U.S. patent application number 10/704919 was filed with the patent office on 2004-05-20 for injectable microspheres for dermal augmentation and tissue bulking.
This patent application is currently assigned to BioSphere Medical, Inc.. Invention is credited to Boschetti, Egisto, Thomas, Richard, Vogel, Jean-Marie.
Application Number | 20040096514 10/704919 |
Document ID | / |
Family ID | 24108049 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040096514 |
Kind Code |
A1 |
Vogel, Jean-Marie ; et
al. |
May 20, 2004 |
Injectable microspheres for dermal augmentation and tissue
bulking
Abstract
The present invention relates to elastic, hydrophilic and
substantially spherical microspheres useful for dermal augmentation
and tissue bulking. The invention provides injectable compositions
comprising the microspheres and a biocompatible carrier for use in
dermal augmentation. The present invention further provides methods
of dermal augmentation and tissue bulking, particularly for the
treatment of skin contour deficiencies, Gastro-esophageal reflux
disease, urinary incontinence, and urinary reflux disease, using
the injectable compositions.
Inventors: |
Vogel, Jean-Marie;
(Boxborough, MA) ; Thomas, Richard; (Lincoln,
MA) ; Boschetti, Egisto; (Gougenot, FR) |
Correspondence
Address: |
JONES DAY
51 Louisiana Aveue, N.W
WASHINGTON
DC
20001-2113
US
|
Assignee: |
BioSphere Medical, Inc.
|
Family ID: |
24108049 |
Appl. No.: |
10/704919 |
Filed: |
November 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10704919 |
Nov 12, 2003 |
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09528991 |
Mar 20, 2000 |
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6660301 |
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09528991 |
Mar 20, 2000 |
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09263773 |
Mar 5, 1999 |
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6335028 |
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Current U.S.
Class: |
424/489 |
Current CPC
Class: |
A61K 49/0442 20130101;
A61P 13/00 20180101; A61K 9/14 20130101; A61L 2400/06 20130101;
A61P 1/04 20180101; A61P 17/02 20180101; A61L 31/005 20130101; A61K
31/785 20130101; A61K 9/1641 20130101; A61K 47/6925 20170801; A61L
31/14 20130101; A61L 27/50 20130101; A61L 27/38 20130101; A61K
9/1635 20130101; A61K 9/0019 20130101; A61K 9/16 20130101; A61K
47/6927 20170801; A61K 49/048 20130101; A61K 31/78 20130101 |
Class at
Publication: |
424/489 |
International
Class: |
A61K 009/14 |
Claims
What is claimed is:
1. A method for dermal augmentation in a mammal comprising
injecting a composition of elastic, hydrophilic, non-toxic and
substantially spherical microspheres in a biocompatible carrier to
said mammal through a needle of about 30 gauge or smaller.
2. The method of claim 1, wherein the composition is a suspension
of said microspheres in said biocompatible carrier.
3. The method of claim 1, wherein there is no aggregation or
clumping of the microspheres prior to or during the
administration.
4. The method of claim 1, wherein the microspheres comprise one or
more elastomers.
5. The method of claim 4, wherein the elastomers are selected from
the group consisting of acrylic polymers, acrylamide polymers,
vinyl alcohol polymers, acrylate polymers, polysaccharides,
silicones, or derivatives and mixtures thereof.
6. The method of claim 1, wherein the composition further comprises
therapeutic agent, radio-pacifying agent, contrast media, or
mixtures thereof.
7. The method of claim 6, wherein said therapeutic agents are bound
to the microspheres.
8. The method of claim 1, wherein the microspheres are capable of
being chemically modified to have therapeutic effects,
anti-inflammatory effects, anti-bacterial effects, anti-histamine
effects, or combinations thereof.
9. The method of claim 8, wherein the chemical modification of the
microspheres are caused by interactions between the microspheres
and neighboring tissues after administration thereof.
10. The method of claim 1, wherein the administration comprises
injecting said composition into an area of said mammal in need of
dermal augmentation.
11. The method of claim 10, wherein the administration comprises
injecting said composition into the subcutaneous layer.
12. The method of claim 1, wherein the dermal augmentation is for
treatment of contour deficiencies of said mammal.
13. The method of claim 12, wherein the contour deficiencies are
caused by aging, environmental exposure, weight loss, child
bearing, surgery, disease, or combinations thereof.
14. The method of claim 13, wherein the disease is acne, skin
cancer, or combination thereof.
15. The method of claim 13, wherein the contour deficiencies are
one or more of the group consisting of frown lines, worry lines,
wrinkles, crow's feet, marionette lines, stretch marks, and
internal or external scars resulted from injury, wound, surgery,
bites, cuts, or accident.
16. The method of claim 1, wherein the mammal is human.
17. The method of claim 1, wherein the administration comprises
injecting said composition extracorporeally into organs, components
of organs, or tissues prior to their inclusion into said mammal's
body, organs, or components of organs.
18. A kit for performing dermal augmentation comprising: (a) a 30
gauge or smaller needle; (b) means for injecting a liquid based
composition through said needle; and (c) biocompatible, elastic,
hydrophilic, non-toxic and substantially spherical microspheres
injectable through said needle and are not capable of being
eliminated by macrophage or other elements of said mammal's immune
system after injection thereof.
19. The kit of claim 18, wherein the means for injection is a
syringe corresponding to said needle.
20. The kit of claim 18, further comprising a liquid based
biocompatible carrier injectable through said needle.
21. The kit of claim 20, wherein the microspheres are suspended in
the biocompatible carrier.
22. The kit of claim 21, wherein the microspheres are associated
with cells.
23. A method of tissue bulking in a mammal comprising injecting a
composition of elastic, hydrophilic, non-toxic and substantially
spherical microspheres in a biocompatible carrier to said mammal
through a needle of about 18 to about 26 gauge.
24. The method of claim 23, wherein the composition is a suspension
of said microspheres in said biocompatible carrier.
25. The method of claim 23, wherein there is no aggregation or
clumping of the microspheres prior to or during the
administration.
26. The method of claim 23, wherein the microspheres comprise one
or more elastomers.
27. The method of claim 26, wherein the elastomers are selected
from the group consisting of acrylic polymers, acrylamide polymers,
vinyl alcohol polymers, acrylate polymers, polysaccharides,
silicones, or derivatives and mixtures thereof.
28. The method of claim 23, wherein the composition further
comprises therapeutic agent, radio-pacifying agent, contrast media,
or mixtures thereof.
29. The method of claim 28, wherein said therapeutic agents are
bound to the microspheres.
30. The method of claim 23, wherein the microspheres are capable of
being chemically modified to have therapeutic effects,
vascularization effects, anti-vascularization effects,
visualization properties, anti-inflammatory effects, anti-bacterial
effects, anti-histamine effects, or combinations thereof.
31. The method of claim 30, wherein the chemical modification of
the microspheres are caused by interactions between the
microspheres and neighboring tissues after administration
thereof.
32. The method of claim 23, wherein the administration comprises
injecting said composition into an area of said mammal in need of
tissue bulking.
33. The method of claim 32, wherein the injection is into the vocal
cord.
34. The method of claim 23, the tissue bulking is for the treatment
of gastroesophageal reflux disease.
35. The method of claim 34, wherein the administration comprises
injecting said composition into the lower esophageal sphincter or
the diaphragm of said mammal.
36. The method of claim 23, wherein the tissue bulking is for the
treatment of urinary incontinence or urinary reflux disease.
37. The method of claim 36, wherein the administration comprises
injecting said composition into the bladder sphincter or urethra of
said mammal.
38. The method of claim 36, wherein the urinary incontinence is
caused by bladder-neck hypermobility.
39. The method of claim 23, wherein the mammal is a human.
40. The method of claim 23, wherein the administration comprises
injecting said composition extracorporeally into organs, components
of organs, or tissues prior to their inclusion into said mammal's
body, organs, or components of organs.
40. A kit for performing tissue bulking comprising: (a) an 18 to 26
gauge needle; (b) means for injecting a liquid based composition
through said needle; and (c) biocompatible, elastic, hydrophilic,
non-toxic and substantially spherical microspheres injectable
through said needle and are not capable of being digested or
eliminated by macrophage or other elements of said mammal's immune
or lymphatic system after injection thereof.
41. The kit of claim 40, wherein the means for injection is a
syringe corresponding to said needle.
42. The kit of claim 40, further comprising a liquid based
biocompatible carrier injectable through said needle.
43. The kit of claim 42, wherein the microspheres are suspended in
the biocompatible carrier.
44. The kit of claim 43, wherein the microspheres are associated
with cells.
Description
[0001] This is a continuation-in-part application of U.S. patent
application Ser. No. 09/263,773, filed Mar. 5, 1999.
1. FIELD OF INVENTION
[0002] The present invention relates to dermal augmentation and
tissue bulking, particularly for the treatment of gastroesophageal
reflux disease, urinary incontinence, urinary reflux disease, or
skin contour deficiencies and wrinkles, using injectable
microspheres.
2. BACKGROUND OF INVENTION
2.1 Gastroesophageal Reflux Disease ("GERD")
[0003] Although gastroesophageal reflux is a normal physiological
phenomenon, in some cases it is a pathophysiological situation that
can result in a variety of symptoms which may become severe in
extreme cases. Gastro-Esophageal Reflux Disease ("GERD"), describes
a backflow of acidic and enzymatic liquid from the stomach to the
esophagus. It causes burning sensations behind the sternum that may
be accompanied by regurgitation of gastric acid into the mouth or
even the lung. Complications of GERD which define the severity of
the disease include esophageal tissue erosion, and esophageal ulcer
wherein normal epithelium is replaced by a pathological tissue.
[0004] Statistical data indicate that about 35% of the American
population suffer from heartburn at least once a month and between
5 to 10% once a day. More importantly for this kind of disease
about 2% of the American population suffer from GERD based on
medical evidence data from endoscopic examination. This disease is
related to the age of individuals and seems to increase after 40
years of age. (Nebel O. T. et al., Am. J. Dig. Dis., 21(11):953-956
(1976)).
[0005] In normal patients, after a meal the lower esophageal
sphincter remains closed, but in patients with GERD, it relaxes and
allows some acidic material to reflux into the esophageal tube as a
result of stomach contractions. Actually GERD can be attributed
primarily to transient relaxation of the lower esophageal
sphincter. In other cases, GERD can be attributed to decreased
resting tone of the lower esophageal sphincter or to congenital
small dimension of the sphincter itself. Other causes also exist
which contribute to varying degrees to the existence and severity
of this disease.
[0006] In addition, there are external factors that contribute to
exacerbate the symptoms of GERD, which conditions include eating
fatty foods, caffeine intake, smoking, tight clothing and certain
medications. Decrease in salivation can also be a factor that
exacerbates GERD, since under normal conditions saliva, which is an
alkaline liquid, aids in neutralizing acidic reflux and therefore
diminishing the duration of the acidic exposure of the
esophagus.
[0007] Erythema is one of the first visible signs of GERD, which
can be seen by endoscopy. Tissue erosion indicates more advanced
disease which can then become deep ulcers and lead to cancer
(adenocarcinoma increases in incidence faster than other types of
cancer). Diffuse ulceration and specific complications occur in
about 3.5% of patients less than 65 years of age with esophageal
obstruction, blood loss, and in some cases, perforation. Ulcerative
situations not only lead to complications, but they are also more
resistant to treatments. Although severe complications are uncommon
in young patients, they occur in about 20-30% of patients over 65
(Reynolds J. C, Am. J. Health-Sys. Pharm 53, (1996)).
[0008] Prior to the present invention, in an attempt to increase
the function of the sphincter, bulking methods using bovine
collagen and Teflon paste have been used in patients. Both methods
have been unsuccessful, however, as these materials migrate over
time from the initial site of implantation.
[0009] At present, GERD is generally managed by over-the-counter
("OTC") antacids or prescription drugs, including proton pump
inhibitors, motility agents and H.sub.2 blockers. In addition, a
portion of GERD patients require surgical intervention; the most
common type of surgery is fundoplication which can be done by
conventional surgical techniques, or using laparoscopic techniques.
However, fundoplication surgery carries the risk of serious side
effects and is only marginally successful in curing GERD.
Respiratory symptoms are also associated with GERD in about 50% of
patients, and in patients undergoing fundoplication, these
respiratory symptoms can even increase (76% reported in a study by
Johnson W. E. et al., Archives of Surgery, 131:489-492 (1996)).
2.2 Urinary Incontinence and Urinary Reflux Disease
[0010] Urinary incontinence is a prevalent problem that affects
people of all ages and levels of physical health, both in the
community at large and in healthcare settings. Medically, urinary
incontinence predisposes a patient to urinary tract infections,
pressure ulcers, perineal rashes, and urosepsis. Socially and
psychologically, urinary incontinence is associated with
embarrassment, social stigmatization, depression, and especially
for the elderly, an increased risk of institutionalization (Herzo
et al., Ann. Rev. Gerontol. Geriatrics, 9:74 (1989)). Economically,
the costs are astounding; in the United States alone, over ten
billion dollars per year is spent managing incontinence.
[0011] Incontinence can be attributed to genuine urinary stress
(urethra hypermobility), to intrinsic sphincter deficiency ("ISD"),
or both. It is especially prevalent in women, and to a lesser
extent incontinence is present in children (in particular, ISD),
and in men following radical prostatectomy.
[0012] One approach for treatment of urinary incontinence involves
administration of drugs with bladder relaxant properties, with
anticholinergic medications representing the mainstay of such
drugs. For example, anticholinergics such as propantheline bromide,
and combination smooth muscle relaxant/anticholinergics such as
racemic oxybutynin and dicyclomin, have been used to treat urge
incontinence. (See, e.g., A. J. Wein, Urol. Clin. N. Am., 22:557
(1995)). Often, however, such drug therapies do not achieve
complete success with all classes of incontinent patients, and
often results in the patient experiencing significant side
effects.
[0013] Besides drug therapies, other options used by the skilled
artisan prior to the present invention include the use of
artificial sphincters (Lima S. V. C. et al., J. Urology,
156:622-624 (1996), Levesque P. E. et al., J. Urology, 156:625-628
(1996)), bladder neck support prosthesis (Kondo A. et al., J.
Urology, 157:824-827 (1996)), injection of crosslinked collagen
(Berman C. J. et al., J. Urology, 157:122-124 (1997), Perez L. M.
et al., J. Urology, 156:633-636 (1996); Leonard M. P. et al., J.
Urology, 156:637-640 (1996)), and injection of
polytetrafluoroethylene (Perez L. M. et al., J. Urology,
156:633-636 (1996)).
[0014] A recent well known approach for the treatment of urinary
incontinence associated with ISD is to subject the patient to
periurethral endoscopic collagen injections. This augments the
bladder muscle in an effort to reduce the likelihood of bladder
leakage or stress incontinence.
[0015] Existing solutions to circumvent incontinence have well
known drawbacks. The use of artificial sphincters for children with
intractable incontinence requires long term surveillance of the
urinary tract because of the potential for renal failure after
device placement (Levesque P. E. et al., J. Urology, 156:625-628
(1996)). While endoscopically directed injections of collagen
around the bladder neck has a quite high success rate in sphincter
deficiency with no significant morbidity, the use of collagen can
result in failures that occur after an average of two years and
considerations need to be given to its cost effectiveness (Khullar
V. et al., British J. Obstetrics & Gynecology, 104:96-99
(1996)). In addition, deterioration of patient continency, probably
due to the migration phenomena (Perez L. M. et al.) may require
repeated injections in order to restore continency (Herschorn S. et
al., J. Urology, 156:1305-1309 (1996)).
[0016] The results with using collagen following radical
prostatectomy for the treatment of stress urinary incontinence have
also been generally disappointing (Klutke C. G. et al., J. Urology,
156:1703-1706 (1996)). Moreover, one study provides evidence that
the injection of bovine dermal collagen produced specific
antibodies of IgG and IgA class. (McCell and, M. and Delustro, F.,
J. Urology 155, 2068-2073 (1996)). Thus, possible patient
sensitization to the collagen could be expected over the time.
[0017] Despite of the limited success rate, transurethral collagen
injection therapy remains an acceptable treatment for intrinsic
sphincter deficiency, due to the lack other suitable
alternatives.
[0018] Urinary reflux disease, or "vesicoureteral reflux" in its
medical term, simply means that urine goes backwards in the ureters
during urination. The disease often occurs in young children. The
ureter is the tube which connects the kidneys with the bladder.
Urine is supposed to go in one direction: from the kidneys to the
bladder. When urine goes up from the bladder to the kidneys, it can
result in health problems for the person.
[0019] Urinary reflux can lead to kidney damage. Refluxing urine
can carry bacteria to the kidney, where it can cause kidney
infection. Children with reflux of urine are much more likely to
have kidney infection than children who do not have reflux. The
combination of reflux and infection can lead to areas of permanent
kidney damage or "renal scarring." This scarring is detected by
doing an X-ray called an intravenous pyelogram (IVP), or
preferably, a renal scan. If it is extensive enough, the scarring
can lead to loss of function of one or both kidneys.
[0020] The key to preventing renal scarring is preventing kidney
infections. This is currently being carried out in two ways. In
most cases, long term prophylactic antibiotics are given. The other
method of preventing urinary tract infections is surgical
correction of the reflux. Both methods, however, have drawbacks.
Long term use of antibiotics may cause unpredictable side effects
and surgical procedures involve unnecessary risks.
[0021] Even though many urinary reflux disease will go away on its
own in children, some cases often lead to severe kidney and urinary
tract infections and even total kidney failure. There is a need,
therefore, for a safe, effective, less intrusive, and long lasting
method of treating urinary reflux disease.
2.3 Skin Deficiencies
[0022] Damage to the skin due to aging, environmental exposure to
the sun and other elements, weight loss, child bearing, disease
such as acne and cancer, and surgery often results in skin contour
deficiencies and other skin anomalies. In order to correct contour
deficiencies and other anomalies of the skin, people often resort
to cosmetic surgery, such as face lifts and skin tucks. Cosmetic
surgery, however, has several drawbacks, in addition to the high
cost associated with it. It is usually an invasive and risky
procedure, having the potential of leaving scars in areas of
operation and affecting normal biological and physiological
functions. Furthermore, cosmetic surgery is often a limited option,
available only for certain skin deficiencies.
[0023] In addition to cosmetic surgery, various other methods are
used to remove or ameliorate the deficiencies with different levels
of success. The use of injectable material for soft tissue
augmentation is a method often used. The advantage of using
hypodermic needles as a delivery device for dermal augmentation
reflects the advantages of using hypodermic needles in general:
easy, precise and, usually, non-invasive deliveries. Yet, the
requirement for such use is also quite strict: the material to be
delivered must be deliverable through the needles, which means the
material must be able to easily pass through the hollow centers of
the needles.
[0024] One method of dermal augmentation using injectable material
is liquid or semi-liquid injections, usually containing collagen.
The best known example is a collagen preparation manufactured by
Collagen Corporation (now part of Inamed Corporation) and marketed
by C. R. Bard. However, collagen is a naturally occurring substance
which the body may enzymatically degrade and eliminate over time,
thus requiring repeat treatments. Also, collagen may be displaced
within the tissue in which it was originally injected, thereby
reducing or eliminating the intended dermal augmentation effect.
Collagen is also digested directly (biochemically), through
macrophages, through the lymphatic system, or by other means. Even
more alarming from a cosmetic perspective, collagen may move from
the initial site of injection, causing unsightly bumps and bulges
under the skin at undesired locations. See, e.g., Millikan, Long
Term Safety and Efficacy with Fibrel in the Treatment of Cutaneous
Scars, J Dermatol Surg Oncol, 15:837-846 (1989).
[0025] Injection of liquid silicone has also been used extensively
to treat skin deficiencies. However, due to long term side effects,
such as nodules, recurring cellulitis, and skin ulcers, the use of
injectable silicone is on the decline. See, e.g., Edgerton et al.,
Indications for and pitfalls of soft tissue augmentation with
liquid silicone, Plast. Reconstr. Surg, 58:157-163 (1976).
2.4 MicroParticles
[0026] Prior to the present invention, microspheres have been
manufactured and marketed for in vitro use in anchorage dependent
cell culture. (Van Vezel, A. L., Nature, 216:64-65 (1967); Levine
et al., Somatic Cell Genetics, 3:149-155 (1977); Obrenovitch et
al., Biol. Cell., 46:249-256 (1983)). They have also been used in
vivo to occlude blood vessels in the treatment of arteriovascular
malformation, fistulas and tumors (See, U.S. Pat. No. 5,635,215,
issued Jun. 3, 1997 to Boschetti et al.; Laurent et al., J. Am.
Soc. Neuroiol, 17:533-540 (1996); and Beaujeux et al. J. Am. Soc.
Neuroial, A:533-540 (1996)).
[0027] Further, direct implantation of cells into living tissues
such as brain or liver to correct specific deficiencies has been
attempted albeit with a number of failures. The major problems
associated with direct cell transplantation are the long term
viability of the cell transplant and the immunopathological as well
as histological responses. Microparticles with cells attached on
their surface have been used in some in vivo applications.
Cherkesey et al., IBRO, 657-664 (1996), described the culture of
adrenal cells on coated dextran beads and the implantation into
mammalian brain to supplant some specific disorders related to
6-hydroxydopamine-induced unilateral lesions of the substantia
nigra. The pre-attachment of cells to dextran microcarriers allowed
for improved functions of the cells implanted into the brain. Also
liver cells transplantation has been used to manage acute liver
failure, or for the replacement of specific deficient functions
such as conjugation of bilirubin or synthesis of albumin. For this
purpose, an intrasplenic injection of hepatocytes grown on the
surface of microspheres was performed (Roy Chowdhury et al., in:
Advanced Research on Animal Cell Technology, AOA Miller ed.,
315-327, Kluers Acad. Press, 1989).
[0028] Most of cell implant results have been, however, largely
disappointing for the designated functions (or have had low levels
of biological function).
[0029] Prior to the present invention, solid microparticles have
also been used for the correction of skin deficiencies and for
tissue bulking. For example, carbon particles, silicone particles,
TEFLON paste, collagen beads and polymethylmethacrylate spheres,
have been used with disappointing results due to, inter alia,
adverse tissue reactions, biological degradation and migration from
the initial implantation location.
[0030] The problems associated with rigid and non-deformable
particles, such as carbon particles and silicone particles, in
tissue bulking or treating skin deficiencies are that they are
either too fragile or too large to be injected, or too small and
are digested or eliminated by the body. Therefore, such particles
all have one or more of the following limitations: (i) too large to
be injected through a 30 gauge or smaller needle; (ii) particles of
irregular shape clump together, making injection difficult; (iii)
particles are too fragile, resulting in breakage during injection
and digestion of the residues; (iv) injected particles are too
small and are digested by macrophages or other components of the
lymphatic system; and (v) injected particles are displaced as they
do not adhere to the surrounding cells.
[0031] Injectable deformable particles, such as Teflon.RTM.
particles, have also been used for tissue bulking and for treating
skin deficiencies. However, Teflon.RTM. particles have one or more
of the following limitations: (1) the particles slide with the
tissue and do not stay in place of injection; (2) the particles
deform during and after injection, reducing the intended tissue
bulking effect; and (3) the particles are digested or eliminated by
the lymphatic system partly due to the fact that their diameters
become smaller as a result of injection.
[0032] Therefore, there is a great need for safe, biocompatible,
stable and effective methods of tissue bulking for the treatment of
GERD, urinary incontinence, and urinary reflux disease and methods
of dermal augmentation for treatment of skin disorders.
3. SUMMARY OF INVENTION
[0033] The present invention encompasses the use of implantable
microparticles, or microspheres or microbeads, in the treatment of
GERD, urinary incontinence, urinary reflux disease and skin
deficiencies such as skin wrinkles. In each use the particles are
implanted into the appropriate tissue, muscle, organ etc. as a
bulking or augmentation agent.
[0034] In a preferred embodiment, the invention provides a method
of dermal augmentation, suitable for the treatment of skin
deficiencies, and a method of tissue bulking, suitable for the
treatment of GERD, urinary incontinence, or urinary reflux disease,
wherein the microspheres used are injectable through needles of
about 18 gauge or smaller, depending on the particular method and
treatment, and are not capable of being digested or eliminated
through the lymphatic or the immune system. Thus, the invention
encompasses injectable compositions and methods for dermal
augmentation or tissue bulking by injecting using syringes,
catheters, needles or other means of injecting or infusing
microspheres in a liquid medium so as to avoid surgical
intervention.
[0035] The microparticles of the invention, whether implantable by
injection or otherwise, are preferably pre-coated, with autologous
cells, for example, muscle cells, fat cells and the like. The
microparticles of the invention are biocompatible non-toxic
polymers coated with, linked to or filled with cell adhesion
promoters. The microparticles preferably contain a positive charge
on their surface by way of a cationic monomer or polymer.
[0036] In one embodiment, the invention encompasses the treatment
of gastroesophageal reflux disease in a human which comprises
implanting hydrophilic biocompatible microparticles comprising (a)
a positive charge and a cell adhesion promoter; and (b) autologous
cells layered on the surface of said beads, into the lower
esophageal sphincter. The microparticles are preferably
microspheres or microbeads which are described in detail herein.
The autologous cells are preferably taken from the area where the
implantation is to be made. Serum or whole blood taken from the
patient can be used to wash the microparticles prior to
implantation. For GERD treatment, implantation is preferably made
by using standard techniques known to the ordinary skilled artisan,
such as injection (or injections) via syringe or other suitable
devices.
[0037] In yet another embodiment, the invention encompasses the
treatment of urinary incontinence in a human which comprises
implanting hydrophilic biocompatible microparticles comprising (a)
a positive charge and a cell adhesion promoter; and (b) autologous
cells layered on the surface of the beads, into the urinary
sphincter. The microparticles are preferably microspheres or
microbeads as described herein. Further, the autologous cells are
preferably taken from the area where the implantation is to be
made. Serum or whole blood from the patient can be used to wash the
microparticles prior to implantation. Implantation is preferably
made using a syringe or other device suitable for the particular
tissue of implantation.
[0038] In another embodiment, the invention encompasses a method of
treating skin wrinkles in a human which comprises the
administration or implantation of microparticles comprising a
hydrophilic copolymer having a positive charge, and a cell adhesion
promoter, which microparticles have been pre-treated with
autologous cells. The microparticles can be simply exposed to the
autologous cells or mixed thoroughly with autologous cells prior to
implantation. The microspheres are preferably injected via syringe
or other suitable device through a needle of 30 gauge or smaller
into the area, or under the area, of the skin deficiencies.
[0039] In yet another embodiment, the invention encompasses the
treatment or amelioration of skin wrinkles which comprises
administering hydrophilic biocompatible microparticles comprising:
(a) a positive charge and a cell adhesion promoter; and (b)
autologous cells, collagen, collagen derivatives or
glucosaminoglycans layered on the surface of the beads, into the
area of or surrounding the skin wrinkles. In other words,
microspheres or microbeads coated with a cell adhesion promoter and
pre-treated with the appropriate tissue bulking cells, are
administered to the area of treatment.
[0040] The present invention additionally provides methods of
dermal augmentation and treatment of skin deficiency. Specifically,
the invention provides a method of causing dermal augmentation in a
mammal by administering a composition of elastic, hydrophilic, and
substantially spherical microspheres in a biocompatible carrier to
the mammal. The composition is injectable through a needle of about
30 gauge or smaller and the microspheres are not capable of being
digested or eliminated by macrophage or other elements of the
mammal's immune system. According to the present invention, a
preferred method of administration is injecting the composition
into an area of the subject mammal that is in need of dermal
augmentation. A more preferred method of administration is
injecting the composition into the subcutaneous layer of the
subject mammal.
[0041] The dermal augmentation method of the present invention is
especially suitable for the treatment of skin contour deficiencies,
which are often caused by aging, environmental exposure, weight
loss, child bearing, injury, surgery, in addition to diseases such
as acne and cancer. Suitable for the treatment by the present
invention's method are contour deficiencies such as frown lines,
worry lines, wrinkles, crow's feet, marionette lines, stretch
marks, and internal or external scars resulted from injury, wound,
bite, surgery, or accident.
[0042] The invention also encompasses the use of the injectable
compositions to treat skin deficiencies caused by diseases such as
acne and cancer.
[0043] The present invention further provides a method of causing
dermal augmentation in a mammal by administering the injectable
suspension extracorporeally into organs, components of organs, or
tissues prior to the inclusion of said tissues, organs, or
components of organs into the body.
[0044] The invention further encompasses method for tissue bulking
in a mammal by administering a composition of elastic, hydrophilic,
non-toxic and substantially spherical microspheres in a
biocompatible carrier to the mammal. The composition is injectable
through needles of about 18 to about 26 gauge, preferably, 22 to 24
gauge, and preferably administered by injection directly into the
site of treatment, e.g., the sphincter.
[0045] Thus, in one embodiment, the tissue bulking method is used
for the treatment of gastroesophageal reflux disease in a mammal;
preferably by direct administration via injection of the
composition into the lower esophageal sphincter or the diaphragm of
the mammal.
[0046] Similarly, the tissue bulking method is used for the
treatment of urinary incontinence or urinary reflux disease via
administration of the composition into the bladder sphincter or the
urethra of the mammal.
[0047] The present invention further provides kit for performing
dermal augmentation or tissue bulking. The kit comprises a syringe
and a 30 gauge or smaller needle for dermal augmentation and an 18
to 26 gauge needle for tissue bulking. The syringe optionally
comprises a composition of elastic, hydrophilic, non-toxic and
substantially spherical microspheres in a biocompatible carrier.
Alternatively, the syringe does not contain a solution or
suspension but is accompanied by (a) dry sterilized microspheres
which are ready for preparation of a suspension; (b) a preformed
suspension of microspheres; and (c) dry microspheres and a
biocompatible solution in separate containers. The final
composition of microspheres is injectable through the needle into a
mammal and the microspheres are not capable of being digested or
eliminated through said mammal's macrophages or other elements of
the immune system or the lymphatic system.
[0048] It should be recognized that both treatments for GERD,
urinary incontinence, urinary reflux disease, and skin deficiencies
described above can be used in combination with conventional
therapies now used to treat these diseases or conditions i.e., oral
diuretics, antacids, suitable drug therapy, cometic surgeries and
the like. Such combination therapy can lead to a faster, safer and
more comfortable recovery for the patient.
[0049] As used herein the terms "administered", "implanted", or
"implantation" are used interchangeably and mean that the material
is delivered to the area of treatment by techniques know to those
skilled in the art and appropriate for the disease to be treated.
Both invasive and non-invasive methods may be used for delivery.
"Injectable" as used in the present invention means capable of
being administered, delivered or carried into the body via needle
or other similar ways.
[0050] As used in the present invention, "microparticles" means
polymer or combinations of polymers made into bodies of various
sizes. The microparticles can be in any shape, although they are
often in substantially spherical shape, in which case the
microparticles are referred to as "microspheres" or "microbeads."
Before injection or being composed into an injectable composition,
the microspheres are sterilized. "Elastic" microparticles or
microspheres refers to microparticles or microspheres comprise
polymers that have elastic properties. Specific to the present
invention, elastic microspheres means particles that are flexible
enough so that they can be easily injected through needles of 18
gauge or smaller, yet the microspheres are not fragile so that they
are not broken during the process of injection.
[0051] The microspheres of the present invention also comprise
particles that are "hydrophilic," which, as used in the invention,
means the particles can dissolve in, absorb, or mix easily with
water or aqueous solution.
[0052] "Substantially spherical" generally means a shape that is
close to a perfect sphere, which is defined as a volume that
presents the lowest external surface area. Specifically,
"substantially spherical" in the present invention means, when
viewing any cross-section of the particle, the difference between
the average major diameter and the average minor diameter is less
than 20%. The surfaces of the microspheres of the present invention
appear smooth under magnification of up to 1000 times. The
microspheres of the present invention may comprise, in addition to
the particles, other materials as described and defined herein.
[0053] "Skin wrinkles," "skin deficiencies," and "skin contour
deficiencies" are used interchangeable in the present invention to
refer to skin conditions that are either abnormal or undesirable
due to various internal or external conditions such as aging,
environmental exposure to the sun and other elements, weight loss,
child bearing, disease such as acne and cancer, surgery, wounds,
accidents, bites, cuts.
[0054] "Dermal augmentation" in the context of the present
invention refers to any change of the natural state of a mammal's
skin and related areas due to external acts. The areas that may be
changed by dermal augmentation include, but not limited to,
epidermis, dermis, subcutaneous layer, fat, arrector pill muscle,
hair shaft, sweat pore, and sebaceous gland.
[0055] "Tissue bulking" in the context of the present invention
refers to any change of the natural state of a mammal's non-dermal
soft tissues due to external acts or effects. The tissues
encompassed by the invention include, but not limited to, muscle
tissues, connective tissues, fats, and, nerve tissues. The tissues
encompassed by the present invention may be part of many organs or
body parts including, but not limited to, the sphincter, the
bladder sphincter and urethra.
[0056] "Cell adhesion promoter" in the present invention means any
material that, because of their presence in or association with the
microspheres, promotes or enhances the adhesiveness of cells to the
surface of the microspheres. These materials are often proteins
that are bound to the surface of the microspheres through covalent
bonds of the proteins and the polymers.
[0057] "Therapeutic agent" in the present invention refers to any
substance that provides therapeutic effects or biological or
physiological responses to the dermal augmentation or tissue
bulking procedure. An example of therapeutic agent is an
anti-inflammation agent that prevents or reduce the effect of
inflammations associated dermal augmentation or tissue bulking
procedure, an anti-inflammatory agent, an anti-bacterial agent, or
an anti-histamine agent.
[0058] "Chemical modification" in the present invention means the
changes of chemical properties and characteristics of the
microspheres, either during their production process or by way of
mixing or contacting them with various agents or tissues, such that
the microspheres have the ability to perform, in addition to dermal
augmentation or tissue bulking, other functions once injected into
the body.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 is a schematic representation of sphincter bulking.
Beads are coated and injected under physiological conditions into
the sphincter. The sphincter volume increases proportionally to the
amount of injected beads and the lumen size decreases. The beads
are progressively and non-reversibly integrated within the
muscles.
5. DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention provides a safe, effective, stable,
and long lasting methods of tissue bulking and dermal augmentation,
which methods are useful for the treatment of gastroesophageal
reflux disease, urinary incontinence, urinary reflux disease, and
skin deficiencies. The invention provides methods of tissue bulking
and dermal augmentation by administrating injectable composition
comprising elastic, hydrophilic, non-toxic and substantially
spherical microspheres and a biocompatible carrier to a mammal in
need of treatment for gastroesophageal reflux disease, urinary
incontinence, urinary reflux disease, or skin deficiencies. The
methods of the present invention are intended to encompass the
following advantages: (1) the injected materials are not easily
displaced within the tissues in which they were originally
injected, thus the intended dermal augmentation or tissue bulking
effect is achieved without repeated administration or causing
adverse effects to the patient, (2) the injected materials are not
readily digested, displaced, or eliminated either biochemically or
through the immune or lymphatic system, thus the method is more
effective and longer lasting, (3) the materials are of sufficient
size to be injected through 18 to 26 gauge needles, preferably 22
to 24 gauge needles, for tissue bulking or 30 gauge or smaller
needles for dermal augmentation, thus the method is more accurate,
efficacious and less intrusive to the patient, (4) the injected
particles are flexible, but not fragile, facilitating easy
injection without being broken, thus providing easy and safe
injection, and (5) the injected particles are not irregularly
shaped and do not clump together, also providing easy and accurate
injection. These benefits, whether alone or in combinations,
enhance the effectiveness of the treatment and are safe, more
convenient and comfortable for patients.
[0061] In one embodiment, the present invention uses
microparticles, particularly microspheres or microbeads, having a
positive charge on its surface and a cell adhesion promoter and
optionally, a cell growth promoting agent, to treat GERD, urinary
incontinence, and skin wrinkles. The microparticles of the
invention are preferably used with autologous cells. In other
words, the microparticles of the invention are colonized with the
appropriate cells prior to implantation. This pre-implantation
(pre-administration) step has been shown to reduce or eliminate
immunological responses and implantation rejection reactions.
Further, the use of non-biodegradable biologically compatible
microbeads with positive charges and autologous cells, whether
tissue-specific or not, improves tissue acceptance and overall
treatment.
[0062] According to the methods of the present invention, treatment
of GERD, urinary incontinence, and urinary reflux disease, and skin
wrinkles is achievable while avoiding or substantially reducing
adverse tissue reactions, including implantation rejection,
degradation of particles, resorption, migration and other adverse
events. The methods of the invention also involve increased
connective tissue response.
[0063] Microbeads or microparticles for use in the present
invention are based on a biocompatible, hydrophilic, substantially
spherical, and non-toxic polymers. The microspheres are injectable
through needle of 18 gauge or smaller and are not capable of being
digested or eliminated through the mammal's immune or lymphatic
system. The polymers may preferably be coated with agents which
promote cell adhesion. Living cells may also attach to the
microparticles forming layered cells therein which link with
surrounding tissues to enhance long term stability of the
beads.
[0064] Microparticles intended to be implanted, preferably through
injection, in various locations of the body according to the
present invention are composed of a non-resorbable hydrophilic
polymer containing the appropriate material for cell adhesion, and
may additionally contain radiopaque molecules or other marking
agents, to facilitate localization by radiology prior to or during
intervention. The microspheres of the present invention comprise
elastomers, preferably elastomers selected from the group
consisting of acrylic polymers, vinyl alcohol polymers, acrylate
polymers, polysaccharides, silicones, or mixtures thereof. More
preferably, the hydrophilic copolymers usable for this application
are those of the acrylic family such as polyacrylamides and their
derivatives, polyacrylates and their derivatives as well as
polyallyl and polyvinyl compounds. All of these polymers are
crosslinked so as to be stable and non-resorbable, and can contain
within their structure other chemicals displaying particular
properties, such as chemotactic effects, promotion of cell adhesion
to cells or tissues, such as cells of the esophagus wall or the
urethra wall, or skin cells, and/or marking agents.
[0065] The microparticles for use in the present invention are
non-toxic to tissues and cells, biocompatible, and adhesive to
various cells and tissues at the site of implantation by means of
the cell growth they promote. In addition, these microparticles are
non-resorbable and non-biodegradable, and thus are stable, durable,
and will maintain their general shape and position once implanted
at a desired site.
[0066] In general, microparticles for use in the present invention
may have any shape, with microparticles which are substantially
spherical in shape being preferred. Microparticles for use in the
present invention may have diameters ranging between about 10 .mu.m
to about 1000 .mu.m. Preferably, microparticles for use in the
present invention which have cells adhered to the surface thereof
will have diameters ranging between 50 .mu.m and 1000 .mu.m.
[0067] For purposes of tissue bulking, the microspheres of the
invention preferably have diameters ranging from about 10 .mu.m to
about 500 .mu.m, more preferably from about 100 .mu.m to about 300
.mu.m. For purposes of dermal augmentation, the microspheres
preferably have diameters ranging from about 10 .mu.m to about 400
.mu.m, preferably from about 50 .mu.m to about 200 .mu.m.
[0068] Possible variations of the present invention include
replacing the microparticles with any biocompatible, non-toxic
non-resorbable polymeric particles, membrane, fibers or other solid
substrates treated with an agent promoting cell adhesion. The
invention also includes linear soluble polymers which, after
injection, crosslink in situ to constitute a solid, cell adhesion
promoting filling agent. Preparation and/or injection of empty
microparticles (microbubbles) that are prepared in advance or are
generated in place via the use of appropriate catheters, are also
contemplated in this invention.
[0069] The microparticles, or other solid substrates, for use in
the present invention are flexible, such that they can easily pass
into and through injection devices and small catheters without
being permanently altered, but the microparticles are also
resistant to the muscle contraction stress generated during and
after the implantation process. They are also thermally stable
which allows for easy, convenient sterilization, and frozen
storage.
[0070] The microparticles, or other solid substrates, for use in
the present invention are also stable in suspension which allows
the microparticles or other solid substrates to be formulated and
stored in suspension and injected with different liquids. More
specifically, the hydrophilic nature of the microparticles permits
placing them in suspension, and in particular, in the form of
sterile and pyrogenic (pyrogen-free) injectable solutions, while
avoiding the formation of aggregates or adhesion to the walls of
storage containers and implantation devices, such as catheters,
syringes, needles, and the like. Preferably, these injectable
solutions contain microparticles or other solid substrates
distributed approximately in caliber segments ranging between about
10 .mu.m and about 2000 .mu.m.
[0071] The microparticles of the present invention are both
hydrophilic and cationic. The microparticles preferably comprise a
copolymer of a neutral hydrophilic monomer, a difunctional monomer,
one or more monomers having a cationic charge, and optionally, a
functionalized monomer capable of rendering the microparticle
detectable. The microparticles may also comprise one or more cell
adhesion promoters and a marking agent.
[0072] The copolymer is preferably a hydrophilic acrylic copolymer
which comprises in copolymerized form about 25 to about 98% neutral
hydrophilic acrylic monomer by weight, about 2 to about 50%
difunctional monomer by weight and about 0 to about 50% by weight
of one or more monomers having a cationic charge.
[0073] By way of example, the copolymers described in French Patent
2,378,808, which is incorporated herein by reference, can be used
in accordance with this invention to prepare the base microparticle
copolymer.
[0074] As hydrophilic acrylic monomer, acrylamide and its
derivatives, methacrylamide and its derivatives or
hydroxymethylmethacrylate can be used.
[0075] Examples of difunctional monomer, include but are not
limited to the N,N'-methylene-bis-acrylamide,
N',N'-diallyltartiamide or glyoxal-bis-acrylamide.
[0076] Further, the monomer having a cationic charge, includes but
is not limited to those carrying a tertiary or quaternary amine
function, preferably diethylaminoethyl acrylamide,
methacrylamidopropyl trimethylammonium or acrylamidoethyl
triethylammonium.
[0077] In a particularly preferred embodiment, a copolymer
comprising about 25 to about 98% methacrylamide by weight, about 2
to about 50% N,N-methylene-bis-acrylamide by weight is used.
[0078] In one particularly advantageous embodiment of the
invention, it is possible to increase the stability of the
microspheres by reticulating the adhesion agent. By way of example,
in the case of gelatin, the reticulating agent can be chosen among
the difunctional chemical agents reacting on the gelatin amines
(e., glutaraldehyde, formaldehyde, glyoxal, and the like).
[0079] The functionalized monomer is generally obtained by chemical
coupling of the monomer with a marker, which can be:
[0080] a chemical dye, such as Cibacron Blue or Procion Red HE-3B,
making possible a direct visualization of the microspheres
(Boschetti, J. Biochem-Biophys. Meth., 19:21-36 (1989)). Examples
of functionalized monomer usable for this type of marking
N-acryloyl hexamethylene Cibacrone Blue or N-acryloyl hexamethylene
Procion Red HE-3B;
[0081] a magnetic resonance imaging agent (erbium, gadolinium or
magnetite);
[0082] a contrasting agent, such as barium or iodine salts,
(including for example acylamino-e-propion-amido)-3-triiodo-2, 4,
6-benzoic acid, which can be prepared under the conditions
described by Boschetti et al. (Bull. Soc. Chim., No. 4 France,
(1986)). In the case of barium or magnetite salts, they can be
directly introduced in powered form in the initial monomer
solution.
[0083] As indicated above it is also possible to mark the
microspheres after their synthesis. This can be done, for example,
by grafting of fluorescent markers derivatives (including for
example fluorescein isothiocyanate (FITC), rhodamine isothiocyanate
(RITC) and the like).
[0084] Various types of cell adhesion promoters well known in the
art may be used in the present invention. In particular, cell
adhesion promoters can be selected from collagen, gelatin,
glucosaminoglycans, fibronectins, lectins, polycations (such
polylysine, chitosan and the like), or any other natural or
synthetic biological cell adhesion agent.
[0085] Preferably, the cell adhesion promoter is present in the
microparticle, or other solid substrate, in an amount between about
0.1 to 1 g per ml of settled microparticles.
[0086] Microparticles are prepared by suspension polymerization,
drop-by-drop polymerization or any other method known to the
skilled artisan. The mode of microparticle preparation selected
will usually depend upon the desired characteristics, such as
microparticle diameter and chemical composition, for the resulting
microparticles. The microparticles of the present invention can be
made by standard methods of polymerization described in the art
(see, e.g., E. Boschetti, Microspheres for Biochromatography and
Biomedical Applications. Part I, Preparation of Microbands In:
Microspheres, Microencapsulation and Liposomes, John Wiley &
Sons, Arshady R., Ed., vol. 2, p171-199 (1999), which is
incorporated herein by reference). Microspheres are prepared
starting from an aqueous solution of monomers containing adhesion
agents such as collagen (gelatin is a denatured collagen). The
solution is then mixed with a non-aqueous-compatible solvent to
create a suspension of droplets, which are then turned into solid
gel by polymerization of monomers by means of appropriate
catalysts. Microspheres are then collected by filtration or
centrifugation and washed.
[0087] Cell adhesion promoters or marking agents are introduced on
microbeads by chemical coupling procedures well known in affinity
chromatography, referred to by the term "ligand immobilization".
Another method of introduction is by diffusion within the gel
network that constitutes the bead and then trapping the diffused
molecules in place by precipitation or chemical cross-linking.
Therapeutic agents, drugs or any other active molecules that are
suitable for transportation by the beads can also be introduced
into the microbeads prior to bead implantation according to this
last method.
[0088] The microspheres of the present invention also can be
chemically modified so that they will "carry" therapeutic effects,
vascularization effects, anti-vascularization effects,
visualization properties, anti-inflammatory effects, anti-bacterial
effects, or anti-histamine effects, or combinations thereof. The
chemical modification of the microspheres of the present invention
is made possible by the fact that the microspheres comprise
particles made of polymers that are crosslinked so that they can
contain chemicals within their structures that possess various
properties and that they possess unique characteristics associated
with surface covalent bonds. The chemical modification of the
microspheres of the present invention may also occur through the
interactions between the microspheres and the neighboring cells and
tissue after the administration.
[0089] The microspheres of the invention can also be obtained by
standard methods of polymerization described in the art such as
French Patent 2,378,808 and U.S. Pat. No. 5,648,100, each of which
is incorporated herein by reference. In general, the polymerization
of monomers in solution is carried out at a temperature ranging
between about 0.degree. C. and about 100.degree. C. and between
about 40.degree. C. and about 60.degree. C., in the presence of a
polymerization reaction initiator.
[0090] The polymerization initiator is advantageously chosen among
the redox systems. Notably, it is possible to use combinations of
an alkali metal persulfate with
N,N,N',N'-tetramethylethylenediamine or with
dimethylaminopropionitrile, organic peroxides such as benzoyl
peroxides or even 2,2'-azo-bis-isobutyronitrile.
[0091] The quantity of initiator used is adapted by one skilled in
the art to the quantity of monomers and the rate of polymerization
sought.
[0092] Polymerization can be carried out in mass or in
emulsion.
[0093] In the case of a mass polymerization, the aqueous solution
containing the different dissolved constituents and the initiator
undergoes polymerization in an homogeneous medium. This makes it
possible to access a lump of aqueous gel which can then be
separated into microspheres, by passing, for example, through the
mesh of a screen.
[0094] Emulsion or suspension polymerization is the preferred
method of preparation, since it makes it possible to access
directly microspheres of a desired size. It can be conducted as
follows: The aqueous solution containing the different dissolved
constituents (e.g., different monomers, cell adhesion agent), is
mixed by stirring, with a liquid organic phase which is not
miscible in water, and optionally in the presence of an emulsifier.
The rate of stirring is adjusted so as to obtain an aqueous phase
emulsion in the organic phase forming drops of desired diameter.
Polymerization is then started off by addition of the initiator. It
is accompanied by an exothermic reaction and its development can
then be followed by measuring the temperature of the reaction
medium.
[0095] It is possible to use as organic phase vegetable or mineral
oils, certain petroleum distillation products, chlorinated
hydrocarbons or a mixture of these different solutions.
Furthermore, when the polymerization initiator includes several
components (redox system), it is possible to add one of them in the
aqueous phase before emulsification.
[0096] The microspheres thus obtained can then be recovered by
cooling, decanting and filtration. They are then separated by size
category and washed to eliminate any trace of secondary
product.
[0097] The polymerization stage can be followed by a stage of
reticulation of the cell adhesion agent and possibly by a marking
agent stage in the case of microspheres rendered identifiable by
grafting after synthesis.
[0098] Microparticles of the present invention which have the
specific properties of cell adhesion and growth promotion can be
used directly for tissue bulking or dermal augmentation. Moreover,
the microparticles of the present invention can have specific
autologous cells grown on their surface in vitro, thereby making
the microparticles particularly useful for tissue bulking or dermal
augmentation.
[0099] Prior to the present invention, the injection of implantable
substances suspended in a physiological solution into a tissue
resulted in the formation of discrete aggregates inside the muscle
mass. These discrete aggregates can constitute various amounts of
the implanted substance which stays together, however, the
substance does not become attached to or a part of the tissue
itself. This detachment allows the implanted substance to move from
the original implantation site.
[0100] According to the present invention, in order to avoid this
problem, the microparticles may be injected individually and
separately, or more preferably, the surface of the microparticles
may be colonized by a layer of cells for better integration and
long term stability of the implant.
[0101] Microparticles of the present invention demonstrate superior
ability to grow cells on their surfaces. For example, primary
muscle cells have been successfully adhered to the surface of the
microparticles of the present invention thereby allowing for a
better integration within a muscle tissue. In addition, since the
ultimate goal of tissue bulking is to artificially increase tissue
mass, preadipocytes have also been used to colonize the surface of
the microparticles prior injection. In this case, the preadipocytes
have a volume similar to any other regular cell, but after
implantation when the preadipocytes are subject to in vivo
physiological conditions, they accumulate droplets of fats thereby
increasing the mass of the implant by more than 10% in volume.
[0102] The present invention provides a method for causing tissue
bulking in a mammal. The method comprises administering a
composition of elastic, hydrophilic, non-toxic and substantially
spherical microspheres in a biocompatible carrier to the mammal.
The composition is injectable through a needle of about 18 to about
26 gauge and the microspheres are not capable of being digested or
eliminated by macrophage or other elements of said mammal's
lymphatic system. The tissue bulking method of the present
invention is suitable for the treatment of various tissue defects
including, but not limited to, dental tissue defects, vocal cord
tissue defects, or other non-dermal soft tissue defects. The
present method is particularly suitable for GERD, urinary
incontinence, or urinary reflux disease.
[0103] The injection method of the present invention can be carried
out by any type of sterile needles of 18 gauge or smaller and
corresponding syringes or other means for injection, such as a
three-way syringe. The injection is preferably made into the area
that needs tissue bulking treatment. The needles, syringes and
other means for injection are commercially available from suppliers
such as VWR Scientific Products (West Chester, Pa.), Becton
Dickinson, Kendal, and Baxter Healthcare. The size of the syringe
and the length of the needle used will dependent on the particular
injection based on factors such as the specific disease or
disorders being treated, the location and depth of the injection,
and the volume and specific composition of the injectable
suspension being used. A skilled practitioner will be able to make
the selection of syringe and needle based on experience and the
teaching of the present invention.
[0104] The present invention additionally provides a kit for
performing dermal augmentation tissue bulking. The kit comprises an
18 gauge or smaller needle and a corresponding syringe (both of
which are sterile), wherein the syringe optionally contains a
composition comprising biocompatible, elastic, hydrophilic,
non-toxic and substantially spherical microspheres and a
biocompatible carrier. The composition is injectable through the
needle and the microspheres are not capable of being eliminated by
macrophage or other elements of said mammal's immune or lymphatic
system. Alternatively, the kit comprises an 18 gauge or smaller
needle, a corresponding syringe, and separate containers containing
the microspheres in dried and sterilized form and the biocompatible
solvent. The dried sterilized microspheres and the solvent are
ready to be mixed for injection either in their respective
containers or in the syringe. These kits are sterile and ready to
use. The kits are designed in various forms based the sizes of the
syringe and the needles and the volume of the injectable
composition contained therein, which in turn are based on the
specific skin or tissue defects the kits are designed to treat.
[0105] According to the present invention, one means of performing
tissue bulking in a patient can be described as follows:
[0106] a) Primary cells are extracted from the patient by a simple
biopsy and isolated;
[0107] b) These cells are grown on the surface of the
microparticles under growth promoting conditions (e.g., possibly
using a nutrient media which contains autologous serum (drawn from
the patient), until confluence);
[0108] c) The microparticles having the patient's cells grown on
the top are injected into the patient's target tissue to be
bulked.
[0109] For the treatment of GERD, the microparticles, or other
solid substrates, are preferably introduced via the esophagus,
either by endoscopic delivery or by laparoscopic technique, and are
injected into the walls of the sphincter where the esophagus meets
the stomach, i.e., the lower esophageal sphincter. This decreases
the internal lumen of the sphincter muscle thus permitting easier
contraction of the muscle with reduced regurgitation of the gastric
fluids into the esophagus. In addition, this treatment reduces the
inflammation of the lower esophagus. The microparticles, or other
solid substrates, may also be loaded with X-ray opaque dye or other
imaging agents for subsequent X-ray visualization.
[0110] In another embodiment, microparticles injected into the
sphincter at the junction of the esophagus and stomach in order to
treat GERD may also include an amount of a drug used to treat GERD,
such as H.sub.2 histamine antagonists including cimetidine,
ranitidine, famotidine and nizatidine; inhibitors of
H.sup.+,K.sup.+-ATPase including omeprazole and lansoprazole;
antacids including e.g., Al(OH).sub.3, Mg(OH).sub.2, and
CaCO.sub.3. As with the treatment of urinary incontinence, urinary
reflux disease, and skin wrinkles, the microspheres may also be
used with anti-inflammatory agents, angiogenesis inhibitors,
radioactive elements, and antimitotic agents.
[0111] Other therapeutic agents to be used in combination with the
microspheres or microparticles of the present invention include
those for the treatment of skin disorders, GERD, urinary
incontinence and urinary reflux disease as reported in Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
McGraw-Hill (1996) and The Physicians's Desk Reference.RTM.
2000.
[0112] The primary advantages of the method of treating GERD
according to the present invention over the prior art methods
are:
[0113] a) Less invasive effects on the patient compared to
surgery;
[0114] b) More accurate and effective delivery of the microspheres
and therapeutic agents;
[0115] c) More permanent effects over antacids or other drugs;
[0116] d) Good biocompatibility with chemotactic effects; and
[0117] e) Ability to use X-ray visualization or MRI to assist in
follow-up evaluation of the patient.
[0118] For the treatment of urinary incontinence and urinary reflux
disease, the microparticles or microspheres of the present
invention are injectable through needles of about 18 gauge to about
26 gauge, preferably, 22 to 24 gauge, and are not capable of being
eliminated through the lymphatic system. The microparticles are
introduced via the urethra and injected into the walls of the
bladder sphincter, decreasing the internal lumen of the sphincter
muscle thus permitting easier contraction of the muscle with
reduced likelihood of incontinence. The microparticles, or other
solid substrate, may also be loaded with X-ray opaque dye, or other
imaging agents for subsequent X-ray visualization.
[0119] In another embodiment, microparticles injected into the
bladder sphincter in order to treat urinary incontinence or urinary
reflux disease may also include an amount of a drug used to treat
urinary incontinence or urinary reflux disease, such as
antidiuretics, anticholinergics, oxybutynin and vasopressins.
[0120] Injected microparticles can generate some transient adverse
reactions such as local inflammation, therefore the microparticles
can contain or be injected with anti-inflammatory drugs, such as
salicylic acid derivatives including aspirin; para-aminophenol
derivatives including acetaminophen; non-steroidal
anti-inflammatory agents including indomethacin, sulindac,
etodolac, tolmetin, diclodfenac, ketorolac, ibuprofen, naproxen,
flurbiprofen, ketoprofen, fenoprofen, oxaprozin; anthranilic acids
including mefenamic acid, meclofenamic acid; enolic acids such as
piroxicam, tenoxicam, phenylbutazone, oxyphenthatrarone;
nabumetone; Vioxx.RTM. and Celebrex.TM.. These anti-inflammatories
are preferably adsorbed on the microparticle's network and released
slowly over a short period of time (a few days). The microparticles
may also be used to release other specific drugs which can be
incorporated within the microparticle network before injection into
the patient. The drug would be released locally at the site of
implantation over a short period of time to improve the overall
treatment.
[0121] Incorporation of active molecules, such as drugs, into the
microparticles of the present invention can be accomplished by
mixing dry microparticles with solutions of said active molecules
or drugs in an aqueous or hydro-organic solution. The
microparticles swell by adsorbing the solutions and incorporate the
active molecule of interest into the microparticle network. The
active molecules will remain inside the microparticle due to an
active mechanism of adsorption essentially based on ion exchange
effect. The microparticles by their nature carry cationic groups
and have the ability to adsorb anionic molecules, such as well
known anti-inflammatory drugs, and these anionic molecules are then
released slowly upon injection into the patient due to the action
of physiological salt and pH. The ability of various types of
microparticles to adsorb drug molecules may be readily determined
by the skilled artisan, and is dependent on the amount of cationic
monomers present in the initial solution from which the
microparticles are prepared.
[0122] Some of the primary advantages of treating urinary
incontinence or urinary reflux disease according to the present
invention over prior art methods are:
[0123] a) More permanent effect than the use of regular viscous
solutions of collagen;
[0124] b) More accurate and effective delivery of the microspheres
and therapeutic agents;
[0125] c) Good biocompatibility with chemotactic effect;
[0126] d) Visualization under X-ray or MRI to assist in follow-up
evaluation; and
[0127] e) Preventing repeated treatments with resorbable naturally
occurring substances like collagen.
[0128] The primary advantages of the method of treating skin
wrinkles according to the present invention are:
[0129] a) less invasive effects on the patient compared to
surgery;
[0130] b) More accurate and effective delivery of the microspheres
and therapeutic agents;
[0131] c) more permanent effects than the use of collagen
injections; and
[0132] d) good biocompatibility with chemotactic effects.
[0133] The dermal augmentation method of the present invention
comprises administering a composition of elastic, hydrophilic,
non-toxic and substantially spherical microspheres in a
biocompatible carrier to a mammal in need of such treatment. The
composition is injectable through needles of about 30 gauge or
smaller and the microspheres are not capable of being digested or
eliminated through macrophages or other elements of the immune
system. The injectable composition is preferably a suspension of
the microspheres in the biocompatible carrier. The microspheres are
preferably injected into the mammal's subcutaneous layer. The
microparticles may also include one or more anti-inflammatory
agents.
[0134] Suitable for treatment using the dermal augmentation method
of the present invention are skin contour deficiencies caused by
various conditions including, but not limited to, aging,
environmental exposure, weight loss, child bearing, surgery,
disease such as acne and cancer, or combinations thereof. The
dermal augmentation method of the present invention is particularly
suitable for skin contour deficiencies such as frown lines, worry
lines, wrinkles, crow's feet, facial scars, marionette lines,
stretch marks, surgical scars, wounds, and cuts and bites due to
injury or accidents.
[0135] The present invention also provides methods of causing
tissue bulking or dermal augmentation by injecting the injectable
composition not directly into the body, but extracorporeally into
organs, components of organs, or tissues prior to their inclusion
into the body, organs, or components of organs.
[0136] The injection of the present invention's method can be
preferably carried out by any type of sterile syringes with needles
of about 18 to 26 gauge. The size of the syringe and the length of
the needle used will dependent on the particular injection based on
factors such as the specific disease or disorders being treated,
the location and depth of the injection, and the volume and
specific composition of the injectable suspension being used. A
skilled practitioner will be able to make the selection of syringe
and needle based on experience and the teaching of the present
invention.
[0137] The invention is further defined by reference to the
following examples that describe in detail the preparation of
microparticles for use in tissue bulking, and the treatment of skin
wrinkles, urinary incontinence, and GERD. The following examples
are illustrative only and should in no way limit the scope of the
present invention. It will be apparent to those skilled in the art
that many modifications, both to materials and methods, may be
practiced without departing from the purpose and scope of this
invention.
6. EXAMPLES
6.1 Example 1
Preparation Of Irregular Hydrogel Particles with Chemotactic
Properties
[0138] 58 grams of sodium chloride and 27 grams of sodium acetate
were dissolved at room temperature in 100 ml of demineralized
water. To this solution 400 ml of glycerol were added, the pH was
adjusted to 6.0 and monomers were then dissolved. More specifically
to this solution 90 gram of methylolacrylamide, 2 g of
methacrylamidopropyl-trimethylammonium-chlo- ride hydrochloride and
10 gram of N,N'-methylene-bis-acrylamide were added and the mixture
was agitated until complete solubilization. The solution was heated
at about 70.degree. C. and 100 ml of a solution of gelatin at a
concentration of 500 mg/ml was added. The total volume of the
mixture was then adjusted to 1000 ml by addition of demineralized
water. Finally 20 ml of 70 mg/ml ammonium persulfate aqueous
solution and 4 ml of N,N,N',N'-tertamethyl-ethylene-diamine was
added. The obtained mixture was stored at 70.degree. C. for about 3
hours until formation of a compact three-dimensional gel. This gel
was totally insoluble in water. It was cut in small pieces and then
ground to get very small particles of a dimension close to 100-200
.mu.m. The particles were then suspended in 1 liter of
physiological buffer containing 5% (w/v) glutaraldehyde and were
shaken for two hours. Finally the particles were extensively washed
to eliminate unpolymerized material, by-products and salts. To
obtain homogeneous particle size distribution the particle
suspension was sieved using an appropriate sieving net.
[0139] These particles possess the characteristics desired for
tissue cell adhesion prior to muscle bulking and include cationic
groups and adhesion agents for an effective cell adhesion
mechanism.
6.2 Example 2
Preparation of Spherical Polyacrylic Hydrogel Gel Particles with
Chemotactic Properties
[0140] The solution of monomers prepared as described in Example 1
above was poured slowly into 1500 ml of stirred and hot paraffin
oil (50-70.degree. C.). After a few minutes a suspension/emulsion
of liquids was obtained (the aqueous monomer solution was dispersed
into oil and forms very small spherical droplets) and the
polymerization occurred in suspension. The microdroplets were
transformed into microbeads. The solid microbeads were recovered by
centrifugation and suspended in 1 liter of physiological buffer
containing 5% (w/v) glutaraldehyde and shaken for two hours.
Finally the particles were extensively washed with water to
eliminate completely the oil traces. Organic solvent extraction can
be used for a more effective oil removal or an extensive washing in
the presence of traces of nonionic detergents. The obtained
microbeads are calibrated if necessary by sieving through a nylon
net and sterilized in an autoclave. These microspheres possess
desired characteristics and properties for cell adhesion prior to
muscle bulking.
6.3 Example 3
Preparation of Hydrophilic Spherical Polystyrene Copolymer
Particles Useful for Tissue Bulking
[0141] 10 gram of styrene is mixed with 60 ml of toluene. 1 gram of
divinylbenzene, 1 gram of dimethyl-aminoethyl-methacrylate and 1
gram of dimethyl-acrylamide are added to the resulting solution.
After complete solubilization the monomer solution is mixed with 1%
of AIBN (2,2'-azobisisobutyronitrile) as a polymerization catalyst
and with 40 ml of paraffin oil as a viscosity inducer agent. The
mixture is poured in an agitated water solution containing 0.5%
Tween 80. In this situation there is formation of droplet
suspension which turns into solid microbeads when the temperature
is raised to 80-90.degree. C. for three to five hours. The
resulting beads are dried and organic solvents extracted. They are
then swollen in an aqueous solution of collagen in phosphate buffer
at neutral pH. Embedded collagen is then crosslinked with
glutaraldehyde as described in Examples 1 and 2. The resulting
beads possess cationic charges to interact with cell tissues and
collagen for cell adhesion, and a chemotactic agent for cell growth
and biocompatibility. They are suitable as tissue bulking
agent.
6.4 Example 4
Preparation of Hydrophilic Silicone Beads for Cell Adhesion and
Tissue Bulking
[0142] 10 gram of silicone beads of a diameter of 20-300 .mu.m are
suspended in 30 ml of a solution of hexadecylamine (10 mg/ml) in
ethylacetate. The suspension is stirred for two hours and 100 ml of
ethanol is added. A 1 M ammonium sulfate or sodium chloride
solution in water is added slowly until a 300 ml suspension is
obtained. The amino-containing silicone beads are then reacted with
a butanedioldiglycydylether in alkaline conditions. Epoxy
derivatives are thus obtained on which gelatin is coupled using a
method well known in the art. The resulting beads have the target
properties of biocompatibility, hydrophilicity,
non-biodegradability and cell adhesion by the presence of cationic
amino groups and of gelatin as a cell growth promoting agent. They
are suitable for tissue bulking in accordance with the present
invention.
6.5 Example 5
Preparation of Beads for Tissue Bulking Containing Adhesion
Factors
[0143] Beads prepared according to Example 2 were chemically
activated with well known reagents used in the preparation of
affinity chromatography sorbents. Activated beads were then used
for the immobilization of cell adhesion agents such as fibronectin
or vitronectin or laminin. Adhesion agents were dissolved at 1-10
mg/ml in a coupling buffer (100 mM carbonate or borate buffer pH 8
to 10) and the solution was mixed with the activated beads. The
resulting beads possess the target properties of cell adhesion and
growth, non-biodegradability and were non-resorbable. They are
suitable for cell adhesion and permanent tissue bulking in
accordance with the present invention. Similarly, beads prepared
according to Examples 3 and 4 can also be used.
6.6 Example 6
Preparation of Spherical Polyacrylic Hydrogel Particles with
Chemotactic Properties
[0144] Microbeads commercially available under the name SPEC-70
(BioSepra Inc., Marlborough, Mass.) are polyacrylic polyanionic
beads with elastic properties suitable for tissue bulking
applications. However, these microbeads are not chemotactic and do
not possess cationic charges. SPEC-70 microbeads are first drained
under vacuum to eliminate water and then suspended in an aqueous
solution of 1% chondroitin sulfate sodium salt in physiological
conditions. Once this compound is absorbed on the bead structure,
the beads are drained under vacuum and suspended in an aqueous
solution containing 20% polylysine by weight. The suspension is
shaken for a few hours and then drained under vacuum and rapidly
washed with distilled water. The beads are then suspended in a
solution of 5% butanedioldiglycidylether in ethanol and shaken
overnight. Under these conditions, the polylysine is crosslinked as
well as chondroitin sulfate. The resulting modified beads possess
properties such as cationic charge for cell adhesion and promoting
agents for cell growth such as polylysine and chondroitin
sulfate.
6.7 Example 7
Preparation of Radiopaque Microbeads with Chemotactic Properties
for Tissue Bulking
[0145] Microbeads from Examples 2 were drained under vacuum and
then suspended in a saturated solution of barium chloride. They
were shaken for two hours at room temperature and then drained
under vacuum to eliminate the excess of barium chloride solution.
The beads were suspended in a saturated solution of ammonium
sulfate and shaken for two additional hours before elimination of
the excess ammonium sulfate by vacuum filtration. This operation of
contact with barium salts and ammonium sulfate can be repeated
several times until the resulting radiopaque precipitate inside the
beads reaches the desired amount. Resulting beads have radiopaque
properties without having lost their initial desirable properties
for tissue bulking. The microbeads from Examples 3, 4 and 6 can be
similarly used.
6.8 Example 8
Preparation of Radiopaque Microbeads with Chemotactic Properties
for Tissue Bulking
[0146] Microbeads from Example 6 coated with polylysine are washed
extensively with distilled water and suspended in a solution of
sodium triazoate. The suspension pH is adjusted at about 7 by
addition of acetic acid and shaken for several hours. The triazoate
which is a radiopaque molecule is adsorbed tightly to the beads and
the remaining reagents are eliminated by washing under vacuum. The
resulting beads still possess cell promotion properties and now
radiopacity as well.
6.9 Example 9
Introduction of Anti-Inflammatory Drugs Inside the Bulking
Beads
[0147] Microbeads described in the previous Examples may generate
local temporary inflammatory reactions when injected in the target
tissue. To avoid or decrease this phenomenon, the microbeads once
coated with autologous cells can be filled with one or more
anti-inflammatory drugs. The microbeads are cationic by their
nature and can absorb anionic drugs by ion exchange effect.
[0148] Prior to injection microbeads are mixed with a 10 mg/ml
anti-inflammatory anionic drug solution in sterile physiological
saline. The suspension is shaken for several hours, and the beads
filled with the drug are recovered by filtration or centrifugation.
The resulting anti-inflammatory containing microbeads may then be
used as tissue bulking agents for use in the present invention.
6.10 Example 10
In Vitro Pre-Adipocytes Adhesion and Growth on Polymeric Beads
[0149] In order to assess the ability of polymeric beads from
Example 2 to allow adhesion and growth of pre-adipocytes, fresh
pre-adipocytes were collected and isolated from Wistar rat
peri-epididymal fat tissue. Pre-adipocytes were then cultured in
the presence of above described microbeads at a concentration of
about 7.1.times.10.sup.5 to about 1.7.times.10.sup.6 cells/ml using
the classical protocol for microcarrier culture in vitro. In a
first phase the cells adhere on the bead surface and then they grow
to totally cover the bead surface. The total colonization period is
about 72 hours.
[0150] Pre-adipocytes from this type of culture show good growth
and specific biological activity associated with differentiation
into adipocytes (accumulation of lipids). Moreover these cells show
the presence of specific enzymatic markers such as
glycerol-3-phosphate-dehyd- rogenase and malate dehydrogenase.
Microbeads having cells adhered thereto are useful for tissue
bulking for use in the present invention. The polymeric beads of
Examples 2 to 5 can be similarly assessed.
6.11 Example 11
Culture of Pre-Adipocytes and Myocytes on Microbeads In Vitro to
Check Their Ability of Integrate Into an In Vivo Tissue
[0151] Preadipocytes and smooth muscle cells were isolated from
Wistar rats according to a classical protocol to eliminate most of
other contaminating cells. Separately these cells were cultured in
a Petri dish in the presence of Dulbecco's Modified Eagle Medium
supplemented with 10% fetal bovine serum. Gelatin-coated cationic
microbeads prepared in accordance with Example 2 were added to
cells cultured in vitro until they covered the surface of the Petri
dish. Initial cell seed concentration was 0.7.times.10.sup.6
cells/ml.
[0152] Repeated observations showed that cells adhered on the
surface of microbeads and further multiplied to cover all the
surface of the beads. After 5 to 7 days of culturing, there was
formation of a solid network of beads where cells acted as a binder
to consolidate the blocks of several beads. In most cases there
were formation of solid non dissociable aggregates comprising beads
and cells.
[0153] When, after a growing period (generally 5 to 7 days), a
differentiating element such as 3,3',5-triiodo-D-thyronine was
added to preadipocytes, the preadipocytes started to accumulate
fats as micro-droplets within the cytoplasm.
[0154] Specific staining with 3,3'-dioctadecyloxacarbocyanine
perchlorate or
2'-[4-hydroxyphenyl]-5-[4-methyl-1-piperazinyl]2,5'-bi-1H-benzimidazol-
e demonstrated good adhesion of the cells on the bead
substrate.
[0155] Staining of the cells with red oil at the beginning of the
differentiating phase evidenced the accumulation of fats inside the
cells.
[0156] In addition, specific enzymatic reactions of malic enzyme
indicated that, at the end of the culture, resulting adipocytes
were functionally viable with their major expressed
characteristics. This enzyme is not expressed at the beginning of
the culture and appeared simultaneously with the accumulation of
fats.
[0157] Smooth muscle cells were also followed in their
proliferation by DNA synthesis assay; their adhesion on the
substrate was followed as per preadipocyte cells. Myocytes also
showed good proliferation as well as adhesion on the beads.
6.12 Example 12
In Vitro Myocyte Adhesion and Growth on Polymeric Beads
[0158] In order to assess the ability of polymeric beads from
Example 2 to allow adhesion and growth of muscle cells, fresh
smooth cell myocytes were collected from rat esophagus according to
classical procedures. Cells were then cultured in the presence of
above described microbeads at a concentration of about 10.sup.6
cells/ml using the classical protocol for microcarrier culture in
vitro. In a first phase the cells adhered on the bead surface and
then they grow until they cover the total bead surface. The total
colonization period was about 72 hours.
[0159] Myocytes from this type of culture showed good growth and
behavior and displayed the specific myosin marker. These microbeads
having cells adhered thereto are useful for tissue bulking in
accordance with the present invention. The beads from Examples 2 to
5 can be similarly assessed.
6.13 Example 13
Preparation of Injectable Suspension of Cell-Microbead Particles
for In Vivo Bulking
[0160] At the issue of cell culture phase, the cell-bead particles
are collected by filtration and washed extensively with blood serum
from the host where the material is to be implanted. This operation
ensures the elimination of foreign material from cell culture. The
microbeads are then suspended in a few ml of autologous serum (a
ratio of beads/serum is about 1:1) and are ready to be injected
within the tissue to be bulked by means of an appropriate syringe
or other injection device.
6.14 Example 14
Preparation of Injectable Suspension of Cell-Microbeads Particles
for In Vivo Bulking
[0161] Microbeads described in Example 2 are colonized with rat
muscle cells according to Example 10 and conditioned according to
Example 13 using rat serum diluted with physiological saline
(50%-50%). The final sterile suspension of cells anchored on beads
(50% of volume is constituted of beads and 50% of physiological
saline) is injected in the right thigh muscle of a rat. Three
months after bead injection the muscle was observed in its shape
and histologically examined. Muscle volume should be larger than
the left thigh muscle upon autopsy. Beads inside the muscle mass
should appear surrounded by fibroblastic cells with no specific
adverse inflammatory or necrosis effects.
[0162] The embodiments of the present invention described above are
intended to be merely exemplary and those skilled in the art will
recognize, or be able to ascertain using no more than routine
experimentation, numerous equivalents to the specific procedures
described herein. All such equivalents are considered to be within
the scope of the present invention and are covered by the following
claims.
[0163] The contents of all references described herein are hereby
incorporated by reference.
[0164] Other embodiments are within the following claims.
* * * * *