Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Injectable compositions conlainin~ollagen microcapsules
FIELD OF THE~ INVENTION
The present invention essentially relates to injectable compositions
5 containing collagen-based microcapsules in suspension, to their biomedical us~
and to pharrnaceutical compositions.
The invention relates more precisely to the preparation of an injectable
l carrier which makes it possible to fill in losses of substance or to conveniently
;¦ introduce active substances in the system of live beings, in particular humans.
10 PRIOR ART
A large number of investigations have been conducted to develop filling-in
materials. Suitable ways of filling in soft tissues are:
¦ - injections of silicones, gelatin, autologous and heterologous collagen
- implantation of threads (catgut, Gore-tex, collagen).
15 It has become clear today that the matèrial giving the best performance, from the
point of view of biocompatibility and stability in vitro is heterologous (of bovine
origin) collagen (injectable and threads).
The most common applications of the materials for filling-up soft tissues
relate essentially to the treatment of skin depressions and losses of facial
20 substances due to age (wrinkles), or to other causes (diseases, traumatissns, surgical
operations, ...) and which cause scars, emaciations, irregularities, etc...
In some of these cases, the use of crosslinked collagen threads such as
described in document No. 91 04518 filed by BIOETICA is especially indicated on
account of the high properties of biocompatibility, stability in vitro and filling-up
25 power of this rnaterial.
'l Nevertheless, although the use of such threads is quite suitable for filling-up
deep wrinkles, it has been found to be less adapted to other situations (such as for
~l example, superficial wrinkles, non-furrowlike skin defects). In those cases, it is
.`i necessary to resort to an injectable form.
Also, besides plastic surgery, there are medical fields (such as
~ l otorhinolaryngology, urology, orthopedics,.. ) for which the nature of the fillings to
; i be performed and/or the accessibility of the implanting sites necessarily require the
use of an injectable form.
- The most recent among the existing inventions are described in documents EP
83868 A1 and EP 89145 A2 filed by the company COLLAGEN CORPORATION,
EP 132979 A2 filed by the company KOKEN CO. LTD. and US-A-3 949 073.
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- The existing injectable collagens arc collagenic solutions. In most cases the
implant should be able to be injected with fine needles (generally having a
diameter of 0.3 to 0.5 mm). To meet this requirement, the solutions have to be
- relatively fluid hence of low concentration (2 to ~.5%). Such limitation raises the
S problem of biodegradability and of stability in time of such implants, the quantities
of active substances administered being relatively low.
- For reasons of solubility and biocompatibility, the collagen used is an
atelocollagen, namely a collagen from which the end regions of non-helical
structure called telopeptides have been eliminated. ~aid telopeptides are indeedwhere cross link-type intra- and inter-molecular bonds occur, which bonds rnake
the collagen non-soluble. Moreover, the telopeptides carry the principal antigenic
determinants of collagen. In the existing products, atelocollagen is obtained byprotease treatment with pepsin which preferentially attacks the telopeptides
without destroying the helical structure. Pepsin, however, has the disadvantage of
settling very firmly on its substrate and is therefore difficult to eliminate. As it
happens, it is a very strongly antigenic molecule of which the presence, even insmall quantity, risks to alter the biocompatibility of the product thus obtained.
- To ensure its filling-up function, the implant should, after injection, retain a
certain volume and it should not spread too much through thc surrounding tissues.
Thus, it constitutes a kind of matrix which, being maintained long enough, settles
in the host tissues and enables a restoration of the treated area. In the existing
products, such characteristic is met by using atelocollagen solutions which
precipitate at body temperature, thus leading to the formation of fibers which
remain at the injection site whereas the excipient is progressively resorbed (see
US-A-3 949 073)
¦ The result is that before being injected, the product has to be kept at low
temperature (below 10-C) so that it can retain its fluidity, which property is
necessary for it eo be administered by injection. Consequently, the injectable
collagen based on this principle require particular precautions of preservation
which complicate the use thereof.
- The rnajor limitation of the prior injectable collagens reside in their resorbability
"in vivo".
Indeed, the degradation of collagen is a normal biological process which
. forms part of the metabolism of the connective tissues. This process involves a
number of enzymes, in particular collagenases which are responsible for the initial
attack of collagen. The biodegradability of collagen raises a particularly important
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problem. Indeed, the filling-up of depressions and of losses of substance should be
- a permanent treatment or in any case a reasonably durable trcatment which hardly
needs a rapid resorption of the implant.
Different physical and chemical treatments exist, which treatments are able
5 to reduce resorbability by increasing the number of crosslinking bonds between the
, molecules of collagen. Glutaraldehyde is the most widely used crosslinking agent
-, (as described in EP-A-C) 089145).
It is particularly efficient but has a major disadvantage which is to be
polymerized when in solution. Accordingly, materials which are crosslinked with
10 this agent release, after a time, glutaraldehyde monomers which are cytotoxicbeyond 10-25 ppm. Therefore, the injectable collagens prepared according to thisprinciple (Patent EP 89 145 A2) are generally less degradable than the non-
~, crosslinked implants (average stability between 1 and 2 yeaIs instead of 6 months
~ to one year, as regards filling-up of wrinkles~. On the other hand, they can cause
.. ~ 15 more frequent adverse reactions which raises a certain amournt of suspicions and
reduces the uses.
. SUMMARY OF THE INVE~NTION
The main object of the present invention is to provide a solution for
. I preparing an injectable material which is easy to inject even with a fine needle
whose diameter is between 0.3 and 0.5 mm, and a high collagen content, in
. particular at least 7% by weight/volume and preferably as high as about 10%.
. Another object of the present invention is to solve the new technical problem
. indicated above by providing a solution whereby a system is proposed for carrying
active substances while allowing the therapeutic optimization of the active
¦ 25 molecules, for example by controlled release into the organism, by screening of the
tissues or organs concerned, and by protection of the natural substances.
Yet another object of the present invention is to solve the new technical
problem indicated above by providing a solution of improved biocompatibility in
which the degradation products do not create residues liable to cause undesirable
. 30 reactions.
A further object of the present invention is to solve the new technical
problems indicated above with a solution whereby the degradation rates of the
material injected in a live being, in particular a human being, are modulated so as
to be able to adjust the life duration of an implant as well as the vehiculated active
substance-release kinetics. !t
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lhe object of the invention is also to provide filling materials whose stabilityin vivo can be at least equal to that of the collagell materials treated with
glutaraldehyde.
All said technical problems have becn solved simultaneously, and in a
S satisfactory, safe, reliable and reproducible manner, usable on an industrial and
medical scale, while being inexpensive.
Thus, in a first aspect, the present invention provides a collagen-based
composition, which is easy to inject, characterized in that it comprises
'~ microcapsules of atelocollagen or of a mixture of atelocollagen with a
10 polysaccharide, in particular a glycosaminoglycan, in suspension in a viscousbiocompatible carrier solution. Preferably, the microcapsules together with the
viscous carrier solution have, as a whole, a viscosity which permits the injection of
said composition, particularly in the form of a continuous thread from a needle.¦ According to a particular variant of embodiment, the carrier solution
15 comprises at least a prote;n substance, such as for example atelocollagen and/or a
polysaccharide substance.
If the carrier solution contains a protein substance, said substance is
advantageously atelocollagen, which may be obtained by enzymatic or chemical
means, preferably by chemical treatment notably with sodium hydroxide, the
20 chemical treatment being particularly advantageous in that it prevents any residual
il presence of enzyme as it happens if an en~ymatic treatment is used, the enzyme
being usually pepsin, which increases the biocompatibility of the material. If the
atelocollagen is obtained by enzymatic treatment, notably with pepsin, or by
chemical treatment, notably with sodium hydroxide, it is possible to modulate the
25 degree of decrosslinking of collagen, hence the viscosity of the carrier material.
According to another particular variant of embodiment, the polysaccharide
substance is used, optionally mixed with the protcin substance in the carrier
., material, and it is selected from the group consisting of a glycosaminoglycan, an
~ alginate, a dextran, a cellulose or a cellulose derivative, xanthan gum, gum alabic,
¦ 30 and mixtures thereof.
Thus, according to an advantageous variant of embodiment, the
atelocollagen in the microcapsules is decrosslinked with a decrosslinking rate
adapted to the proposed application.
The polysaccharide which can be used in mixture with atelocollagen for the
35 preparation of said microcapsules, is in particular a glycosaminoglycan selected
among the group consisting of the structural glycosaminoglycans selected among
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chondroitine-4-sulfate, chondroitine-6-sulfate, dermatan-sulfate, heparan-
sulfate, keratan-sulfate, as well as heparin and its derivatives, particularly the
heparins of low molecular weight having a molecular weight comprised between
about 2,000 and about 10,000; and a polyholoside, and in particular dextran.
The process for preparing the microcapsules is based on that described in
Applicant's prior patent FR-A-2 6~2 329 incorporated herein by way of reference.Advantageously, the proportion of polysaccharide with respect to
atelocollagen is comprised between 10 and 70 %, and better still between 15 and
50 % by weight.
The preparation of the microcapsules consists in crosslinking the
atelocollagen and optionally the polysaccharides by interfacial crosslinking, this is
done by producing an emulsion of the solution of atelocollagen and
polysaccharides as phase dispersed in a hydrophobic liquid forming the continuous
phase, in which the atelocollagen and/or the polysaccharides are essentially non-
soluble.
For the crosslinking, a crosslinking agent is added to the emulsion of the
solution of atelocollagen and optionally of the polysaccharides, said crosslinkiDg
agent comprising reactant groups capable of reacting with the acylable groups ofthe atelocollagen and of polysaccharides so as to cause an interfacial crosslinking
reaction. A preferred crosslinking agent is an acid dichloride or an acid anhydride
or a di- or polybasic carboxylic acid. Other preferred crosslinking agents are,
terephtaloyl chloride, phtalic acid chloride, sebacic acid chloride, succinic acid
chloride, the chloride of a tricarboxylic acid such as citric acid or an anhydride of
an acid such as succinic anhydride.
As hydrophobic liquid, in which the atelocollagen and/or polysaccharides are
non-soluble, it is preferred to use fatty acid esters such as selected among those
which can normally be administered by parenteral route, such as triglycerides,
ethyl oleate; or which have no toxic properties, such as isopropyl myristate, 2-hexyl ethyl cocoate.
The concentration of polysaccharides in the polysaccharide solution is
advantageously between 0.5 and 4 %, and better still between 0.5 and 2 %, and
even preferred around 1 %.
The atelocollagen solution for the preparation of the microcapsules is an
aqueous solution of atelocollagen having a concentration cornprised between 0.5
and 2 % by weight.
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, For preparing the microcapsules, the atelocollagen may have been obtained
' by enzymatic digestion of collagen, notably using pcpsin, or else by chemical
treatment using sodium hydroxide.
For the achlal process for preparing the microcapsules, reference will be
made to document FR-A-2 642 329, incolporated herein by way of reference, and
in particular to Examples 1 to 6.
According to an advantageous embodiment, the size of the microcapsules is
less than or equal to 500 ,um. In some cases, the size of the microcapsules is less
than 250 ,~an and can even be comprised between 20 and 50 ,um, in particular in the
case where the composition is supposed to be injected with a fine needle having a
diameter comprised between ().3 and 0.5 mm.
According to a particular variant, the relative proportion of the
microcapsules with respect to the viscous biocompatible carrier solution is
, cornprised between 0.5-15/99.5-85 by weight/volume.
'l 15 According to a particularly advantageous embodiment, the microcapsules
contain at least an active substance which is, either encapsulated within, or in the
mass of the walls of the microcapsules, or grafted on the internal and/or external
surface. Said active substance may be biologically, cosmetically or
pharmaceutically active. By biologically active substance is meant a substance
having a biological effect on the system which is not necessarily cosmetic or
therapeutical. A currently preferred active substance is hydroxyapatite, particularly
in particle form, for example of size equal to about sn,um.
In a second aspect, the present invention also provides a use of said
composition as carrier agent of biologically, cosmetically or pharmaceutically
active substance.
The present invention further relates to a pharmaceutical composition,
characterized in that it contains a collagen-based composition which is readily
l injectable as defined hereinabove.
The invention finally relates to an implant, characterized in that it is prepared
f~om an injectable composition as defined hereinabove.
Moreover, the invention further relates to special embodiments of
compositions. A preferred special embodiment is an embodiment in which the
microcapsules are based on atelocollagen optionally mixed with a
glycosaminoglycan such as chondroitine-4-sulfate, said microcapsules containing
granules of hydroxyapatite in suspension in a viscous biocompatible carrier
solution of a gel of atelocollagen optionally mixed with a glycosaminoglycan, in
~2~3~c-3
particular chon~roitine-4-sulfate. When ~he granules of hyclro~yapaiite havc a
dimension of about 50 ,um, the microcapsules havc a dimension o~ about 2()0 ~m.
According to a particularly advantagcous embodiment, the viscous
biocornpatible carrier solution is adapted to the physiological conditions of pH and
5 of osmolic pressure, having preferably a pH comprised bctween 6.8 and 7.5 and an
osmotic pressure comprised between 260 and 32() mOsm/l H20 Such a pH and
osmolarity of the carrier solution can be obtained by adjusting with a phosphatebuffer containing for example a mixture of phosphates, of mono- and disodic
sodium and/or potassium, or of a mixture of phosphate buffer and sodium chloride10 for completing the solution carrying the suspension of microcapsules.
Owing to the nature of the constituents of the microcapsules, atelocollagen
optionally mixed with polysaccharides, the microcapsules are soft, deformable, so
that when in suspcnsion in thc viscous biocompatiblc carricr solution according to
the invention, the composition has, as a whole, flowing properties such that it is
15 possible to easily inject, with a needle having a diameter of 0.3 to 0.5 rnm, a
material containing as much as 10 % collagen by weightlvolume. This possibility
represents an increase of 35 % with respect to the most concentrated prior products
(6.5 %)~
Thus, the compositions according to the invention are particularly suitable to
20 be used as filling-in materials, either for filling-in soft tissues, or as bone fillings
for bone reconstruction. In such applications, it is obvious that any increase of the
active substance conccntration causes a de facto incrcase of the lifc of the implant
and of the efficiency of the filling.
The composition according to the invention thus combines the biological
25 properties of collagen, in its atelocollagen form, with those of any other substances
that may be used (polysaccharides crosslinked together with the atelocollagen,
active substances in solution or in suspension in thc sphercs, molccules grafted on
their surface).
Thus, the compositions according to the invention have a wide range of
30 applications as carrier systems for active substances. This galenic form is
particularly advantageous in that it enables, depending on the uses:
- therapeutic optimization of the active molecules by controlled release into
the system, screening of the target tissues or organs, protection of the natural parts;
- access through percutaneous route to areas which are normally only
35 accessible by surgical route (for e~ample, the bone areas), thus limiting thetraumatism suffered by the paticnt and the hospitalization timc and costs.
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Obviously, it is also possible to envisage a number of applications in which
the function of filling-in is advantageously combined with the usc of ~ particularly
advantageous galenic fonn.
The composition according to the invention is particularly adapted to the use
~, S as a carrier insofar as its degradation does not create residues liable to cause
undesirable reactions. This constitutes a major advantage over certain synthetic
polymers of the prior art.
Degradation of the microcapsules can be modulated within very wide limits,
on the one hand by a variable degree of decrosslinking of ~he atelocollagen by
chemical or enzymatic means; and on the other hand, by adjusting the rate of
interfacial crosslinking. It is thus possible to adjust the speed of resorbability from
a fast speed to a speed higher than that obtained with materials crosslinked with
glutaraldehyde, while avoiding the disadvantages linked to the toxicity of that
agent.
Thus, the range of application of the compositions according to the invention
is multiplied, by relying on the duration of the composition, particularly in implant
fonn, whether it is intended to stay the longest possible time in the implanting area
or to be resorbed and replaced more or less rapidly by the host tissues; and thekinetics of the release of the active substance.
It is therefore understandable that the present invention provides a technical
solution which has definite technical advantages over the prior art.
Other objects, characteristics and advantages of the invention will be more
clearly understood from the explanatory description given with reference to several
examples of embodiment of the present invention and just by way of illustration
and which could not possibly limit the scope of the invention in any way.
However, the examples form an integral part of the invention and the man skilledin the art will, on reading them, appreciate the new general characteristics resulting
therefrom.
In the ex~unples, all the percentages are given by weight, except where
otherwise stated.
Example 1: Suspellsion of ateloeollagen microcaPsules of ~0 to 50 ,um in an
atelocolla~en ~el.
This suspension constitutes a product intended to ensure a filling-in
function, particularly for soft tissues.
a) Preparation of atelocollagen by treatment with pepsin
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Cal~fs skin from an animal just slaughtered is subjected to a chemical
-. depilatory treatment in a bath containing 3 % sodium sulfide and 4 % lime, the
proportion being 1200 g of skin for 200 ml of solution. The dermis is then isolated
from the rest of the skin by a ripping operat;on with a rotating band saw.
, 5 The obtained tissue is ground and extruded through a screen having 4 mm
`~ holes.
The ground material is dispersed in a solution of acetic acid 0.5 M containing
pepsin in the proportion of 1 g enzyme for 10 g of dry collagen, the pH of the
mixture being adjusted if necessary to obtain a pH of between 2 and 3. After
10 stirring, the mixture is left to incubate for 36 hours at 10-C. After filtration, the
.l collagen is precipitated by the addition of NaCl 0.7 M. After stirring and
incubation (4 hours), the precipitated fibers are rccovered by centrifuging (14 000
, rpm). The supernatant is eliminated and the pepsin is inactivated by dispersion of
f the residue in a Tris buffer 0.05 M - NaCl 0.15 M of which the pH is adjusted,
15 after dispersion of the fibers, to 8.5. After 3 days' incubation at 10DC, the dispersion
f is dialyzed against deioni~ed and sterile water using dialysis membranes,
.`ff preferably formed by tubings of which the cutting threshold is between 6,000 and
.¦ 8,000 daltons.
b) Preparation of the ate3Ocollagen solution in a buffer medium of pH 8-8~2
. 20 The atelocollagen coming from the dialysis tubings is diluted with a solution
of sodium bicarbonate, of volume and concentration such that thc final
concentrations are as follows:
¦ - atelocollagen..................... 1 %
l - anhydrous bicarbonate of sodium .. ..4 %
1 25 It is checked that the pH of the obtained solution is definitely comprised
bet~veen 8 and ~.2. 1 kg of such solution is prepared.
. c) Preparation of the crosslinkiog agent
;1 100 g of terephtaloyl chloride are added to 4 l of a hydrophobic solvent,
preferably fatty acid esters selected among those which can normally be
30 administered by the parenteral route (such as triglycerides, ethyl oleate), or among
f those which have no toxic properties (such as isopropyl myris~ate, ethyl-2-hexyl
cocoate). The mixture is stirred mechanically.
d) EmulsificatioD
3 l of the hydrophobic solvent selected in accordance with the aforecited
. 35 criteria are introduced into a container. The previously prepared buffered
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atelocollagen solution is added under stirring to the Ultra Turax (~) tuming at 7,200
;, rpm.
e) Crosslin}ung
~ 5 The crosslinking agent solution is added to the resulting emulsion. Stirring is
-¦ kept up for 30 mins.
Microcapsules are thus obtained.
f~ Washes
.j Three washes are carried out with 10 1 of hydrophobic solvent and the
10 microcapsules are collected by decantation with a Robatel decanter turning at
¦ 1,000 rpm.
Five to eight washes in ethanol (a total of 15 to 201) are performed in the
same way, followed by three to five washes in deionized and sterile water.
g) Lyophilization
A Iyophilization of the washed product containing the microcapsules is
conducted in the conventional way.
h) Sterilization
After packing the dry spheres in a sealed package, the microcapsules are
sterilized by being exposed to gamma rays (25 kGy).
20 i) Preparation of the suspension medium
The atelocollagen is decrosslinked by treatment with soda in the conditions
¦ explained hereafter.
:¦ After depilation, ripping and grinding of the skins such as described in a), the
`1 ground material undergoes two washes with a phosphate buffer of pH 7.8 ~21.7 g/l
25 of NA2HP04 and 0.78 g/l of KH2P04 ) and then two washes in deionized and
sterile water.
~¦ The ground material is then placed for eight days in contact with a solution
of soda in the proportion of 1 kg of ground material for 4 1 of solution of
concentration such that the final quantity of soda is 8 % (w/v). After this treatment,
30 the atelocollagen is precipitated by the addition of hydrochloric acid concentrated
-¦ reaching a pH of between 2 and 2.5. The resulting suspension is introduced in
~ dialysis tubing (having a cutting threshold of between 6~000 and 8,000 daltons)
.~ and dialyzed against deionized and sterile water.
The obtained atelocollagen is then Iyophilized, sterilized by gamma rays and
j 35 placed in solution in sterile conditions. The pH and osmolarity of the solution are
adjusted by using a phosphate buffer (mixture of mono- and disodic sodium
,
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and/or potassium phosphates) or a mixture of phosphate buffer and sodium
chloride so as to obtain a pH comprised between 6.~ and 7.5 and an osmotic
pressure comprised between 260 and 320 mOsm/l H2O.
Example of composition of the suspension medium:
.1 5 - Atelocollagen .......................... ..1 cr/o
- Na2HPO4................................. 0.02 M
- NaCI ................................... 0.13 M ..
l - pH ..................................... 7.3
j) Placing the micr~apsul~s in suspensiorl
.~ 10 The microcapsules are placed in suspension in the atelocollagen suspension
medium obtained in step i) is sterile conditions in the proportion of 100 mg of dry
.~ spheres per ml of suspension medium. The suspension is stirred so as to obtain a
, perfectly homogeneous mixture. The pH of the mi~ture is chccked.
k) Preparation of the syring~s
Thc mixture is introduced in sterile conditions, in syringes of 1 ml equipped
. with a luer-lock type endpiece and needles of diameter between 0.3 and 05 mm.
. I) Evaluation of the obtaine~ product
Ihermal stability by Programmed Differential Scanning Calorimetry:
. The parameters recorded on the microcapsules after Iyophilization are as
20 follows:
. - beginning-oE-denaturation temperature: 65-C
, - peak-of-denaturation temperature: 72.6-C
- end-of-denaturation temperature: 80-C.
. These values are particularly high and are of the same magnitude thaD with
25 the products obtained by crosslinking with glutaraldehyde for example.
. $ Evaluation in vivo:
The tests were conducted on Wistar female rats weighing between 180 and
200 g.
The material was implanted on the rats' back, in sub-cutaneous position.
. 30 Each implanting site (2 per rat) received about 0.1 ml of the suspension.
The biopsies and histological controls conducted after 1 and 7 days of
implantation have shown:
! - a high filling-in power
~¦ - a perfect biocompatibility
35 - a total absence of degradation of the implant.
These results ensure for the implant a particularly high long-term stability.
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-¦ Example 2: Suspension of microca~?sules of atelocolla~en and of chondroitin-
4-sulfate of abou O uim containing ~ranules of hydroxyapatite in a ~el of
atelocolla~en and chondroitine-4-sulfate.
In this example, granules of hydroxyapatite (a mineral chemical composition
S close to the bone mineral and used in bone rcconstrl ctions) are incorporated in
microcapsules. This system makes it possible to carry the granules of
hydroxyapatite and makes them injectable through a trocar. They can thus be
implanted in a bone area by percutaneous route.
a) Preparation of the atelocollagen by treatment with soda
This step is performed in the same conditions as those described in Example
-¦ 1, paragraph i).
;¦ b) Preparation of the chondroitine-4-sulfate
Nasal septa from lambs, wherefrom the muscular and adipous tissues have
been removed are chopped and ground by extrusion through a screen having 4 mm
hoies; the ground material is placed ~or 24 hours at a temperature of 6-C in a buffer
of potassium chloride (11.8 g/l of KCI; 78.8 mg/l of cysteine, 180 mg/i of EDT~)containing I % of "MERCK" papain. The proportion being of 130 g of ground
;l material for 11 of buffer.
The supematant is separated from the residue by continuous centrifuging
J 20 using a decanter turning at 4.000 rpm. 40 g/l of trichloroacetic acid are then added
to the supernatant. The precipitate is eliminated by continuous centrifuging
: according to the preceding technique. The supernatant is neutralized with pellets of
: soda. The mixture is then dialyzed against deioni7ed and sterile water using tubing
having a cutting threshold between 6 and 8,000 daltons. The dialyzed solution isl 25 liophylized. The chondroitine-4-sulfate is obtained in dry state.c~ Preparation of the solution of atelocoliagen and oî chondroitin-4-sulfate in
a buffer medium of pH 8.9
The atelocollagen in fiber form coming from the dialysis tubing is diluted
with a solution containing chondroitin-4-sulfate and sodium carbonate of volume
and concentration such that the final concentrations are as follows:
- Atelocollagen: 1.6 %
- Chondroitin-4-sulfate: 0.6 %
- Anhydrous sodium carbonate: 4.8 %
The pH of the mixture is adjusted to 8.9 with concentrated hydrochloric acid.
- 35 1 kg of this solution is prepared.
d) Incorporation of hydroxyapatite
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The hydroxyapatite is incorporated in thc forrn of spherical granules of about
SO,um diameter to the preparation described hereinabove, in the proportion of 5 %
'j (w/v).
e) Preparation of the crosslinking agent
180 g of terephtaloyl chloride are added to 41 of isopropyl myristate. The
mixture is stirred mechanically.
Emulsification
lSû ml of Span 85 (ICI Company) are mixed with 3 1 of isopropyl myristate.
The collagen solution containing hydroxyapatite is poured into the mechanically
10 stirred mixture. The whole is stirred for a few minutes in order to obtain the
emulsion.
g) Crosslinking
3 The crosslinking agent solution is added to the resulting emulsion. Stirring is
kept up ~or 30 minutes.
The microcapsules containing the granules of hydroxyapatite are thus
obtained.
h) Washes
The microcapsules are washed in the same conditions as those described in
Example 1.
20 i) Preparation of the suspension medium
The atelocollagen coming from the dialysis tubing is diluted with a solution
of chondroitin-4-sulfate of volume and concentration such that the final
concentrations are as follows:
- Atelocollagen ......................... 0.8 %
- Chondroitin-4-sulfate.................. Ø2 %
j) Placing the microcapsules in suspension
The micIocapsules are placed in suspension in the medium described
hereinabove in proportions such that in the final mixture 90 % of the mixture
composed of atelocollagen and chondroitin-4-sulfate come from the
30 microcapsules and 10 % come from the suspension medium.
A composition according to the invention is thus obtained~ which
composition can be used as is or it can be treated as described hereunder.
k) Lyophil;zatioll
The composition obtained in step j is Iyophilized in conventional manner.
35 !) Sterilization
Same conditions as in Example 1.
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m) Rehydration in bu~er medium
The Iyophilisate is placed in suspension in sterile conditions in the
proportion of 500 mg of Iyophilisate per ml of phosphate buffer 0.1 M. The pH ofthe phosphate buffer is fixed so that the pH of the mixture is between 7 and 7.3.
S o) Preparation of the syringes
The mixture is introduced, in sterile conditions, in S ml syringes equipped
with needles of 1.5 mm diameter.
