FR2667789A1 - Cicatrising composition and method for preparing it - Google Patents
Cicatrising composition and method for preparing it Download PDFInfo
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- FR2667789A1 FR2667789A1 FR9012742A FR9012742A FR2667789A1 FR 2667789 A1 FR2667789 A1 FR 2667789A1 FR 9012742 A FR9012742 A FR 9012742A FR 9012742 A FR9012742 A FR 9012742A FR 2667789 A1 FR2667789 A1 FR 2667789A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/495—Transforming growth factor [TGF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/49—Platelet-derived growth factor [PDGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
La présente invention concerne un procédé de préparation d'une composition à activité cicatrisante. The present invention relates to a method for preparing a composition with healing activity.
La cicatrisation des plaies endothéliales chez l'homme et/ou l'animal met en oeuvre un processus complexe impliquant des interactions entre de nombreuses substances de type enzymes de sérum, facteurs de croissance, plaquettes, monocytes et macrophages notamment. The healing of endothelial wounds in humans and / or animals involves a complex process involving interactions between numerous substances such as serum enzymes, growth factors, platelets, monocytes and macrophages in particular.
I1 est aujourd'hui reconnu que ce sont les plaquettes et les macrophages qui sont les cellules les plus impliquées dans ce processus de cicatrisation. Les plaquettes initient la cicatrisation en libérant au niveau de la plaie des facteurs angiogénique et de croissance qui agissent sur l'angiogénèse et stimulent la synthèse en collagène. It is now recognized that platelets and macrophages are the most involved cells in this healing process. Platelets initiate healing by releasing angiogenic and growth factors at the wound level that act on angiogenesis and stimulate collagen synthesis.
Ces facteurs, et plus particulièrement le facteur de croissance
PDGF, ont déjà fait l'objet de nombreuses études compte tenu de leur importance dans ce processus de cicatrisation. (Journal of Cellular
Physiology, vol. 113, p. 261; Blood, vol.64, N" 2, 1984, p.458).These factors, and more specifically the growth factor
PDGF, have already been the subject of many studies given their importance in this process of healing. (Journal of Cellular
Physiology, vol. 113, p. 261; Blood, Vol. 64, No. 2, 1984, p.458).
On sait aujourd'hui qu'ils sont présents au sein des alphagranules plaquettaires, et qu'ils circulent sous cette forme jusqu'à ce que les plaquettes soient induites, par une activation extérieure, à se dégranuler. It is now known that they are present in platelet alpha-granules, and that they circulate in this form until the platelets are induced, by external activation, to degranulate.
Ainsi, il a été démontré, in vitro (Stakenow et al., Exp. Cell. Res., 1981, 136, 321-25; Burke JM et al., Exp. Cell. Res., 1977, 107-387) et in vivo (Knighton D. et al., Annal of Surgery 1982, vol. 196, n"4, p. 379) que l'activation des plaquettes par de la thrombine libérait un mitogène ou encore facteur de croissance pour les fibroblastes, et stimulait la synthèse du collagène par ces cellules.Thus, it has been demonstrated, in vitro (Stakenow et al., Exp Cell Res, 1981, 136, 321-25, Burke JM et al., Exp Cell Res, 1977, 107-387) and in vivo (Knighton D. et al., Annal of Surgery 1982, vol 196, No. 4, p.379) that platelet activation by thrombin releases a mitogen or growth factor for fibroblasts, and stimulated the synthesis of collagen by these cells.
Le rendement de cicatrisation est par conséquent limité par la biodisponibilité de ces facteurs de croissance qui contrôlent la migration et la prolifération cellulaires. Healing efficiency is therefore limited by the bioavailability of these growth factors that control cell migration and proliferation.
Aujourd'hui il est intéressant de disposer de compositions à base de facteurs de croissance qui puissent être administrées à des patients souffrant d'une carence en ce type de composés, ou simplement présentant des difficultés de cicatrisation, tels les diabétiques et les personnes âgées. Today it is interesting to have compositions based on growth factors that can be administered to patients suffering from a deficiency in this type of compounds, or simply having difficulty in healing, such as diabetics and the elderly.
Le brevet WO 86 03122 décrit l'utilisation d'un surnageant total de plaquettes activées à la thrombine pour améliorer le processus de cicatrisation. WO 86 03122 discloses the use of a total supernatant of thrombin-activated platelets to enhance the healing process.
La présente invention concerne notamment un procédé de préparation d'une composition cicatrisante améliorée obtenue à partir d'un surnageant de plaquettes d'origine humain ou animal, activé in vitro, caractérisé en ce que ledit surnageant subit au moins une étape de traitement acide à pH inférieur à 5 pendant un temps suffisant pour obtenir le TGFPw, puis en ce que l'on récupère le surnageant traité et on élimine le précipité obtenu après neutralisation. The present invention relates in particular to a method for preparing an improved healing composition obtained from a platelet supernatant of human or animal origin, activated in vitro, characterized in that said supernatant undergoes at least one acid treatment stage at pH less than 5 for a time sufficient to obtain TGFPw, then that the treated supernatant is recovered and the precipitate obtained after neutralization is removed.
Dans ce procédé, l'activation des plaquettes libère le contenu des *-granules, c'est-à-dire des protéines plasmatiques et des peptides à caractère hormonal ou de facteur de croissance. In this method, platelet activation releases the content of granules, i.e., plasma proteins and hormonal or growth factor peptides.
On a ainsi pu mettre en évidence - de l'albumine, - du fibrinogène - du facteur v.W, - de la sérotonine, - de l'histamine, - du Connective Tissu Activating Peptide III (CTAP III), - de la 3 thromboglobine (P.T.G.), - du Platelet Factor IV (P.F.IV), - du Platelet Derived Growth Factor (P.D.G.F.), - de l'Epidermal Growth Factor (E.G.F.), - de l'Insuline like Growth Factor I et II (I.G.F.I et II), - du Transforming Growth Factor et (T.G.F. (ss). Thus, albumin, fibrinogen, vW factor, serotonin, histamine, Connective Tissue Activating Peptide III (CTAP III), thromboglobin, were detected. PTG), - Platelet Factor IV (PFIV), - Platelet Derived Growth Factor (PDGF), - Epidermal Growth Factor (EGF), - Insulin like Growth Factor I and II (IGFI and II) - Transforming Growth Factor and (TGF (ss).
C'est donc tout un cocktail de produits biologiquement actifs qui est ainsi libéré. Cependant, certains facteurs de croissance, liés à une protéine porteuse, sont inactifs (Furlanetto et Marino, Meth in Enz, Vol. It is therefore a cocktail of biologically active products that is released. However, some growth factors, linked to a carrier protein, are inactive (Furlanetto and Marino, Meth in Enz, Vol.
146, P. 216-226).146, p. 216-226).
C'est le cas notamment du T.G.F, qui est présent sous une forme latente dans l'extrait des ? -granules (Pircher et al. B.B.R.C., Vol 136, P. 36-37 (1986)). This is particularly the case of T.G.F, which is present in a latent form in the extract of? granules (Pircher et al., B.B.R.C., Vol 136, P. 36-37 (1986)).
Grâce au procédé selon l'invention qui comporte une étape de traitement acide, le surnageant obtenu contient notamment le TGF à l'état actif et conduit à des résultats de cicatrisation très supérieurs à ceux obtenus pour les extraits non traités. Cette amélioration de l'activité est due en grande partie à l'activation du TGFA . Thanks to the method according to the invention which comprises an acid treatment step, the supernatant obtained contains TGF in the active state and leads to healing results much higher than those obtained for untreated extracts. This improvement in activity is due in large part to the activation of TGFA.
De préférence, ce traitement acide est conduit à un pH inférieur à 3, par exemple 2,5, pendant une durée de quelques minutes, notamment une dizaine de minutes. Bien entendu, ce traitement peut être optimisé en fonction du surnageant utilisé. Preferably, this acid treatment is conducted at a pH of less than 3, for example 2.5, for a period of a few minutes, in particular about ten minutes. Of course, this treatment can be optimized depending on the supernatant used.
Le surnageant à traiter sera de préférence obtenu de la façon suivante - séparation des plaquettes de la fraction plasmatique et déleucocytation, - activation dans une solution tampon, en présence d'un activateur, - homogénéisation du mélange, et - récupération du surnageant. The supernatant to be treated will preferably be obtained in the following manner - platelet separation of the plasma fraction and leukocyte depletion, - activation in a buffer solution, in the presence of an activator, - homogenization of the mixture, and - recovery of the supernatant.
Certaines opérations mises en oeuvre dans les différentes étapes de ce procédé font appel à des techniques familières de l'homme du métier, notamment la séparation des plaquettes et la déleucocytation; elles ne seront pas rappelées ici en détail, mais apparaîtront plus clairement à la lecture des exemples ci-après. Certain operations carried out in the different stages of this process make use of techniques familiar to those skilled in the art, in particular the separation of platelets and leukocyte depletion; they will not be recalled here in detail, but will appear more clearly on reading the examples below.
L'activation est conduite de préférence sur une suspension de plaquette mise en présence d'un activateur. Parmi les activateurs on préfère utiliser la thrombine, mais l'ADP calcique est un activateur qui présente certains avantages. The activation is preferably carried out on a platelet suspension placed in the presence of an activator. Among the activators, it is preferred to use thrombin, but calcium ADP is an activator which has certain advantages.
En effet, les activateurs du type thrombine, collagène inactivé sont d'origine humaine ou animale, et peuvent présenter des inconvénients liés à ces origines. Indeed, activators of the thrombin type, inactivated collagen are of human or animal origin, and may have drawbacks related to these origins.
Au contraire, 1'ADP calcique est un produit qui peut être synthétisé chimiquement, ce qui garantit son inocuité. In contrast, calcium ADP is a product that can be synthesized chemically, which ensures its safety.
Après l'activation, le surnageant obtenu subit une inactivation virale, de préférence par pasteurisation. Cette étape de pasteurisation permet de se débarrasser de toute trace de contamination bactérienne ou virale éventuellement présente dans le surnageant. Elle permet en outre d'inactiver l'activateur contenant la thrombine éventuellement présente. After activation, the resulting supernatant undergoes viral inactivation, preferably by pasteurization. This pasteurization step makes it possible to get rid of any trace of bacterial or viral contamination possibly present in the supernatant. It also makes it possible to inactivate the activator containing the thrombin that may be present.
La pasteurisation est conduite de préférence par chauffage du surnageant pendant environ 10 heures à une température de l'ordre de 60"C. Le pH est un pH neutre ou légèrement acide. Pasteurization is preferably carried out by heating the supernatant for about 10 hours at a temperature of about 60 ° C. The pH is a neutral or slightly acidic pH.
C'est ce surnageant obtenu après l'inactivation virale, éventuellement sans avoir été séparé du précipité formé par l'étape de chauffage, qui est soumis au traitement acide. It is this supernatant obtained after viral inactivation, possibly without having been separated from the precipitate formed by the heating step, which is subjected to acid treatment.
Le surnageant traité obtenu par la mise en oeuvre du procédé selon l'invention présente l'avantage, compte-tenu de sa pureté protéique, d'être stable et de pouvoir être stocké dans de bonnes conditions. The treated supernatant obtained by carrying out the process according to the invention has the advantage, given its protein purity, of being stable and of being able to be stored under good conditions.
Il est également possible de prévoir que la composition cicatrisante est préparée extemporanément à partir des plaquettes du sujet qui doit être traité, ce qui écarte tout risque biologique, réaction ou contamination. It is also possible to provide that the healing composition is prepared extemporaneously from the platelets of the subject to be treated, which eliminates any biological risk, reaction or contamination.
La présente invention se rapporte également aux compositions cicatrisantes obtenues à partir de ce surnageant. The present invention also relates to healing compositions obtained from this supernatant.
Il s'agit notamment de telles compositions contenant plus de 1 ug/ml de TGF activé, de préférence 3 ,ug/ml. These include such compositions containing more than 1 μg / ml of activated TGF, preferably 3 μg / ml.
Compte-tenu de sa bonne stabilité, le surnageant peut être conservé sous forme lyophilisée par exemple, disponible pour un mélange extemporané au moment de son application sur la plaie avec un agent support, ou encore pour l'élaboration de compositions cicatrisantes. Given its good stability, the supernatant can be stored in lyophilized form for example, available for extemporaneous mixing at the time of its application to the wound with a carrier agent, or for the preparation of healing compositions.
Les compositons selon l'invention peuvent bien entendu contenir d'autres principes thérapeutiquement actifs, qui pourront être choisis parmi des composés antiseptiques, antibiotiques, et/ou anesthésiques par exemple, ou bien d'autres substances actives connues pour leur activité dans la cicatrisation des plaies. The compositions according to the invention may of course contain other therapeutically active principles, which may be chosen from antiseptic, antibiotic and / or anesthetic compounds for example, or else other active substances known for their activity in the healing of wounds.
Il est également possible de prévoir des compositions contenant des colles biologiques à base d'un mélange extemporané de fibrinogène/fibronectine et thrombine, notamment la colle biologique décrite dans les demandes de brevet français 90 07505 et 89 10355. It is also possible to provide compositions containing biological glues based on an extemporaneous mixture of fibrinogen / fibronectin and thrombin, in particular the biological glue described in French patent applications 90 07505 and 89 10355.
Ces compositions peuvent comporter également des excipients pharmaceutiques acceptables, choisis parmi des composés présentant une bonne compatibilité avec les principes actifs et le surnageant, contenus dans ladite composition. These compositions may also comprise acceptable pharmaceutical excipients, chosen from compounds having good compatibility with the active ingredients and the supernatant, contained in said composition.
Ces excipients pharmaceutiquement acceptables pourront ainsi être choisi parmi des polymères hydrosolubles de type polymère naturel, tels les polysaccharides (gomme xanthane, gomme de caroube, peptine...) ou polypeptides, des dérivés cellulosiques type méthylcellulose, hydroxypropylcellulose, hydroxypropylméthylcellulose ou encore des polymères synthétiques, polaxamère, carbomère, PVA ou PVP. These pharmaceutically acceptable excipients may thus be chosen from water-soluble polymers of the natural polymer type, such as polysaccharides (xanthan gum, locust bean gum, peptin, etc.) or polypeptides, cellulosic derivatives such as methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or else synthetic polymers. , polaxamer, carbomer, PVA or PVP.
Bien entendu, de telles compositions devront posséder un pH de même qu'une osmolarité dans les limites de la physiologie. Of course, such compositions should have a pH as well as an osmolarity within the limits of physiology.
Enfin, il est à la portée de tout homme de l'art d'ajouter dans ces compositions divers excipients type cosolvant comme l'éthanol, le glycérol, l'alcool benzylique, des humectants (glycérol), des agents facilitant la diffusion (transcurol, urée), ou encore des conservateurs anti-bactériens. Finally, it is within the scope of any person skilled in the art to add in these compositions various cosolvent type excipients such as ethanol, glycerol, benzyl alcohol, humectants (glycerol), diffusion-promoting agents (transcurol , urea), or anti-bacterial preservatives.
Ces compositions pourront se présenter sous une forme de liquide, de lait, de poudre, de spray, et de façon préférée sous forme d'hydrogel. These compositions may be in the form of liquid, milk, powder, spray, and preferably in the form of a hydrogel.
Les exemples présentés ci-après à titre non limitatif permettront de mettre en évidence d'autres caractéristiques de la présente invention. The examples presented below in a non-limiting way will make it possible to demonstrate other characteristics of the present invention.
EXEMPLE 1
Les plaquettes sont extraites du plasma par double centrifugation. Elles sont déleucocytées par filtration sur filtre PALL et lavées en tampon Tris 0,04 M pH 7.4.EXAMPLE 1
The platelets are extracted from the plasma by double centrifugation. They are leukylated by filtration on a PALL filter and washed in 0.04 M Tris buffer, pH 7.4.
On récupère ainsi environ 3 x 1010 plaquettes par don standard de sang total, en suspension dans 5 à 10 ml de tampon. Approximately 3 x 10 10 platelets are recovered per standard blood donation, suspended in 5 to 10 ml of buffer.
On ajoute à cette suspension de la thrombine à raison de 5
U/ml et on laisse agir 10 min.; le surnageant obtenu est ajusté à pH 7,0 et chauffé pendant 10 h à 60"C au bain-marie.To this suspension is added thrombin 5
U / ml and allowed to act for 10 minutes; the resulting supernatant is adjusted to pH 7.0 and heated for 10 h at 60 ° C in a water bath.
On obtient ainsi un produit qui peut être traité par le procédé de l'invention. This produces a product that can be treated by the process of the invention.
EXEMPLE 2: ACIDIFICATION
L'extrait suivant l'exemple 1 est porté à pH 2,5 par addition d'HCl, maintenu 10 min. avec une agitation douce, puis il est neutralisé à pH 7,0 par addition de NaOH.EXAMPLE 2: ACIDIFICATION
The extract according to Example 1 is brought to pH 2.5 by addition of HCl, maintained for 10 min. with gentle stirring and then neutralized to pH 7.0 by the addition of NaOH.
Le précipité, formé pendant la pasteurisation et l'étape de traitement à pH acide sont centrifugés et le surnageant est filtré si nécessaire. The precipitate formed during pasteurization and the acidic pH treatment step are centrifuged and the supernatant is filtered if necessary.
EXEMPLE 3
Les plaquettes sont extraites du plasma par double centrifugation.EXAMPLE 3
The platelets are extracted from the plasma by double centrifugation.
Les plaquettes sont déleucocytées par filtration sur filtre
PALL et lavées en tampon TRISS 0.02 M pH 7.4.Platelets are depleted by filter filtration
PALL and washed in 0.02 M TRISS buffer pH 7.4.
On récupère ainsi environ 3.10 exp 10 plaquettes par don standard de sang total, en suspension dans 5 à 10 ml de tampon. About 3.10 exp 10 platelets are thus recovered per standard blood donation, suspended in 5 to 10 ml of buffer.
On ajoute à cette suspension de l'ADP à hauteur de 2.5 micromole par litre et 19 millimole/litre de calcium. La suspension est homogénéisée, puis laissée au repos pendant 20 mn. L'agrégat plaquettaire est alors centrifugé. ADP is added to this suspension at a level of 2.5 micromoles per liter and 19 millimoles / liter of calcium. The suspension is homogenized and then left standing for 20 minutes. The platelet aggregate is then centrifuged.
L'extrait plaquettaire est chauffé à pH 7.0 pendant 10 heures à 60"C, au bain-marie. The platelet extract is heated to pH 7.0 for 10 hours at 60 ° C in a water bath.
Le précipité important qui se produit pendant le chauffage est centrifugé et le surnageant récupéré. The large precipitate that occurs during heating is centrifuged and the supernatant recovered.
Ce surnageant peut être traité comme dans l'exemple 2. This supernatant can be treated as in Example 2.
EXEMPLE 4
L'extrait selon l'exemple 2 est dilué au 1/5ème dans une solution d'hydroxyméthylcellulose à 2 g/l ou dans une solution de collagène à 20 g/l.EXAMPLE 4
The extract according to Example 2 is diluted to 1 / 5th in a hydroxymethylcellulose solution at 2 g / l or in a solution of collagen at 20 g / l.
On réalise sur un rat Wistar femelle de 200 g une série de 6 punchs dorsaux circulaires de diamètre 6 mm et profondeur 2 mm environ symétriques par rapport à la colonne vertébrale. A series of 6 circular dorsal punches with a diameter of 6 mm and a depth of approximately 2 mm which are symmetrical with respect to the spine is produced on a 200 g female Wistar rat.
Une série de 3 puits est remplie par 10 ul d'extrait dilué, l'autre série par 3 puits de placébo. A series of 3 wells is filled with 10 ul of diluted extract, the other series by 3 wells of placebo.
Trois lots de rats sont ainsi réalisés - 1 lot utilisant de l'extrait selon l'exemple 1, - 1 lot utilisant de l'extrait selon l'exemple 2, - 1 lot comparant l'extrait acidifié versus l'extrait non acidifié (2 VS 3). Three batches of rats are thus made - 1 batch using the extract according to Example 1, - 1 batch using the extract according to Example 2, - 1 lot comparing the acidified extract versus the non acidified extract ( 2 VS 3).
Les orifices sont ensuite recouverts par un film plastique, lui-même recouvert de tulle gras, le tout maintenu en place par une bande auto-adhésive. The holes are then covered with a plastic film, itself covered with tulle gras, all held in place by a self-adhesive tape.
Les rats sont sacrifiés à j4 et j7 après le traitement, la plaie est ensuite prélevée et fixée pendant 48 h dans du paraformaldéhyde à 4% contenant du saccharin (0,4M), du CaCl2 1% et du Nacl (0,15M), puis lavée à l'eau courante. The rats are sacrificed on days 4 and 7 after the treatment, the wound is then removed and fixed for 48 hours in 4% paraformaldehyde containing saccharin (0.4M), 1% CaCl 2 and NacI (0.15M). then washed under running water.
Pour l'histologie, les pièces fixées sont enrobées dans du tissu
Teck, coupées ou cryotome et colorées au trichome de Masson.For histology, fixed parts are embedded in tissue
Teak, cut or cryotome and colored with a trichome of Masson.
RESULTATS
Voici les résultats obtenus à partir d'une suspension à 5 x plaquettes/ml.
Here are the results obtained from a suspension at 5 × platelets / ml.
<tb><Tb>
<SEP> Extrait <SEP> Suivant <SEP> Extrait <SEP> Suivant <SEP> Extrait <SEP> Suivant
<tb> <SEP> Exemple <SEP> 1 <SEP> * <SEP> Exemple <SEP> 1 <SEP> Exemple <SEP> 2
<tb> <SEP> P.D.G.F. <SEP> 350 <SEP> ng/ml <SEP> 300 <SEP> ng/ml <SEP> 280 <SEP> ng/ml <SEP>
<tb> T.G.F.ssactif <SEP> 30 <SEP> ng/ml <SEP> 50 <SEP> ng/ml <SEP> 2,8 <SEP> g/ml <SEP>
<tb> T.G.F.sstotal <SEP> 3,0 <SEP> g/ml <SEP> 3,0 <SEP> g/ml <SEP> 3,0 <SEP> g/ml <SEP>
<tb> * avant pasteurisation
Le dosage du T.G.F.ssest fait suivant la technique du clonage en agar de Pircher et al. (BBRC, Vol 136, P. 30-37)
Le T.G.F.ss actif est la quantité mesurée spontanément dans l'échantillon.<SEP> Preview <SEP> Next <SEP> Preview <SEP> Next <SEP> Preview <SEP> Next
<tb><SEP> Example <SEP> 1 <SEP> * <SEP> Example <SEP> 1 <SEP> Example <SEP> 2
<tb><SEP> PDGF <SEP> 350 <SEP> ng / ml <SEP> 300 <SEP> ng / ml <SEP> 280 <SEP> ng / ml <SEP>
<tb> TGFssactif <SEP> 30 <SEP> ng / ml <SEP> 50 <SEP> ng / ml <SEP> 2,8 <SEP> g / ml <SEP>
<tb> TGFsstotal <SEP> 3.0 <SEP> g / ml <SEP> 3.0 <SEP> g / ml <SEP> 3.0 <SEP> g / ml <SEP>
<tb> * before pasteurization
The assay of TGFss is done according to the agar cloning technique of Pircher et al. (BBRC, Vol 136, P. 30-37)
Active TGFss is the amount spontaneously measured in the sample.
Le T.G.F. ss total est la quantité mesurée après activation complète de l'échantillon. T.G.F. ss total is the quantity measured after complete activation of the sample.
La différence entre les deux correspond au T.G.F.ss latent. The difference between the two corresponds to the latent T.G.F.ss.
On constate donc que l'activation obtenue persiste malgré la neutralisation suivant l'exemple 2. It is therefore found that the activation obtained persists despite the neutralization according to Example 2.
Résultats sur l'animal: effets observés après 7 jours
Le tissu cicatriciel du témoin placebo s'organise en une trame fibrillaire, lâche, faiblement cellularisée.Results on the animal: effects observed after 7 days
The scar tissue of the placebo control is organized into a fibrillated, loose, weakly cellularized weft.
En présence d'extraits selon l'exemple 2 ou 3, le tissu de régénération se différencie à plusieurs fibres - la densité cellulaire extérieure beaucoup plus élevée, - la matrice collagénique est mieux définie, son organisation générale est dense. Elle s'exprime sous forme de dépôts fibrillaires annonçant l'organisation classique du derme, - la couche cornée est mieux différenciée pour l'extrait suivant l'exemple 3 que pour l'extrait suivant l'exemple 1 et la maturation est entamée comme en atteste la différenciation de follicule pileux. In the presence of extracts according to Example 2 or 3, the regeneration tissue is differentiated to several fibers - the outer density of the cell much higher, - the collagen matrix is better defined, its general organization is dense. It is expressed in the form of fibrillar deposits announcing the classical organization of the dermis, the stratum corneum is better differentiated for the extract according to Example 3 than for the extract according to Example 1 and the maturation is started as in attests the differentiation of hair follicle.
On constate donc que les fractions acidifiées sont plus efficaces que les fractions non acidifiées, notamment par une différenciation cellulaire plus précoce. It is therefore found that the acidified fractions are more effective than the non-acidified fractions, in particular by an earlier cell differentiation.
Les effets à j4 sont similaires, mais moins accentués. The effects on day 4 are similar, but less pronounced.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9012742A FR2667789A1 (en) | 1990-10-16 | 1990-10-16 | Cicatrising composition and method for preparing it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9012742A FR2667789A1 (en) | 1990-10-16 | 1990-10-16 | Cicatrising composition and method for preparing it |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| FR2667789A1 true FR2667789A1 (en) | 1992-04-17 |
Family
ID=9401255
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| FR9012742A Withdrawn FR2667789A1 (en) | 1990-10-16 | 1990-10-16 | Cicatrising composition and method for preparing it |
Country Status (1)
| Country | Link |
|---|---|
| FR (1) | FR2667789A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5422340A (en) * | 1989-09-01 | 1995-06-06 | Ammann; Arthur J. | TGF-βformulation for inducing bone growth |
| EP1091735A4 (en) * | 1998-06-22 | 2004-05-06 | Cytomedix Inc | Application for utility patent for improved enriched platelet wound healant |
| US7112342B2 (en) | 1998-06-22 | 2006-09-26 | Cytomedix, Inc. | Enriched platelet wound healant |
-
1990
- 1990-10-16 FR FR9012742A patent/FR2667789A1/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5422340A (en) * | 1989-09-01 | 1995-06-06 | Ammann; Arthur J. | TGF-βformulation for inducing bone growth |
| EP1091735A4 (en) * | 1998-06-22 | 2004-05-06 | Cytomedix Inc | Application for utility patent for improved enriched platelet wound healant |
| US7112342B2 (en) | 1998-06-22 | 2006-09-26 | Cytomedix, Inc. | Enriched platelet wound healant |
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