Methods for wound treatment

A method and a device for the treatment of wounds and promotion of wound healing and muscle regeneration. The method includes applying a composition including a non-biodegradable microsphere with a substantial surface charge to the subject. The device further includes a pharmaceutically acceptable carrier and a container for containing the composition and the carrier. The microspheres used in the present invention have been shown to promote wound healing and muscle regeneration both in vivo and in vitro.

FIELD AND BACKGROUND OF THE INVENTION 
The present invention relates to wound treatment and, in particular, it 
concerns a device and methods for accelerating wound healing and 
enhancement of muscle regeneration with microspheres as a therapeutic 
agent. 
Wound healing is a complex process involving such factors as cells, 
extracellular matrix (ECM) components and the cellular microenvironment. 
Essentially, all wound healing involves the repair or replacement of 
damaged tissues. The precise nature of such repair or replacement depends 
upon the tissues involved, although all such processes involve certain 
basic principles. To illustrate these principles, cutaneous, or skin, 
wound healing will be described, it being understood that the discussion 
could also extend to all types of wound repair. 
Skin has multiple layers, including keratin, epidermis and dermis. If only 
the epidermis is damaged, as in most minor injuries, keratinocytes migrate 
from the edge of wound and eventually cover it, reforming the epidermis 
and keratin D. R. Knighton and V. D. Fiegel, Invest. Radiol., 26:604-611, 
1991!. 
If all skin layers are damaged or destroyed, new connective tissue, called 
granulation tissue, must first fill the wound space. This tissue is formed 
by deposition of ECM components by fibroblasts, which migrate into the 
wound space D. R. Knighton and V. D. Fiegel, Invest. Radiol., 26:604-611, 
1991!. The deposition of these ECM components, such as collagen, is 
currently believed to be important for healing of the wound. Indeed, the 
prior art teaches that the strength of the healing wound is ultimately 
dependent upon collagen deposition Haukipuro, K., et al., Ann. Surg., 
213:75-80, 1991!. Thus, collagen deposition must be present at a 
sufficiently high level to give the healing wound strength and support. 
This entire multi-step process must be completed for successful wound 
healing. If one or more of these components is missing, healing does not 
take place, the skin is not repaired and the wound remains open. Such open 
wounds can easily become infected, further retarding the process of 
healing and leading to the formation of ulcers and sores on the skin. The 
process of wound healing is further inhibited in many patients by the 
presence of other complicating conditions, such as diabetes and old age. 
Patients with such conditions often have skin wounds which ulcerate and 
refuse to heal, or only heal slowly after an extended period of time has 
elapsed. 
Various treatments have been used in order to accelerate the rate at which 
wounds heal. For example, U.S. Pat. No. 4,772,591 discloses a method of 
accelerating the rate of wound healing by applying a combination of 
ascorbic acid, calcium, tyrosine or phenylalanine, and anti-inflammatory 
substances to the wound. Similarly, U.S. Pat. No. 4,590,212 discloses a 
method of applying acetaminophen to the wound. Many other patents have 
focussed upon other methods of accelerating the rate of wound healing. 
However, none of these methods has proven broadly effective. 
In an attempt to improve treatments for wounds, various pharmaceutical 
carriers have been employed to deliver chemotherapeutic agents to the 
wound. Such carriers are particularly required for skin wounds since they 
are generally either exposed to air, or covered by bandages or clothing. 
In either case, the therapeutic agent can easily be removed by rubbing, 
for example. Thus, various creams, gels and powders have been used as 
pharmaceutical carriers, in an attempt to overcome this problem. 
One interesting group of pharmaceutical carriers employs microspheres, 
which are small, microscopic particles made of various materials, 
including plastics and long-chain carbohydrates. Many prior art 
applications are known for microspheres as carriers for various 
therapeutic agents. For example, U.S. Pat. No. 5,264,207 discloses 
microspheres as a carrier for a pharmaceutical or cosmetic substance. A 
composition containing the microspheres and the active substance is 
applied cutaneously, with the microsphercs in effect enabling such a route 
of administration for the active substance. However, this reference does 
not teach or suggest using the microspheres themselves as a therapeutic 
substance. 
Similarly, PCT Application Nos. WO 96/13164 and WO 94/13333 both disclose 
microspheres made of a material which catalyzes the production or release 
of certain therapeutic substances. PCT Application No. WO 96/13164 
discloses polymeric nitric oxide adducts which release nitric oxide when 
directly applied to damaged tissue. PCT Application No. WO 94/13333 
discloses particles which are chemically modified to have free radical 
activity in the wound environment. Again, neither reference teaches or 
suggests using the microspheres themselves as a therapeutic substance, 
without chemical modification of the microsphere material. 
However, certain properties of the microspheres used as pharmaceutical 
carriers were shown to influence the effect of the therapeutic substance 
itself. For example, the activation of cytotoxic T lymphocytes by class I 
alloantigen immobilized on latex microspheres was studied. Although the 
class I alloantigen was clearly providing the stimulus itself, the extent 
of cell stimulation was increased by using particle sizes of 4 to 5 
microns M. F. Mescher, J. Immunol., 149:2402-2405, 1992!. Such increased 
stimulation may demonstrate surface contact requirements for cytotoxic T 
lymphocytes. In other words, the optimum particle size may have increased 
the effect of class I alloantigen by providing an optimum surface area for 
cell contact. It should be emphasized, however, that these beads were 
still only carriers for the active substance. 
Attempts have been made to exploit the apparent ability of certain 
particles to enhance the efficacy of active substances to promote wound 
healing. For example, U.S. Pat. No. 3,842,830 discloses glass 
microparticles which act to promote wound healing when directly applied to 
damaged tissue. U.S. Pat. No. 5,092,883 discloses biodegradable 
positively-charged dextran beads with a similar ability to promote 
osteogenesis and healing of soft tissue injuries. However, none of these 
references teaches or suggests the promotion of regeneration of muscle by 
administration of microspheres to the wound. Furthermore, none of these 
references teaches microspheres which initially promote more rapid cell 
metabolism and proliferation, yet which have a limited, finite effect, so 
that rapid cell metabolism and proliferation are not permanently induced. 
Such a limited effect is especially important in promoting wound healing, 
which requires an initial increase in cell metabolism and proliferation, 
followed by a cessation of such cell activation after healing has 
occurred. Without an induction of such activation, wound healing will not 
occur. However, if cell activation does not cease after healing is 
substantially complete, abnormal scar formation can result, as in the 
formation of keloids. Thus, there must be a balance between promotion and 
inhibition of cell metabolism and proliferation during wound healing. 
There is therefore an unmet medical need for a particulate substance which 
can be directly applied to damaged tissue in order to promote healing, yet 
which has self-limited effects and which is substantially non-toxic, and 
which can also promote muscle regeneration. 
SUMMARY OF THE INVENTION 
According to the teachings of the present invention, there is provided a 
method and device of treating a wound of a subject. The method includes 
the step of administering a composition to the wound of the subject, the 
composition consisting essentially of an agent capable of forming a 
multi-point contact with a cellular membrane, the agent being 
substantially non-biodegradable during the period of treatment. 
Preferably, the agent is a microsphere having charged surface groups. 
According to one embodiment, the composition consists essentially of an 
agent with charged surface groups, wherein the charge can be negative or 
positive. 
According to particular embodiments of the present invention, the 
microsphere material is selected from the group consisting of polystyrene, 
derivatized polystyrene, polymethylmethacrylate (PMMA), silicone, 
polylysine, poly-N-ethyl-4-vinylpyridinium bromide and latex. According to 
certain embodiments of the present invention, the charged surface groups 
are selected from the charged groups consisting of polystyrene, 
derivatized polystyrene, sulfate, poly-N-ethyl-4-vinylpyridinium bromide, 
protamine, protamine sulfate, protamine salts, polylysine and carboxyl. 
Also preferably, the microsphere has a diameter in a range of from about 
0.01 microns to about 200 microns, more preferably in a range of from 
about 0.1 to about 100 microns, and most preferably from about 0.1 to 
about 20 microns. According to another embodiment of the present 
invention, the composition also includes a pharmaceutically acceptable 
carrier for the microsphere. 
According to yet another embodiment of the present invention, there is 
provided a method of promoting muscle regeneration in a subject, including 
the step of administering a composition to the subject, the composition 
including an agent capable of forming a multi-point contact with a muscle 
cell, preferably a microsphere having a surface group with a substantial 
charge which may be either positive or negative. 
According to still another embodiment of the present invention, there is 
provided a device for treating a wound, including: (a) a composition 
including an agent being capable of forming a multi-point contact with a 
cellular membrane and a pharmaceutically acceptable carrier in which the 
agent is substantially insoluble; and (b) a container for containing the 
composition. As exemplified, the carrier is preferably selected from the 
group consisting of aqueous medium, aerosol carrier, ointment and bandage. 
According to yet another embodiment of the present invention, there is 
provided a device for promoting muscle regeneration, including: (a) a 
composition including an agent being capable of forming a multi-point 
contact with a cellular membrane and a pharmaceutically acceptable carrier 
in which the agent is substantially insoluble; and (b) a container for 
containing the composition. 
The method of the present invention may also be used cosmetically, to 
prevent excess scar formation in a cut or other wound to the skin such as 
the skin of the face, and to treat acne. 
DETAILED DESCRIPTION 
The present invention relates to a device and a method for promoting wound 
healing by using microspheres. Unexpectedly, microspheres of the 
particular size range described herein are able to promote wound healing 
without the further addition or inclusion of any drug or other therapeutic 
substance. Indeed, as described below, these microspheres do not degrade 
or undergo other chemical alteration in order to produce their therapeutic 
effect. 
The structure of these microspheres includes a core material and at least 
one type of charged surface group which is present at least on the 
exterior of the microsphere. Examples of materials include long-chain 
polymers such as polystyrene, latex, poly-.alpha.-alanine, 
polymethylmethacrylate (PMMA), silicone and derivatized polystyrene. 
Examples of surface groups include sulfate, poly-N-ethyl-4-vinylpyridinium 
bromide, protamine, protamine sulfate, protamine salts, polylysine, 
carboxyl and polystyrene. These surface groups may be present as part of 
the core material, or may be added later by such chemical processes as 
derivatization of the long-chain polymer. Hereinafter the term 
"derivatization" refers to the process of chemically altering, modifying 
or changing a molecule or a portion thereof. The microspheres produced 
from the polymer should be substantially insoluble in aqueous media, 
instead forming a suspension or dispersion in such media. 
In order to further clarify the parameters of the present invention, a 
number of terms should be defined. Hereinafter, the term "wound" includes 
any injury to any portion of the body of a subject including, but not 
limited to, acute conditions such as thermal burns, chemical burns, 
radiation burns, burns caused by excess exposure to ultraviolet radiation 
such as sunburn, damage to bodily tissues such as the perineum as a result 
of labor and childbirth, including injuries sustained during medical 
procedures such as episiotomies, trauma-induced injuries including cuts, 
those injuries sustained in automobile and other mechanical accidents, and 
those caused by bullets, knives and other weapons, and post-surgical 
injuries, as well as chronic conditions such as pressure sores, bedsores, 
conditions related to diabetes and poor circulation, and all types of 
acne. Areas of the body which can be treated with the present invention 
include, but are not limited to, skin, muscle and internal organs. 
Hereinafter, the term "subject" refers to a human or lower animal on whom 
the present invention is practiced. 
Hereinafter, the term "promoting" includes accelerating and enhancing. 
Hereinafter, "reducing scarring" includes preventing or decreasing excess 
scar formation such as keloids and hypertrophic scars, as well decreasing 
the extent of scar tissue formation both externally such as on the skin of 
the subject, and internally such as adhesions. Finally, it should be noted 
that the method of the present invention may also be used cosmetically, to 
prevent excess scar formation in a cut or other wound to the skin such as 
the skin of the face, and to treat acne. In a cosmetic sense, the term 
"excess scar formation" includes any scarring which is cosmetically 
undesirable or unacceptable. 
Although the discussion below refers to specific types of microspheres, it 
should be noted that this is not intended to be limiting in any way. It 
will be appreciated to those skilled in the art that these microspheres, 
more generally described as "agents", can be beads, particles or globules 
which are either solid or hollow. In preferred embodiments of the present 
invention, these agents are dispersed in a pharmaceutically acceptable 
carrier medium in which the agents are substantially insoluble, as a 
suspension in aqueous medium for example, or in a non-aqueous medium such 
as an ointment, aerosol spray, or a bandage which may be occlusive or 
non-occlusive. The shape of the agents can be regular, such as spherical 
or elliptical, or regular non-spherical shapes; or the shape of the 
particles can be non-regular, so that the surface is not a single 
continuous curve or so that the surface is not smooth. 
Furthermore, the agents can be a mixture of different polymers and can also 
be a mixture of different particles, beads or globules of different sizes. 
The agents can also have pores of different sizes. 
By way of example, the long-chain polymer forming the agents, such as 
poly-.beta.-alanine, can be cross-linked, which particularly favors the 
spherical shape of a microsphere, although such a shape can be obtained 
without cross-linking. An example of a method of manufacture for a 
cross-linked poly-.beta.-alanine microsphere is given in U.S. Pat. No. 
5,077,058, although it should be noted that this material would require 
further derivatization to obtain an overall surface charge of the 
microsphere. 
Alternatively, the particles can have chaotic irregular forms, particularly 
if the polymer is not cross-linked. The particle can have any form, such 
as coiled, globular, extended and random coil. Preferably, the polymer 
should not be biochemically reactive and should be non-biodegradable. Most 
preferably, the polymer is non-biodegradable substantially during the 
treatment period, so that it would remain undegraded during the period 
required for healing of the wound. Hereinaftcr, the term 
"non-biodegradable" refers to agents which are not biodegradable during 
the treatment period, which is the period required for treatment of the 
wound. 
At the very least, the agents should have the following properties: 
1. They should be capable of forming multi-point contacts with cells or 
portions of cells thereof, such as the outer cell membrane and molecules 
on this membrane; 
2. They should be able to promote wound healing without significant 
chemical alteration or degradation; and 
3. They should be substantially insoluble in aqueous media such as bodily 
fluids, and instead should form a suspension. 
These characteristics are important because as further discussed below, the 
effect of the agents of the present invention appears to be directly 
linked to the formation of multi-point contacts between the material of 
the agents and a portion of the cell such as the outer cell membrane, 
thereby forming an adherent surface for the cells to attach to. Such 
multi-point contacts are possible with many different polymers which 
permit charged groups to be accessible for interaction with molecules and 
portions of the outer cell membrane. Thus, although the description below 
focuses on one type of agent, microspheres, it is understood that the 
present invention covers any material capable of forming such multi-point 
contacts. 
As noted above, preferably the microsphere has a diameter in a range of 
from about 0.01 microns to about 200 microns, more preferably in a range 
of from about 0.1 to about 100 microns, and most preferably from about 0.1 
to about 20 microns. Without desiring to be bound by any mechanism, it 
should be noted that these preferred ranges are the best size for enabling 
uptake of the microspheres by macrophages infiltrating the wound area. The 
microspheres appear to actually attract and activate the macrophages 
through contact with at least a portion of the macrophages, probably the 
molecules of the outer cell membrane of the macrophage. The 
anti-inflammatory and anti-bacterial effects observed for the microspheres 
are thus presumably indirect effects, obtained through the activation of 
the macrophages or other cells. 
Another important property of the microspheres is the charge of the surface 
groups. The overall charge carried by certain preferred examples of 
microspheres was measured as a Z or zeta potential by electrophoretic 
mobility (milliVolts) by a ZetaMaster (Malvern Instruments, United 
Kingdom). The range of Z potentials measured in certain embodiments 
exemplified herein was from -29.58 mV to -79.76 mV. Hereinafter, the term 
"charged" refers to a Z potential with an absolute value of at least about 
1 mV, and preferably of at least about 10 mV, whether negative or 
positive. 
The microspheres in the suspensions tested did not aggregate, coalesce, 
clump or undergo irreversible caking. Although the microspheres did settle 
somewhat over time, they were easily resuspended with gentle agitation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is exemplified herein by the use of microspheres 
which can be used to promote wound healing in general, as well as muscle 
regeneration. Wound healing and muscle regeneration both involve the 
repair of damaged tissue and the replacement of missing tissue. The 
migration and proliferation of specific types of cells must occur in an 
orderly and structured manner, which can be easily differentiated from the 
unrestrained growth of malignant tissues such as solid tumors. In 
particular, cells involved in wound healing and muscle repair must first 
become activated in order to perform their required roles in the healing 
process. Although the exact mechanism is not known, the orderly, 
structured cell growth in proliferation which occurs in wound healing 
clearly demonstrates the presence of a highly organized regulatory 
process. 
As demonstrated in the Examples given below, the microspheres of the 
present invention do not appear to interfere with this complex, organized 
and structured process, since these microspheres clearly only quicken the 
pace of the overall healing process, as well as of specific steps within 
that process. However, unexpectedly the microspheres of the present 
invention do not cause the cells to exhibit a state of continuous, 
unrestrained metabolic activation, indicating that normal regulatory 
processes are not affected. Thus, the microspheres of the present 
invention do not cause unrestrained cellular activation. 
Without limiting the present invention to a particular mechanism, the 
addition of microspheres with negatively charged groups may have a 
therapeutic effect on wound healing by serving as an additional surface 
for the attachment and plating of cells. One hypothesis for the efficacy 
of the microspheres of the present invention is that the negatively 
charged groups enable the creation of multiple links between the solid 
surface of the microsphere and the cell membranes, which represent 
multi-point contacts between the material of the microsphere and the cell 
membrane. The formation of these links causes changes in the distribution 
and state of membrane ligands, cytoskeletal reorganization, activation of 
intracellular signal transduction and other biochemical changes, 
eventually leading to activation of the cell. Cell activation then leads 
to cell proliferation and production of growth factors, and of collagen 
and other components of the extracellular matrix. It should be noted that 
the present invention need not rely on any particular mechanism, since as 
demonstrated below, these microspheres clearly had a beneficial effect for 
wound treatment and healing in vivo. 
A number of different types of microspheres were tested in the Examples 
below. These microspheres were made of polystyrene, either with carboxyl 
or amino surface groups or without additional surface groups. The 
diameters of the microspheres ranged from about 0.1 to about 20 microns. 
The zeta potential of certain microspheres was also tested and 
demonstrated that the size of the sphere and the type of surface groups 
clearly had an effect on the amount of overall charge carried by each 
microsphere, which could have important effect on the ability of the 
microsphere to promote wound healing. 
Although certain specific types of microspheres are illustrated, it is 
understood that many other related types of microspheres could be used if 
the following characteristics were fulfilled. 
1. They should be capable of forming multi-point contacts with cells or 
portions of cells thereof; 
2. Their mechanism of action should not require chemical alteration or 
degradation; and 
3. They should be substantially insoluble in aqueous media such as bodily 
fluids, and instead should form a suspension. 
Other preferable attributes include the following. First, the microspheres 
should preferably be made from material which is non-biodegradable during 
the treatment period, most preferably polystyrene. Second, the 
microspheres should preferably carry a substantial charge, more preferably 
an overall negative charge. Although the size of the microspheres is less 
critical, preferably the microspheres should be from about 0.1 to about 20 
microns in diameter. Preferably, the microspheres should be derivatized 
with carboxyl surface groups, although other negatively charged groups may 
also be used. Thus, these types of microspheres are given for illustrative 
purposes only and are not meant to be limiting in any way. 
The principles and operation of microspheres according to the present 
invention may be better understood with reference to the Examples, 
drawings and the accompanying description. 
EXAMPLE 1 
Effect of Microspheres on Creatine Phosphokinase 
The microspheres of the present invention clearly induced an initial 
increase in creatine phosphokinase (CPK) activity of cultured myoblasts, 
as shown in FIG. 1. However, after eight days, the untreated and treated 
cells both demonstrate the same level of CPK activity, indicating that the 
induction of increased CPK activity by the microspheres of the present 
invention is temporary. The experimental method was as follows. 
A primary culture of rat embryo skeletal muscle was prepared as described 
by Freshney R. J. Freshney, Culture of Animal Cells, Willey, 1986, p. 
117, 170-172!. Briefly, the muscles were dissected tree of skin and bone 
and desegregated by warm trypsinization (0.25% trypsin at 36.5.degree. 
C.). Contamination by fibroblasts was reduced by preplating cells for 1 
hour in an incubator with 5% CO.sub.2, 37.degree. C., since fibroblasts 
adhere to tissue culture plates first. Myoblasts were then seeded on 35 mm 
Petri dishes at a concentration of 50,000 cells per ml with 2 ml of media 
(Dulbecco modified Eagle medium: medium 199 at a 1:4 ratio), enriched by 
antibiotics, 10% vol/vol horse serum and 4% vol/vol chick embryonic 
extract. The chick embryonic extract was prepared from 10 day-old chick 
embryos according to R. J. Freshney, Culture of Animal Cells, Willey, 
1986. The antibiotics included amphotericin and gentamicin, diluted as 
1:1000 from the standard initial concentration of 2.5 mg/ml. After 24 
hours, the media was decanted and replaced with new media containing 20% 
vol/vol foetal horse serum and 1% vol/vol chick embryonic extract. 
The cultured cells were then either treated with microspheres, starting at 
the time of plating, in media for 4-8 days or with media alone. The 
microspheres were either carboxylated polystyrene of 1, 2 or 4.5 microns 
in diameter, or polystyrene alone at 4.5 microns in diameter. The 
concentration of microspheres was either 10.sup.6 or 10.sup.7 per m1 of 
media, with similar results obtained for both concentrations (not shown). 
After 4, 5, 6, 7 or 8 days of treatment, creatine phosphokinase activity 
was measured by a standard assay ("Creatine Kinase", Worthington Enzyme 
Manual, Worthington Biochemical Corporation, Freehold, N.J., USA, 1972, 
pp. 54-55). Results are shown in FIG. 1, as Units of CPK activity per mg 
of total cellular protein. 
FIG. 1 clearly demonstrates the ability of the microspheres of the present 
invention to induce an initial increase of creatine phosphokinase 
activity, as compared to control cells. After 4 days of treatment, 
microsphere-treated cells show an initial increase of CPK activity as 
compared to control cells. This increase is particularly pronounced at 
days 5 and 6 of treatment. However, by day 7, CPK activity in control 
cells is beginning to achieve parity with that of microsphere-treated 
cells. By day 8, both control and microsphere-treated cells show similar 
levels of activity. Clearly microspheres promoted an initial increase of 
CPK activity in myoblasts, which leveled off after 8 days of treatment. 
Such increased CPK activity is correlated with biochemical maturation of 
myogenic cells. Thus, the microspheres promoted biochemical maturation of 
the cultured myoblasts. 
EXAMPLE 2 
Effect of Microspheres on Cell Proliferation and Fusion 
The microspheres of the present invention were demonstrated to induce an 
initial increase in both cell proliferation and myoblast fusion, as 
compared to control (untreated) cells, as shown below. 
Primary cultures of rat myoblasts were prepared as described in Example 1 
above, except that the cells were grown on cover slips. Treated cells were 
incubated with microspheres in media, as further described below, while 
control cells were only given media. 
To determine the extent of cell proliferation, cells were fixed in 
ethanol/acetic acid (3:1) and then stained by hematoxilin-eosin. The 
stained cells were then counted in a light microscope. The mitotic index 
was calculated as the proportion of cells in mitosis counted per 1000 
cells. 
For the examination of cell proliferation, polystyrene microspheres which 
had sulfate surface groups were used, with a diameter of 0.18 microns, and 
a concentration of 10.sup.7 microspheres/ml of media. A 20-fold increase 
in the mitotic index was observed after treatment for 24 hours with 
microspheres as compared to control cells. Specifically, the mitotic index 
of control cells was 1.25.+-.0.7%, while that of microsphere-treated cells 
was 24.6.+-.1.0%. Thus, clearly microspheres promoted a large increase in 
the mitotic index of the myoblasts. 
The effect of microspheres on myoblast fusion was also examined. Results 
are given in Table 1. Generally, cells treated with microspheres exhibited 
about 150% fusion rate as compared to controls. However, the extent of 
this effect depended upon the type of microspheres and the length of 
treatment. 
The types of microspheres tested are given in Table 1. The diameter of the 
microspheres is given in microns under "Diameter". The surface groups on 
the polystyrene beads are given under "Surface Group". Polystyrene beads 
without any further derivatization are "polystyrene". Beads derivatized 
with either carboxyl or amino surface groups are described as "carboxy" 
and "amino", respectively. The concentration of beads is given as number 
of beads per ml of media under "Conc." 
Cells were prepared, fixed and stained as for determining the rate of 
proliferation of myoblasts, described above. Cells were initially plated 
at the density given in Table 1 as cells per ml media, under the column 
"Initial Cells". The measurements of myoblast fusion were made after the 
given number of days after treatment under "Days after Treatment". 
The extent of fusion is calculated as the proportion of nuclei within 
multinuclear cells, or myosimplasts, related to the total amount of nuclei 
within the microscopic field, given as "Proportion of Fusion" for 
microsphere treated cells, and "Control Fusion" for control, untreated 
cells. At least 400 nuclei were counted for each experimental condition. 
The ratio of the extent of fusion in microsphere treated cells and 
control, untreated cells is given as "Relative Effect". If no value is 
given for a particular slot in Table 1, the value is the same as that in 
the row above. 
TABLE 1 
______________________________________ 
Effect of Microspheres on Fusion of Myoblasts 
Days Pro- 
After portion Relative 
Dia- Surface Initial 
Treat- 
of Control 
Enhance- 
meter 
Group Conc. Cells ment Fusion 
Fusion 
ment 
______________________________________ 
0.22 poly- 10.sup.7 
3 * 10.sup.4 
6 0.75 .+-. 
0.58 .+-. 
1.29 
styrene 0.06 0.10 
7 0.82 .+-. 
0.64 .+-. 
1.28 
0.09 0.09 
0.49 6 0.86 .+-. 
0.58 .+-. 
1.48 
0.06 0.10 
7 0.91 .+-. 
0.64 .+-. 
1.57 
0.07 0.09 
0.91 carboxy 10.sup.8 
5 * 10.sup.5 
5 0.84 .+-. 
0.64 .+-. 
1.31 
0.06 0.09 
4 0.63 .+-. 
0.51 .+-. 
1.23 
0.09 0.06 
1.12 amino 3 * 10.sup.4 
6 0.69 .+-. 
0.58 .+-. 
1.18 
0.15 0.10 
0.73 .+-. 
0.58 .+-. 
1.25 
0.08 0.10 
2.01 carboxy 10.sup.7 
5 * 10.sup.5 
5 0.84 .+-. 
0.64 .+-. 
1.31 
0.06 0.09 
4 0.63 .+-. 
0.51 .+-. 
1.23 
0.06 0.06 
4.58 10.sup.6 5 0.72 .+-. 
0.64 .+-. 
1.12 
0.09 0.09 
4 0.67 .+-. 
0.51 .+-. 
1.31 
0.09 0.09 
10.85 5 0.68 .+-. 
0.64 .+-. 
1.06 
0.08 0.09 
4 0.60 .+-. 
0.51 .+-. 
1.17 
0.10 0.09 
______________________________________ 
As can be seen from Table 1, all of the different types of microspheres 
promoted myoblast cell fusion, although the extent of the effect depended 
upon the diameter of the microsphere, the surface group on the 
microsphere, the number of days after treatment and the concentration. 
Myoblast fusion occurs when muscle tissue is formed during embryogenesis, 
and is also a very important step in muscle regeneration and repair of 
damaged muscle tissue. Thus, the ability of microspheres to promote such 
fusion clearly indicates the potential of these microspheres to promote 
muscle regeneration, as demonstrated in Example 5 below. 
EXAMPLE 3 
Effect of Microspheres on Collagen Synthesis and Deposition 
As noted above in the Background section, collagen synthesis and deposition 
is an important step in the process of wound healing. Furthermore, the 
amount of collagen deposited in the wound is an important determinant of 
wound strength. Thus, although the microspheres of the present invention 
clearly have a variety of effects on different cell types, as demonstrated 
in the preceding and following Examples, clearly one important determinant 
of the ability of a composition to promote wound healing is its effect on 
collagen synthesis and deposition. 
As shown in FIGS. 2A and 2B, the microspheres of the present invention 
clearly promote collagen synthesis by cultured fibroblasts. The largest 
effect is seen with Type I and Type II microspheres. Type I microspheres 
had a diameter of 4.5 microns, was made of carboxylated polystyrene and 
had a Z potential of about -29.96 mV. Type II microspheres had a diameter 
of 0.49 microns, were made of polystyrene alone and had a Z potential of 
about -34.5 mV. Type III microspheres had a diameter of 1.0 microns, were 
made of carboxylated polystyrene and had a Z potential of about -53.34 mV. 
The experimental method was as follows. 
Foreskin fibroblast cultures were grown in 75 cm.sup.2 plastic flasks 
(Corning Glass Works, Corning, N.Y.) in Dulbecco's modified Eagle medium 
(DMEM) containing 4.5 mg/ml glucose supplemented with 10% vol/vol fetal 
calf serum, 2 mM L-glutamine, 50 .mu.g/ml gentamycin sulfate and 2.5 mg/ml 
amphotericin B. The cultures were incubated at 37.degree. C. in 5% 
CO.sub.2 until confluent. Fibroblasts were harvested using 0.25% 
trypsin/0.05% EDTA solution and subcultured in 24-well plates at a density 
of 200,000 cells/well with the same media for 24 hours, at which time 
treated cells were incubated with Type I, II or III microspheres. Control 
cells were incubated with media alone. 
Collagen synthesis was measured as follows. The cultured fibroblasts were 
preincubated in DMEM supplemented with 0.5% dialyzed fetal calf serum for 
24 hours. Cells were labeled with 3 .mu.Ci 2,3-.sup.3 H-proline or 
3,4-.sup.3 H-proline solution containing .beta.-aminopropionitrile 
fumarate (BAPN) at a final concentration of 100 .mu.M, in the presence 
(FIG. 2A) or absence (FIG. 2B) of 1 0 .mu.M ascorbic acid as indicated. 
Ascorbic acid promotes collagen synthesis in fibroblasts and is an 
important stimulation factor. 
After 24 hours of incubation the reaction was terminated and collagen was 
extracted from each well by the addition of 30 .mu.l cold acetic acid 
(0.5M) containing pepsin (final concentration 0.5 mg/ml), followed by 
gentle shaking at room temperature for 4 hours. After centrifugation, the 
cellular debris was discarded and 80 .mu.l of collagen solution in 0.5M 
acetic acid was added to each supernatant, with a final collagen 
concentration of about 200 mg/mi. Collagen was precipitated from each 
supernatant by the addition of 0.4 ml of 5.2M NaCl solution in 0.5M acetic 
acid. After standing for 2 hours, precipitated collagen was separated by 
centrifugation for 15 minutes at 15,000 rpm. Next, the pellet was 
resuspended in 750 .mu.l of 10 mM TRIS buffer, pH 7.4 containing 1M NaCl. 
Collagen was precipitated by the addition of 750 .mu.l TRIS buffer, pH 7.4 
containing 5M NaCl. After 2 hours the collagen was separated by 
centrifugation, redissolved in 0.5M acetic acid and each sample was 
measured in a scintillation counter. Results are shown in FIGS. 2A and 2B, 
given as cpm per well. Data presented are an average of quadruplicate 
samples. 
Both Type I and Type II microspheres were able to stimulate collagen 
synthesis above the level seen in control (untreated) fibroblasts, both in 
the presence (FIG. 2A) and absence (FIG. 2B) of ascorbic acid. Type I 
microspheres had a greater effect relative to Type II microspheres in the 
presence of ascorbic acid, although both types had a similar effect in the 
absence of ascorbic acid. Type III microspheres did not have a detectable 
effect on collagen synthesis either in the presence or absence of ascorbic 
acid. 
One particularly interesting finding is that both Type I and Type II 
microspheres had an effect, while Type III microspheres did not, 
indicating that the specific size and material of the microspheres is 
important. Furthermore, both Type I and Type II microspheres elicited an 
effect even in the absence of ascorbic acid, indicating that these two 
types of microspheres can potentiate collagen synthesis even in the 
absence of other stimulatory factors. Thus, clearly both Type I and Type 
II microspheres have a substantial stimulatory effect on collagen 
synthesis. 
EXAMPLE 4 
Effect of Microspheres on Myoblast Shape 
Primary cell cultures of rat myoblasts were prepared as described in 
Example 1 above. Cells were then incubated with polystyrene microspheres 
(treated cells) or without (control cells) for 48 hours. Cells were then 
fixed in 1% glutaraldehyde in phosphate buffered saline for 1-4 days, and 
rinsed in PBS. Cells were then transferred to a solution of 1% tannic acid 
and 1% guanidine HCl(1:1 ratio) in PBS for 1 hour. Specimens were 
post-fixed in 1% OsO.sub.4 for 1 hour and dehydrated in graded ethanol and 
Freon 113 at room temperature. Specimens were then mounted on slides, 
coated with gold and examined in a JEOL T-300 scanning electron microscope 
at 2 kV. 
FIGS. 3A-3C illustrate the effect of the microspheres of the present 
invention on myoblast shape. The cell in FIG. 3A has grown over the 
microsphere, so that part of the cell surface is convex rather than flat. 
FIGS. 3B and 3C show cells extending pseudopodia from a portion of the 
cell on which the microsphere rests. The pseudopod of the cell in FIG. 3C 
is particularly pronounced, showing that the microspheres clearly 
influence myoblast shape. Furthermore, the formation and extension of a 
pseudopod clearly requires changes in the cytoskeletal structure, 
demonstrating that the microspheres also affect the cytoskeleton of the 
cell. The formation of such pseudopodia may be important for the migration 
of cells into the wound area. Thus, the stimulation of such pseudopodia by 
the microspheres indicates their ability to promote another important step 
in the wound healing process. 
EXAMPLE 5 
Device and Method for Application 
The following description is a general device and method for application of 
the agents for wound healing. The agents, such as microspheres, are 
preferably applied repeatedly to the wound to be treated. The frequency of 
application, and the concentration applied, is dependent on the severity 
of the symptoms and on the responsiveness of the subject to the treatment. 
Persons of ordinary skill in the art can easily determine optimum 
concentrations, dosing methodologies and repetition rates. In the present 
study, the microspheres were applied to the wound to be treated about once 
per day, although of course other application rates are possible. 
The method includes the step of administering the agents such as 
microspheres, in a pharmaceutically acceptable carrier in which the agents 
arc substantially insoluble, to a subject to be treated. Examples of 
pharmaceutically acceptable carriers include aqueous media for a 
suspension of agents, non-aqueous media such as ointments, creams and 
aerosol-forming material, as well as bandages soaked in, or otherwise 
containing, media with the agents. The bandages can be occlusive or 
non-occlusive. In any case, the agents which are in a pharmaceutically 
acceptable carrier can be described as a dispersion of agents. 
The agents are administered according to an effective dosing methodology, 
preferably until a predefined endpoint is reached, such as the absence of 
clinical symptoms in the subject. The closure of the wound to be treated 
is an example of such an endpoint. 
The device of the present invention includes a composition with one or more 
agents and a pharmaceutically acceptable carrier for the agents, and a 
container for containing the composition. Examples of suitable containers 
include aerosol-dispersing pumps and spray cans. One of ordinary skill in 
the art could easily select suitable containers for the composition. 
Regardless of the particular device used, the agents, such as microspheres, 
are preferably applied in a two step procedure. The microspheres are first 
applied in a dispersion to the wound, by dripping, spraying, painting, 
washing or by any other suitable method of topical application. 
Preferably, 30 sec to 2 minutes are allowed to elapse before the second 
step, in order to allow the microspheres to form initial contact with the 
wound. Preferably, the second step includes applying an occlusive or 
non-occlusive bandage, or other suitable covering soaked in the liquid 
suspension containing the microspheres, to the wound. This substantially 
reduces or eliminates absorption of the microspheres by the bandage or 
covering. This method was used both in rats and humans for wound healing, 
as described in the Examples below. 
The microspheres in the suspension did not aggregate, coalesce, clump or 
undergo irreversible caking. Although the microspheres did settle somewhat 
over time, they were easily resuspended with gentle agitation. 
EXAMPLE 6 
Promotion of Wound Healing in Rats by Microspheres 
As noted above in Examples 1-4, the microspheres of the present invention 
promote various in vitro cell processes which are important for wound 
healing. However, in vitro and in vivo effects do not always correlate. 
Therefore, in vivo experiments were performed to assess the ability of the 
microspheres to promote wound healing in rats. As shown in FIGS. 4A-4D, 
the microspheres of the present invention clearly promote wound healing in 
rats. FIG. 5 is a graph of the rate at which the wound area decreases, 
showing that the microspheres of the present invention increase the rate 
at which such a decrease occurs. Finally, Table 2 shows that the 
microspheres promote muscle regeneration in rats. The experimental method 
was as follows. 
Male Wistar rats, weighing between 300 and 400 g, were anesthetized by 
nembutal (5 mg/kg of body weight). An excision injury to the lateral parts 
of the Tibialis anterior muscle was performed as follows. First, a 
longitudinal incision was made in the skin to expose the Tibialis anterior 
muscle. Next, the partial excision of this muscle was made by a transverse 
cut of the muscle fibers, along about half of the muscle width. The 
excised piece was then cut out of the muscle, leaving a gap of about 5 mm 
by 5 mm in the muscle. In all rats the same amount of excised tissue 
(80.+-.10 mg) was removed from precisely the same location in the muscle. 
The wound area was then dressed with 2 micron polystyrene microspheres in 
saline for treated rats, and saline alone for control rats. The wound area 
was measured for between 3 and 15 days Following injury. 
FIGS. 4A-4D show pictures of wound areas prepared as described above. FIG. 
4A shows the wound of the control rat immediately after injury, while FIG. 
4B shows the equivalent wound of the rat to be treated. FIGS. 4C and 4D 
show the same rats five days after injury. The wound of the control rat 
was treated with saline alone, and still has not completely healed. By 
contrast, the wound of the treated rat, treated with microspheres, has 
completely healed. Thus, clearly the microspheres of the present invention 
promote faster wound healing. 
FIG. 5 further illustrates the promotion of wound healing by the 
microspheres of the present invention. The wounds of control rats 
eventually heal, but at a much slower rate than the wounds of treated 
rats. Thus, the microspheres clearly increase the rate at which the wound 
area decreases and the wound heals. 
Slides were prepared for histological analysis by making a biopsy punch of 
the wound area. Rats were sacrificed 4, 5, 6, 7, 8, 9, 13 or 14 days after 
injury and biopsies were taken for histological examination. The number of 
specialized myogenic cells incorporated into the newly formed or repaired 
muscle fibers was counted by determining the number of "new" nuclei, which 
represent activated myogenic cells. The nuclei of these cells are large, 
basophilic nuclei with dispersed chromatin and can be easily 
differentiated from the nuclei of existing myoblasts. Results are given in 
Table 2. 
TABLE 2 
______________________________________ 
Promotion of Muscle Regeneration by Microspheres 
"New" "New" "New" 
Post-surgical 
Nuclei Per 
Nuclei Per 
Nuclei Per 
Treatment 
Day Slide Field Fiber 
______________________________________ 
M 4 422 .+-. 67 
52.8 .+-. 22 
9.5 .+-. 3.5 
C 4 117 .+-. 37 
14.6 .+-. 10 
5.9 .+-. 1.4 
M 5 350 .+-. 84 
43.8 .+-. 13.5 
8.6 .+-. 1.8 
C 5 110 .+-. 31 
14.1 .+-. 4.6 
4.8 .+-. 1.2 
M 6 1221 .+-. 180 
94 .+-. 25 
11.9 .+-. 5 
C 6 676 .+-. 120 
52 .+-. 11 
4.9 .+-. 0.9 
M 7 762 .+-. 110 
95 .+-. 51 
9.4 .+-. 3.5 
C 7 169 .+-. 47 
21.1 .+-. 4.8 
4.5 .+-. 0.8 
M 8 715 .+-. 140 
89.4 .+-. 36 
11 .+-. 2.2 
C 8 126 .+-. 32 
18.6 .+-. 12 
5.2 .+-. 1.5 
M 9 299 .+-. 75 
42.7 .+-. 19 
7.4 .+-. 1.3 
C 9 235 .+-. 84 
33 .+-. 12 
6.5 .+-. 2.8 
M 13 747 .+-. 129 
53.3 .+-. 15 
9.7 .+-. 1.5 
C 13 582 .+-. 140 
42 .+-. 24 
5 .+-. 1.7 
M 14 665 .+-. 143 
83 .+-. 24 
9.4 .+-. 1.9 
C 14 491 .+-. 124 
61 .+-. 36 
5.5 .+-. 2.7 
______________________________________ 
As shown in Table 2, the microspheres of the present invention clearly 
promoted muscle regeneration, as measured by the number of "new" or 
incorporated nuclei in muscle fibers. The fact that such measurements were 
made on histological samples taken from rats treated in vivo also 
indicates that the microspheres promote muscle regeneration in vivo as 
well as in vitro. 
Finally, FIG. 6 compares the effect of the microspheres of the present 
invention on wound healing with tissue culture media and saline in rats. 
Wounds were induced in rats as described above, and the rats were treated 
with saline alone (FIG. 6A, .chi.), tissue culture media alone (FIG. 6B, 
.chi.), saline plus microspheres (FIG. 6A, !) or tissue culture media plus 
microspheres (FIG. 6B, !). The rats were then photographed 4 days after 
wounding occurred. As can be seen from FIGS. 6A and 6B, the microspheres 
were able to induce a much more rapid rate of wound healing regardless of 
whether the carrier was saline or tissue culture media. Thus, tissue 
culture media was not responsible for any part of the effect of the 
microspheres of the present invention on wound healing. 
EXAMPLE 7 
Toxicity Studies of Microspheres 
No toxic effect of a preparation containing microspheres was observed. 
Preliminary examination of treated rats 65 and 180 days after injury 
showed that none of the following organs exhibited signs of pathological 
changes: heart, liver, lungs, kidney, blood vessels, stomach, lymph nodes 
and brain. Experiments with fluorescently-labeled microspheres showed that 
no signs of pathology were observed in treated rats. Furthermore, the 
microspheres did not penetrate into any of the above-referenced organs. No 
new growth was detected in the above-referenced organs. Finally, the 
microspheres were dispersed within the wound area but did not penetrate 
into regenerating muscle fibers. 
EXAMPLE 8 
Effect of Microspheres on Wound Healing in Humans 
The in vivo experiments described in Example 6 above clearly demonstrate 
that the microspheres of the present invention can promote wound healing 
and muscle regeneration in rats. Furthermore, the results of the toxicity 
studies in rats described in Example 7 show that the microspheres are 
substantially non-toxic. Therefore, studies were performed to determine 
the effect of the microspheres of the present invention on wound healing 
in humans. As described in detail below, case studies demonstrated that 
the microspheres clearly promoted wound healing in humans. 
The first case study was that of a 66-year old female with ulcers in the 
left leg which refused to heal. The patient also had cellulitis of the 
left leg and varicose veins in both legs. Ulcers on the inner thigh of the 
patient were treated with Milton 2% which is a corrosive chlorine salt in 
water. Ulcers on the outer thigh of the patient were treated with 4.5 
micron microspheres of the present invention made from polystyrene in 
tissue culture medium. FIG. 7A shows the control wound at day 0, while 
FIG. 7B shows the control wound after 4 months of treatment. FIG. 7C shows 
the treated wound at day 0, while FIG. 7D shows the treated wound after 4 
months of treatment. 
Both the wounds treated with the microspheres of the present invention and 
those treated with Milton exhibited signs of infection and other 
difficulties healing during the next four months. However, at the end of 
the treatment period, the wounds treated with microspheres had shown a 
significant improvement. The wound size had decreased and the wounds were 
clean, without signs of infection. Thus, even for wounds which were 
difficult to heal, due to complications such as infection, the 
microspheres of the present invention exhibited greater efficacy in wound 
healing promotion than currently available treatments. 
As a further proof, the wound which had served as a control for FIG. 7 
above (FIGS. 7A and 7B) was treated with the same microspheres as those 
used to treat the wound in FIGS. 7C and 7D. The results are shown in FIGS. 
8A and 8B. FIG. 8A shows the wound at day 0 of treatment with 
microspheres, while FIG. 8B shows the wound after 21 days of treatment. 
Clearly, the extent of the wound has decreased, even after such a short 
time period. Furthermore, the wound was superficial and clean, and was no 
longer producing exudations. 
The second case study was that of a 52-year old female who had a year-old 
infected wound on the front side of the left thigh. The wound was treated 
with 1% Milton for a week, debrided and then treated with the microspheres 
of FIGS. 7 and 8 for 10 days. FIG. 9A shows the wound at day 0 of 
treatment, while FIG. 9B shows the wound after 10 days of treatment. 
After 10 days, the wound showed a significant improvement. It had decreased 
in extent to a small size, was clean and was no longer producing 
exudations, as can be seen from FIG. 9B. Although the wound did not fully 
close during the relatively short treatment period, its effects had been 
significantly ameliorated. 
The third case study was that of a 19-year old male who was injured by a 
chemical spill in an industrial workplace accident. The chemicals in 
question, sulfurides, caused severe burns and blistering on the right side 
of his neck and right hand. For the first two days, all wounds were 
treated with Silverol, a hydrogel with strong absorptive properties. Next, 
the wounds on the right forearm were treated with the microspheres of case 
studies 1 and 2, while the remaining wounds were treated with Silverol. 
The results are shown in FIGS. 10A and 10B (control wound at day 0 and day 
5, respectively), and in FIGS. 10C and 10D (treated wound at day 0 and day 
5, respectively). 
After 5 days of treatment with microspheres, the condition of the treated 
wound on the forearm had improved significantly over that of the remaining 
wounds, which were not treated with microspheres. The wound on the forearm 
had completely healed after 5 days of treatment with microspheres. By 
contrast, the remaining wounds which were treated with Silverol had not 
healed completely. Thus, the microspheres clearly promoted wound healing, 
demonstrating a greater efficacy than currently available treatments. 
The fourth case study was of a 52-year old female who had sustained 
second-degree burns on the buttocks from a hot bath. Wounds on the left 
buttock were treated with Silverol, while those on the right buttock were 
treated with microspheres of the previous case studies. The results are 
shown, for microsphere-treated wounds only, in FIGS. 11A (day 0) and 11B 
(day 7) of treatment. 
Seven days after beginning treatment, the wounds on the right buttock, 
which were treated with microspheres, had completely healed with good 
epithelial growth. By contrast, the wounds on the left buttock, which were 
treated with Silverol, had not completely healed and were closing 
relatively slowly. Thus, the microspheres were able to promote wound 
healing at a more rapid rate than conventional treatments. 
The fifth case study was of a 28 year old female who had suffered extensive 
and severe sunburn (data not shown). She was treated with the microspheres 
of the previous case studies. The patient reported both a significant 
reduction in discomfort and rapid healing of the sunburn. Thus, the 
microspheres used in the method and device of the present invention can 
both relieve discomfort and promote wound healing, although it should be 
noted that the relief of discomfort is probably a highly indirect effect 
of the microspheres rather than direct analgesia. 
Indeed, it is worth mentioning that the above patient report can only be 
inferred to include the apparent reduction in the sensation of discomfort 
from the sunburn. Such decreased discomfort probably does not demonstrate 
any ability of the microspheres to have a direct effect on the 
transmission of nerve impulses, or indeed to directly alter any of the 
many factors which lead to the sensation of discomfort. Instead, this 
effect is probably highly indirect, occurring as a result of the 
activation of macrophages, which in turn has anti-inflammatory effects, 
leading to the decreased sensation of discomfort by the patient. 
From these five case histories, coupled with the extensive evidence 
obtained from studies in rats, the use of the agents, such as 
microsphercs, according to the present invention has clearly been shown to 
have greater efficacy for the promotion of wound healing and muscle 
regeneration than currently available, prior art treatments. The method 
and device promotes, accelerates and enhances wound healing, as well as 
diminishing discomfort experienced by the subject. 
With regard to the diminished discomfort, it should be noted that the 
patients in the above case studies also reported local reduction in pain 
and discomfort from the treated wounds, particularly the patient suffering 
from sunburn, probably an indirect effect of the microspheres through 
their (also indirect) anti-inflammatory action. 
Finally, although the data is not shown, an indirect bacteriostatic effect 
against infections of the wounds by Pseudomonas species was also noted in 
humans. The mechanism for both the indirect anti-inflammatory action and 
the indirect bacteriostatic effect is not clear, but is probably a result 
of a cellular effect involving the attraction and activation of 
macrophages. Regardless of the exact mechanism, the use of the 
microspheres according to the present invention clearly represents a 
significant improvement in the treatment of wounds. 
It will be appreciated that the above descriptions are intended only to 
serve as examples, and that many other embodiments arc possible within the 
spirit and the scope of the present invention.