Note: Descriptions are shown in the official language in which they were submitted.
CA 02320135 2000-08-11
Preparation composed of calcium hydroxide, a dihydric
or polyhydric alcohol and a fixed oil of vegetable or
animal origin and its use for collagen regeneration
The invention relates to a preparation composed
of a fixed oil of vegetable or animal origin, calcium
hydroxide, a dihydric or polyhydric alcohol and, where
appropriate, pharmaceutically acceptable excipients, to
the preparation of such a mixture, to the use of such a
mixture for collagen regeneration, and to the use of
such a mixture for producing a medicine for promoting
collagen regeneration in vivo.
Bone consists of about 60s mineral substance
(hydroxyapatite, calcium phosphate) and about 40%
organic material, principally collagen. Bone metabolism
is determined mainly by the interplay of lbone-
constructing cells (osteoblasts) and bone-degrading
cells (osteoclasts and osteocytes), whose activities in
healthy bone are in a balanced relationship.
Bone formation can be divided into two main
phases, (a) the synthesis of organic tissue (collagen
synthesis) and (b) the following incorporation of
mineral substance in the previously provided organic
matrix mediated by so-called matrix vesicles.
The connective tissue protein collagen accounts
for most of the organic substance in bone. The protein
consists of three helically coiled polypeptide chains
whose amino acid composition may vary, which leads to a
diversity of individual types of collagen. It is common
to all types of collagen that the collagen fibers have
exceptionally great mechanical strength. This strength
is based on a multiplicity of intra- and intermolecular
linkages of the collagen fibers which, in this way,
form the dense collagen fiber network of connective
tissue. Bone tissue is - as already mentioned - formed
by incorporation of mineral substances (hydroxyapatite
and calcium phosphate) into this network. Bone
construction as a result of growth or regeneration
processes is always preceded by collagen biosynthesis.
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To date, in cases of bone injury of any cause,
the bone regeneration process has been left to itself,
at the most assisted by antibiotics and corticoids in
order to prevent any risk of infection compromising the
healing process.
Several factors able to influence bone
formation and regeneration have also been described.
These are mainly physical factors (mechanical and
electrical forces), hormones (for example parathyroid
hormone, calcitonin, insulin, glucocorticoids, 1,25-
(OH)zD3) and a not firmly defined group of growth
factors with protein characteristics (osteocalcin,
osteonectin, "insulin-like growth factors") - cf.
S. Wallach, L.V. Avioli, J.H. Carstens jun. "Factors in
Bone Formation", Calcified Tissue International 45: 4-6
(1989)). The effect of the hydrogen ion concentration
(pH) on the metabolic processes in bone regeneration
has not as yet been adequately investigated.
Dietz describes in DE-A-42 40 713 the use of a
mixture of calcium hydroxide and neatsfoot oil for
collagen regeneration following bone injuries. This
preparation of calcium hydroxide and neatsfoot oil
suffers, however, from the fact that its stability is
very limited as a consequence of saponification. This
may impair the effect of the mixture.
The invention was accordingly based on the
object of providing an improved mixture with stability
for a long time for the specific external influencing
of the bone regeneration process by stimulating or
initiating of collagen regeneration.
It has now surprisingly been found that it is
possible by using a preparation composed of calcium
hydroxide, a dihydric or polyhydric alcohol and a fixed
oil of vegetable or animal origin and, where
appropriate, pharmaceutically acceptable excipients to
improve the stability of the preparation markedly and
thus by using this preparation in or for bone injuries,
the extent of collagen regeneration in vivo is
improved.
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The present invention thus relates to
preparations which comprise calcium hydroxide, a
dihydric or polyhydric alcohol and a fixed oil of
vegetable or animal origin and, where appropriate,
pharmaceutically acceptable excipients.
The present invention further relates to a
process for producing such a preparation by mixing the
calcium hydroxide and the dihydric or polyhydric
alcohol and, where appropriate, pharmaceutically
acceptable excipients into a fixed oil of vegetable or
animal origin.
The present invention further relates to the
use of such a preparation for collagen regeneration.
The present invention further relates to the
use of such a preparation for producing a medicine for
promoting collagen regeneration in vivo.
Barium sulfate-containing mixtures of calcium
hydroxide and neatsfoot oil have been used in dentistry
as root-filling paste (DE-C 29 32 738). Mixtures of
carboxylate cement, calcium hydroxide and neatsfoot oil
have likewise been used in dentistry as temporary
fixing means for provisional copings (DE-C 34 13 864).
The task of the calcium hydroxide in the former case is
to convert the acidic environment in the root canals
into an alkaline one, resulting in the elimination of
inflammations and gradual formation of a hard tissue
barrier. In the latter case, the pulpitis-prophylactic
effect of calcium hydroxide is utilized. The neatsfoot
oil serves in both cases as a pasting auxiliary in
order firstly to ensure simple and complete filling of
the root canals with the actual active ingredient
calcium hydroxide (and the contrast agent barium
sulfate), and secondly to slow down the setting of the
temporary fixing means for provisional copings so that
the calcium hydroxide is also able to penetrate through
the fine dentinal tubules to the pulp and display its
effect there. Neither of the two references gives the
slightest hint that the mixture according to the
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invention is able to induce extensive collagen
regeneration as prerequisite for bone regeneration.
The term "preparation", which is novel
according to the invention and is used hereinafter,
refers to a pharmaceutical preparation (sometimes also
referred to as mixture hereinafter) which contains at
least the above-mentioned ingredients. It is
particularly suitable for administration to humans or
animals for research in collagen regeneration as a
prerequisite for bone regeneration.
The ingredients of the mixture according to the
invention are described in more detail below:
The fixed oils of vegetable origin which can be
used may comprise one or more ingredients of the
following vegetable oils:
Soybean, sunflower, rape seed, cottonseed,
linseed, castor, palm, palm kernel, coconut and olive
oils.
The fixed vegetable oils which can be used are
preferably fixed vegetable oils with high stability on
heating, such as soybean, sunflower and olive oils, in
particular olive oil.
The fixed animal oils which can be used may
comprise one or more ingredients of the following
animal oils:
Fish oils, animal foot oils and tallows.
The animal oils which can be used are
preferably animal foot oils, in particular neatsfoot
oil.
The dihydric or polyhydric alcohols which can
be used may comprise dihydric alcohols such as ethylene
glycol, propylene glycol, butylene glycol, pentylene
glycol, hexylene glycol and polyethylene glycols such
as diethylene glycol, triethylene glycol, polypropylene
glycols such as dipropylene glycol, trihydric alcohols
such as glycerol, tetrahydric alcohols such as
threitol, erythritol, pentahydric alcohols such as
arabitol, adonitol, xylitol, hexahydric alcohols such
CA 02320135 2000-08-11
as sorbitol, mannitol, dulcitol, or higher polyhydric
alcohols.
Preference is given to the use of dihydric and
trihydric alcohols, such as ethylene glycol, propylene
glycol, butylene glycol, pentylene glycol, hexylene
glycol and polyethylene glycols such as diethylene
glycol, triethylene glycol, polypropylene glycols such
as dipropylene glycol, and trihydric alcohols such~as
glycerol, in particular glycerol as dihydric or
polyhydric alcohol.
LVithout wishing to adhere to a theory, we
assume that the dihydric or polyhydric alcohol prevents
saponification of the vegetable or animal fixed oil.
This makes it possible to keep the mixture in a
kneadable or creamy consistency for longer, so that
collagen biosynthesis can be increased and improved.
A creamy, kneadable preparation is produced
according to the invention from the individual
ingredients. The calcium hydroxide is added to the
preparation in amounts of preferably 1-90o by weight,
more preferably expediently 10-70o by weight, most
preferably 20-60o by weight based on the total weight
of the preparation, respectively.
The fixed oil of vegetable or animal origin is
added to the composition to result in a creamy,
kneadable consistency in amounts of preferably 9-90o by
weight, more preferably 10-60o by weight, most
preferably 20-40o by weight based on the total weight
of the preparation, respectively.
The dihydric or polyhydric alcohol is added to
the composition to result in a creamy, kneadable
consistency usually in amounts of preferably 1-40o by
weight, more preferably 10-40o by weight, most
preferably 20-30o by weight based on the total weight
of the preparation, respectively.
A preferred embodiment of the present invention
relates to an above-mentioned mixture which
additionally comprises MgO. Mg0 can be added for this
purpose in amounts of 1-90o by weight, preferably
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10-70o by weight, more preferably 20-60% by weight
based on the total weight of the preparation,
respectively. However, it is assumed at present that
Mg0 will preferably be added in smaller amounts of
10-20o by weight. Mg0 acts as an antacid in the bone
material to counteract the acidic environment in the
bone.
The ratio of calcium hydroxide to vegetable or
animal fixed oil in the mixture according to the
invention can be 5/1 to 1/5, preferably 5/1 or 1/1.
However, a deviation from the preferred mixing ratio
may be necessary owing to the specific circumstances of
the wound trauma.
If the mixture according to the invention is to
have a particularly soft and smooth consistency, it is
also possible to incorporate white petrolatum into it.
It is usually possible to add white petrolatum in
amounts of 1-60$ by weight, preferably in amounts of
10-60o by weight, more preferably in amounts of 20-400
by weight based on the total weight of the preparation,
respectively.
Although it is not normally necessary to
monitor the bone healing or regeneration process
radiologically, this may nevertheless be indicated in
some cases. In this case, it is also possible to
incorporate barium sulfate as an X-ray contrast agent
into the mixture according to the invention. However,
since the collagen regeneration resulting with barium
sulfate is somewhat less good, barium sulfate is added
to the mixture according to the invention if necessary
in an amount just sufficient (for example 10-20o by
weight based on the total weight of the preparation)
for the mixture just to be radiographically visible.
Application of the mixture according to the
invention onto or into the bone injury can take place
depending on its consistency - by use of syringes,
spatulas or brushes.
There are numerous possible uses of the mixture
according to the invention in general surgery and oral
CA 02320135 2000-08-11
surgery, orthopedics, implantology, traumatology and
the like, because the mixture according to the
invention can be applied onto or into bone tissue
injuries such as fracture surfaces, drillings, cavities
and the like and immediately induces in vivo collagen
regeneration at the particular site of application.
Since, as is well known, metallic fixing means
are used in some relevant medical disciplines, it~ is
advisable in this case to fill the drilling prepared
for insertion of the fixing means with the mixture
according to the invention before insertion of the
fixing means, and only then to introduce the fixing
means. It is possible in this way to counter the
primary osteolysis unavoidable with such procedures and
thus speed up the fitting or adaptation of the fixing
means in or to the surrounding bone tissue and the
fixing of the fixing means itself by the bone tissue.
Excess mixture moreover does not interfere in
this case because on introduction of the fixing means
into the drilling filled with the mixture it is either
forced out again or diffuses into the spongiosa.
It ought to be self-evident that the mixture
according to the invention and its ingredients must be
both packaged and applied under sterile conditions.
It has also emerged in an extremely surprising
manner that the mixture according to the invention
counteracts, even without antibiotic and/or corticoid
assistance, an inflammatory reaction caused by the bone
injury, and rapidly causes it to subside. Its simple
composition and pronounced efficacy for in vivo
collagen regeneration with simultaneous inhibition of
inflammation make the mixture according to the
invention a composition which will be indispensable in
future in bone traumatology.
The following examples are intended to
illustrate the invention in detail.
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Example 1
It is firstly intended to elucidate the
interaction between the mixture according to the
invention and tissue. Thus, data on the distribution of
the mixture according to the invention in bone tissue
are the prerequisite for it to be possible to propose
theories about a possible mechanism of action of the
medicine. Experiments on tissue cultures are therefore
more sensible than those on cell cultures because only
in a tissue culture is it possible to study cell-cell
interactions.
1. Material and methods
1.1 Tissue material
Human bone tissue resulting from osteotomies
was made available by hospitals.
Embryonic bone tissue was obtained from 10 to
17 days old chicken embryos (callus domesticus).
1.2 Tissue culture
The tissue was transferred into the transport
medium immediately after removal. Bone fragments about
2 mm3 in size were prepared under sterile conditions
and, after determination of the weight, employed
directly for the experiments.
Earl's modified Eagle's minimal essential
medium (MEM) with 20 mM Hepes buffer was used for the
tissue culture.
Before the start of the experiment, 4o fetal
calf serum and to antibiotic solution
(penicillin/streptomycin/amphotericin B) are added to
the medium and, for the labeling experiments,
additionally 1 mM beta-aminopropionitrile, 2 mM
Na ascorbate and 2 to 10 ~g isotopes (14C-proline).
Cultivation is performed in 25 ml Erlenmeyer flasks at
37°C in a shaking water bath at the lowest frequency.
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1.3 Determination of the respiratory activity
The respiratory activity is a sensitive marker
for the metabolic activity of the tissue. Even very
small changes in the physiological condition of the
tissue are reflected by a measurable change in the
respiratory activity.
A Clark sensor (platinum/silver electrodes in
saturated potassium chloride solution) was used to
determine the respiratory activity. On applying a
voltage of 0.8 V to the electrode, the oxygen reduction
current is directly proportional to the partial oxygen
pressure in the measured solution (culture medium). The
supply of 02-saturated medium where the oxygen partial
pressure falls below a particular value, and the data
analysis take place with computer control.
Bone tissue typically has a respiratory
activity of 2-3 ~1 of Oz x min-1 x g-1. The respiratory
activity is thus in the region of the order of
magnitude of the respiratory activity of resting muscle
tissue. A typical respiratory curve for bone tissue is
shown in Fig. 1. The sawtooth-like course of the
respiratory curve shown in Fig. 1 derives from the fact
that when the Oz partial pressure in the measured
solution falls below a particular value fresh
02-saturated medium is supplied.
Fig. 2 shows average OZ consumption values from
three measurements. The oxygen consumption by embryonic
bone tissue (callus domesticus) was determined in a
tissue culture using the Clark sensor. The oxygen
consumption is between 3 and 5 ~l of OZ x min-1 x g-1.
The respiratory activity falls by about 500 over the
course of time, which is perfectly normal for tissue
cultures.
1.4 Enzyme assays
An enzyme which is thought to be closely
connected with the mineralization of bone tissue is
alkaline phosphatase. This enzyme was characterized
some time ago but there is still discussion about the
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function of the enzyme in mineralization. Since there
is a close connection between osteoblast activity and
the activity of alkaline phosphatase it is possible to
regard alkaline phosphatase as a marker of osteoblast
activity. Elevated levels of alkaline phosphatase
activity in blood serum are found during skeletal
growth in childhood, during bone regeneration and in
disorders of bone metabolism.
The activity of alkaline phosphatase was
determined in a crude extract. For this purpose, 500 mg
of tissue were mixed with 1 ml of disruption buffer and
cut up with a knife. Subsequently 500 mg of grinding
beads were added and disruption was carried out for
min. After centrifugation, the crude extract was
15 employed for the measurements.
Alkaline phosphatase is detected on the basis
of the conversion of p-nitrophenyl phosphate into
nitrophenol and phosphate. The nitrophenol produced in
the hydrolysis is yellow and can therefore be detected
20 in a photometer at a wavelength of 410 nm.
Fig. 3 shows the pH dependence of the alkaline
phosphatase activity. The activity of alkaline
phosphatase from bone was determined on the basis of
the conversion of p-nitrophenyl phosphate. The activity
maximum is at pH 10.5. At the physiological pH of 7,
alkaline phosphatase has only about 1% of the maximum
activity.
1.5 pH determination
A mixture according to the invention containing
calcium hydroxide, glycerol and neatsfoot oil in the
weight proportions 30% by weight, 30% by weight and 40%
by weight, respectively, based on the total weight of
the preparation, or an aqueous calcium hydroxide
suspension was covered with 30 ml of imidazole/HC1
buffer (1 mM, pH 7), after which the pH in the solution
was continuously followed using a pH electrode.
It emerged from these measurements that a
mixture of calcium hydroxide and glycerol in neatsfoot
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oil has fundamentally different properties from calcium
hydroxide in aqueous suspension. Fig. 4 shows that an
aqueous calcium hydroxide suspension causes an
immediate pH jump to a pH 12, and that with the
glycerol/calcium hydroxide/neatsfoot oil mixture
according to the invention there is a slow rise to a pH
above 10.
1.6 Collagen determination
The major part of the organic substance in bone
consists - as already mentioned - of collagen, a
connective tissue protein. The bone growth and
regeneration processes are associated with new collagen
synthesis. The synthesis is followed by further intra-
and extracellular collagen processes. It is possible by
using radioactive collagen precursors (14C-proline) to
quantify accurately the rate of collagen synthesis in a
tissue culture. This makes qualitative and quantitative
recording of the effect of medicines on collagen
synthesis possible.
The total collagen content is determined by the
so-called hydroxyproline assay. The amino acid
hydroxyproline occurs mainly in collagen, and the
hydroxyproline content in other proteins can be
neglected. After liberation of the amino acids from the
proteins by hydrolysis, (16 h at 116°C, 22% HC1) and
after chemical modification (oxidation of the
4-hydroxyproline to pyrrole), the total hydroxyproline
content in the assay mixture is quantified by a
specific color reaction with p-dimethylamino-
benzaldehyde.
1.7 Determination of the rate of collaaen synthesis
Collagen in tissue and medium is obtained by
Proff's (1991) modification of the method of Miller and
Rhodes (c. f. E.J. Miller and R.K. Rhodes 'Methods
Enzymol." 82:33 (1982)). Via several precipitation
steps with subsequent centrifugation, collagen is
quantified by SDS gel electrophoresis and subsequent
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scintillation measurement (determination of the
specific radioactivity). To calculate the rate of new
synthesis, the collagen content determined through the
incorporation of 19C-proline is related to the total
collagen content determined by the hydroxyproline
assay.
It is possible by quantifying the collagen
biosynthesis to investigate the effect of calcium
hydroxide products on bone formation.
Collagen biosynthesis is quantified - as
indicated in 1.7 - by the technique of radiolabeling of
the collagen. One constituent of the collagen fiber is
the amino acid proline. An exactly defined amount of
19C-labeled proline is added to the culture medium. This
proline is incorporated into the proteins newly
synthesized during incubation of the tissue. After
separation of the collagen from other proteins it is
possible, by determining the specific radioactivity, to
make an accurate quantitative statement about the rate
of new collagen synthesis.
The collagen is isolated by several
precipitation steps with subsequent centrifugation and
SDS gel electrophoresis. In the specific precipitation
of the collagen, the collagen fibers are separated from
other proteins by adjusting, by addition of sodium
chloride, to a suitable salt concentration at which the
non-collagen proteins mainly remain in solution but the
collagen separates out of the solution as a
precipitate. The collagen is sedimented by subsequent
centrifugation. In the SDS gel electrophoresis,
proteins are separated from one another in a size-
dependent manner. The proteins migrate in an electric
field through a matrix of a highly crosslinked polymer
(acrylamide). Small proteins migrate rapidly through
this matrix because it offers less resistance to the
small molecules, while large proteins migrate more
slowly because their mobility is greatly impeded by the
matrix. After staining, proteins are visible in this
gel as so-called "bands". It is possible in this way to
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identify proteins on the basis of their size using
internal size standards.
Proteins are made available for further
analysis, for example radioactivity measurement, by
cutting bands of interest out of the gel.
Extraction of collagen:
The tissue culturing (cf. 1.2) was stopped by
adding 3% acetic acid. Collagen which had dissolved was
precipitated by 2 M sodium chloride at 4°C overnight
and then recovered by centrifugation (1 h, 24,000 x g,
4°C). The sediment was taken up in 10 ml of 3o acetic
acid. Newly synthesized collagen present inside the
tissue block was included in the analysis by mechanical
disintegration of the tissue blocks. Tissue residues
were recovered by centrifugation (1 h, 45,000 x g,
4°C). The sediment was fractionated by gel
electrophoresis after solubilization. To check the
fractionation of the proteins, they were stained in the
gel.
The gel was cut up after the run into 5 mm wide
strips perpendicular to the direction of migration, and
the gel fragments were transferred into scintillation
vials and counted in a scintillation counter.
Fig. 5 shows the comparison between a vital and
a heat-denatured tissue.
This comparison is depicted in Fig. 5 on the
basis of the radioactivity distribution in the gel.
Collagen , as a relatively large protein, is found in
the region at a distance of 2 cm from the start.
A specific radioactive band can be assigned t
the collagen band detectable through Coomassie
staining.
Fig. 5 label I, head of femur, spongiosa, male,
45 years:
Difference in collagen synthesis output between vital
and heat-denatured tissues (CPM: counts per minute).
This shows the distribution of the radioactivity in the
gel. The collagen band is found in a region at a
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distance of about 2 cm from the start. A collagen band
occurs only for vital tissue, which means that the
radioactivity detectable in the gel corresponds to new
collagen synthesis during the incubation.
The vital tissue shows a detectable output of
collagen synthesis whereas the dead tissue no longer
shows any metabolic activity. This shows that the
detected radioactive collagen is in fact attributable
to new collagen synthesis in the tissue culture and not
to nonspecific binding of radioactive proline to
proteins of the bone tissue. A comparable amount of
tissue material was employed for all the experiments
(about 100 mg).
Other radiolabeled bands of smaller size can be
detected, possibly being collagen degradation products.
Degradation of collagen in tissue culture is observed
in particular in labeling experiments with an
incubation time of more than 4 days. Radiolabeled
proline is also present in the gel to a small extent,
it not having been possible to remove this completely
by the precipitations.
The tolerance of bone tissue to weakly alkaline
pH is evident from an experiment paralleling the
experiment illustrated in Fig. 5. In the parallel
experiment, the physiological Hepes buffer in the
culture medium (pH 7.4) was replaced by a bicarbonate
buffer (pH 8.0). It emerged from this that
alkalinization of the culture medium to pH 8.0 resulted
in no measurable difference in the collagen synthesis
from pH 7.4. Above pH 8.5 any increase in collagen
regeneration relative to spontaneous collagen
regeneration can no longer be seen.
Figures 6 to 9 show the amount of newly
synthesized collagen in the test batches with various
mixtures according to the invention compared with
control batches without this mixture or in the absence
of a dihydric or polyhydric alcohol. The difference in
the amount of newly synthesized collagen in the control
and in the test batch is expressed in percent. 0% means
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that there is no increase in the amount of newly
synthesized collagen compared with the control; 1000
means collagen regeneration increased two-fold compared
with the control under the influence of a mixture
according to the invention.
It is evident from Fig. 6 that in four
experiments collagen synthesis is increased by between
100 and 1200 compared with the control under the
influence of a glycerol/calcium hydroxide/neatsfoot oil
mixture (composition: 30o by weight Ca(OH)2, 30% by
weight glycerol, 40% by weight neatsfoot oil). This
increase is significant because experimental variations
in collagen synthesis output are in the range from 10
to 20%, whereas the increases determined under the
influence of the glycerol/calcium hydroxide/neatsfoot
oil mixture are between 100 and 120%. In addition,
there is evidently also an increase in collagen
synthesis output, compared with a mixture containing no
glycerol.
It is evident from Fig. 7 that in four
experiments collagen synthesis is increased by between
100 and 1200 compared with the control under the
influence of a propylene glycol/calcium
hydroxide/neatsfoot oil mixture (composition: 30s by
weight Ca(OH)2, 30o by weight propylene glycol, 40°s by
weight neatsfoot oil). This increase is significant
because experimental variations in collagen synthesis
output are in the range from 10 to 20%, whereas the
increases determined under the influence of the
propylene glycol/calcium hydroxide/neatsfoot oil
mixture are between 100 and 1200. In addition, there is
evidently also an increase in collagen synthesis
output, compared with a mixture containing no propylene
glycol.
It is evident from Fig. 8 that in four
experiments collagen synthesis is increased by between
100 and 120s compared with the control under the
influence of a glycerol/calcium hydroxide/olive oil
mixture (composition: 30o by weight Ca(OH)2, 30% by
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weight glycerol, 40% by weight olive oil). This
increase is significant because experimental variations
in collagen synthesis output are in the range from 10
to 20%, whereas the increases determined under the
influence of the glycerol/calcium hydroxide/olive oil
mixture are between 100 and 1200. In addition, there is
evidently also an increase in collagen synthesis
output, compared with a mixture containing no glycerol.
It is evident from Fig. 9 that in four
experiments collagen synthesis is increased by between
100 and 1400 compared with the control under the
influence of a glycerol/calcium hydroxide/magnesium
oxide/neatsfoot oil mixture (composition: 20o by weight
Ca(OH)2, 20o by weight glycerol, 20o by weight
magnesium oxide, 40o by weight neatsfoot oil). This
increase is significant because experimental variations
in collagen synthesis are in the range from 10 to 20 0,
whereas the increases determined under the influence of
the glycerol/calcium hydroxide/magnesium oxide/
neatsfoot oil mixture are between 100 and 1400. In
addition, there is evidently also an increase in
collagen synthesis, compared with a mixture containing
no glycerol and MgO.
Example 2
It was found in a stability test in which the
mixtures indicated below after formulation to a
kneadable or creamy mass were stored at room
temperature and ambient humidity that retention of the
desired consistency of the mixture can be extended by
at least 50% of the stability period in the presence of
a dihydric or polyhydric alcohol compared with samples
without the alcohol.
Mixtures used:
1) Glycerol/calcium hydroxide/neatsfoot oil mixture
(30°s by weight/30o by weight/40% by weight) and
2) Propylene glycol/calcium hydroxide/neatsfoot oil
mixture (30o by weight/30o by weight/40s by weight) and
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3) Glycerol/calcium hydroxide/olive oil mixture (30o by
weight/30% by weight/40o by weight)
1st mixture without glycerol with glycerol
kneadable 6 months 12 months
consistency
creamy 12 months 18 months
consistency
2nd mixture without propylene with propylene
1 col 1 col
kneadable 6 months 12 months
consistency
creamy 12 months 18 months
consistency
3rd mixture without olive oil with olive oil
kneadable 6 months 12 months
consistency
creamy 12 months 18 months
consistency
