctors and cDNA jus t be fore implantation.. To s tart the indus trial valoriz ation of the s e re s ults , the proof of conce pt re garding the as s ociation of the ge ne tic mate rials and the de vice is the firs t s te p and has s till to be pe rforme d. II. Biological e valuations : in vitro te s ts 1) Ce ll adhe s ion [tas k T3.1] A firs t crite rion for a de dicate d hos ting ce lls s caffold is to allow ce lls to s pre ad on its s urface . Human me s e nchymal s tromal ce lls we re s pre ad onto MBCP s caffolds and vis ualiz e d by SEM. It obvious ly appe are d that ce lls attache d and s pre ad into concavitie s of macropore s . 2) Ce ll viability [tas k T3.1] Ce lls afte r s pre ading on ce ramic granule s have to be able to prolife rate ; it will de pe nd on the biocompatibility of biomate rials . So as to e valuate this viability, a bioche mical te s t (MTS) was pe rforme d us ing murine bone ce lls (murine bone ce lls ). Re s ults highlighte d a ve ry good viability in comparis on with a pos itive and ne gative control, ove r 90% viability. 3) Ce ll diffe re ntiation [Tas k T3.1] Whe n s te m ce lls are re cruite d from blood or bone marrow, the y adhe re to the s urface of ce ramic granule s , the n prolife rate and if the re is a favorable e nvironme nt ce lls diffe re ntiate into bone ce lls . So as to as s e s s the os te oinduction of our mate rials , a re ve rs e trans cription polyme ras e chain re action (RT-PCR) analys is was pe rforme d. Re s ults s hown os te oge nic diffe re ntiation pote ntial was hundre d time s highe r whe n ce lls we re s pre ad on os te oconductive ce ramic granule s than along bioine rt polyme r fibe rs (without granule s ). 4) Active s ubs tance ads orption/re le as e [Tas k T3.1] Active s ubs tance ads orption/re le as e quantification has be e n done in a comparis on s tudy be twe e n round de ns e granule , MBCP granule and ne w lowe r de ns ity granule s . Albumin prote in involve d in ce ll adhe s ion was te s te d, as we ll as s pe cifically de s igne d Pe ptide 8 from ABS company and vancomycin antibiotic (CI-IT Borde aux). It was notice d that MBCP granule s are e ligible for drug de live ry purpos e , thanks to inte rconne cte d poros ity and highe r s pe cific s urface are ra, allowing gradual re le as e ove rtime without burs t e ffe ct at s hort time . Conclus ion The joint e fforts of Gamba cons ortium partne rs made it pos s ible to de ve lop multiple pote ntial s olutions to the challe nge s rais e d by ge ne the rapy and bone tis s ue e ngine e ring s take s . MBCP te chnology de mons trate d all its as s e ts to ans we r os te oconduction and os te oinduction obje ctive s . As to ne w granule s formulation with large inte rnal concavity and lowe r de ns ity, the y are highly promis ing in orde r to offe r favorable e nvironme nt to patie nt ce lls by acting as phos phocalcic niche s , prote cting ce lls and providing the m with ions pre curs or of biological apatitic pre cipitations le ading to mine raliz ation of os te oid tis s ue . Compos ite hydroge l or fibe r polyme r bas e d-s caffold have als o be e n s ucce s s fully de ve lope d thanks to one -s te p proce s s . Combine d prope rtie s of the s e ine rt 3D polyme r me s he s and highly os te opromotive ce ramic granule s cons titute an e xciting outcome obtaine d thanks to Gamba Europe an proje ct. P a ge 9 o f 23 Research and Innovation
To go furthe r, the as s ociation of the de vice s (granule s , compos ite s bas e d on hydroge l or fibe rs ) with the ge ne tic mate rials has to be te s te d in vivo to validate the ir the rape utic pote ntial. WP4: Atte nuation of inflammation for os te ochondral de fe ct re pair by immune modulation Le ad be ne ficiary: NUI Galway WP le ade r: Mary Murphy Os te oarthritis (OA) is a de ge ne rative dis e as e that re pre s e nts an e normous me dical and s ocio-e conomic burde n worldwide . De s pite inte ns ive re s e arch ove r many ye ars , tre atme nts for the dis e as e re main an unme t ne e d and the re is no acce pte d way to re pair damage due to os te oarthritis or pre ve nt the ons e t of this dis e as e . One of the major challe nge s to addre s s in e s tablis hing nove l the rapie s for OA is inflammation. Although damage to articular cartilage is cons ide re d a primary fe ature the re are als o change s to the s ubchondral bone and s ynovium. It is known that inflammation is a major drive r of dis e as e progre s s ion in OA acting in an inhibitory manne r on re ge ne rative mole cular proce s s e s . In fact s ynovial inflammation occurs in ve ry many patie nts and is a pre dictive factor in dis e as e progre s s ion. The obje ctive of this work package was to e s tablis h optimal conditions for s patiote mporal, controlle d inducible cytokine re le as e to achie ve immune modulation in orde r to facilitate re pair of pathologie s in the injure d os te oarthritic joint. Inte rle ukin 10 (IL-10) has both anti-and pro-inflammatory functions ; howe ve r, the viral analog (vIL-10) is known to have an atte nuating influe nce on inflammation. The GAMBA conce pt anticipate d the incorporation of a ge ne s huttle with the coding information for vIL-10 in the uppe r compartme nt of the GAMBA module s . The propos e d ge ne tic s witch was the cyclooxyge nas e -2 (COX-2) promote r which has be e n s hown to re s pond to inflammatory s ignals . The goal was to imple me nt a fe e d-back me chanis m whe re inflammatory mole cule s re le as e d by the s ynovium would activate production of vIL-10 through the COX-2 promote r, the re by re ducing the inflammation and s ile ncing the s ignalling s witch in a cyclical manne r. Tas k 1: Se le ction of optimal anti-inflammatory ve ctor s ys te m Plas mid DNA was clone d into COX-2 inducible promote r containing cas s e tte s . Non-viral and ade noviral s huttle ve ctors containing a s hort (-372/+59) and long form (-1432/+59) of the COX-2 promote r we re cons tructe d for as s e s s me nt of inducible e xpre s s ion of vIL-10. In addition the y provide d ve ctors with a re porte r ge ne . For this tas k the s e le cte d re porte r was s e cre te d Me tridia longa lucife ras e (METLuc). Initial as s e s s me nt indicate d s ucce s s ful functionality of the inducible cons tructs in ce ll line s . Following purification of the virus e s , a validate d me thod for the viral trans duction of human me s e nchymal s te m ce lls (hMSCs ) us ing lanthanum chloride was e s tablis he d. Furthe rmore , an in-hous e ELISA for the as s e s s me nt of vIL-10 re le as e from the ce lls was optimis e d and the ability of hMSCs to re s pond to optimis e d le ve ls of tumour ne cros is factor (TNF)-α and IL-1β as the inflammatory s ignals by incre as e d e xpre s s ion of COX-2 e s tablis he d (Fig. 4.1). De s pite inte ns ive and de taile d as s e s s me nt it was found that the COX-2 promote r cons tructs we re not functional at re gulating vIL-10 in hMSCs whe the r afte r a primary (Fig. 4.2) or s e condary inflammatory challe nge : induce d COX-2 e xpre s s ion or production of anti-inflammatory vIL-10 was not found. We als o inve s tigate d whe the r re gulate d vIL10 re le as e could be achie ve d following non-viral trans duction with COX-2 promote r drive n vIL-10 plas mids . Although vIL10 re le as e at low le ve ls was found with the cons titutive CMVIL-10 plas mid following trans fe ction of hMSCs with lipofe ctamine , no vIL-10 was de te cte d with the COX-2 inducible promote r plas mids (Fig. 4.3). The us e of the cons titutive CMV promote r, which was obs e rve d to ge ne rate high le ve ls of vIL-10 re le as e from hMSCs , was s e le cte d for furthe r in vitro me chanis tic analys e s and as s e s s me nt of immune modulation in a mous e mode l of OA. In te rms of alte rnative s to a ge ne tic s witch for controlle d re le as e of vIL-10, the pharmacological s witch, doxycycline , was found to be ve ry e ffe ctive in inducing re le as e of vIL-10 from both human and mous e MSCs trans duce d with a te tracycline (te t)-inducible vIL-10 virus (Fig. 4.4). Tas k 2: Sys te matic as s e s s me nt of the anti-inflammatory e ffe ct of re le as e d vIL-10 An in vitro mode l of inflammation us ing LPS activation of the monocyte ce ll line THP1 was e s tablis he d to as s e s s the anti-inflammatory ability of vIL-10 conditione d me dium (CM) from hMSCs . Re le as e profile s for vIL-10 we re e s tablis he d pos t hMSC trans duction us ing CMVIL-10 for the s e me chanis tic s tudie s . It was s hown that vIL-10 re producibly re duce d the le ve ls of TNFalpha re le as e and ge ne e xpre s s ion in THP1 ce lls following s timulation with LPS (Fig. 4.5). Mainte nance of cartilage inte grity or promotion of a pro-chondroge nic e nvironme nt is important for e s tablis hme nt of a re parative proce s s in OA. The re fore , inve s tigations with EMC we re pe rforme d to as s e s s the pote ntial of vIL-10 in dampe ning inflammation in vivo us ing an e x vivo mode l with chondroge ne s is of hMSCs achie ve d on an alginate ge l modulate d by CM from os te oarthritic s ynovial tis s ue (SCM). Factors s e cre te d from the os te oarthritic s ynovial tis s ue we re cle arly s hown to inhibit chondroge ne s is by de cre as e d ge ne e xpre s s ion for collage n II and aggre can. Additionally, e xpe rime nts we re pe rforme d to as s e s s the ability of vIL-10 to pre ve nt inflammation.. Synovium and fat pad we re obtaine d from patie nts unde rgoing total joint re place me nt with informe d cons e nt and e thical approval from local e thics committe e s . The following obje ctive s we re addre s s e d: to inve s tigate whe the r 1) inflammatory me diator production by os te oarthritic s ynovium and polaris e d macrophage s can be modulate d by tre atme nt with vIL-10, 2) the inhibition of MSC chondroge ne s is by os te oarthritic s ynovium is due to macrophage s , and ide ntify a role of macrophage phe notype in the inhibition of MSC chondroge ne s is , and 3) whe the r chondroge ne s is may be re s cue d by modulating macrophage s ubtype with vIL-10. P a ge 10 o f 23 Research and Innovation
Re combinant vIL-10 was not found to re producibly dampe n inflammatory me diator production by e nd s tage os te oarthritic s ynovium as as s e s s e d by quantification of pro-inflammatory IL-6, cytokine , or anti-inflammatory CCL18 [Che mokine (C-C motif) ligand 18]. The e ffe ct of SCM and vIL-10 tre ate d SCM on collage n type 2 ge ne e xpre s s ion in hMSCs was als o as s e s s e d to de te rmine the pote ntial of vIL-10 to modulate chondroge ne s is . Although vIL-10 had a pos itive e ffe ct on chondroge ne s is in the abs e nce of tre atme nt with SCM, this e ffe ct was ablate d with SCM pre s e nt. Again donor variability was a fe ature of the re s pons e to vIL-10. Le ve ls of collage n type 2 mRNA we re s ignificantly incre as e d in Donors 2 and 4 afte r v-IL-10 tre atme nt in the pre s e nce of SCM. No e ffe ct was s e e n in Donor 1 but vIL-10 was inhibitory in Donor 3. vIL-10 s timulation of M1 and M2 macrophage s did not s ignificantly alte r the re le as e and ge ne e xpre s s ion of inflammatory me diators . Human monocyte s we re is olate d from pe riphe ral blood and induce d to the M1 phe notype by tre atme nt with 100ng/ml LPS and 10ng/ml IFNgamma and to the M2 phe notype by e xpos ure to 10ng/ml IL-4. Morphological analys is s howe d the characte ris tic appe arance of M1 and M2 ce lls . M1 macrophage s de mons trate d characte ris tic e xpre s s ion of IL-6 and cytokine s e cre tion with M2 macrophage s s howing incre as e d e xpre s s ion and s e cre tion of CCL18. Expos ure to 100ng/ml vIL-10 had no e ffe ct on the s e parame te rs and als o did not impact e xpre s s ion of TNFalpha, CD206 and IL-10 its e lf. Finally, the e ffe ct of M1 and M2 conditione d me dia on chondroge ne s is of hMSCs and the impact of vIL-10 on this proce s s was as s e s s e d. Expre s s ion of type II collage n and aggre can was s ignificantly inhibite d by e xpos ure to M1 CM and vIL-10 was not able to re s cue this e ffe ct. M2 CM had a s light inhibitory e ffe ct on the e xpre s s ion of the chondroge nic factors . vIL-10 did promote e xpre s s ion of collage n type II mRNA but not aggre can. Joint fat pad CM als o de cre as e d the collage n type II and aggre can e xpre s s ion of MSCs . Tas k 3: In vivo trans fe ction and cytokine e xpre s s ion As a s trate gy to achie ve e xpre s s ion of vIL-10 in the joint, we inve s tigate d the e fficacy of dire ct intra-articular ve ctor inje ction in a s mall animal mode l. This s trate gy for in vivo trans fe ction and cytokine e xpre s s ion re quire d the us e of a murine collage nas e -induce d mode l of os te oarthritis . CMVIL-10 was us e d to as s e s s whe the r in vivo trans duction could be achie ve d and an anti-inflammatory e ffe ct of re le as e d vIL-10 could be de mons trate d in vivo. At Time 0, all mice re ce ive d two inje ctions of 1U collage nas e ove r 2 days . One we e k afte r the firs t inje ction, an e quivale nt amount of CMVIL-10 virus us e d to trans duce hMSCs at MOI 100 was inje cte d alone . All mice we re humane ly s acrifice d at s e ve n we e ks following collage nas e inje ction. Poplite al and inguinal lymph node s we re harve s te d and dige s te d to ge ne rate a s ingle ce ll s us pe ns ion for analys is of T ce ll and macrophage populations us ing flow cytome try. A tre nd of de cre as e d le ve ls of CD11b+ and CD11b+Ly6C hi inflammatory monocyte le ve ls in both the inguinal and poplite al lymph node s was obs e rve d 6 we e ks pos t intra-articular inje ction with AdIL-10. Cytokine e xpre s s ion in vivo was as s e s s e d following analys is of harve s te d s e rum us ing a multiple x ELISA as s ay to inve s tigate re le as e d le ve ls of vIL-10 following in vivo trans duction, as we ll as othe r inflammatory me diators . Howe ve r, available re age nts we re not s ufficie ntly s e ns itive . Tas k 4 Ex vivo trans duction and immune modulation The re afte r, in vivo as s e s s me nt of AdIL-10 e xpre s s ing hMSCs unde r the control of the CMV ve ctor was als o pe rforme d. Five groups of 8 C57BL/6 mice , 8-10 we e ks old we re te s te d. Groups we re as follows : Ve hicle (s aline ), AdIL-10, MSCs , Adnull MSCs and AdIL-10 MSCs . A dos e of 20 x 104 hMSCs was inje cte d into the tre ate d joint, ide ntifie d as the optimal ce ll numbe r re quire d for the production of 100 ng/ml vIL-10, following in vitro trans duction s tudie s . hMSCs we re trans duce d with CMVIL-10 or Ad-Null virus at MOI of 100, us ing Lanthanum Chloride bas e d trans duction. Furthe rmore , an e quivale nt amount of CMVIL-10 virus us e d to trans duce hMSCs at MOI 100 was inje cte d alone . Blood was harve s te d prior to collage nas e inje ction, 1 we e k pos t-tre atme nt inje ction and again at s acrifice . Se rum was is olate d and froz e n for cytokine analys is until re quire d. Cytokine e xpre s s ion analys is , and lymph node harve s t and analys is we re pe rforme d as de s cribe d above . Tre ate d kne e joints we re harve s te d, fixe d in 10% formalin ove rnight and de calcifie d in EDTA for 2 we e ks prior to paraffin e mbe dding for his tological analys is of OA progre s s ion. The le ve l of OA damage and de gre e of s ynovial inflammation in all tre ate d joints was grade d by thre e blinde d re vie we rs . As s e s s me nt of inflammatory ce ll s ubs e ts was pe rforme d to inve s tigate whe the r vIL-10 e xpre s s ion re s ulting from in s itu trans duction, or re le as e from trans duce d hMSCs has an e ffe ct of inflammatory ce ll phe notype within the lymph node s . Pre liminary analys is indicate d a de cre as e in the total numbe r of CD4+ and CD8+ T ce lls locally in the poplite al lymph node in re s pons e to intraarticular inje ction of hMSCs trans duce d to e xpre s s vIL-10. At the optimal time s us e d for analys is of immune modulation by vIL-10, as s e s s me nt of s ynovium damage and os te oarthritis progre s s ion was not s ignificantly diffe re nt be twe e n groups and additional e xpe rime nts will ne e d to be pe rforme d to s e e if the change s that are s e e n in te rms of the inflammatory e nvironme nt re s ult in prote ction from OA at late r time points . Conclus ions : COX-2 inducible ade noviral ve ctors we re not found to be functional at re gulating vIL-10 in hMSCs although controlle d re le as e of the cytokine was achie ve d with chondrocyte s in vitro. The inducible re le as e of vIL-10 from human and mous e MSCs following doxycycline s timulation of ce lls that we re co-trans duce d with AdCMVrtTA and AdTREtvIL-10 ve ctors , was achie ve d. OA s ynovium and infrapate llar fat pad inhibit chondroge nic diffe re ntiation of MSCs , and tre atme nt of the s ynovium with vIL-10 doe s not counte ract this e ffe ct. The ne gative e ffe ct of OA s ynovium can be addre s s e d to the pre s e nce of M1 macrophage s and the cytokine s that are re s pons ible for the ne gative e ffe ct of OA s ynovium s ignal act, at le as t partially, via the inflammatory pathways TAK1 and JAK. The s e re s ults indicate that vIL-10 has the pote ntial to impact the os te oarthritic joint pro-inflammatory e nvironme nt but this e ffe ct may be limite d by the le ve l of inflammation. In vivo trans fe ction utilis ing AdCMVIL-10 viral ve ctors s e rve d as an alte rnative s trate gy to achie ve vIL-10 re le as e and s ubs e que nt immune modulation. A tre nd in de cre as e d le ve ls of CD11b+ and CD11b+Ly6Chi inflammatory P a ge 11 o f 23 Research and Innovation
monocyte le ve ls was achie ve d in both the inguinal and poplite al lymph node s , 6 we e ks pos t intra-articular inje ction with AdIL-10, s ugge s ting modulation of inflammatory proce s s e s . In vivo immune modulation following intra-articular inje ction of hMSCs trans duce d with AdvIL-10 was als o e vide nt, with re duce d CD4+ and CD8+ T ce ll le ve ls de te cte d following tre atme nt with AdIL-10 MSCs . A s ignificantly highe r proportion of CD4+CD62L- ce lls in the AdvIL-10 MSC tre ate d group compare d to hMSC alone was als o e vide nt, which may s ugge s t an incre as e of e ffe ctor me mory ce lls in re s pons e to this tre atme nt. Obs e rve d tre nds in modulation of the de ve lopme nt of os te oarthritic s ymptoms ne e d to be addre s s e d at a late r time point, and with mous e MSCs . WP5 Spatiote mporal control of growth factor ge ne de live ry for cartilage re pair Le ad be ne ficiary: EMC WP le ade r: Ge rjo van Os ch We have de mons trate d that inflammatory factors s e cre te d by s ynovium from os te oarthritis patie nts pre ve nt the formation of cartilage by s te m ce lls originating from bone marrow. Spe cific drugs can partially counte ract this e ffe ct: the y pre ve nt the biological communication of the inflammatory factors into the ce lls , but the timing of adminis tration appe are d to be an important is s ue . The inflammatory factors are mainly produce d by ce rtain immune -ce lls pre s e nt in s ynovium: macrophage s . To favor cartilage formation in a patie nt with os te oarthritis , changing the s e macrophage s from producing inflammatory factors towards producing pos itive factors is of gre at importance . So far, IL10, a prote in that can have s uch an e ffe ct was te s te d for this purpos e . Unfortunate ly this was not s ufficie nt. Furthe r e xpe rime nts us ing othe r s timulants to change the be haviour of s ynovial macrophage s are ne ce s s ary. In orde r to le t s te m ce lls originating from bone marrow form cartilage unde r laboratory conditions , a s timulant calle d TGFb is ge ne rally e s s e ntial. The TGFb s hould be pre s e nt the whole time in s ufficie nt quantitie s . We we re not able to re ach s ufficie ntly long e xpre s s ion us ing plas mids to trans fe ct human s te m ce lls . Howe ve r, in a mode l that we de ve lope d, the pre s e nce of additional TGFb appe are d to be no longe r ne ce s s ary. Joints that are le ft ove r from s laughte re d cows we re us e d to obtain cartilage -bone biops ie s : pie ce s of bone that are cove re d with cartilage on top. In this cartilage we can make de fe cts to s imulate cartilage damage . Us ing this mode l we te s te d the capacity of s te m ce lls originating from bone marrow to form cartilage . Factors produce d by the bone we re s ufficie nt to s tart cartilage formation. Initially we hypothe s iz e d that TGFb produce d by the bone would be re s pons ible for this e ffe ct. Howe ve r we have prove n us ing multiple ways to pre ve nt TGFb from having its e ffe ct on s te m ce lls that the bone mus t produce othe r factors that are s timulating the s te m ce lls to form cartilage . We have not ye t ide ntifie d which factor or which combination of factors is re s pons ible for this . The pos itive e ffe ct of the bone was als o influe nce d by the type of mate rial that was us e d to e ncaps ulate the ce lls . Whe n we us e d HA-pNIPAM or alginate , the s te m ce lls s tarte d to form cartilage , whe re the y did this to le s s e r e xte nt whe n we us e d fibrin. This e ffe ct was e ve n s tronge r whe n we ins te ad of culture in the laboratory, trans fe rre d this mode l s ys te m of cartilage -bone biops ie s unde r the s kin of mice : cartilage was forme d by the s te m ce lls in alginate and not in fibrin. The us e of fibrin e ve n re s ulte d in unwante d bone formation, a phe nome non that is known to occur in s te m ce lls . Alginate appe are d to pre ve nt this . Altoge the r, we have de ve lope d a mode l s ys te m that is more re pre s e ntative for the s ituation in a joint than conve ntional laboratory mode ls . This mode l can be us e d to s cre e n ce ll type s and/or mate rials for the ir us e in cartilage re ge ne ration. This s cre e ning is of vital importance towards the de ve lopme nt of tre atme nts for patie nts with os te oarthritis as we have found that the s pe cific mate rial us e d to e ncaps ulate ce lls can have major e ffe cts on the outcome . WP6 Spatiote mporal growth factor de live ry for bone formation Le ad be ne ficiary: IRCCS (FORMERLY INRC OR USMI) WP le ade r: Chiara Ge ntili/ Ranie ri Cance dda Workpackage 6 is de dicate d to s patiote mporal growth factor de live ry for bone formation. One of the initial obje ctive s was to de fine the te mporal re quire me nts for BMP2 e xpos ure to obtain os te oge nic diffe re ntiation. While the re combinant prote in is approve d for clinical us e in s ome applications , major re s e arch is re quire d to e s tablis h the us e of its pre curs or, the corre s ponding ge ne tic cons tructs have be e n accomplis he d by partne r 1 (TUM), that the y had cons tructe d non-viral and ade noviral ve ctors for e xpre s s ion of TET-on BMP-2. We have achie ve d inducible BMP2 e xpre s s ion in human MSC following Doxycycline s timulation of ce lls . We obs e rve d that at e arly pas s age the BMP-2 MSC incre as e os te oge nic diffe re ntiation in vitro, but BMP-2 trans fe ction probably inte rfe re with the prolife ration and diffe re ntiation of the MSC in culture , re s tricting the ability to e xpand ce lls in vitro. Combining s caffolds and ve ctors
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To te s t the e fficacy of e ndochondral-bone formation us ing cons tructs /ve ctors de ve lope d in GAMBA cons ors ortium, in WP6 we e valuate d with diffe re nt type of ce lls , human bone marrow s tromal ce lls (BMSCs ) and human os te oblas ts (OB) the capacity to form bone in vivo. We loade d the s caffolds that we re ce ive d from Biomatlante partne r, only with ce lls and we implante d the m in mice to e valuate the e fficacy of bone formation of the s caffold plus and minus ce lls . Human and s he e p BMSCs we re is olate d from adult bone marrow and culture d in vitro. Human OB we re harve s te d from bioptic pie ce s of large bone s by s e ve ral me chanical and e nz ymatic dige s tions and culture d according to (Torte lli e t al. Tis s ue Eng Part A. 2009 Se p;15(9):2373-83). We us e d two micro/macroporous biphas ic calcium phos phate ce ramics (MBCP) with diffe re nt compos ition; MBCP A, compos e d of 60% of hydroxyapatite and 40% of tricalcium phos phate be ta and MBCP +, compos e d of 20% of hydroxyapatite and 80% of tricalcium phos phate be ta. To e valuate the e ffe ct of the diffe re nt calcium phos phate ce ramics compos ition on e ctopic bone formation, we us e d hMSCs and hOB and pos itive control ce lls s he e p MSC, we s e e de d 2 x 106 ce lls at pas s age 1 into MBCP (A and +) s caffolds . Immunode ficie nt mice (CD-1 nu/nu; Charle s Rive r, Calco, Italy) we re ane s the tiz e d and four s ubcutane ous pocke ts cre ate d. Scaffolds of e ach of the calcium phos phate ce ramics we re implante d for 1, 4 and 8 we e ks . At the e nd of time points , animals we re s acrifice d and implants we re harve s te d for his tological analys is to acce s s bone formation. As re quire d by the Italian Minis try of He alth, all animals we re maintaine d in accordance with the s tandards of the Fe de ration of Europe an Laboratory Animal Scie nce As s ociations (FELASA). The re s ults s hown that us ing diffe re nt MBCP s caffolds loade d with no trans fe ct BMSC a good bone formation occur. More ove r, we analyz e d s e ve ral his tological s e ctions to compare bone s iz e and dime ns ion for both s caffolds and re s ults that hBMSCs de pos it more bone e xtrace llular matrix in the s caffolds made with MBCP plus granule s (Fig.6.1). His tomorphome tric analys is was pe rforme d to quantify bone and blood ve s s e l amounts . The re s ults s hown an ove rall bone formation in both s caffolds loade d with hMSC. We als o obs e rve d a s ignificantly large r amount of blood ve s s e ls in the condition with more bone forme d. The induction of large and mature bone as s ociate d with vas t pre s e nce of organiz e d blood ve s s e l s tructure s might be re late d not only to diffe re nt microporos ity grade but mainly to Ca2+ ion flux product of CaP dis s olution/re -pre cipitation e ve nts occurring in bone microe nvironme nt. From the in vitro s tudie s , pre vious de s cribe d, we obs e rve d that BMP-2 trans fe ction inte rfe re with the prolife ration and diffe re ntiation of the MSC in culture , re s tricting the ability to e xpand ce lls in vitro. For the s e re as ons , we have not obtaine d the corre ct numbe r of ce lls to load on MBCP s caffolds , and pe rforme d the in vivo s tudie s us ing BMP-2/MSC combine d with MBCP s caffolds . More ove r, in collaboration with WP1 (OZB, TUM) we pe rforme d in vivo trans fe ction s tudie s with lipople xe s /GFP cDNA and Microporous Biphas ic Calcium Phos phate (MBCP) granule s provide d by BIM (WP3). We us e d DOGTOR lipid, ide ntifie d in pre vious e xpe rime nts as a favourable trans fe ction re age nt for hMSC and we combine d this lipid comple x and MBCPs s caffolds to pe rform an in vivo 3D s caffold trans fe ction. The s e cons tructs s hould be us e ful for following dire ctly the in vivo trans fe ction of the ce lls re cruite d from the s caffolds . Te s ting the e fficacy to re pair de fe cts in s mall animal mode ls Since biphas ic calcium phos phate (BCP) granule s are known to be os te oconductive , INSERM, Biomatlante , TUM and OZB have e valuate d the os te oinductive e ffe ct of MBCP+™ micro-macroporous s ubs titute s us ing he at-induce d mode l of rabbit os te one cros is in fe moral e piphys is s ite s . The re fore , afte r as s e s s ing the the rape utic os te oge nic pote ntial of the s e BCP granule s , the ultimate obje ctive of this work was to combine MBCP+™ granule the rapy with s pe cifically de s igne d ge ne ve ctors (Dogtor, OZbios cie nce , Mars e ille , France ) whos e plas mid is coding for BMP2 growth factor. Critical s iz e de fe cts (5–6 mm in diame te r and 8 mm in le ngth) we re induce d in fe moral e piphys e s of 36 Ne w Ze aland rabbits . A the rmocouple , which was controlle d by a te mpe rature probe , was he ate d to 80°C. Rotation of the the rmocouple was pe rforme d in orde r to improve homoge ne ity of the the rmal tre atme nt, which las te d for 45 s e conds . Following implantation of loade d/unloade d bioce ramics s caffolds with ve ctor/plas mid, the animals we re confine d for 3, 6, and 12 we e ks (Figure 6.2). Re s ults de mons trate d the ne cros is mode l induce d by focal he ating ins ults was e ffe ctive as a ve ry highly s ignificant diffe re nce was obs e rve d be twe e n e mpty-ne cros e d s ite and ce ramic fille d s ite s . A highly os te oinductive pote ntial of MBCP+™ granule s in os te one cros e d s ite s was prove n s ys te matically by his tological analys is (Figure 3). The addition of Dogtor/plas mid s ys te m to MBCP+ granule s s hown at le as t as good re s ults in te rms of ne w bone volume but not s tatis tically s ignificant diffe re nce s we re highlighte d. The s e re s ults are the n promis ing s ince optimiz ation of ve ctor/plas mid conce ntration on granule s and improve me nt of the as s ociation protocol could be done thanks to furthe r inve s tigations . WP7 Large animal mode ls for OA Le ad be ne ficiary: ARI WP Le ade r: Mauro Alini The aim of this Workpackage was to pe rform a proof of conce pt of one or more module s cre ate d by the pre vious WPs P a ge 13 o f 23 Research and Innovation
in a large animal mode l, the goat. Although the is olation and e xpans ion of the e quivale nt ce ll populations de ve lope d in the workpackage 3-5 was e s tablis he d for the goat mode l, the large animal pre -clinical trial (WP-7) was not pe rforme d as the the rape utic s olutions e nvis ione d could not be fully prove n in pre liminary s mall animal s tudie s . The re fore , the cons ortium could not e thically and s cie ntifically approve the trial. Howe ve r, s mall animal s tudy was pe rforme d by ARI to de mons trate the biocompatibility of the biomime tic hyaluronan hydroge l. The rabbit mode l us e d was re le vant for the GAMBA proje ct aiming to re pair both cartilage and bone tis s ue s . It has the advantage to be a s imple r in vivo mode l, us e ful for pre -s cre e ning biomate rials and tis s ue e ngine e ring s olutions in comparis on to the more clinically re le vant but complicate d and e thically more de manding goat mode l. The in vivo mode l cons is te d in an os te ochrondral de fe ct on the we ight-be aring s urface of the me dial fe moral condyle of a rabbit with a diame te r of 2.7 mm and a de pth of 4 mm and cre ate d us ing a drill bit. The s tudy was approve d by authoriz e d e thical commis s ion. In this de fe ct a the rmore s pons ive hydroge l was inje cte d or le ft e mpty re s pe ctive ly. During the s tudy radiographs we re take n be fore (e ntry te s t) and imme diate ly afte r s urge ry. We ights we re controlle d pre ope rative ly, 3 days and 7 days afte r s urge ry. The rabbits we re e uthaniz e d 1 or 12 we e ks afte r s urge ry. Pos t morte m radiographs we re take n. All rabbits re cove re d we ll from s urge ry and all clinical e xams are within normal limits for the duration of the e ntire s tudy. During the s tudy no high burde n for the animals was de te cte d as s hown with the individual s corings during the whole s tudy duration. His tological findings indicate d that the biomime tic hydroge l was biocompatible and could the re fore be us e d as a de live ry carrie r in vivo. WP8: Linking GAMBA to s ocie ty through s cie nce -s ocie ty dialogue s on chance s , ris ks and e thical as pe cts Le ad be ne ficiary: SCID WP Le ade r: Katharina Zölle r Workpackage 8: Socie tal Vie ws : Patie nt and Citiz e n Pane ls on GAMBA What is a lay pane l? In a lay pane l, a group of 12-25 inte re s te d participants de ve lop a joint s tate me nt on a s ocially re le vant topic. Participants re ce ive information unde rs tandable to lay pe ople , and inte r¬vie w e xpe rts s ome of whom the y can choos e the ms e lve s . The y are s upporte d by a te am of profe s s ional, ne utral facilitators . The te rm “lay pane l“ is de rive d from “citiz e ns ' pane l“, which is a form of participation ins pire d by the me thods of “citiz e ns jury” and cons e ns us confe re nce s . The goal is to de ve lop a joint lay opinion in the form of a “lay re port“. The GAMBA lay pane ls took place be twe e n May 2011 and July 2012 on 3,5 re s p. 4 days pe r pane l (two we e ke nds with an inte rval of thre e we e ks ) in Munich/Ge rmany, Galway/Ire land and Zollike rbe rg ne ar Zurich/Switz e rland with altoge the r 71 participants age d 19 to 82. The participants of the citiz e ns ' pane ls we re partly re cruite d random¬ly. In this way we hope d to avoid re cruiting only participants who are alre ady (ve ry) active ly involve d in s ocie tal de bate s . Anothe r advantage of re cruiting at random is that participants from diffe ring s ocial backgrounds can be re ache d. The lay pane ls are a type of „microcos m“, re fle cting a s malle r image of s ocial re ality. The whole group’ as s e s s me nt, and not – as in a s urve y – the s um of the uninforme d individuals ' opinions , is le ading to the re s ults , as the y are bas e d on inte ns ive dis cus s ions and ne gotiations . One ce ntral e le me nt in the proce dure of the lay pane ls is a wide varie ty of e xpe rt input. It s e ts out to inform the participants on the whole range of s ubje ct-s pe cific ins ights and as s e s s ¬me nts . This e ns ure s that opportunitie s as we ll as unce rtaintie s and ris ks are dis cus s e d, and e thical que s tions are rais e d. This innovative form of public participation in s cie nce in profe s s ionally facilitate d lay pane ls e nable s the participants to take a s tand on the comple x s ubje ct matte r of GAMBA (ge ne and s te m ce ll the rapie s ). The y we re able to advis e the s cie ntis ts , the EU as our s pons or, re s e arch politics and othe r age nts on s ocie tal is s ue s on GAMBA in a knowle dge able way. Ove rvie w of the lay pane l proce s s : In the lay pane ls , participants we re as ke d to as s e s s the GAMBA fie ld of re s e arch from the ir various pe rs pe ctive s as patie nts or inte re s te d citiz e ns . To pre pare the m for this , the y firs t re ce ive d an introduction to GAMBA as a fie ld of re s e arch: in the run-up to the pane ls , participants re ce ive d the “Manual” and “Compe ndium” brochure s which had be e n writte n in layman’s te rms by the s cie nce journalis t, Be atrice Lugge r, in collaboration with the proje ct te am. The pane ls lis te ne d to pre s e ntations (on os te oarthritis , the propos e d mode of ope ration of the GAMBA approach, pos s ible ris ks and the e thical as pe cts of ge ne and s te m ce ll the rapie s ) and que s tione d s e lf-s e le cte d e xpe rts dire ctly in a he aring. Equippe d with this information, the y the n dis cus s e d the opportunitie s , pos s ible ris ks and e thical as pe cts of GAMBA, and afte r an e xte ns ive dis cus s ion in bre akout groups and in ple nary the y dre w up module s for s ubmitting the ir opinions (lay re port in Ge rman and Englis h: http://www.wis s e ns chafts dialog.de /inde x.php/download). Participants pre pare d the ir “Lay State me nts ” in two s e s s ions (of two days e ach) on the is s ue s re lating to GAMBA. The pane ls ’ firs t s e s s ion s e rve d to “e mpowe r” participants in the s ubje ct and me thodology:
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• Day 1: Afte r participants had be e n give n an opportunity to ge t to know e ach othe r and we re provide d with an outline of the s che dule , the y lis te ne d to an introductory pre s e ntation on os te oarthritis , we re give n an ove rvie w of the GAMBA re s e arch proje ct, put the ir que s tions to the s pe ake rs and dis cus s e d the is s ue s with the m. • Day 2: On the s e cond day, participants cons ide re d GAMBA in de pth with the local re -s e arch te ams . The lay participants als o lis te ne d to pre s e ntations on the pos s ible ris ks re late d to the GAMBA fie ld of re s e arch and on the e thical as pe cts . The s e s s ion conclude d with participants s e le cting e xpe rts for a he aring on the s e cond we e ke nd and the y we re give n the opportunity to e xte nd the ir knowle dge of the individual as pe cts of GAMBA in te rms of e thical and s ocial factors be twe e n the two s e s s ions through the cre ation of “ambas s adors hips ”, i.e . individual participants adopting an is s ue to purs ue on be half of the pane l. The s e cond s e s s ion (thre e we e ks late r) was wholly ge are d towards the de ve lopme nt of as s e s s me nts , opinions and the s ugge s te d wording of the lay re port: • Day 3: On the third day, thos e participants who had pre pare d a “ambas s adors hip” pre s e nte d or dis cus s e d the ir re s ults with the ir fe llow pane llis ts . The n the he aring with the e xpe rts s e le cte d by the participants took place . • Day 4: The fourth and las t day was us e d for an in-de pth dis cus s ion and for the as s e s s me nt of the GAMBA fie ld of re s e arch. The pane llis ts pre pare d module s of te xt cove ring the opportunitie s , ris ks and e thical as pe cts of GAMBA which we re the n compos e d by the Scie nce Dialogue te am and – afte r the pane ls - fe d back to the participants for comme nts and approval. Two e le cte d s poke s pe rs ons pre s e nte d the re comme ndations at the final e ve nt in e ach country. Each day e nde d with a “s naps hot re vie w”, i.e . a brie f apprais al of the re s pe ctive day. The facilitation te am was fle xible in addre s s ing the ne e ds of the individual groups , acce pte d criticis m and s ugge s tions and made change s . The re fore , e ve n though the proce s s is comparable ove rall, the re may be minor local variations . Summary of the re comme ndations of lay participants in the GAMBA pane ls : s e e attachme nt Quality crite ria for s cie ntific lay pane ls The following quality crite ria are important for public participation proce s s e s : Cle ar mandate : From the s tart it is cle arly de fine d what will be done with the re s ults and who the inte nde d audie nce is compris e d of. The s e addre s s e e s s hould commit to making a qualifie d public s tate me nt and include the lay re comme ndations in the ir work whe re ve r pos s ible . Me thodical e mpowe rme nt of participants : This is s upporte d by an inclus ive , e xpe rie nce d and appropriate me thod of facilitation, which is committe d to upholding the quality crite ria (be low). The facilitators e mpowe r the s e lf-confide nce and the as s e s s me nt s kills of the participants , allowing a dis cus s ion be twe e n laype rs ons and e xpe rts at e ye le ve l. Qualifie d input of information: This mus t be e ns ure d by a qualifie d information bas e (brochure s and othe r s ource s of information) which is balance d and unde rs tandable for laype rs ons , by e xpe rt pre s e ntations from various pe rs pe ctive s , as we ll as influe nce on the choice of e xpe rts and pos s ibilitie s for own re s e arch. Trans pare ncy: It is important to provide information about pane l obje ctive s including cre ative le e ways and limits . Furthe rmore , a continuous vis ualiz ation and docume nt¬ation of the proce s s s upports the proce s s ing of information and the achie ve me nt of re s ults . Outcome ope nne s s and impartiality: Participants mus t be able to influe nce the infor-mation proce s s through the s e le ction of the matic as pe cts and e xpe rts as we ll as the ir own re s e arch; outcome s are ge ne rate d as the s ole re s pons ibility of the participants . WP9: Dis s e minating and e xploiting GAMBA Le ad be ne ficiary: TUM WP Le ade r: Chris tian Plank, Martina Anton Dis s e mination of knowle dge has be e n on s e ve ral le ve ls : Inte rnal dis s e mination - re gularly s che dule d progre s s me e tings , phone confe re nce s , e le ctronic communication - e le ctronic communication, cre ating and continuous ly updating common knowle dge bas e s - the we bpage contains a s e cure d are a with acce s s re s tricte d to partne rs for e xchange and dis s e mination of information amongs t partne rs . - the we bpage contains a lis t of proje ct as s ociate d publications of partne rs and othe rs . - lab vis its of s tude nts in orde r to le arn ne w te chnique s Exte rnal dis s e mination:
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Exte rnal dis s e mination has be e n on s e ve ral le ve ls : to the inte re s te d lay-public and to the s cie ntific community. - a we bpage was cre ate d to allow for information on GAMBA - pre s s re le as e s : on ge ne ral is s ue s like granting of the proje ct by the EC, on s e le cte d re s ults - publications in non-s cie ntific journals to attract maximal atte ntion to GAMBA outputs and impact on the the rapie s availability and s afe ty for patie nts s uffe ring from arthritis - publications in s cie ntific journals - pre s e nce on inte rnational me e tings : pre s e ntation of s cie ntific re s ults - organis ation of s ympos ia and s ate llite me e tings on inte rnational congre s s e s - citiz e n and patie nt pane ls we re part of the GAMBA WP8 - the pre s e ntation of the proje ct obje ctive s , re s ults and impacts in works hop s e s s ions ope n for profe s s ionals - e ffe ctive communication with the me dia (te le vis ion, radio and pre s s ) has be e n e s tablis he d in orde r to be tte r inform the public, be more vis ible and incre as e citiz e n’s aware ne s s and inte re s t in arthritis ’ re s e arch. Nume rous pre s s article s and broadcas ts made the wide r public aware of the proje ct. Scie ntific community dis s e mination (including clinicians ). A public progre s s re port has be e n pos te d on the we bpage at re gular inte rvals . As part of the GAMBA proje ct, 20 pape rs we re acce pte d for publication in s cie ntific journals and 9 mas te r and PhD the s e s we re comple te d by s tude nts participating in the proje ct. Partne rs als o atte nde d inte rnational me e tings to communicate the ir findings . Educational mate rial has be e n pre pare d and made available to s tude nts to s tre ngthe n the ir e xpe rtis e in nanome dicine (WP8). Thre e comme rcial ge ne de live ry products for tis s ue e ngine e ring we re launche d and one pate nt application is unde r way. WP10 Manage me nt Activitie s Le ad Be ne ficiary: TUM Work package le ade r: Martina Anton, Chris tian Plank Obje ctive : The obje ctive of this WP was to e ns ure the following: that all budge tary actions are pe rforme d corre ctly and according to the rule s and re gulations e s tablis he d by the Europe an Commis s ion and in the cons ortium agre e me nt; that the re ce ive d funds are corre ctly dis tribute d and accounte d for, including inde pe nde nt auditing; that the work and tas ks are comple te d in time , within budge t, and s atis fy high-quality re quire me nts ; that re porting is done on a pe riodic bas is , in the mos t e fficie nt and pragmatic way, according to Commis s ion guide line s to provide all cons ortium me mbe rs with all important and impacting information that can influe nce the proje cts ’ outcome ; to imple me nt an authority e ns uring that knowle dge and innovation are prope rly manage d and re s ults e xploitation promote d; to pre pare me as ure s for pate nt application The cons ortium s tarte d with the Kick-off me e ting organiz e d by the coordinating partne r TUM in Ste ine bach Wörths e e , Ge rmany (ne ar Munich) on 16.-17.09.2010. We had a total of 23 participants with e ve ry partne r s e nding at le as t the PI and one more re pre s e ntative . The re the s te e ring committe e as highe s t body of de cis ion making within GAMBA was e s tablis he d with one re pre s e ntative of e ach partne r. The s te e ring committe e was chaire d by Martina Anton as coordinator. Chris tian Plank (TUM) was appointe d as e xploitation manage r. The s ix-month me e ting was he ld on 24.-26.02.2011 in Davos , Switz e rland. This me e ting was atte nde d by the PIs and additional inve s tigators of all partne rs . Additionally, the proje ct office r Dr. Johan Ve iga-Be ne s ch and the EU appointe d proje ct te chnical advis or Dr. Roxana Pitice s cu atte nde d the me e ting. Additionally the following phone confe re nce s took place : 20.12.2011 be twe e n TUM and NUI Galway;25.01.2012 be twe e n ARI, TUM, OZB, BIM, INSERM The midte rm GAMBA me e ting was he ld in Nante s , FR: 26.-27.03. 2012. The 24 months me e ting was he ld in Vie nna, AT, 08.-09.09.2012 on the occas ion of th TERMIS confe re nce . The 30 months me e ting was he ld in Ge nova, IT, 11.-12.2013 and the final me e ting was he ld in Munich, DE, 11.-12.07.2013. All me e tings we re atte nde d by at le as t one re pre s e ntative of e ach partne r and all me e tings include d a s te e ring committe e me e ting as we ll. The final me e ting in Munich was als o atte nde d by the Proje ct Office r Dr. Jaime Ibane z de Ele jalde and the proje ct te chnical advis or Dr. Roxana Pitice s cu. All me e tings we re ve ry e ffe ctive in te rms of s cie ntific e xchange and in te rms of imple me nting the work plan. An e xploitation plan had be e n compile d by the e xploitation manage r as we ll as the final plan of us e and dis s e mination of fore ground. The s e plans we re approve d by all partne rs and we re s ubs e que ntly s ubmitte d to the Commis s ion. Ethical is s ue s : No e xpe rime nts we re pe rforme d without obtaining pe rmis s ions from the appropriate re gulatory bodie s be fore hand. Ethical approvals had be e n s ubmitte d to the EC and update s have be e n obtaine d and communicate d during re gular re porting.
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The mos t challe nging tas k was , like in all EU proje cts , the re porting bus ine s s . Accordingly, this was the mos t time cons uming tas k to WP10. All re ports we re s ubmitte d in a time ly or clos e to time ly manne r. Pote ntial Impact: Impact: The GAMBA proje ct has addre s s e d s e ve ral ke y topics of the call [NMP-2009-2.3-1] as we ll as of the ge ne ral work programme s ummariz e d be low. Ce ll bas e d the rapie s : Me s e nchymal s te m ce lls have be e n activate d by ge ne s in the mate rials to re pair damage d tis s ue s . Os te oarthritis /Os te oporos is : GAMBA te chnologie s have be e n validate d in mode ls of os te oarthritis . Re s ults will be wide ly applicable in tis s ue re pair and have implications for future application in os te oporotic bone fracture s . Us e of bioactive mole cule s couple d to e ngine e re d biomate rials : The bioactive mole cule s are ge ne ve ctors . The biomate rials are e ngine e re d to allow for s patiote mporal control. Advance d multidis ciplinary approache s : The cons ortium has be e n a multidis ciplinary te am uniting biomate rials s cie nce , ge ne the rapy approache s , me dical s cie nce s , immunology, s te m ce ll re s e arch, trans lational re s e arch, s ocial s cie nce s and e conomic e xploitation. Local tis s ue re pair: Our mate rials we re de s igne d for tis s ue re pair. Inhibition of inflammation: The multicompone nt s ys te m compris e s ge ne ve ctors re s ponding to inflammatory mole cule s with the e xpre s s ion of anti-inflammatory mole cule s . Mate rials which can match and e xchange s timuli with the natural biological e nvironme nt and s timulate he aling: The s ys te m was de s igne d to adopt anatomical s tructure s , to re s pond to biological and phys ical s timuli and to induce he aling. Bio-ins pire d mate rials bas e d on natural or s ynthe tic biomime tic ge ls and polyme rs : The mate rials us e d he re e ntire ly conforme d to this re quire me nt. Radical innovation in s tate of the art biomate rials : Imple me ntation of s patiote mporal control of s pe cific ce llular actions to orche s trate tis s ue re ge ne ration. Mate rials with e le ctromagne tic prope rtie s : Supe rparamagne tic nanoparticle s we re include d to allow for s patiote mporal control. Pote ntial for me dical applications : Pote ntial for future me dical applications is cle arly e vide nt and has be e n dis cus s e d with affe cte d patie nts and the ge ne ral public. Ge nde r and age -re late d: Patie nt pane ls with mainly fe male and e lde rly patie nts and works hops with fe male e mploye e s : curre nt s ituation, care e r e xpe ctations and de mands . Within GAMBA, mate rials have be e n de ve lope d to re s ult in biome dical implants having characte ris tics clos e to thos e of natural tis s ue s . The innovative approach within GAMBA is e xpe cte d to contribute to an incre as e d compe titive ne s s of the Europe an biomate rial and biome dical indus try by giving ris e to pate ntable innovations to be e xploite d by the e xis ting ne tworks of the SME and acade mic Partne rs . The GAMBA proje ct was comple me ntary to othe r pre vious and on-going Europe -wide and national re s e arch programme s . Notably, Partne r ARI is re pre s e nte d by an inte rnational ne twork of more than 10’000 s urge ons s pe cializ e d in tre atme nt of trauma and dis orde rs of the mus culos ke le tal s ys te m. Eras mus MC participate s in all large national cons ortia in the fie ld of re ge ne rative me dicine and os te oarthritis (Dutch Platform for Tis s ue Engine e ring, Trans lational Exce lle nce in Re ge ne rative Me dicine ; “Smart-Mix”, BioMe dical Mate rials programme , Top Ins titute Pharma). In addition Eras mus MC is coordinator of a Marie Curie Fe llows hip program on bone re ge ne ration. Ranie ri Cance dda’s laboratory has be e n involve d in many EU s pons ore d proje cts , s uch as Angios caff, Purs te m, Join(e d)T, and ERISTO IV. The Re ge ne rative Me dicine Ins titute (REMEDI) is a biome dical re s e arch ce ntre with a ce ntral focus on the de ve lopme nt of nove l the rapie s for human dis e as e s us ing adult s te m ce ll the rapy and ge ne the rapy. GAMBA re s e arche rs have be e n involve d in additional EU-s pons ore d proje cts including Purs te m ADIPOA, BioSte m and ANGIOSCAFF, ERISTO. INSERM, EMC, ARI and IRCCS we re partne rs of cos t proje ct (Namabio, Franco Rus tiche lli coordination). More s pe cifically INSERM and Biomatlante are partne rs in bone re ge ne rative me dicine (REBORNE proje ct, 7th frame work program HEALTH-2009-1.4.2 of the Europe an Commis s ion on Re ge ne rative Bone de fe cts us ing Ne w biome dical Engine e ring approache s ) Among the GAMBA partne rs have be e n two SME’s which can comme rcializ e mate rials and re age nts e me rging from the GAMBA proje ct. OZ Bios cie nce s , France , is active in the re age nts bus ine s s . Biomatlante , France , is a we ll-known manufacture r of implantable biomate rials for re ge ne rative me dicine . The whole cons ortium and the companie s in particular contribute to re inforcing the Europe an compe titive ne s s and innovation as s ummariz e d in the table s be low. P a ge 17 o f 23 Research and Innovation
Dire ct e conomic be ne fits : • De cre as e time and cos t of nove l tre atme nts (matrix-bas e d; activate d on command and de mand) de ve lopme nts for cartilage and bone dis e as e s upporte d by the high e fficie nt te chnological package and as s ociate d proce s s e s de ve lope d by the GAMBA Partne rs , • Improve Europe an compe titive ne s s in the os te oarthritis fie ld, • Additional pate nts and compe titive pos itioning of GAMBA Partne rs on the te chnologie s and mode ls to be de ve lope d, • Be tte r manage me nt of age ing population s uffe ring from os te oarthritis (nove l tre atme nt s trate gie s ). Indire ct e conomic be ne fits : • • • •
Ne w tre atme nts for os te oarthritis , Bone re ge ne ration us ing bone s ubs titute s Significant re duction of Europe an He alth care cos ts as s ociate d to os te oarthritis , Improve d knowle dge and compe titive ne s s of Europe in os te oarthritis , bone re ge ne ration us ing bone s ubs titute s Pro-active campaign for acce ptance of te chnology by the ge ne ral public
GAMBA outputs : • Ne w e ngine e re d multifunctional ge ne ve ctors , • Nove l trans fe ction s trate gie s us ing non-viral or ade noviral ve ctors with fe e dback re s pons e e le me nts driving trans ge ne e xpre s s ion • Ne w biomime tic the rmo-re s pons ive hyaluronan hydroge l • Multiphas ic as s ociation of Biomime tic Calcium Phos phate granule s • Innovative granule s to e nhance bone re ge ne ration • Ne w Scaffolds for Drug De live ry Sys te ms • Ne w in vitro mode ls for e valuation of the rapie s to re pair os te ochondral de fe cts • Ne w dialogue tool (Pane l conce pt) in include s ocie tal/e thical as pe cts /value s into re s e arch Short-te rm innovation: • Ne w ge ne ve ctor carrie rs , • Innovative matrice s forme d by MSCs ce lls , biomate rials and ge ne ve ctors • Spatiote mporal control of chondroge ne s is and os te oge ne s is of MSCs e mbe dde d in ge ne -activate d mate rials and de ve lopme nt of a mode l that has prope rtie s clos e to natural tis s ue • Pate ntable de s ign of hollow granule s for be tte r os te oconduction • Nove l s trate gie s for immune modulation in a controlle d s patiote mporal manne r re s ulting in atte nuation of in vivo joint re pair for os te ochondral de fe cts and os te oarthritis Long-te rm innovation: • Innovative matrice s providing re me die s and innovative mode l to s tudy and tre at os te oarthritis dis orde r. • Enlarge me nt of GAMBA te chnologie s s pe ctrum of action to othe r cartilage and bone dis orde rs e .g. os te oporos is , de ntal implants . • Exte ns ion of GAMBA to wound he aling (s kin de fe cts ), he aling of te ndon e tc. • Ge ne ral application of GAMBA to tis s ue e ngine e ring by s e le ction of appropriate promote rs and cDNAs This is conne cte d to s ignificant marke t pe rs pe ctive s . The os te oarthritis marke t is pre dicte d to grow s te adily, and will re ach $7 billion by 2015*. *From the re port: Comme rcial Ins ight: Os te oarthritis - Marke t s e e s s te ady growth (2006) Furthe rmore , GAMBA may have a pronounce d s ocie tal impact in a long-te rm pe rs pe ctive . In Europe , around 6 pe r ce nt of the Europe an population s uffe r from fre que nt kne e pain and radiographic os te oarthritis . It has be e n e s timate d that around 35 to 40 million Europe an s uffe r from os te oarthritis in 2007. It is e s timate d that around 25 pe r ce nt of pe ople with age 60 and above s uffe r from dis ability due to os te oarthritis . The e conomic impact of mus culos ke le tal dis e as e s in indus trialis e d countrie s varie s from 1 to 2.5% of Gros s Dome s tic Product, with re fe re nce to local s ituations and to diffe re nt s ocial policie s . As os te oarthritis (OA) is the mos t common joint dis orde r, it abs orbs mos t of the re s ource s among mus culos ke le tal dis e as e s . GAMBA propos e s ne w the rape utic approache s and tre atme nts for os te oarthritis thus making pos s ible for the s uffe ring population to s ignificantly e nhance the ir quality of life and to re duce morbidity-re late d cos ts . GAMBA may have an impact on s tandards as we ll. With the long-te rm goal of e xploiting re s ults of the proje ct in clinical application, Europe an re gulatory authoritie s will be e ngage d in the future . The nove lty of the involve d te chnologie s will re quire adaptation of curre nt re gulatory provis ions . GAMBA has for the e ntire funding pe riod or parts of it ge ne rate d a numbe r of pos itions : 12 pe rs ons (full time or part time ) we re hire d s pe cifically for the GAMBA proje ct at the diffe re nt partne rs , thus incre as ing work force at the partne rs ins titutions . Additionally to pe rs ons paid by the GAMBA proje ct s cie ntific as we ll as te chnical or adminis trative pe rs onne l P a ge 18 o f 23 Research and Innovation
was involve d in GAMBA. Scie ntis ts include d mas te r s tude nts and inte rns hips that contribute d e s s e ntially to the proje ct although typically not be ing paid pos itions . Thus the GAMBA proje ct contribute d to s cie ntific e ducation at the partne r unive rs itie s as we ll. In total thre e doctoral the s is and s ix mas te r the s e s have be e n produce d in the conte xt of GAMBA. Additionally to e ducation of unive rs ity s tude nts GAMBA re s e arche rs of TUM pre s e nte d the GAMBA proje ct in a life vide o pre s e ntation broadcas te d to highe r e duction s tude nts in the “De uts che s Mus e um, Münche n” Munich during the “Nanoday” 09.02.2011. Furthe rmore the GAMBA manual and compe ndium to the GAMBA manual are we ll s uite d as s cie nce e ducation manuals e xplaining the inte gral topics of ge ne the rapy, adult s te m ce lls , nano-particle s , re gulation of ge ne e xpre s s ion. The GAMBA proje ct has give n ris e to an e xte ns ive collaboration of 3 biote ch companie s , 2 of the m having be e n me mbe rs of the GAMBA cons ortium (OZB and Biomatlante ). The third company, e thris GmbH in Munich, Ge rmany, is a s pin-off of TUM and works on bone re ge ne ration with me s s e nge r RNA e ncoding bone -inducing factors . It is inte nde d to continue this collaboration be yond the GAMBA proje ct. Wide r s ocie tal implications : In orde r to active ly involve lay pe ople a whole WP (WP8) was de dicate d to e ngaging civil s ocie ty in the GAMBA proje ct. For this purpos e citiz e n and patie nt pane ls we re pe rforme d in thre e countrie s (Ge rmany, Ire land and Switz e rland). The aim of the s e pane ls was to inform participants in lay language about the aims , chance s and ris ks of GAMBA. To allow an ope n ne utral dis cus s ion, mode rators we re involve d. Scie nce Dialogue (SCID), who are s ocial s cie ntis ts and profe s s ional mode rators , but we re not involve d in the natural/me dical s cie nce s /e ngine e ring was a partne r to the cons ortium. With the he lp of SCID participants produce d a lay re port on the s e s ubje cts with the main e mphas is on e thics , chance s and ris ks of the approach. The s e re ports we re publis he d in the original language s (Ge rman and Englis h) and als o we re trans late d. Furthe rmore the s e lay re ports we re communicate d to policy make rs , by s e nding the print ve rs ions and publis hing on the inte rne t. In orde r to incre as e public aware ne s s about the GAMBA conce pt in the wide r community and to addre s s re le vant s ocie tal is s ue s citiz e n and patie nt pane ls we re inte gral part of the GAMBA proje ct. With the he lp of partne r SCID in WP8 lay pe rs ons (citz e ns and patie nts ) we re involve d and e ncourage d in ge tting to know, dis cus s and actually give input into GAMBA. As can be s e e n in WP9 e xte ns ive s cie ntific as we ll as non-s cie ntific dis s e mination of GAMBA conce pt and re s ults have be e n achie ve d. TUM and SCID ge ne rate d pre s s re le as e s re late d to GAMBA and e s pe cially to the pane ls . SCID publis he d the lay re ports on the inte rne t as we ll as s e nt print ve rs ions to the EC and policy make rs . Additionally SCID s e nt the final lay-re ports to the participants of the pane ls , thus us ing the m as multiplie rs to othe r lay pe rs ons . With the s e me as ure s , the y contribute d to maximiz ing the aware ne s s of the public of the GAMBA proje ct. The Re ge ne rative Me dicine Ins titute at NUI Galway has a core outre ach e thos and aims • to incre as e public aware ne s s of re s e arch carrie d out at the ce ntre • to incre as e aware ne s s and inte re s t in s cie nce among young pe ople and to e ncourage the m to cons ide r furthe r e ducation or a care e r in this fie ld • to e ngage the public, including patie nt groups and care rs , in dis cus s ions of future applications and e thical implications of conte mporary s cie ntific re s e arch This e ffort is ne ce s s ary to cre ate the ability to be come promine nt as a re s ource for s te m ce ll re s e arch comme nt and analys is in the Iris h me dia. GAMBA’s s ignificant e ffort in addre s s ing s ocie tal is s ue s as a core e le me nt of the proje ct is ins trume ntal in achie ving the s e aims both locally in all partne r countrie s but als o in a broade r Europe an conte xt. Dis s e mination and re al outre ach/e ngage me nt e fforts targe te d to patie nts and the ge ne ral public have e ns ure d this . This e ffort will e ns ure maximum aware ne s s of the contribution of GAMBA to advance s in nanome dicine , incorporating s te m ce ll and ge ne the rapie s as we ll s mart biomate rials , le ading to more e ffe ctive the rapie s in the future . By e ngaging with patie nts and the public s o e ffe ctive ly throughout the cours e of the proje ct, GAMBA has als o incre as e d aware ne s s of the s e nove l the rapie s and promote d ultimate acce ptance of nove l the rapie s that may re s ult from GAMBA output. NUI Galway contribute d to achie ving aware ne s s as follows : NUI Galway built on the GAMBA pre s s re le as e by TUM in Munich and adapte d the information for a re le as e of the launch of the proje ct on the NUI Galway we bs ite . An invitation to the work-package le ade r, Mary Murphy, to participate in a radio Inte rvie w pos t pre s s re le as e was re ce ive d as a re s ult. NUI Galway als o had s ignificant e xpos ure as s ociate d with WP8 outre ach activitie s : s pe cifically, dis s e mination of GAMBA occurre d as a re s ult of activitie s in WP8 and the imple me ntation of Tas k 8.4, “Patie nt and citiz e n pane l in Ire land”. P a ge 19 o f 23 Research and Innovation
In re s pons e to a NUI Galway pre s s re le as e , publis he d on the NUIG we bs ite , to promote the Patie nt Pane l in an e ffort to initiate re cruitme nt, many me dia hits e ns ue d. The s e include d a radio inte rvie w and s ubs e que nt daily promotion of GAMBA by Galway Bay FM for ove r 7 days . Additionally, s e ve n online me dia hits and 3 article s in the local print me dia we re note d. Additional e ffort conce ntrate d on liais ing with Arthritis Ire land and the ir acce s s to me mbe r de tails and dis s e mination of le afle ts through local hos pitals , doctor’s office s and clinics , librarie s , pharmacie s and he alth s tore s . In addition to me dia e xpos ure for the Patie nt Pane l re porte d in the re porting pe riod cove ring the firs t 18 months of the proje ct, an additional 3 ne ws pape r article s focus ing on the GAMBA pane l he ld in Fe bruary and March 2012. As the re are no compre he ns ive vote r databas e s in Ire land due to the ability of citiz e ns to opt out of dis s e minate d re gis tration databas e s , we initiate d a s ignificant e ffort to adve rtis e the Citiz e n Pane l in orde r to re cruit participants for the pane ls he ld in May and June 2012. Efforts include d dis s e mination through le tte rs to the local county council, s e condary s chools , 20 Active Re tire me nt Groups , Galway branch of the Gre e n Political Party, Lite rary and De bating Socie ty at the NUI Galway, local librarie s , Galway City Community Forum all s tude nts and s taff at NUI Galway, volunte e r organis ations , a half page adve rtis e me nt in the local ne ws pape r (Galway Adve rtis e r) and a pre s s re le as e on the NUI Galway we bs ite . Se ve n me dia hits re s ulte d from this e ffort to adve rtis e the citiz e n pane l in the pe riod to the e nd of May 2012. The launch of the combine d Citiz e n/Patie nt pane l re port was he ld on Nov 2, 2012 and furthe r e licite d a numbe r of re ports online and in the print me dia. Re ce ntly, one of the GAMBA patie nt pane llis t was fe ature d on a TV pie ce on s te m ce lls and a ne w initiative re ce ntly launche d at NUI Galway on production of s te m ce lls for clinical trials . All me dia e xpos ure is lis te d in De live rable 10.2 (T9.3: Public re lations campaign). Ge nde r is s ue s : A Ge nde r work s hop was he ld during the Ge nova me e ting 11.-12.02.2013 with fe male participant from the following partne rs : TUM, NUI Galway, IRCCS, EMC and SCID. The s e re s e arche rs als o contribute d to the ge nde r works hop to e mphas is e ge nde r e quity in GAMBA organiz e d by SCID. To pre pare the ge nde r work s hop a que s tionair pre pare d by SCID had be e n pre vious ly s e nt to all fe male me mbe rs of the cons ortium and the WP le ade r pre s e nte d the outcome of the s urve y. An e xce rpt of the re s ults of the ge nde r work s hop was pre s e nte d to the whole cons ortium during the following GAMBA me e ting, highlighting the role s , re quire me nts and wis he s of wome n in the s cie ntific re s e arch world. Dis s e mination and Exploitation Dis s e mination of knowle dge has be e n on s e ve ral le ve ls : Inte rnal dis s e mination: • re gularly s che dule d progre s s me e tings , phone confe re nce s , e le ctronic communication • e le ctronic communication, cre ating and continuous ly updating common knowle dge bas e s • the we bpage contains a s e cure d are a with acce s s re s tricte d to partne rs for e xchange and dis s e mination of information amongs t partne rs . • the we bpage contains a lis t of proje ct as s ociate d publications of partne rs and othe rs . • lab vis its of s tude nts in orde r to le arn ne w te chnique s • Exte rnal dis s e mination • Exte rnal dis s e mination has be e n on s e ve ral le ve ls : to the inte re s te d lay-public and to the s cie ntific community. • a we bpage was cre ate d to allow for information on GAMBA • pre s s re le as e s : on ge ne ral is s ue s like granting of the proje ct by the EC, on s e le cte d re s ults • publications in non-s cie ntific journals to attract maximal atte ntion to GAMBA outputs and impact on the the rapie s availability and s afe ty for patie nts s uffe ring from arthritis • publications in s cie ntific journals • pre s e nce on inte rnational me e tings : pre s e ntation of s cie ntific re s ults • organis ation of s ympos ia and s ate llite me e tings on inte rnational congre s s e s • citiz e n and patie nt pane ls we re part of the GAMBA WP8 • the pre s e ntation of the proje ct obje ctive s , re s ults and impacts in works hop s e s s ions ope n for profe s s ionals • e ffe ctive communication with the me dia (te le vis ion, radio and pre s s ) has be e n e s tablis he d in orde r to be tte r inform the public, be more vis ible and incre as e citiz e n’s aware ne s s and inte re s t in arthritis ’ re s e arch Scie ntific community dis s e mination (including clinicians ). A public progre s s re port has be e n pos te d on the we bpage at re gular inte rvals . Nume rous manus cripts have be e n publis he d in s cie ntific journals , s e ve ral are unde r re vie w and s ome are in pre paration. Partne rs als o atte nde d inte rnational me e tings to communicate the ir findings . Educational mate rial has be e n pre pare d and made available to s tude nts to s tre ngthe n the ir e xpe rtis e in nanome dicine P a ge 20 o f 23 Research and Innovation
(WP8). The following pe e r re vie we d pape rs have be e n publis he d by me mbe rs of the cons ortium during the above me ntione d funding pe riod: Cé dric Sape t, e t al. 2012. Magne tic nanoparticle s e nhance ade novirus trans duction in vitro and in vivo. Pharm Re s . 29(5): 1203-18. (Date of publication: 01.05.2012) Chris tian Plank, e t al. 2012. Ge ne Activate d Matrice s for Bone and Cartilage Re ge ne ration in Arthritis GAMBA – an EUFunde d Proje ct. Europe an Journal of Nanome dicine 4(1): 17-32 (Date of publication: 20.05.2012) Matte o D’Es te e t al. 2012. Single s te p s ynthe s is and characte riz ation of the rmore s pons ive hyaluronan hydroge ls . Carbohydrate Polyme rs . 90(3): 1378-85. (Date of publication: 13.07.2012) Thomas Miramond e t al. 2012. In vivo Comparative s tudy of two inje ctable /moldable Calcium phos phate Bioce ramics . Ke y Engine e ring Mate rials 529-530: 291-5. (Date of publication: 29.11.2012) Thomas Miramond e t al 2012. Compos ite bioce ramics /polyme r e le ctros pun s caffolds for re ge ne rative me dicine . Ke y Engine e ring Mate rials 529-530: 441-6. (Date of publication: 29.11.2012) Guy Daculs i e t al. 2012. Calcium Phos phate Bioce ramic s caffold for bone tis s ue e ngine e ring. Ke y Engine e ring Mate rials 529-530: 19-23. (Date of publication: 29.11.2012) Matte o D’Es te e t al. 2013. Hydroge ls in calcium phos phate moldable and inje ctable bone s ubs titute s : Sticky e xcipie nts or advance d 3-D carrie rs ?. Acta Biomate rialia 9(3): 5421-5430. (Date of publication: 01.03.2013) Rui C. Pe re ira e t al. 2013. Dual e ffe ct of plate le t lys ate on human articular cartilage : a mainte nance of chondroge nic pote ntial and a trans ie nt pro-inflammatory activity followe d by an inflammation re s olution. Tis s ue Eng Part A. 19 (11-12): 1476-1488. (Date of publication: 30.01.2013) Markus Be rninge r e t al. 2013. Tre atme nt of Os te ochondral De fe cts in the Rabbit's Kne e Joint by Implantation of Alloge ne ic Me s e nchymal Ste m Ce lls in Fibrin Clots . Journal of Vis ualiz e d Expe rime nts 75: e 4423. (Date of publication: 21.05.2013) Sape t e t al. 2013. 3D-fe ction: ce ll trans fe ction within 3D s caffolds and hydroge ls . The rape utic De live ry. 4 (6): 673-685. (Date of publication: 01.06.2013) Zölle r, Katharina: Laie n dis kutie re n und be we rte n Wis s e ns chaft: Partiz ipation be i Wis s e ns chafts the me n. Erfahrunge n und Qualitäts anforde runge n. In: UMID 2/2013. 67-74. Zölle r, K., s cie nce Dialogue . 2013. GAMBA Manual and Compe ndium, In Pre s s Bioce ramics De ve lopme nt and Applications . BDA 203, Journal of the Inte rnational Socie ty for Ce ramic in Me dicine , ISCM. Thomas Miramond e t al. Os te opromotion of Biphas ic Calcium Phos phate granule s in critical s iz e de fe cts afte r os te one cros is induce d by focal he ating ins ults . IRBM (acce pte d for publication 24.07.2013) M.L. de Vrie s – van Me lle , R. Narcis i, N. Kops , J.L.M. Koe voe t, P.K. Bos , J.M. Murphy, J.A.N. Ve rhaar, P.M. van de r Kraan, G.J.V.M. van Os ch. Chondroge ne s is of me s e nchymal s te m ce lls in an os te ochondral e nvironme nt is me diate d by the s ubchondral bone . (Acce pte d for publication in Tis s ue Engine e ring Part A – 27.08.2013) Barry F, Murphy M.. 2013. Me s e nchymal s te m ce lls in joint dis e as e . Nat Re v Rhe umatol. [e pub ahe ad of print Jul 23, 2103; DOI 10.1038/nrrhe um.2013.109] Zölle r, Katharina: Scie nce and the Lay Pe rs pe ctive : Lay Pe ople s ’ involve me nt in as s e s s ing Tis s ue Engine e ring Is s ue s . 2013. Acce pte d for publication at the journal Tis s ue Engine e ring Part C. The following manus cripts are unde r pe e r-re vie w: De Vrie s -van Me lle e t al. Europe an Ce lls and Mate rials Van Be uninge n, de Vrie s -van Me lle e t al. Tis s ue Engine e ring A Fahy, de Vrie s -van Me lle e t al. Os te oarthritis and Cartilage The following book chapte r has be e n publis he d: G. Daculs i, T. Miramond. Calcium phos phate de rive d biomate rials . Encylope dia of Biophys ics (Robe rts , GCK, e d.). 01.03.2013: 206-211.
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The following doctoral the s e s have be e n produce d: T. Miramond. Dé ve loppe me nt de matrice s cé ramique s e t compos ite s pour l’ingé nie rie tis s ulaire os s e us e . (Ph.D the s is de fe nce , date : 27.11.2012; INSERM/BIM) M.L. de Vrie s -van Me lle . In vitro mode ls for ce ll-bas e d cartilage re ge ne ration. (Anticipate d de fe nce : s pring 2014; EMC) Pe re ira, Rui. Ins ights into the role of bioche mical factors and e xtrace llular matrix e nvironme nt during chondrocyte culture : re le vance on articular cartilage re pair s trate gie s . Ph.D the s is in pre paration (IRCCS) The following mas te r the s e s have be e n produce d: Ste fan Sandke r: Os te ochondral culture mode l. (2010, Te chnical Me dicine Unive rs ity Twe nte /EMC) Re ne van de r Be l. A highly adaptable os te ochondral culture mode l towards s timulating microfracturing in vitro. (2011 Te chnical Me dicine Unive rs ity Twe nte /EMC) Liz e tte Utomo. Optimiz ation of the pre paration, ce ll e ncaps ulation and analys is of hydroge ls (2011 Te chnical Me dicine Unive rs ity Twe nte /EMC) Johanne s Le hmann: Modulating Synovial Macrophage phe notype s to improve s te m ce ll bas e d cartilage re pair. 2012 (EMC) Maria Tihaya: Comparing the ability of thre e hydroge l ce ll carrie rs to s upport re pair of os te ochondral de fe cts by me s e nchymal s te m ce lls in an in vitro mode l 2012 (EMC). M. Hillre ine r. Evaluation von Ge n-aktivie rte n Matrice s z ur Kontrolle de r Ge ne xpre s s ion. (Evaluation of ge ne -activate d matrice s for the control of ge ne e xpre s s ion). (2012; TUM) Pate nts : A pate nt application for hollow granule s by BIM and INSERM is unde rway. Lis t of We bs ite s : http://www.gamba-proje ct.e u/
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