Note: Descriptions are shown in the official language in which they were submitted.
~S5589
Technical Field
The invention is in the field of body treating
compositions and methods. More particularly it concerns
an injectable, cross-linked collagen implant material
for augmenting soft tissue in mammals.
Back~round Art
Collagen has been used as a pharmaceutical
carrier, as a surgical prosthesis (sutures and wound
dressings), and as an implant material. Chvapil, et al,
Intl Rev of Connective Tissue Res (1973) 6:1. In many
instances the collagen was cross-linked with chemical
agents, radiation, or other means to improve its mechanic-
al properties, decrease its immunogenicity, and/or
increase its resistance to resorption. This prior
cross-linked collagen was solid in nature. Implants
made from solid cross-linked collagen were implanted
surgically (i~e., were emplaced through incision).
Oliver, et al, Clinical Orthopaedics & Re-lated
Research (1976) 115:291~302, Br J Exp Path (1980) 61:544-
549, and Conn Tissue Res (1981) 9:59-62, describe implants
made by treating skin with trypsin followed by cross-
linking with an aldehyde. The resulting solid collagen
implants were reported to maintain their original mass
after prolonged implantation. A main problem with such
solid implants is that they must be implanted surgically.
Other disadvantages are that they are not as deformable
as injectable implants and residual glutaraldehyde may
cause the implant to lose its flexibility due to continu-
ing in situ cross-linking.
Schechter, et al, Br J Plas Surq (1975) 28:
198-202 disclose glutaraldehyde cross-linked skin that
was soaked in L-alanine after cross-linking. ~he article
postulates that the exposure of the skin to L-alanine
blocked residual reactive groups o~ the aldehyde, thereby
preventing the release of toxic molecules generated by
such groups.
iz~cj58~
--2--
U.S. Patent No. 3,949,073 describes the use
of ate~opeptide solutions of collagen as an injectable
implant material for augmenting soft tissue. According
to the patent, the collagen is reconstituted before
implantation and forms a fibrous mass of tissue when
implanted. The patent suggests adding particles of
insoluble collagen microfibrils to control the shrink-
age of the fibrous mass formed at the augmentation site.
ZYDERM ~ collagen implant is a commercial embodiment of
the material described in the patent and is composed of
reconstituted atelopeptide collagen in saline that con-
tains a small amount of a local anesthetic. While this
commercial material is remarkably effective, it may
shrink in volume after implantation due primarily to
absorption of its fluid component by the body. Thus,
if volume constancy, sometimes called "persistency", is
essential, an additional injection or injections of
supplemental implant material is required.
Commonly owned U.S. Patent No. 4,424,208 des-
cribes an injectable dispersion of cross-linked
atelopeptide collagen and reconstituted atelopeptide
collagen fibers in an aqueous carrier. The dispersion
exhibited improved persistence in an animal model as
compared to ZYDERM ~ collagen implant.
A principal object of the present invention is
to provide a cross-linked collagen implant material that
is useful for dermal augmentation and that (1) is uniform
i.e., it contains only a single physical form of collagen
as compared to the two physical collagen forms contained
in the implant material of U.S. Patent No. 4,424,208,
(2) has improved injectability as compared to the dis-
persions of U.S. Patent No. 4,424,208, and (3) has
- improved persistence (reduced solubility, enhanced
resistance to proteolytic degradation) and resistance
to physical deformation relative to Z~DERM ~ collagen
implant.
- i~2SSS~39
Disclosur~ of the Invention
In accordance with one aspect of the inven-
tion, there is provided a novel cross-linked atelo-
peptide collagen for use as an injectable a~ueous
suspension for augmenting soft tissue that:
(a) is substantially free of residual
cross-linking agent;
(b) has greater than about 15 free lysine
residues per 1000 amino acid residues, and
(c) is composed substantially of fibrous
particles which when in suspension in physiological
saline at a concentration of 35 mg/ml exhibit a shear
viscosity whose log varies linearly with the log of
the shear rate and is approximated by the formula
log ~ < -0.96 log y ~ 2.3 (1)
where ~ is the shear rate in sec 1, log y is in the
range of -6 to +2 and ~ is the shear viscosity of
the suspension in Pascal-sec.
According to another aspect of the invention,
there is also provided a collagen implant material for
use in augmenting soft tissue, comprising an aqueous
dispersion of a cross-linked atelopeptide collagen as
defined above.
According to yet another aspect of the inven-
tion, there is provided a process for preparing a crosslinked atelopeptide collagen as defined above, comprising
reconstituting at.elopeptide collagen:from an..acidic -
aqueous so:Lution by neutraliæing the solution at a
reduced temperature and at a hypotonic ionic-strength,
cross-linklng the reconstituted atelopeptide collagen
in an aqueous medium at a concentration of about 0.1 to
10 mg/ml with a cross-linking agent at a concentration
of about 0.,001% to about 0.05% by weight that forms
covalent bonds with the collagen under conditions suffi-
~5589
--4--
cient to produce a fibrous cross~linked collagen thathas greater than about 15 free lysine residues per
1000 amino acid residues and that when in suspension
in physiological saline at a concentration of 35 mg/ml
exhibits a shear viscosity whose log varies linearly
with the log of the shear rate and is approximated by
the formula
log rl -0.96 log y + 2.3
where y is the shear rate in sec 1, log y is in the
range o~ -6 to ~2 and ~ is the shear viscosity of
the suspension in Pascal-sec; optionally quenching the
cross-linking reaction with a quenching agent that
reacts with the cross-linking agent; and separating the
cross-linked atelopeptide collagen from the reaction
mixture~
Still another aspect of the invention is a
method for augmenting soEt tissue in a living mammal
comprising injecting the above described collagen
implant material into the mammal at the augmentation
site.
Modes for Carryinq Out the Invention
The cross-linked collagen used in the invention
may be derived from collagen collected from any number
of mammalian sources. The donor need not be genetically
similar to the host into which the material is ultimately
implanted. Because of their availability, bovine or
porcine corium will usually be employed. The ~irst step
in making the cross-linked collagen is to prepare atelo-
peptide collagen in solution from the corium. The animal
skin is soEtened by soaking it in a mild acid and then
scraping it to remove hair, epidermis, and fat. The
depilated skin is then soaked again in mild acid and then
comminuted by grinding, mincing, milling or like physical
treatment. The comminution prepares the skin ~or solubili-
zation.
The divided tissue may be solubilized under non-
i~5558~
denaturing conditions by dispersing it in an aqueousmedium and digesting it with a proteolytic enzyme
other than a collagenase, preferably an enzyme that
is active at acidic pHs. Dilute acid solutions at low
temperatures will normally be used to avoid denatura-
tion. Mineral acids such as HCl or carboxylic acids
such as acetic, malonic or lactic acids may be used.
The pH will normally be in the range of about 1.5 to
5, depending on the enzyme used, and the temperature
about 5C to 25C. A preferred procedure is to dis-
perse the comminuted tissue in HCl to a concentration
of 1 to 5 g/l at a p~I of about 2 at 20C. After the
tissue is dispersed the enzyme is added and the mixture
is incubated to permit the enzyme to digest the telo-
peptide and other solubilizable components of thetissue. Enzymes that attack the telopeptide portion
of the collagen while not denaturing the helical por-
tion are used. Examples of such enzymes are pepsin and
papain. Pepsin is preferred because it is relatively
easil~ deactivated and removed from the solubilized
collagen. The enzyme concentration will usually be in
the range of about 0.1% to 10% by weight based on the
collagen. The incubation period will typically vary
from about two days to two weeks. The progress of the
solubilization may be monitored by determining the vis-
cosity of the solution. Once the viscosity reaches a
substantially constant level, the solubilization is
complete. At this point, the enzyme is deactivated
(denatured~ and removed.
The enzyme may be deactivated by raising the
pH of the solution to at least about 7 by adding an
alkaline material such as sodium hydroxide. After the
enzyme has been denatured the solution is treated to
remove denatured enzyme and the portions of the tissue
that were digested during the solubilization. Variousdialysis, sedimentation, and filtration techniques may
.~S5S~
be used to effect such removal. See U.S. Patents Nos.
3,949,073 col 3, lines 10-22 and 4,140,537 col 5, line
48 to col 6, line 3~. A preferred procedure is to
first lower the pH by adding acid and then clarify the
solution by diatomaceous earth sedimentation. The
sediment is filtered and the filtrate is concentrated.
The concentrate is then fractionated by ion exchange
chromatography and further concentrated to produce a
substantially pure atelopeptide collagen solution that
may be used to make the cross-linked collagen.
The next step in making the cross-linked
collagen is to reconstitute the atelopeptide collagen
from solution. The reconstitution is preferably done
by neutralizing the solution at reduced temperatures,
preferably about 10C to 25C. The ionic strength of
the neutralized solution is preferably hypotonic rela-
tive to physiological conditions. Ionic strengths in
the range of about 0.03 to about 0.1, preferably about
0.06, will typically be used. The neutralization
involves raising the pH of the solution by adding an
appropriate base or buffer, such as ~a2HPO4 or ~aOH,
to a level at which the collagen in solution reaggregates
into fibrils. Fiber formation occurs under these con-
ditions at pHs in the range of about 4.9 and about 10Ø
The final pH is preferably in the range of about 5 and 80
Within this range pHs below about 7 favor formation of
fine, soft fibrils whereas pHs above about 7 favor forma-
tion of coarser fibrils. Such texture makes the soft
fibril dispersion easier to inject. The duration of the
fibril formation step will normally be in the range of
about l/2 to about 18 hr.
The injectability of the ultimate cross-linked
product ma~ be enhanced by forcing the suspension of
collagen fibers through a screen of defined pore size
after or cluring the reconstitution step. This procedure,
called "screening", provides a preferred starting material
1~2S5S89
for the cross-linking step. Sereening breaks up any
fibrillar aggregates that may be in the reconstituted
fiber suspension and gives a more uniform fiber size
distribution. A preferred screening protocol is to
repeatedly pass the fiber suspension through a 60
mesh stainless steel screen at about 5C and a flow
rate of ahout 7-7.5 l/min about 2-3 hr after the
fibers precipitate. The suspension is reeirculated
through the sereen for about 4-5 hr, with about 35
passes through the sereen being optimum. After the
sereening the fiber suspens:ion is incubated in the
reconstitution medium for about 6 to 15 hr.
The resulting reconstituted atelopeptide fibrous
collagen geL suspension is then eross-linked with a eross-
linking agent that forms eovalent bonds between itselfand the collagen. Usually the agent will be polyfunction-
al, and more usually bifunctional. The cross-linking
conditions are sueh as to produee a eo~alently cross-
linked collagen that may be formulated as an injeetable
fluid and that provides an implant that has improved
persistenee relative to an implant made from a eomparable
formulation of non-eross-linked fibrous atelopeptide
collagen. When this degree of cross-linking has been
reaehe~ the cross-linking reaetion is optionally quenehed
by adding a quenching agent. The quenehing agent forms a
water soluble adduet with the eross-linking agent. The
eoneentration of collagen in the suspension at the time of
eross-linking, the concentration of cross-linking agent,
and the duration of cross~linking reaction are important
proeess eonditions as regards obtaining the kind and
degree of cross-linking that provides a product having
enhanced injectability. The coneentration of collagen in
the suspension must be sufficiently low to cause the
l~S55~
cross-linking to be substantially intrafibrillar rather
than interfibrillar. With intrafibrillar cros~-linking
the collagen particles can still flow, whereas at high
collaqen concentrations ther~e is significant
interpartilcle cross-linking and the product becomes
solid or too vi~cous to flow. The collagen
concentration at the time of cross-linking will usually
be in the range of 0.1 to 10 mg/ml, more u6ual~y 1 to 5
mg/ml. Aldehydes are preferred cross-linking agents.
Examples of aldehydes that may be u~ed to cross-link the
collagen are formaldehyde, aaetaldehyde, glyoxal pyruvic
aldehyde, and dialdehyde starch. Glutaraldehyde i8
particularly preferred. Compounds that have function~l
groups that react with the functional groups of the
cross-linking agent (e.g., aldehyde group~ to form water
~oluble adducts may be used to guench the cros6-linking
reactlon. Quenching agents that have free amino groups
such as amino acids are preferred. Glycine is
particularly preferred. The concentration of glutar-
aldehyde in the reaction mixture will typically be about0.001% to about o.os% by weight. The glutaraldehyde
reacts with ly~ine residues of the collagen fiber6
thereby reducing the number of free lysines p~r lOOo
amino acid residues in the collagen. At the
glutaraldehyde concentrations mentioned above, the
number of free lysine residues per 1000 residues
remaining after cross-linking will be greater than about
15 per loO0, more usually greater than about 20 per
loO0. Lysine conten~ may be mea~ured by reducing tha
cross-linked collagen with borohydride and hydrolyzing
the reduced material under vacuum in 5 . 7 N HCl for 24 hr
at 100C. Amino acid analysis may be performed with
available analyzers (e.g., a Durrum Model D-500
analyzer) and the lysine residues guan~itated by
lZS55~9
9
comparing the lysine/alanine ratio to those ob6erved in
noncrofis-linked controls.
The duration of the cross-linking reaction will
usually be in the range of one-half hr to about one
week. The reaction will norl~ally be carried out at
about 10~ to about 35~C. Tlhe quenching agent i8 added
in at least stoichiometric proportions relative to the
cross-linking a~ent.
A particularly preferred cros~-linking protocol
0 i6 about 3 mg/ml collayen concentration; abaut 0.01%
by weight glutaraldehyde for about 16 hr at
approximately Z2C.
A~ter the cro66-linking reaction has been
terminated the cross-linked atelopeptidH collagen
product may be wa~hed with an aqueous buffer solution to
remove unreacted aldehyde~ aldehyde polymers, and, i~
quenching was employed, unreacted guenching agent and
aldehyde-quenching agent adduct6. A 60dium
phosphate-~odium chloride bufer solution, pH 6.9 to
7.4, is preferred. The particle 6ize of the
cross-linked collagen i8 normally sub~antially less
and, when a Rcreened 6tarting material i~ u~ed, more
uniform than that of the cro6~-linked collagen of U.S.
Patent No. 4,4Z8,208. The particle size of cross-linked
collagen made with un~craened starting material i8
nominally (large~t dimen~ion) less than about 750
microns, usually between about 75 and 750 micron~. The
wa~hed product may be soncentrated, ~uch as by
filtration or centrifugation, to a suitable pro~ein
concentration range, typlcally about Z0 to 65 mg~ml,
more usually about ~5 to 40 mg/ml. Protein
concentration may be adju6ted to thi~ ran~e by addition
of buffer or further concentration, as the case may be.
The wa~hed product will have a free aldehyde content
lZSSS8~
- 10--
below about 20 ppm and a visco~ity ln the range of about
700 to about 3000 cp at Z2C, mea6ured by an oscillating
di~k viscometer which mea6ures dynamic, not steady flow
Vi8CoBity, (Nametre Co., model 7.006 P~D). A more
definitive way of expressing the rheology oP aqueous
su~pen~io~s of the cro~s-linked collagen is by sh~ar
vi6cosity as mea6ured with a Contrave~ ~heomat Model 135
(Contraves AG, Zurich, Switzerland~ viscometer ~itted
with a Couette cell. At a concentration of 35 my/ml in
phy~iological saline the shear viscosity of the cross~
linked collagen of the invention measured in this manner
i8 approximated ~y formula (1) above.
The shear vi~cogity of similar 6uspension6 of
the cross-linked collagen of U.S. Patent No. 4,428,200
i8 2 to 2.5 time6 greater than the shear viscosity of
the su~pension6 of the present invention made with
unscreened ~tarting materials.
Final formulation o~ the aqueous 6uspen~ion o~
cross-linked, quenched collagen will typically involve
adjusting ~he ioni~ 6trength of the 6uspen6ion to
isotonicity (i.e., about 0.15 to about 0.2) and adding a
local anesthetic, ~uch ae lidocaine. to a ~oncentration
of about 0.3% by weight to reduce local pain upon
in~e~tion. The suspen~ion i6 then loaded into syringe~
~5 fitted with a ~25 gauge or larger gauge needle for
injection. In the case of formulations used ~or dermal
augmentation the term "injectable" means the formulation
can be dispensed from syringe~ having a gauge as low a~
#25 under normal conditions under normal manual presfiure
without substantial spiking. ~he abovs de~cribed stepfi
in the proce~s for preparing the novel injectable
cross-linked collagen are preferably carried out in
sterile conditions using sterile materials.
... . .. .. .
lZSSS~
The above de6cribed collagen implant material
may be in~ected intradermally or subcutaneously to
augment 80~t ti6~ue, to repair or correct congenital
anomalie~, acquired de~ects Ol cosmetic defects.
Example~ og 6uch conditions are congenital anomalies
~uch as hemifacial micro~omia, malar and zygomatic
hypoplasia0 unilateral mammary hypoplasia, pectu~
excavatum, pectoraliæ agenesis (Poland' 8 anomaly) and
velopharyngeal incompetence secondary to cleft palate
repair or ~ubmucous ~left palate (as a retropharyngeal
implant); acquired defects (post traumatic, po~t
surgical, post infectious) such as depre~ed scars,
subcutaneous atrophy (e.g., secondary to di~coid lupi8
erythemato~is), keratotic le~ions, enophthalmos in the
enucleated eye (also superior sulcus syndrome), acne
pitting of the face, linear scleroderma with
subcutaneous atrophy, ~addle-nose deformity, Romberg's
disease and unilateral vo~al cord paraly6i6; and
co~metic defect~ ~uch a~; glabellar frown lines, deep
na~olabial creases, circum-oral geographical wrinkles,
sunken cheeks and mammary hypoplasia.
The ~ollowing examples illustrate the
cros~-linked ~ollagen, implant material~ made therefrDm,
the method by which the materials used, and the merits
oP implant~ made of these materials. These examples are
not intended to limit the invention in any manner.
Preparation of AteloPePtide_Bovine Collagen Solution
Bovine hide wa~ ~oftened and depilated by
treatment wit~ HCl. The hide wa~ then comminuted and
di~per~ed in HCl, pH 2, at 8-11 g~l. Pep~in was added
to the dispersion at 0.1% by weight ba~ed on total
protein and the ~ixture was allowed to incubate for
about 100-300 hr at 15C to Zo~C. NaO~ was then added
l~S~89
-12-
to raise the pH of the incubation medium to about 7 and
thereby terminatQ the digestion. The denatured enzyme
was removed from the reaction mixture by sedimentation
at reduced pH. The solution was then purif~ed and
concen~rated by filtration and chromatography to form a
3 mg/ml ~clution of atelopeptide bovine collagen in
dilute agueous HCl, pH 1-4. This ~olution i8
hereinafter referred to as CIS.
Reconstitution of Fibrous Collaqen from CIS
Fibrous collagen was reconstituted from CIS by
adding 0.02 M NazHPO4 to the CIS at 15C to 22C to
increase its pH to 7.4 + O.Z or 5.8 to 6.5. Fibers were
allowed to form for 1-2 hr.
Preparation o~ Cro~-Linked Viscou~ Collaqen
A. Using Collagen Reconstituted at pH 7.4 ~ 0.2.
To one hundred-sixty ml of the neutral
reconstituted fibrous collagen 6uspension wa~ added 1.62
ml oP 1.0~ aqueou~ glutaraldehyde at pH 3. The
glutaraldehyde 601ution wa~ added gradually with
stirring to attain a ~inal level of 0.01%. After a
reaction period of 16 hr the reaction was guenched by
adding 3 M gIycine to 0.2 M. The guench time was one
hr. The cross-linked collagen was then washed three
timefi with approximately 100 ml of buffer, 0.02 M
NazHPO4, 0.13 M NaCl, pH 7.4, with centrifuging at
17000 x g for 5 to 7 min between each wash. The
dyanamic ~iscosity of the collagen was mea~ured by an
oscillating disc device (Nametre Co., model 7.006 P~D~
measuremen~ at a~6hear rate of about 5000 sec 1) and
found to be approximately 700 cp at 22C. After the
final wash and centrifugation the collagen wa~
resuspended in the buffer to a protein concentration of
l~S~S~3~
-13-
about 1~.~ mg~ml. Thi6 di~persion was loaded into
~yringes ~itted with a #Z7 gauge needle. This collagen
preparation i~ hareina~ter designated preparation C.
Preparations of injectable cro66-linked
collagen ~luid were carried out a6 above using differing
cross-linlcing reaction time~3, glutaraldehyde
concentration6 and final protein concentrations. Thes~
preparation& are listed below.
Final Protein
10 Preparation % Glutaral-Reaction Concentration
De6i nation _ dehydeTime (hr
E 0.051 29.9
G 0.0516 ll.4
B. Using Collagen Recon~tituted at pH 5.8 to 6.5.
one % aqueous glutaraldehyde, pH 3, wa~ added
to the reconstituted fibrou6 collagen di6persion
gradually with 6tirring to give a final glutaraldehyde
concentration of 0.005%-0.01%. Cros~-linking wa~
allowed ~o proceed with ~tirring for 16 hr. The
reaction wa6 then quenched by adding ZM glycine with
stirring to a final concentration of 0.2 to 0.3M.
Quenching ~ontinued with 6tirring for 2-3 hr. The
quenched, cros~-linked collagen wa6 then centrifuged at
25 16,000 x g for 5 t~ 10 min, harvested, and re6u6pended
in 10 to ~0 vol of 0.02M Na~HP04, 0.13M NaCl, pH
7.4. This su6pen6ion was centri~uged at 16,000 ~ g for
5 to Z0 mi,n and the ~inal viscous cro~6-linked collagen
product wa~ harvested. After the final centrifugation
the protein con~entration wa~ 45 ~ 5 mg~ml.
In Viv Testin~l of Collaqen Preparation~
Sprague-Dawley female rat6 45-50 days old
weighin~ 120 ~ 20 g were u~ed as implant recipients.
l~S~S~3~
-14-
Groups of three rats were implanted with one of
preparations A, C, E and G and with ZYDERM0 collagen
implant a~ a control material. Each animal wa~
implanted in two ~ites: the cross-linked ~ollagen
~ 5 preparaeion in the right suprascapular region: and the
con~rol material in the left supra6capular region.
Approximat,ely one cc of material per site was injected
into the subcutaneum.
All materials were explanted in the fifteenth
day post implantation. Host ti6sues were care~ully
dissected from the explants, and the wet weights were
recorded. The percent weight recovery (psrsistence) was
then calculated from the weight implanted. Weighed
- ~pecimens were then embedded, sectioned, and stained for
histological examination. Stains used included
hematoxylin~ eosin, and Gomori trichrome.
Results of Testinq
Summaries of ~he histological examinationæ of
preparations A, C, E, and G follow:
Preparation A:
This material presented a fairly uniform lacey
appearance as compared to implants of more highly
cross linked non~glycine guenched preparations. The
latter formulations were generally organized into large
densely-packed ~egments with intervening clef tR ~ Cle~
also occurred within the preparation ~ implants but they
were smaller and fewer in number. Fibroblast
inf iltration was excellent throughout the substance o~
~he preparation A implants as well a~ within the small
intervening cleftE. New collagen synthesi~ appeared to
be occurring wlthin the clefts. Very few round cell6
were observed. Vascular channels were good to moderate
.. . 1~5S.r~8
-15-
ln the peri~heral one-half of the preparation A implants
and were not limited to zone~ of new collagen
synthe~i~. No evidence of encap~ulation was observ~d.
Epithelioicl ~e11B and multinucleate~ were absent.
Preparation C:
Thi~ material wa~ even more lacey and porous
than preparation A. It ~howed excellent diPfu~e cell
inflltration along with area~ of new collagen
syntha~is. These characteri~tics, along with the
general paucity of round cells, make thi6 material the
best of the four preparation~ from a hi6tological
viewpoint.
Preparation E:
Thi6 preparation wa6 generally less lacey than
those cro6s-linked with 0.01% glutaraldehyde.
Fibro~la~ts were diffu6ely di6tributed but ~ewer in
numbers and uascularization was generally limited to the
peripheral one-third of the implants. Area~ of new
collagen synthesis were al80 ob6erved le~s frequently.
Preparation G:
Thi6 preparation exhibited the most uniform
lacey porous appearance of all the material~
cross-l~nked with 0.05% glutaraIdehyde. Although cell
migration into thi6 material wa6 good, focal areas of
multinucleates and an increa6e in the number of round
cells were al~o apparent: thi6 had not been ob~erved in
preparations cross-linked with 0.01% gluta~aldehyde.
The following table report6 the re~ult6 of the
persistence (percent wet weight recovery of car~ully
dis~ected explanted ~aterial relative to wet weight of
.... . . .. .
:12S55t~{3
-16-
implant) evaluations of the peeparation~ and the control
mateeials measurQd at 17 days post implantation.
Preparation Persistence
h 77%
C 68 + 5%
E 82 ~ Z%
G - 64 ~ 5%
Control ~avg) 30-40%
PreParation of CrOB~-Lillked V:i8cou8 Collagen ~ithout
Ouenchinq
Neutral (pH 7.4 + O.Z) reconstituted ~ibrou~
collagen prepared a~ above was used. One % aqueou~
glutaraldehyde was added to two par~s by volume of the
reconstituted fibrous collagen to a final glutaraldehyde ~,
concentration of 0.01%. The mixture was mixed for 10
min and incubated Por 16 hr at room temperature. For
comparison, one part by volume of the mixture wa6
withdrawn~ 3 ~ glycine was added to it to a ~inal
concentration of O.Z M, and it was incubated for 2 hr.
Poth aliquots (glycine-quqn~hed and nonguenched) were
then cen~rifuged at 17,000 x g for 5 min. The re6ulting
pellets ware each resuspended in 250-~00 ml bufer, 002
M NazHPO4, 0.13 ~ NaCl, pH 7.4, for 30 min at room
temperature and centrifuged at 17,000 x g for 25 min.
After centrifugation the protein concentrations were
checked and adjusted to 30-~0 mg/~l by recentrifuging or
addition of buffer. The dispersions were homogeni~ed
and loaded into syrinqe6 fitted with #Z7 or ~30 gauge
needles.
S55~3~3
-17-
In vivo Te~tinq of Nonnuenched and Ouenched PreParatlons
The nonguenched cro~s-linked disper~ion and the
comparison quenched cross-linked collagen disper~ion
desceibed above were subjected to in vivo testing in
rats and guinea pigs by the general procedure described
ahove except that 0.5 cc of material per site was
injected. Th~ re6ultB of the histologic~l and weight
persi~tance analyses indicated there was no significant
difference in the biocompatibility of the nonquenched
and guenched materials.
Physico-chemical Testing of_Non uen_bed and Ouenched
Materials
Qualitative evaluation~ of the ea6e of
extrudability of the materials were made by extruding
the material6 after storage for up to four weeks at 37C
through #30 gauge needles and counting the degree and
- magnitude of ~piking (number of spikes > Z x minimum
force in newtons). These tests indicated that the
extrudability of the quenched material was 61ightly
better than the nonquenched material.
Evaluations of the color change of the two
material6 after storage at 37C indicated that the
nonquenched material demonstrated 6ignificantly les6
color change than the guenched material.
As~ays for protein concentration, pH, free
aldehyde, eoluble amine, ~oluble protein and ly6ine
content 6howed minimal dif~erence6 be~ween the two
materials except for concentration~ of free aldehyde
(18% le6s in quenched material) and soluble amine
(~ubstantially higher in quenc~ed material).
The conclusion reached from the in vivo and
physico-chemical te6ts on the quenched and nonquenched
materials was that the two materials were sub6tantially
. .
1~55~
.
18-
eguivalent functionally and that the quenching ~tep i8,
tharefore, optional.
Rheoloqy a~d Characterization of Cro6s-lin~ed ~ollaae~
Suspension~
S Nonquenched ~uspensions of fibrous cros6-linked
collagen were prepared by the general procedur~
described above. The buffered noncross-linked collagen
fiber suspension was not screened. The-pellet was
resuspended in physiological saline at 35 mg/ml.
For compari60n cross-linked collagen was
prepared a~ described in U.S. Patent No. 4,42~,20B and
suspendeld at 30 mg/ml in physlological salina and in
physiological saline in admixture with ~ibrous collagen
(15%), total protein 30 mg/ml, as described in ~he
patent.
Shear vi6cosities of the6e Bu~pen6ions were
determined using a Contrave~ Rheomat Model 135
viscomete~ (per manufacturer' B instructions) over a log
shear rate range of ~6 to +2 ~ec 1. The log of 6hear
viscosity varied linearly with the log ~hear rate vver
this range. The 6hear visco6ities of the patent
suspen~ion~ were approximately 2 to 2.5 greater-than
that of the suspen~ion of the invention material.
Sample~ oP each of the suspensions were
~ompressed between microscope 61ides and examined at
10-?5 x in a dis~ecting micro6cope. Both ~uspen6ion6 of
U.S. Patent No. 4,428,Z08 expressed 1/4 to 1/3 of their
volume as free liquid under mild compres~ive force and
the remaining ~emi-solid residue included fibrou~
particles hlving nominal diameters from 117 to ZoO0
microns. Much les~ free liquid expressed from the
invention suspension at a comparable compre6sive force
~ZS5S~9
--19--
with the compressed particles having a fibrous globular
shapa~ 88 to 710 microns nominal diameter.
Modification~ of the above described
embodlments of the invention that are obvious to thosa
5 . o~ skill in the biochemical, medical. and/or surgical
arts are intended to be within the scope of the
following claims.
