| WO/2009/070722 | CELL CULTURE HYDROGEL WITH pH INDICATOR |
| WO/1999/064435 | THE HUMAN KIDNEY SPECIFIC GENE KK86 |
| WO/2007/134220 | A GENERAL METHOD FOR GENERATING HUMAN ANTIBODY RESPONSES IN VITRO |
MASTROGIACOMO, Maddalena (Via Sturla 2/2, Genova, I-16132, IT)
SCALA, Marco (Via Salgari 69/27, Genova, I-16162, IT)
CANCEDDA, Ranieri (Via Nizza, 11/20 sc.A, GENOVA, I-16145, IT)
MASTROGIACOMO, Maddalena (Via Sturla 2/2, Genova, I-16132, IT)
SCALA, Marco (Via Salgari 69/27, Genova, I-16162, IT)
CLAIMS
1. A bio-membrane essentially constituted b ' y , mesenchymal 'stem cells arid/or mesenchymal precursor cells and by a gel able to provide support' and growth factors and/or differentiation factors and/or angiogenic factors for the t futl in vivo functionality of the cells, in which said mesenchymal cells grow within or above said 'gel.
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2 The bio-membrane as claimed in claim 1, wherein the mesenchymal stem cells and/or mesenchymal precursor cells are dermogenic cells. ', <' '
3 The bio-membrane as claimed in claim 1, wherein the mesenc ;hymal stem cells arid/or mesenchymal precursor cells are chondrogenic cells. • * t
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4 The bio-membrane as claimed in claim 1, wherein the mesenchymal stem cells 'and/or mesenchymal precursor cells are osteogenic cells. ( • ;
5, The bio-membrane as claimed in claims 1 through 5 wherein the cells are obtained from bone man ow. ■ ' 6 The bio-membrane as_ claimed in claim 4, wherein the cells i are obtained from peuosteum. > " ■ >, ',
7 The bio-membrane as claimed in claim 4, wherein the bio-membrane is'pre-treated in culture medium with osteogenic factors. < i , - >
8 The bio-membrane as claimed in one of the previous claims w ( herein * the cells are autologous. 1 I i '
9. The bio-membiane as claimed in one of the claims from 1 through 7 wherein the cells are allogeneic t \ '
10 The bio-membrane as claimed in bhe of the previous claims wlierein the gel is >a platelet gel. . i 1 1 The bio-membrane as claimed in one of the claims 1 through 9 wherein the gel is essentially constituted by reabsorbable synthetic, natural or recombinant polymers ' , supplemented with growth and/or differentiation and/or angiogenic ,factors for the full functionality of the cells. , ' !
12 The bio-membrane as claimed in one of the previous claims,' further comprising micro and/or nanoparticles able to release growth factors and/or differentiation factors and/or angiogenic factors, '
13 The bio-membrane as claimed in one of the previous claims being partially dehydrated ^
14. An implant device for reconstructive surgery of bone tissue, essentially constituted by a porous scaffold and by the bio-membrane as claiiηed in claim 4 or 6, wherein the bio- membrane envelops the support. , ' «
15 The implant device for reconstructive surgery of bo'ne tissue as claimed in claim,. J, 4,, wherein the bio-membrane is pre-treated in culture medium 1 with osteogenic factors. < , 16. The implant device for reconstructive surgery of bone .tissue as claimed in claim 14 or
15, further comprising an additional gel membrane ( with growth and/or differentiation and/or angiogenic factors, wherein said additional 1 gel membrane ( is enveloped shortly " before implanting ' ' ! ' ,' ' ' , , 17 The implant device for reconstructive surgery of bone tissue as claimed ln'claim.jl 6, wheiem said additional gel membrane is a platelet gel. 1 , , '
18. Use of a platelet gel for the preparation of a medication for repairing skin a ' nd soft tissue lesions. ' ' ' ' ! > " i t 1 '
19. Use of a platelet gel as claimed in claim 18, wnerein'the 1 repair of skin a'nd soft tissue
' ' ' ' . ' > . i * lesions comprises chronic ulcers, difficult wounds, bedsores, chinks, tendon lacerations, , soft tissue substance loss. ' ' / ' *
20 An adhesive plaster for the repair of 'skin and soft tissue lesions ' comprising a 1 platelet <
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22 An adhesive plaster for the repair of skin and SQfI 1 tissue lesions comprising as' a theiapeutically active substance micro and/or nanoparticles able' to 'release growth and/or diffeientiation and/or angiogenic factors. , ^ 1 ' ( 4
23. A method for obtaining a bio-membrane as claimed in one of the claims 1 through 13, '
' ' • ' ' ' •, ! essentially comprising the following steps: < a) obtaining a platelet gel from mixing a platelet concentrate and, a cryoprecipitate obtained fiom peripheral blood, in appropriate conditions; , , p • , > ,, b) obtaining and growing on said gel, or within said gel,, mesenchymal stem 1 cells and/or mesenchymal precursor cells.
24. The method for obtaining a bio-membrane 1 as claimed ^n. claim 23 wherein trie mesenchymal stem cells and/or mesenchymal precursor cells are aμtdlogous or allogeneic with respect to the subject to be implanted. |
BIO-MEMBRANE FOR TISSUE REGENERATION
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an engineered " tissue bio-membrafib, ' an- implant device, for , tissue regeneration and repair as bone reconstruction; repair of lesions of the skin " and -.of soft tissues, e.g. chronic ulcers, difficult wounds, bedsores,/ chinks,- tendon lacerations, soft ' tissue substance loss, and methods for the production thereof ,
BACKGROUND ART In clinical practice and in surgery, it is ever more needed ' to identify ' a valid System to repair large tissue lesions associated with substance losses.
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Another object of the invention is an implant device for reconstructive surgery of bone tissue, essentially constituted by a porous suppoϊt (scaffold) 1 and by the bio-membrane according to the invention, in which the bio-membrane envelops the support and it is preferably pre-treated in culture with osteogenic factors, for a variable time period, such as 1-2 weeks In an embodiment, the micro and/or nano-particles >with gradual release of growth factors can be associated to the porous support. ' , , , '
In a preferred embodiment, the implant device for the reconstructive surgery of bone tissiie according to the invention comprises an additional gel membrane ,with growth and/or diffeienliation and/or angiogenic factois, in which said additional gel membrane 1 is enveloped just before implanting. Preferably, said additional membrane is a platelet' gel. Anothei object of the invention is the use of a platelet gel 'for the preparation of a medication for the repair of skin and soft tissue lesions, preferably comprising chrpnic υlceis, difficult wounds, bedsores, chinks, tendon lacerations, soft tissue 'substance loss. \ Foi the repair of skin lesions, the invention proposes an adhesive planter that includes only platelet gel. The adhesive plaster is constituted by three essential elements: the pad, the support and the adhesive. The pad can be constituted by cotton mixed with acrylic with high absorption capacity or by a material with similar characteristics and covered by a thin film of polyester or by a material with similar characteristics, loaded with platelet gel rich in active biological factors which, in contact with the wound, accelerates healing. A further object of the invention is an adhesive plaster for the repair of skin and soft tissue lesions comprising a platelet gel as a therapeutically active. substance. • ' ' < >
Alternatively, the adhesive plaster for the repair of skin and soft ti,ssue lesions comprises as a thei apeutically active substance a gel constituted essentially by reabsorbable synthetic, natural or recombinant polymers supplemented with growth ,and/or differentiation and/or angiogenic factors. < 1 M - ' > '
Alternatively, the adhesive plaster for the repair of skin and soft tissue lesions comprises as a therapeutically active substance micro and/or nanoparticles able to release growth and/or differentiation and/or angiogenic factors. . , ■
A fiulher object of the invention is a method for, obtaining a bio-membrane according to the invention, essentially comprising the following steps'i i ' . , ι' a) obtaining a platelet gel from mixing a platelet concentrate and a cryoprecipitate obtained from peripheral blood, in appropriate conditions;
The CP and the cryoprecipilate were mixed in plate in a 1 : 1 ratio, r l ml of autologous , thiombin and 1 ml of 10% calcium gluconate on a total vojumfe of.10 ml were added to initiate the gel polymerization process. ', ' , ' »
To assess the effect of the platelet gel on human BMSC,' the cells' were, grown in the presence of culture medium complete with supplements' ajnd ( with different concentrations , of Platelet Lysate (LP) (5%, 10% and 20%), obtained from the, OP^ as described below. ' The cells were plated in wells at high density (10,000 cells/well) and at low concentration (2,000 cells/well) in the presence or absence of LP. Cell proliferation was evaluated, in , the diffeient conditions, by cell count when the culture had reached serhicoηfiuence (lβ days). JFor the preparation of the Platelet Lysate, the protocol described by Dbucet C et al. (2005) was followed. The LP is obtained after subjecting the GP, to 3 freezing/thawing cycles to promote complete platelet lysis and total release of all growth factors contained therein ' (PDGF-bb, PDGF-aa, EGF, IGF etc. .) and in the presence, of Jow EDTA concentration.
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The LP was added to the cultuie at different concentrations. > Prepaiation of Platelet Gel from an Animal (Horse) ' '
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The day before the intervention, two units of 450 ml of blood are drawn from the horse, by means of a standard triple bag for the withdrawal of human blood containing ACD ((citric acid + sodium -citrate + dextrose) as an anticoagulant (Fresenius HemoCare CODE T2375). The bags were centrifuged in an ALC PM980R centrifuge (BICase, Italy) for 8 minutes at 500 x g ; the blood is then separated into red cells, and Platelet Rich Plasma (PRP), paitially entering into the Buffy-Coat©. ' ,
The PRP must be re-centrifuged at 5,000 x g for 7 minutes to obtain the Platelet Concentiate (CP) that must be re-suspended in about 80 mL of autologous plasma adjusting platelet count between 0.5 and 3x10 microliter. f ' , ' ' ' The bag containing the CP is placed in an agitator thermostated at + 22° until the time of \ use. - , ' ' i
The day of the intervention, the CP is drawn under stdrile hood from the tags, with syi inges labeled with the identifying data of the horse.
The CP is ready to be injected into the site of the lesion to be repah'ed
« • If the product is to be used in the form of semi-solid gel, at the time of use some sterile plastic Petri dishes of about 10 cm diameter are prepared adding 10 mL of platelet concentrate, 1 mL of Calcium Gluconate (ind. Farmaceutica Senese, Italy, Lot. No.
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After a few minutes, the transformation occurs from fibrinogen. to fibrin with "geϊificatiori" , of the Platelet Concentrate which can be applied topically on 1 large' surfaces. i 1
In case of tendon lesions, the product will be injected noα 'gelifjed- into the site of 'the > lesion, under echographic guidance. > ' < , , ( ,. Bone Tissue Foimation in Vivo , ' ' , ' •
To evaluate bone tissue formation in vivo, a small animal model was used, UQ. the ' immunodeficient mouse (Nu/Nu strain or SCID strain). 'Ceramic, scaffolds of , different ' sizes and breakdown (Engipore®, 100 % HA, Fincerarnioa, Fa'enza, , Italy and Slcelite®, TCP70/HA30, Millenium Biologix) were implanted subputaheoϋsly into the back of immunodeficient mice after enveloping them with a biό'-membrane of platelet gel and human or sheep BMSC. ' r '
The BMSC were layered on the gel (pN) or included in the gel (IN) directly during the i ; f polymerization phase. The bio-membrane of platelet gel, (obtained with the ON method or with the IN method) was kept in complete medium but without FGF-2' for 1-3 days, before ' being enveloped around cubic scaffolds (60-70 mm 3 ) of 'HA 100% (EngiPore®). In some i expeπmenls, a few minutes before the implant, the sample was 1 envelo'ped by an additional membiane of fresh platelet gel without cells, to assure a greater, supply of growth factors. In each animal, 4 scaffolds were implanted including a control implant,' ,in which the BMSC were loaded directly into the scaffold using fibrin glue (Tissucol®, Baxter) as an
. 1 i ' adjuvant of the adhesion of the cells to the ceramic. s ,
In a second series of experiments, larger size (hollow • cy ; li .nde ! rs " of 2000-2500 m ' m ' 3 )' ' rcabsorbable ceramic scaffolds made of skelite® (TCP ' 70%, < HA 30%, MiUeniψn Biologix, Ontario, Canada) were used. In this case,' a single sample, was implanted ι per animal. ' , , In some experiments, the platelet gel conjugated to BMSC was partially dehydrated by superposing absorbent, sterile filter paper, thereby formiηg a more consistent and more i ' . ' ' ' ,' ' easily handled bio-membrane. In the partially dehydrated gel, the cells proliferated noi mally, maintaining their osteogenic potential after implant in the ariimal.
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In some experiments, the platelet gel conjugated to human or sheep BMSC was pre-treated in vitro with osteogenic medium. 24 hours after preparation, the platelet gel membranes were transferred in culture medium supplemented with factors inducing osteogenic differentiation: 10 "8 M dexamethasone, 10 mM b-glycerol-phosphate (BGP), and 50 mg/ml
decalcified samples, paraffin-enclosed samples, it was possible to observe that the cells,/ both enmeshed in the gel (IN, a,c) and kept on the surface of, the gel '(ON, b,d) are able to differentiate into osteoblasts and to deposit osteogenic matrix ( into the ceramic, pores already during the first four weeks of implant. A significant line of osteoblasts at the edge of the newly laid bone indicates an intense bone matrix 'laying activity. After 8 weeks of
, . M / ' ' ' implant, a greater quantity of bone fills the pores of the ceramic. No significant difference was observed in the formation of bone tissue' both in the samples enveloped by bio- ' membranes with layered cells in the surface (IN. method} and in those with bio-membranes
, ' ' . ' with cells enmeshed in the fibrin mesh (ON method). > > ' ■ Though the model of the ID mice is one of the most accredited, in v t ιvo models to test the osteogenicity of cells and biomateπals, the authors deemed it appropriate to repeat the experiment under test conditions that would more closely approach the "real" condition^ to be found in clinical practice. ! ' , ' v <
For this purpose, a porous, reabsorbable ceramic scaffold was used (fytastrogiacomo et al., 2006), with a greater presence of Tricalcium phospliate and a smajler presence of hydioxyapatite (TCP 70%, FIA 30%). Hollow cylinders ,of about 2,000 mm 3 were enveloped with platelet gel bio-membranes, alone or associated with cells with the ι JN method or with the ON method and implanted in ID mice for,' 8 weeks Panel a) in Fig. 3 shows no bone tissue foimation in samples enveloped by platelet .gel without cells. Only
1 < . fibrous tissue together with fatty tissue populates theφores of the ceramic. However, some vascularization can be observed, confirming the important role played by the platelet gel in the vascularization of the lesion site (Rhee JS, 2004). -When the scaffold is wrapped t by film of platelet gel associated with cells obtained with the IN method or with the ON method (Fig 3 b-c), bone tissue is observed ini the pores of 'the' ceramic Abundant osteoblasts are distributed at the surface of the laid bone'. Upon ' microscopic observation of i the ample colored section, it is possible to note a distribution of newly formed bone tissue that goes fiom the periphery to the center of the bcaffpld. , ' . , » , In the attempt to generate a bio-membrane with the, highest osteogenic and angiogenic potential starting from a bio-membrane of platelet gel and BMSC (human Or 1 srjdep) obtained both with IN method and with ON method, the bio-membranes were pre-treated in vitro for a period of 2 weeks with osteoinductive culture medium (see methods), to promote an initial laying of matrix prior to the transfer on- scaffold. After 8 weeks of in
vivo implant, the samples (Fig. 4) exhibited good bone tissue formation distributed from , the periphery to the center of the samples. , ' ' !
The prolonged maintenance of BMSC in platelet gel in vitro, reduces their osteogenic . , t potential. However, the bio-membranes can be pre-treated with osteoinductive medium for a period of two weeks, assuring the maintenance of the full osteogenic potential. • ' ' In Fig.5 we show the dehydration of the bio-membrane by me'ans of a continuous i gupci position of disks of sterile absorbent paper that completely removes thς soluble part of the membrane and water. This 'procedure generates a membrane that is more elastic and easier to handle during the surgical procedure without altering, the vitality of the cells included therein. ' ' ', , f ' " , \
With respect to the repair of skin lesion, an example of treatment of skin lesion in a horse is ieported (Fig. 6). In all treated animals, it was sufficient to apply the platelet gel once to ■ tπgger the regenerative process (Fig. 6a-b). The figure clearly shows the reduction of th,e lesion at 15 days from the treatment (Fig. 6 c) and restitutfo ad integntm after thirty days (Fig 6d) when the horse resume its sports-competition activity.
BIBLIOGRAPHY
1 ) Quarto et al , N Engl. J. Med., 2001, 344 (5):385-86.'
2) Mastiogiacomo et al., , Tissue Eng. 2006, 12(5).l'261-73. 3) Rhee et al., Thromb Haemost. 2004, 92(2):394-402. ,
4) Scala M et al., Proc. Am Acad Maxillofacial Prosth. International Congre'ss . Maxillofacial Prosthetics in the 21 Century, Kauai - Hawaii; November 11-14, 2000; 132. '
5) Warnke PH et al., Lancet 2004, 364: 766-70. '