WO2000046599A1 - System for producing morphological and functional changes in adult mammalian cells - Google Patents

Abstract

A system for changing the morphology and function of adult mammalian cells in vitro and in vivo to produce embryonic stem cell types, including cells made by the method and devices for implementing the method. The system is implemented by contacting selected mammalian cells (18) with a suitable silver-containing substrate (16) (preferably a substrate containing crystalline silver). Free silver ions from the substrate contact the cells, inducing dedifferentiation of at least a portion of the cells into the unspecialized, embryonic type known as stem cells. These stem cells are believed to be capable of redifferentiating to other selected cell types.

Description

SYSTEM FOR PRODUCING MORPHOLOGICAL AND FUNCTIONAL

CHANGES IN ADULT MAMMALIAN CELLS

TECHNICAL HELD OF THE INVENTION

The present invention relates to a system for changing the morphology and function of mammalian cells to produce selected cell types in vitro and in vivo. In particular, the present invention relates to a method and devices for changing the morphology and function of adult mammalian cells to produce dedifferentiated cells (including stem cells), and to producing cells of useful types from such dedifferentiated cells. The invention also relates to cells made by the method and devices for implementing the method. BACKGROUND ART

The growth and healing processes of living organisms are among the most complex of all biological functions. Physicians have long wished for the ability to control these processes, to speed up healing or, in the case of malignancies, to stop growth. Some success has been achieved by application of biochemical concepts based upon metabolic inhibition, particularly for control of some malignancies. Elsewhere, electπcal techniques have proved useful for stimulation of fracture healing in nonunions. However, there has been little if any progress in understanding (or controlling) normal healing processes.

Most living organisms exhibit three classes of growth processes, each class having its own unique characteπstics. The first process is embryonic growth, which results in a complex, highly organized living entity arising from a seemingly formless, unstructured egg cell.

The second process is healing or reparative growth, in which an organism effects a repair of traumatic wounds or tissue loss, thereby enabling it to continue its life functions. This process is tπggered by the occurrence of an injury. Although its exact mode of action is not yet understood, it is clear that a feedback mechanism monitors the extent of tissue damage and adjusts cellular activity in the injured area to produce the exact amount of healing needed. (As used herein, the terms "wound" and "injury" reler to tissue damage or loss ol any kind, including but not limited to cuts, incisions (including surgical incisions), abrasions, lacerations, fractures, contusions, burns, and amputations.)

Healing processes can be classified into three types based on how the cells in the injured area react to the injury: regeneration, tissue replacement, and the simplest type of healing, scarification. Regeneration is a process in which lost or damaged cells and tissues are replaced by normal-type cells and tissues appropriate lor the anatomical area. Regenerative healing is present to a limited extent in human embryos, being replaced primarily by scarification (healing by formation of scar tissue) after birth. Almost all adult human tissues heal by scarification.

Some human tissues demonstrate a modest amount of regrowth following injury, as a result of the activity of a small residue of more pπmitive cells of the same type that are present in the pnmary tissue (these tissue-specific stem cells retain the ability to multiply and return to a mature type that is the same as the pnmary tissue). This type of repair is referred to as replacement healing in contrast to true regeneration. The skin, liver, and portions of the gastrointestinal tract heal by replacement. This type of healing is effective only if enough stem cells of the needed types are present in the injured area, and only for the particular types of cells that are capable of healing in this manner.

The most effective— and most complex— type of healing is regeneration. This type of healing is capable of replacing entire limbs and internal organs, and even portions of the brain and heart. In some animals, particularly the amphibians, true multi-tissue regeneration is present throughout life. It is almost totally lacking in humans, except in the fetus and in very young children (who may regenerate the distal finger tip if the wound is left open). In adults, regeneration is largely limited to parts of the fracture healing process.

Regeneration is accomplished by transforming all or most types of mature cells at the site of injury back to their embryonic, unspecialized state via a process known as dedifferentiation. Such transformed cells, vaπously known as dedifferentiated cells, embryomc cells, pπmitive cells, and pluπ-potent stem cells, are capable of transforming into any cell type that may be required to regenerate the missing tissues and structures appropπate to the anatomical area of the injury. The dedifferentiated cells multiply rapidly to form a blastema (a mass of such primitive cells) which provides the biological raw material needed for rebuilding the missing tissues. Formation of an adequate blastema results in complete regeneration of the missing tissues, whereas if the blastema is inadequate in size, only partial or incomplete regeneration takes place (formation of a stunted or incomplete part, oi merely regeneration of individual tissue types that are not fully organized into the desired structure). The embryonic- type cells of the blastema then respecia ze ("re-differentiate") into the vaπous types of cells needed to rebuild the missing tissues and organized structures in complete anatomical detail. The rebuilding process is essentially a recapitulation (albeit on a local scale) of the oπginal embryonic development of the tissues being replaced. Clearly, it would be beneficial if humans could regenerate other damaged tissues, both in terms of more cost- effective treatment modalities and improved outcomes for patients.

The third major growth process is malignant transformation, in which normal tissues become unresponsive to normal controls and begin to grow in an uncontrolled fashion, ultimately causing the death of the host organism. It should be noted that malignant cells (commonly termed cancer cells) always aπse from pre-existing normal cells in the body of the host organism. However, malignant cells have a number of characteristics that enable a pathologist to distinguish them from normal cells. A pπme characteristic of malignant cells is that they are almost always more pπmitive in appearance than the normal cells from which they arose. In general, the more "pπmitive" the appearance of the malignant cells, the faster growing and more difficult to treat the cancer; the closer the malignant cell approximates the tissue of origin, the less aggressive the cancer. All growth processes, whether embryonic growth that results in formation of the organism, reparative growth which heals or restores injured tissues, or uncontrolled malignant growth, are based upon the actions of cells, the basic living entities from which all organisms are constructed. In embryonic development, the final organism is the result of a precisely programmed sequence of cell division that increases the bulk of the organism accompanied by the gradual differentiation and maturation of the embryomc cells into specific tissue types in a highly orgamzed fashion. Scarification healing results from an increase in fibrogenesis stimulated by an injury. Replacement healing results from an increase in the normal rate of cell division in response to the injury. Regenerative healing is characteπzed by the dedifferentiation (that is, a return to pπmitive, embryomc characteπstics resulting from a return of the cell's genetic program to its embryomc state) of certain cells at the site of tissue loss, resulting in a mass of primitive cells (the blastema) which then divides and matures in a replication of oπginal embryomc growth on a local scale resulting in the regrowth of the missing structures. Malignant transformation is thought to involve partial dedifferentiation of the cells, permitting them to divide in an uncontrolled fashion unrelated to the rest of the organism.

Some general rules can be derived from these observations. First, while cell division is the basic process required for increases in the mass of an organisms, not all cells are capable of this process. For example, in the mature human, only those cells specifically preprogrammed to serve as germinal layers for replacement healing such as skin, gut, blood, etc., normally display typical mitotic behavior. Other mature cells, such as muscle, nerve, bone, etc., must first dedifferentiate to a pπmitive, embryomc type before they can engage in mitosis. Clearly, embryonic cells, either derived from dedifferentiation in regenerative growth situations, or in the embryo itself, have the capacity to undergo mitosis. The two basic cellular processes involved in growth, therefore, are mitosis or cell division, and dedifferentiation, a necessary prelude to mitosis for most cells in the adult organism.

Recent research indicates that many forms of cancer are the result of activation or de- repression of previously inactive oncogenes, suggesting a link with cellular dedifferentiation processes. Since dedifferentiation may be viewed as the total deprogramming of the entire genetic code of a cell, it may also permit subsequent reprogramming to take place in the normal configuration of oncogene suppression. Thus, dedifferentiation of cancer cells is a necessary prerequisite to redifferentiation into normal cells.

Mature cells of all somatic types contain a full complement of genes to reconstitute all of the different genes to reconstitute the original organism in its entirety. In the mature cell, however, all genes except for those that specify that particular cell type are repressed. For example, a muscle cell contains the full genetic complement, but only those genes that specify muscle are active. The process of dedifferentiation therefore involves the "de-repression" or "activation" of the remaining genes so that all are available for use by the cell as required.

In ammals possessing major regenerative capability, the pπmitive cells needed for tissue or organ regeneration are provided by dedifferentiation of all normal, mature tissue cells in the vicinity of the wound. This process is the most efficient method of wound healing, resulting in the early formation of an adequate blastema, and does not require preexisting collections of stem cells.

Furthermore, fetal cells from very young embryos can act as pluπ-potent stem cells. This is possible because the genetic alterations which take place as cells mature into specific tissue types has not yet occurred. For example, when neural stem cells from a donor mouse are injected into a host mouse, the neural cells find their way to the host's bone maπow and start producing various types of blood cells beaπng the genetic tag of the donor. It has been postulated that human fetal stem cells that are implanted or injected into the body in areas that require new cells of an appropriate type to regenerate missing or damaged structures would be influenced in some manner by the local cell population to redifferentiate into the appropπate types to bring about such a local regeneration process.

The human body contains a small population of stem cells, particularly in that portion of the bone maπow that is involved in the production of blood cells. Smaller populations are present in a few other tissues, including muscle. A variety of techmques have been developed to separate human stem cells from the pnmary tissue population. However, these techniques are laborious and expensive, and the small number of cells obtained must be expanded in tissue culture to provide adequate amounts for clinical use. Other techmques for obtaining human stem cells have involved using human fetal tissues directly for transplantation into patients, harvesting embryomc cells from early human fetuses and growing them in cell culture, and implanting nuclei from normal human cells into egg cells of other species which have had their nuclei removed. All of these methods raise serious ethical considerations that, to date, have limited their application to laboratory research. All of these methods are time consuming, technically involved, and costly. When used to treat human patients, presently- available methods are productive of immune incompatibility, leading to rejection or requinng the long-term use of anti-rejection drugs with accompanying side effects.

In U.S. Patent No. 4,528,265 (incorporated herein by reference), Becker disclosed a process for stimulating mammalian fibroblasts to assume a simpler, relatively unspecialized form that resembles dedifferentiated or embryonic cell types. The process involves subjecting mammalian cells to the influence of electπcally-generated silver ions. Explants of silver- treated human wound tissue demonstrated profuse clonal-type expansion of cytologically pπmitive cells in culture. In U.S. Patent No. 5,814,094 (incorporated herein by reference), Becker, et al. disclose an lontophoretic system for promoting tissue healing processes and inducing regeneration. The system is implemented by placing a flexible, silver-containing anode in contact with a wound, placing a cathode on intact skin at a distant site, and applying an appropπate DC voltage between the anode and the cathode. Electrically-generated silver ions from the anode penetrate into the adjacent tissues and undergo a sequence of reactions leading to formation of a silver-collagen complex, which acts as a biological inducer to cause the formation in vivo of an adequate blastema-like structure to support regeneration.

Research into the potential therapeutic uses of dedifferentiated cells and stem cells has been severely limited by the lack of reliable sources of such cell types. There have been numerous, largely unsuccessful, attempts to produce useful amounts of stem cells in vitro. While there has been some limited success in producing stem cells from embryomc tissue, there are serious ethical questions that impact any work that involves, even to a limited extent, research on cell lines deπved from human embryos.

The availability of useful amounts of stem cells would further research into the possibility of improved treatment modalities for restoring damaged or missing tissue. Such treatments may ultimately prove useful for augmenting healing processes, and even, in appropπate instances, the potential for inducing true legenerative healing in organisms (including humans) that have largely lost this capability. Similarly, research on malignant cells might ultimately lead to improved treatment modalities for human and animal malignancies. (To avoid incompatibility, any clinical treatments would preferably use stem cells derived directly from the tissues of the patient to be treated, not cells deπved from human embryomc tissue.) However, without a reliable technique for producing, harvesting, and adequately expanding a population of mammalian stem cells (including human stem cells), further studies into the possibility of augmenting the human capacity for healing are probably not feasible. There is a need for a reliable, cost-effective system for producing clinically-useful amounts of such cells in vivo and in vitro, preferably a system that produces cells specific for each patient.

DISCLOSURE OF THE INVENTION

According to its major aspects and broadly stated, the present invention in its broadest embodiments includes a method for changing the morphology and function of mammalian cells in vivo and in vitro to produce embryomc (i.e., dedifferentiated) cell types. The invention also includes cells made by the method, and devices for implementing the method

The method is implemented in vitro by introducing a quots of selected mammalian cell types to petπe dishes or other suitable vessels containing culture medium, placing the cells on glass substrates, and contacting the cells with an appropriate silver-containing substrate (preferably a substrate containing crystalline silver). (A sufficient cell population for use with the invention may be produced by adding an aliquot of the selected mammalian cells to a vessel that contains a suitable culture medium, then incubated until the desired cell population density is obtained.) When the cultuie medium is in vivo, the method is implemented by contacting cells in the treatment area with a suitable silver-containing substrate. Free silver ions from the substrate migrate into the culture medium and the cell population, causing morphological changes that, in at least a portion of the cell population, include dedifferentiation into the unspecialized, embryomc type known as stem cells. The population of these desired cells can then be expanded by further appropπate treatment in culture. These cells can then be harvested and used for research and treatment purposes.

Silver ions released by the substrate may undergo a variety of reactions in vitro or in vivo. Some of these ions may combine with proteins, peptides and various other chemical species normally present in solution in the tissues; others may combine with bacteria, fungi or viruses that may be present, with resulting beneficial effects. At least some of the silver ions may associate with cells in the treated region (including but not necessaπly limited to fibroblasts and epithelial cells), resulting in the above-described morphological changes (formation of dedifferentiated, embryonic cells, including stem cells). If sufficient quantities of silver ions are released into the cultuie medium, it is believed that some of the ions may attach to specific defect sites on collagen fibers in the medium, thereby forming a silver- collagen complex that acts as a biological inducer to further clonal expansion of the embryonic cells that result from direct association with silver ions.

The changing of cell morphology according to the invention is accomplished by contacting mammalian cells with a device that contains sufficient quantities of an appropπate form of silver, preferably silver in crystalline form. Stem cells can be produced in vitro for later in vivo implantation; alternatively, the cells can be produced directly in vivo at the site where hea ng/regrowth of tissues is desired, simply by contacting the cell population at the site with a suitable silver-containing substrate under appropriate conditions. These embryonic cells redifferentiate under the influence of local factors to form cells of the appropriate tvpe(s) to replace the missing tissues (essentially any type of cell that is present in the organism from which the oπginal cells were obtained) Thus, dedifferentiated normal cells have applications in enhancing or restoring healing processes; stimulation of dedifferentiation in malignant cells has potential applications in cancer treatment, with the advantages of no side effects such as are found with chemotherapy.

An important feature of the present invention is its simplicity and cost-effectiveness Use of the method results in production of useful quantities of embryomc cells, including stem cells, with readily available mateπals, with suitable cell culture techmques or directly in situ in the patient, without the need for complex procedures or costly apparatus.

Another important feature of the piesent invention is biocompatibihty. Not only does the invention use biocompatible (in some cases, bioabsorbable) devices, but it avoids the well-known problems resulting from immune rejection of transplanted organs: both dedifferentiated (stem) cells and redifferentiated cells produced by the method are specific to each individual organism to ensure compatibility on the cellular level.

Another feature of the present invention is its broad applicability The method has been found to dedifferentiate both normal and malignant cell types in vitro. Based on these results, it is believed that the method can be used to change the moφhology and function of many mammalian cell types to produce a population of changed cells, at least some of which are dedifferentiated or stem cells.

The devices for implementing the method constitute still another feature of the invention. Cell culture devices are useful for producing dedifferentiated cells in vitro. A flexible, conformable silver-containing substrate is useful for application to surface wounds such as burns, abrasions, and lacerations. Other devices include light-weight, flexible fabπc substrates, including stretchable and expandable substrates, for treating internal organs such as the heart and liver, and, to a lesser extent, skeletal muscle and portions of the intestine and colon. Tubular substrates are used for applications that involve reconnecting severed nerves or tendons; biodegradable substrates are useful for implants.

Other features and advantages of the present invention will be apparent to those skilled in the art from a careful reading of the Best Modes for Carrying Out the Invention piesented below and accompanied by the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

Fig. 1A is a plan view of a culture dish for exposing mammalian cells to silver in vitro;

Fig. IB shows the culture dish of Fig. 1A provided with a silver-containing substrate; Fig. 2 is a schematic illustration of the effects of treatment with a silver-containing substrate in vitro;

Figs. 3A and 3B illustrate additional cell culture systems usable with the invention; Figs. 4A-F are photomicrographic views illustrating the effects of treatment with a silver-containing substrate on normal human dermal fibroblast cells (NHA CC 2565);

Figs. 5A-F are photomicrographic views illustrating the effects of treatment with a silver-containing substrate on human epidermoid carcinoma cells (ATCC CRL-1555);

Figs. 6A and 6B are photomicrographic views of control and silver-treated, respectively, human astrocyte cells (NHA CC 2565) after 4 hours incubation;

Figs. 7A and 7B are photomicrographic views of control and silver-treated, respectively, human fibrosarcoma cells (ATCC HT1080) after 24 hours incubation;

Figs. 8A and 8B are photomicrographic views of cells from a human foot wound at 100X and 200X magnification, respectively; Figs. 9A-C are photographic views illustrating treatment of a human foot ulcer; and

Fig. 10 is a flow chart illustrating the effects of silver ions according to the invention.

BEST MODES FOR CARRYING OUT THE INVENTION In the following descπption of best modes for carrying out the invention, reference numerals are used to identify structural elements, portions of elements, surfaces or areas m the drawings, as such elements, portions, surfaces or areas may be further descπbed or explained by the entire wntten specification. For consistency, whenever the same numeral is used in different drawings, it indicates the same element, portion, surface or area as when first used. Unless otherwise indicated, the drawings are intended to be read together with the specification, and are to be considered a portion of the entire wntten descπption of this invention as required by 35 U.S. C. § 112. As used herein, the terms "horizontal," "vertical," "left," right," "up," "down," as well as adjectival and adverbial derivatives thereof, refer to the relative oπentation of the illustrated structure as the particular drawing figure faces the reader. Previous studies have shown that lontophoretically produced silver ions emitted from a silver anode and directed into a wound site produce stem cells in amounts exceeding the requirement for local tissue regeneration. These studies have relied upon the use of iontophoresis via power supplies, electπcal contacts, and so forth; precise control over the electrical factors of voltage and current are required. Now, suφπsingly, it has been discovered that simple contact with an appropπate form of silver, under appropriate conditions, induces similar moφhological changes in a wide range of mammalian cell types (including human cells), in both normal cells and malignant cells. The end result of these changes is the formation of a population of moφhologically- altered cells, at least some of which are dedifferentiated, embryonic stem cells. As will be described further below, the method can be implemented in vitro and in vivo. The method is implemented in vitro as follows:

1. A quantity of cells of a selected type is placed in a flask, tube, or other suitable vessel that contains an appropriate culture medium. The vessel is incubated until the desired cell population density is reached. 2. Ahquots of the cultured cells are transferred to cell culture plates of any convenient type, each plate having an appropπately-sized glass microscope cover slip (or similar glass plate) inserted into the bottom prior to cell transfer to provide a substrate for attachment of the cells and facilitate their removal. For example, Fig. 1A shows a cell culture device 10 for growing cells in vitro, consisting of a petπe dish 12 containing any suitable culture medium, and a glass substrate 14 (such as a microscope slide or other suitable item). The dimensions of petπe dish 12 and glass slide 14 may vary widely, depending on such factors as the types of cells to be treated, the desired cell population density, and so forth. Standard petπe dishes, multi-well culture dishes, and other cell culture devices such as are known in the art may also be used, as will be described further below. 3. After a sufficient period of time for cell growth has elapsed and the desired cell population density is attained, a silver-containing substrate 16 is moistened with steπle saline solution and placed over glass slide 14 so as to cover a portion of a cell population 18 (Fig. IB). A sufficient number of devices 10 are produced so that ahquots of cell population 18 can be recovered at selected exposure times. 4. After a sufficient period of time has elapsed, substrate 16 is removed from glass slide 14, the slide is removed from culture dish 12, and at least a portion of cell population 18 is harvested.

Examples of additional cell culture devices usable with the invention are shown in Figs. 3A and 3B. A cell culture device 30 includes a plastic well 32 containing a culture medium 34 (well 32 may be one of a multi-well culture dish having an array of such wells, of standard size or indeed any useful size). A glass substrate 14 (a circular glass cover slip), somewhat smaller in diameter than well 32, is placed in the well and seeded with cells of a selected type (not shown). Then, a silver-containing substrate 16 is placed on glass substrate 14. A cell culture device 40 includes a plurality of individual wells 32, each containing a suitable quantity of culture medium 34, a glass substrate 14, and silver-containing substrates 16.

In vitro tests of the above-described method were conducted using equal numbers of two sets of control cultures. The first set of controls was identical to the treated cultures, except that substrate 16 was replaced with a non-silvered substrate (otherwise of identical size and shape to silver-containing substrate 16). The second control set consisted of an equal number of untreated cultures (i.e. , cell population 18 was placed on glass slip 14 as illustrated in Fig. 1A, without the addition of a substrate 16).

After harvesting, the cells were fixed and stained (with Wright's stain or other suitable technique) to highlight cellular detail and morphology. The cells were then examined by standard light microscopy at magnifications of 40X, 100X, 200X, and 400X, and any changes in moφhology were noted. Multi-well dishes (when used) were examined for residual cells using an inverted stereomicroscope at 45X magnification. Photographs of representative fields were taken using an Olympus OM-1 35-mm camera with a photomicrographic attachment and Kodak Tπ-X black-and-white film. Negatives were developed, processed, and pπnted according to standard procedures.

A number of moφhological changes were found, including the following: 1. Umformity of cell population density. In general, untreated cells in culture were firmly attached to glass substrate 14, forming a fairly uniform population density across the entire substrate.

A non-uniform population density was indicative of exposure to silver. Silver- exposed cells tended to stop multiplying within the first few hours of exposure to substrate 16. By 8-72 hours after the start of exposure, a definite, grossly visible difference between the silver-treated area of each glass substrate and the remaining untreated portion became evident (in some instances, there was a transition region due to the diffusion of silver ions into the growth media). As illustrated schematically in Fig. 2, cell population 18a in Zone 1 (exposed to silver-containing substrate 16) appeared lighter in density than cell population 18b in transition Zone 2 or cell population 18c in unexposed Zone 3. The silver-exposed cell population was lighter due to the cessation of growth in Zone 1 compared to Zones 2 and 3 , and the continuing growth of the cells in Zones 2 and 3.

2. Presence of free-floating and/or clumped cells. Dedifferentiated cells lose adhesion to glass substrates and become free floating, often exhibiting a degree of moti ty. As compared to cells in Zones 2 and 3 (and untreated controls), treated cells in Zone 1 tended to either separate from cover slip 14 and become free-floating, or become less adherent and form clumps of cells.

3. Cell contour. Untreated cells tended to retain their normal configuration (most cells used in the studies described herein were spindle-shaped), whereas treated cells tended to become more rounded or oval in form. If the oπginal tissue in culture produced an organized substrate matrix such as keratin, treated cells tended to vacate the lacunae and become free floating; the organic substrate deteπorated rapidly thereafter.

4. Clumping. Treated cells tended to clump together to form floating masses of vanable size 5. Multiple nuclei. Treated cells frequently contained two nuclei, possibly due to being in the inteφhase stage of mitosis when contacted by the silver ions from substrate 16

6. Changes in the nuclear cytoplasmic ratio. In silver-treated cells, this ratio may change, with the nucleus assuming a larger size than in untreated cells.

7. Basophilic appearance. Treated cells (that is, silver-exposed cells) had basophihc staining characteπstics, as compared to the eosinophilic staining of untreated cells or cells exposed to a non-silver-containing substrate.

8. Degree of nuclear extrusion. Nuclei of silver-treated cells frequently became extruded from the cells along with a thin πm of cytoplasm; the free nuclei thus produced became free floating (the free-floating nuclei were frequently vanable in size). These cells also tended to form clumps.

While there are no known chemical markers that positively identify dedifferentiated human cells, the presence of at least three of the above factors in a culture was deemed to be indicative of the presence of dedifferentiated cells.

Any silver-containing material capable of releasing useful amounts of free silver ions into the culture medium is broadly suitable for the practice of the present invention. Such matenals preferably contain at least approximately 2 wt.% silver metal, more preferably, at least approximately 5-10 wt.% silver. However, silver concentrations outside this range may also be useful for some applications.

Suitable matenals include fabrics made by weaving, knitting, felting, blowing, or other convenient process, and consisting of silver-coated or silver-impregnated fibers. Such fabrics are dimensionally stable, flexible, conformable, at least somewhat moisture- absorbing, nonallergenic, and non-adhering to body tissues. For clinical applications, the fabnc is preferably manufactured of biologically inert, nonconductive, nonimmunogenic fibers (since many people are sensitive to certain fibers, the substrate is preferably also nonallergenic or hypoallergemc). Suitable silver-impregnated or silver-coated warp knit nylon fabric, nylon pile fabnc, and other silver-containing fabrics are made by Omnishield, Inc., Swift, Inc, Sauquoit Industπes, and others. Alternatively, other silver-containing or silver-coated matenals (plastics and other polymers, metal, paper, collagen, biocompatible and absorbable matenals, and so forth) may be useful for the practice of the invention.

In general, fabπcs that contain more silver by weight tend to have larger useful amounts of silver than those that contain less silver, whether the additional silver is in the form of a thicker coating, a greater admixture of silver-coated fibers, or the use of smaller- diameter fibers that provide a larger silver-coated surface area. However, there is a practical upper limit to the amount of silver that can be deposited on any type of fabric, due to the difficulty of producing mechanically stable silver deposits on fabric. No matter what the manufactuπng technique, fabrics with more than approximately 20 wt.% silver tend to be mechanically unstable, in that the silver deposits are subject to flaking and powdering so that the silver is easily lost when handling the fabnc and the ability of the coating to release biologically-effective silver ions is diminished. These types of fabrics are not suitable for use with the present invention, which requires a mechanically stable silver-containing substrate 16 from which the silver is transferred to the culture medium in ionic form.

Substrate 16 contains silver in a mechanically stable form. That is, the silver is preferably in a form that is firmly attached or bound to the substrate when dry, and that, when contacted with a suitable aqueous liquid (saline, wound exudate, water, culture medium, etc.), is at least somewhat releasable in ionic form. Thus, at least a portion of the available silver migrates into the culture medium over a period of time. While not wishing to be bound by theory, it is believed that metallized (i.e., metal -containing, metal-coated, metal-plated) matenals that exhibit this property are especially suitable for the practice of the present invention. For example, a useful material for the practice of the invention contains silver (or other suitable metal) in the form of small crystals which tend to easily release free silver ions under the appropπate conditions, that is, when wetted by saline, water, or other suitable liquid. Crystalline silver deposits of this type are believed to have a greater effective surface area than conventional silver-plated coatings, and therefore the capability of releasing more silver ions per unit weight, in shorter periods of time. Recent developments in silver coating technology have led to a coating that emits free silver ions in large quantities without the need for adjunct electrical treatment. This coating may be applied to a variety of matenals including but not limited to fabric, plastic, metal, paper, collagen, and other materials.

The present invention is further illustrated by the following non-limiting examples. In vitro studies included three sets of cultures prepared as described above: a test series with a substrate 16 of silver-containing nylon fabric, a first control series with a substrate of plain nylon fabric, and a second control series with no fabric. Except for the mouse fibroblast 3T3 cells (Example 1), all cell types utilized in the following examples were human in origin. All exhibited normal culture charactenstics, i.e., forming monolayers of cells with firm adhesion to glass substrate 14; the culture growth rate and moφhological characteristics of the two control cultures were identical. The moφhological characteπstics of the control cells are listed for each cell type tested.

EXAMPLE 1

Selected types of cells were grown on thin, circular glass microscope cover slips 14 (approximately 1.25 cm in diameter) placed in individual wells 32 (approximately 1.9 cm in diameter) of multiwell plastic plates 40. Each plate 40 contained 18 such wells 32. Each cell type used was initially grown in standard culture to determine its growth rate; precautions were taken to prevent contamination of the culture media. This data was then used to seed the individual wells with cells at various times so that all wells had a uniform population density at the actual start of the tests.

The following cell types were studied: normal human astrocyte (NHA CC 2565) and normal human dermal fibroblast (NHDF-AD CC 2511) obtained from the Clonetics corporation (San Diego, CA); mouse 3T3 fibroblast (ATCC CCL-163), human epidermoid carcinoma (ATCC CRL-1555), and human g oblastoma multiforme (ATCC CRL-2020) obtained from the Amencan Type Culture Collection (ATCC). The mouse 3T3 cell line was included because this is a standard test bed mammalian cell type for moφhology and toxicology studies.

Three sets of wells 32 were used for each cell type studied: a first control series of 3 untreated wells (Control 1), a second control series of 3 wells treated with plain nylon fabnc (Control 2), and a test series of 3 wells treated with a silver-containing nylon fabnc 16 (in the form of 1/4" (about 0.6 cm) squares). At the start of each test, the appropnate fabnc squares were moistened with culture medium and placed on substrates 14 in wells 32. Each well 32 was incubated for a penod of 1 hour, 4 hours, 8 hours, or 24 hours. After incubation, substrates 14 with cell populations 18 were fixed in situ, removed from wells 32, stained with Wright's stain, and dπed for examination. This 24-hour time sequence permitted observation of the moφhological changes in the cell population immediately in contact with the silver nylon substrates 16 and the establishment of a boundary zone (i.e., Zone 2, Fig. 2) between the changed cells and the unaltered cells as the diffusion boundary of the free silver ions progressed across the culture on substrates 14. No differences in cell moφhology or population density were observed between Control 1 and Control 2 cultures for any of the tested cell types. Results for normal human dermal fibroblast cells (NHA CC 2565) are shown in Figs.

4A-4F (unless otherwise indicated, all photomicrographs are at 100X magnification). After 1 hour, the predominant morphological type in both Control 1 and Control 2 cultures was standard spindle cell moφhology with an over-all swirling pattern (Fig. 4A, Control 2 culture). In the test culture (Fig. 4B), silver- treated nuclei were noticeably smaller, densely staining basophihc, and with a tendency to clump together.

After4 hours, the cell moφhology and pattern of the control cultures were essentially unchanged (Fig. 4C, Control 2). The silver-treated cultures had variable-size, mostly ovoid cells with less clumping than the Control 1 or Control 2 cells, densely stained nuclei, and basophihc cytoplasm (Fig. 4D).

After 8 hours, the control cultures showed essentially normal fibroblast patterns (Fig. 4E, Control 2, 200X magnification). Surprisingly, the silver-treated cultures had an increased cell population with a markedly changed moφhology (Fig. 4F). The cells were smaller than normal and more ovoid-shaped than spindle-shaped, with densely basophihc staining nuclei and cytoplasm indicating increased RNA/DNA content. In some instances, some of the cells in wells 32 had migrated to beneath substrates 14. Similar results were observed after 24 hours.

Results for human epidermoid carcinoma cells (ATCC CRL-1555) are shown in Figs. 5A-F. Alter 1 hour, cells in both Control 1 and Control 2 cultures generated a matnx of keratin, growing in sheets in lacunae within the matrix (Fig. 5A, Control 2). In contrast, silver-treated cells appeared to exit the lacunae, leaving the keratin matnx to dissolve with the cells floating free. At50X magnification, these cells appeared as small dots (Fig. 5B); sheets of empty lacunae (with a few cells still attached) were observed at higher magnifications. After 4 hours, the cell sheets in Control 1 and 2 cultures were well orgamzed within the matrix. These cells had an essentially normal appearance (Fig. 5C, Control 2, 200X magnification), whereas the treated cells (Fig. 5D, 200X magnification) formed loose clumps of small, contracted, densely stained cells.

After 8 hours, Control 1 and Control 2 cultures appeared similar to the 4- hour control cultures described above (Fig. 5E, Control 2). In the silver-treated cultures, the matrix was present as small, granular, disorganized clumps that did not form lacunae, and appeared to contain the cell nuclei clumped together within an abnormal matrix (Fig. 5F). These cells were deeply stained basophihc; details such as the presence of thin cytoplasm rims were not visible. Similar results were observed after 24 hours exposure. Silver exposure resulted primarily in the exit of the cells out of the matrix lacunae, with subsequent dissolution of the oπginal orgamzed matrix. The free-floating cells were small and tended to clump together initially. With continued exposure to silver, the cells tended to form smaller and denser clumps, with the cells within a newly formed abnormal type matnx. Results for the remaining cell types paralleled those described above. For normal adult human astrocyte cells (NHA CC 2565), the predominant morphological type was ovoid- spindle, with the cells growing separated from each other with long projections. Control 1 and Control 2 cultures exhibited increases in pleomorphic appearance and cell population dunng incubation that were roughly proportional to incubation time (Fig. 6A shows a Control 2 culture after 4 hours incubation). Silver-treated cells showed fewer spindle cells, and changes in moφhology suggestive of dedifferentiation. A few spindle cells were still visible in the test cultures after 4 hours incubation (Fig. 6B). There was an apparent increase in cell numbers along with the loss of spindle moφhology. The cells were composed of vanable sized, densely staining nuclei with the appearance of a thin cytoplasmic πm. It is believed that nuclear extrusion causes the appearance of small nuclei that become more abundant with increasing exposure time.

EXAMPLE 2

Human fibrosarcoma (ATCC HT 1080) cells were seeded on standard glass microscope slides approximately 1" x 3" (about 2.5 x 7.6 cm) in size. The slides were placed in plastic petne dishes about 4" (10 cm) in diameter filled with appropriate culture media

After a monolayer of cells had grown and were adhered to the slides, three sets of cultures were prepared. One set (Control 1) received no further treatment except for an additional 24 hours of incubation. The second set (Control 2) received an approximately 1 " x 1" (about 2.5 cm square) of plain nylon material moistened with the culture medium and placed over approximately one third of each slide. The third set was similarly treated with a 1 " x 1" square of silver-containing nylon fabnc. All three sets of cultures were incubated for 24 hours, after which the nylon fabnc discs were removed. All slides were fixed in situ with an appropriate cell fixative, removed from the petne dishes, stained with Wright's stain, dried, and examined.

The Control 1 and Control 2 cells grew within 24 hours to confluence across the entire glass slides (Fig. 7A, Control 2, 100X magnification). All slides showed highly cellular cultures. The cells present were in dense monolayers, most exhibiting numerous mitoses and marked degrees of cellular and nuclear pleomoφhism consistent with that of a neoplastic cell line. Cell moφhology was ovoid, with basophihc expanded nuclei and eosinophilic cytoplasm. In contrast, the cell population of the silver-treated cultures was less than that of the initial population (Fig. 7B, 100X magnification) with fewer mitoses than those of either Control 1 or Control 2 cultures. Some areas of the treated slides showed changes in nuclear moφhology and decreases in cellular and nuclear pleomoφhism. In addition, the treated cell nuclei were deeply basophihc and free floating, having apparently extruded from the cytoplasm. There was evident residual, disorganized cytoplasm with scattered nuclei residual within it.

EXAMPLE 3

A 37-year-old male computer programmer (nondiabetic) incurred blunt trauma to the medial border of the left great toe with subsequent development of an open infected wound involving the nail bed, the medial border of the nail, and the adjacent soft tissues with pain, edema, redness, and drainage. Conservative treatment was instituted, including foot soaks, application of peroxide solution, and debπdement. One month after the initial injury, the open lesion had not healed but rather had increased in extent. Treatment with an appropπate silver-containing substrate was instituted for 8 hours per day. The lesion was packed with a plurality of thin strips of silver-containing nylon fabric, and covered with an external sheet dressing over the affected area.

After 2 weeks of treatment, the infection was controlled. The lesion was decreasing in size, as was the accompanying inflammation. Microscope slide blot preparations from the innermost silver nylon fabnc packing were made and stained with Wright's stain. Clumps of typical dedifferentiated cells were present in the exudate blots (Figs. 8A and 8B), similar in appearance to those described in Example 1 above. Crenellated erythrocytes and some normal leukocytes were visible; the majonty of the cells were strongly basophihc mononuclears with scanty cytoplasm (Fig. 8A, 200X magnification). The mononuclear nature of the cells is clear at 400X magnification (Fig. 8B); some cells how a thin πm of cytoplasm. The cytology of these cells and their clumped appearance are compatible with those of dedifferentiated cells.

The patient maintained a normal work schedule and wore normal dress shoes throughout treatment. Full asymptomatic healing occurred after approximately 8 weeks of treatment.

EXAMPLE 4

Several volunteer patients with non-healing, necrotic foot wounds were successfully treated with an appropπate silver-containing substrate. (Silver-containing wound dressings are descnbed in my copendmg application Serial No. 09/431,991 entitled "Multilayer Antibacteπal Treatment Device," filed November 3, 1999, the disclosure of which is incoφorated herein by reference.) Most patients were diabetic; all had previously been treated unsuccessfully with massive antibiotics (including, in some cases, administration of hyperbanc oxygen and predmsone). In all cases, a suitable quantity of silver-containing nylon material was wetted with distilled or deiomzed water and applied directly to the wound (several such layers were used for deep wounds). Several vaneties of silver-containing nylon fabnc were used, including smooth-surfaced fabnc and pile-type fabnc (all contained equivalent amounts of silver). The silver nylon fabnc was covered with a standard gauze pad (also moistened with water), and finally with a mild compression dressing. Dressings were initially changed every 12 hours. The wound was soaked in 25% peroxide solution with gentle agitation for 10-15 minutes between dressing changes. After any infection present at the site was controlled, the dressings were applied for 8-12 hours per day, with a peroxide soak and application of a stenle moist gauze dressing in the inteπm. Patient 4A, a 75- year-old male retired farmer with a history of moderate arthritis and adult onset diabetes (satisfactoπly controlled with insulin) developed acute-onset local tissue necrosis on the ulnar aspect of the πght hand. Nerve sensation had been progressively inadequate on all peπpheral extremities for the previous few years; however, the hand condition was not preceded by injury. Despite massive antibiotics and administration of hyperbanc oxygen accompanied by predmsone, the necrosis spread to involve the 3rd, 4th, and 5th metacarpals and adjacent soft tissues. A complete amputation of all ulnar aspect structures of the hand through uninvolved tissues finally resulted in halting the spread of the necrosis. A year later, the patient developed an ulcer on the dorsum of the left foot. Within 24 hours, the affected area had become necrotic and was expanding in extent; bactenal cultures were negative. After approximately 10 days of treatment with antibiotics, predmsone, and hyperbanc oxygen, the ulcer was still expanding and amputation was advised. At that time, the ulcer was approximately 4 x 6 cm in extent, ovoid shaped, with complete skin necrosis and with visible extensor tendons embedded in a tough yellowish eschar (Fig. 9A). The proximal half of the ulcer was actively producing additional tissue necrosis.

After debπdement of the eschar, treatment with an appropπate silver-containing substrate was instituted as descnbed above. No further antibiotic treatment was given.

The patient reported prompt relief of local pain and reduction in peripheral edema within 2 days of starting treatment. Over the next month, progression of the lesion stopped and some reduction in size was noted along with gradual disappearance of the abnormal eschar tissue and the appearance of more normal subdermal tissues. There was no loss of major extensor tendons. Granulation tissue began appearing 6 weeks after the start of treatment, and progressed to completely cover the lesion 2 weeks later (Fig. 9B). No exudate was visible. The cavity was filled with healthy granulation tissue with obvious skin ingrowth at the proximal end ands some at the distal end; the size of the ulcer was reduced by approximately 1 cm in all dimensions. Dunng this period, nerve sensation in the 2nd, 3rd, and 4th toes (corresponding to the ulceration) returned to normal.

By the end of the 3rd month of treatment, the ulcer was completely healed with full skin coverage and normal sensation, although slight scarnng and cavitation remained at the central portion of the oπginal lesion (Fig. 9C). The evident depression remaining in the central portion slowly resolved with restoration of normal surface contour over the next 6-9 months. Approximately 2 years after the start of treatment, the scarring and cavitation had resolved and little evidence of the oπginal lesion remained. The patient has had no recurrences of necrosis and has remained healthy, with excellent diabetes control and full use of the foot with good peπpheral sensation.

Patient 4B, a 64- year-old retired male construction worker developed a pressure ulcer over the medial head of the right first metatarsal. The patient was insulin-dependent; his diabetes was poorly controlled at the time of entry into the program. The patient developed a pressure ulcer over the medial head of the πght first metatarsal which progressed with entry into the first metacarpophalangeal joint. Despite antibiotic treatment, infection and tissue necrosis progressed into the local bone structures and the soft tissue of the forefoot. A below- knee amputation was done with resolution of the pathology and satisfactory healing. A year later, patient 4B developed a similar lesion on the intact left foot. The lesion was treated for two weeks with massive antibiotics coupled with surgical incision and debπdement. Amputation was recommended due to spread of the necrosis and infection into the first metacarpophalangeal joint and the first metatarsal bone (a second amputation would have left the patient dependent on a wheelchair). On admission, the patient reported considerable pain in the left foot, accompanied by a fever of 101-102° F (about 38.3-38.8° C). There was local redness and swelling, some of which was progressing up the anteπor aspect of the lower leg. The patient's diabetes was uncontrolled; sensation was totally lacking in the forefoot.

The wound was irrigated, packed with strips of silver-containing nylon fabnc completely into the cavitation in the dorsal aspect of the first metatarsal, and covered with silver-containing nylon fabnc in contact with the packing materials. The dressing was changed every 24 hours with a peroxide foot soak at the times of change. No antibiotics were admimstered.

The patient's body temperature was normal within 24 hours; his diabetes was controlled within 48 hours. One week after the start of treatment, the wound was clean, without visible local swelling or inflammation Two weeks after the start of treatment, the cavity had begun closing with normal granulation tissue. After one month of treatment, the metatarsal cavitation was completely closed and dressing were left in place for 8-12 hours per day. By the 6th week, the wound was completely granulated, with no residual cavitation and restoration of normal contour. Two weeks later, the wound had completely epithelialized with normal appearance and only minimal scarnng. On follow-up 2 years later, the patient had had no recurrences, his diabetes was under control, and the foot appeared anatomically normal. The patient had full use of the foot and leg, and was using his nght below -knee prosthesis for full ambulation. Patient 4C was a 63-year-old female, retired nurse, with adult-onset diabetes and spinal stenosis with severe peripheral neuropathy. The patient incurred non-penetrating trauma to the right dorsal midfoot (navicular area) approximately a year before presentation. She developed an open ulcer at the site accompanied by edema, redness, and decreased circulation. The ulcer increased in extent despite standard treatment (including surgical debπdement and multiple antibiotics) ; amputation was considered.

The ulcer was treated with silver-containing nylon dressings as descnbed above. Improved sensation and circulation were evident after 2 weeks of treatment, and the ulcer had healed after 6 weeks of treatment. The patient subsequently returned with a recurrence of the ulcer after 2 months. Retreatment again resulted in healing within a few weeks; however, with a residual draimng sinus. X-rays revealed that the infection and necrosis had penetrated the deeper tissues with osteomyelitis of the navicular bone.

Surgical debπdement of the navicular bone was performed accompanied by complete local debndement. The wound was left open. A suitable silver nylon-containing fabnc was immediately applied to the entire wound; dressings were changed daily. The patient reported little or no post-operative pain and no problems with diabetes control.

Patient 4C's wound healed completely with granulation tissue after 5 weeks of treatment. Coverage with normal-appearing, full thickness skin and only mimmal scarnng occurred spontaneously, with no drainage and with greatly improved circulation and sensation, within a few additional weeks. Six months post-operatively, the patient was fully ambulatory, with no recurrences, and with excellent nerve sensation and diabetes control.

The clinical results of treatment with silver-containing fabric, without adjuvant lontophoretic electrical current, are comparable to the clinical results reported with silver- based lontophoretic treatment. Results include excellent control of infection, and the occurrence of an obvious growth stimulation process charactenzed by the appearance of abundant granulation tissue accompanied by an exudate containing collections of mononuclear dedifferentiated cells (see Figs. 8A and 8B). Final skin coverage occurs spontaneously with no requirement for skin grafting.

The healing process observed in Examples 3 and 4 appeared to be one of local tissue regeneration with the return of all destroyed or damaged tissues to a normal state. For diabetic patients, healing included the return of normal sensation to the treated area, and peripherally to the structures innervated by the nerve fibers traversing the treatment site. Areas outside of the treatment area retained their oπginal peπpheral neuropathy, indicating that the restoration of nerve sensation was due to the direct action of the free silver ions on the local nerve fibers These results show that contact with free silver ions spontaneously emitted from a suitable silver-containing substrate caused useful moφhological and functional changes in a vaπety of mammalian cell types, both in vitro (Examples 1 and 2) and in vivo (Examples 3 and 4). In vitro results included both normal and malignant cell types. At least some cells of the resulting populations of moφhologically-changed cells were dedifferentiated to form embryonic- type stem cells; the above-descπbed clinical studies indicate restoration of normal tissue types accompanied by normal sensation. Thus, the above-described method is effective for producing stem cells that can then be used for further expenmentation, treatment, and so forth.

Dedifferentiation of mammalian cells in vitro to an embryonic, stem cell state was accomplished simply by contacting the cells with active silver ions under appropπate conditions. Once in this state, the stem cells have the capability of redifferentiating to essentially any type of cell that is present in the organism from which the oπginal cells were obtained. The observed effects may be at least partly due to the following reactions which take place in vitro and/or in vivo. Some of the silver ions released from the substrate may combine with proteins, peptides and various other chemical species normally present in solution in the tissues (or in a tissue culture medium), while others may combine with any bacteria, fungi or viruses that may be present with resulting beneficial effects. The antibactenal action of silver ions is due to this type of process, with an onset typically at about 20-30 minutes following exposure of the bacteria to the ions.

If sufficient quantities of free silver ions are available, at least some of these ions associate with cells in the treated region (including but not necessaπly limited to fibroblast cells), resulting in the above-descnbed moφhological changes. After dedifferentiation, the changed cells remain quiescent when in an in vitro culture medium (as described in U. S. Patent No. 4,528,265). In the wound environment, however, these cells are induced to multiply as dedifferentiated cell types (i.e., stem, pnmitive, or blast cells) in a process known as "clonal expansion." Granulation tissue explants from a wound treated with electncally- generated silver ions wherein such clonal expansion is evident continue to produce large numbers of new dedifferentiated cells when placed into a culture medium.

Finally, some of the silver ions may attach to collagen fibers in the region to form a silver-collagen complex that acts as a biological inducer causing clonal expansion of stem cells (Fig. 10). (As used herein, the term "inducer" refers to a substance that does not actually take part in the process and is not used up in that process. Biological induction usually requires contact between the cells and the inducer.) Human collagen has sites of two specific sizes that can be filled with correspondingly-sized hydrated ions (including silver ions). In mammalian wounds (including human wounds) treated according to the invention, the formation of such a silver-collagen complex may result in activation of the dedifferentiated embryomc cells formed by the action of the silver ions on the pre-existing mature cells. While the mechanism of action of such a complex is presently unknown, it is believed that the specific electπcal field of the silver-collagen structure may act as a biological inducer.

The occurrence of these effects, particularly in vivo, depends on the availability of an adequate supply of silver ions dunng the window wherein the above-described reactions can occur. It is believed that dedifferentiation does not occur unless sufficient amounts of silver ions are present: the key element in implementation of the invention is the production of dedifferentiated cells, which in turn depends on the availability of adequate amounts of silver ions.

In organisms that possess regenerative capability, healing of injunes involves the redifferentiation of stem cells to form the types of cells needed for repair or replacement. Salamanders, for example, are capable of regenerating full, complex anatomical structures composed of vaπous cell and tissue types arranged in a precise, correct anatomical configuration. While the mechanism for this effect is not yet known, there is some evidence that the local normal, mature cell types in the injured area influence the redifferentiation process.

By way of example, cells can be obtained from the patient being treated using standard biopsy techniques. The biopsy specimens are then cultured to produce a cell population having a desired quantity of cells, then contacted with a suitable silver-containing substrate to induce morphological changes (including dedifferentiation of at least some of the cells to stem cells). The resulting population of stem cells can then be implanted directly into the patient's body where they redifferentiate into the cell types required. Alternatively, treatment devices are produced by applying stem cells to appropπate earner matenals such as collagen, absorbable gels, biocompaUble plastics and polymers, absorbable matenals, and so forth. If needed, the stem cell population can be expanded by any technique known in the art prior to implantation or application onto a carrier.

The availability of stem cells and dedifferentiated cells produced according to the invention is believed to further research into healing processes and treatment modalities for restoπng a wide range of tissues, perhaps ultimately the potential for inducing true regenerative healing in appropriate instances.

With respect to the above descπption of the invention, it is to be realized that the optimum dimensional relationships for the parts of the invention, to include vanations in size, matenals, shape, form, function and manner of operation, assembly and use, are deemed readily apparent and obvious to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and descnbed in the specification are intended to be encompassed by the present invention. Therefore, the foregoing descnption is considered as illustrative only of the pnnciples of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention. Thus, it will be apparent to those skilled in the art that many changes and substitutions can be made to the preferred embodiment herein described without departing from the spint and scope of the present invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for changing the moφhology of mammalian cells, said method compπsing the steps of: providing a culture medium containing mammalian cells; and contacting at least some of said cells with a silver-containing material for a sufficient period of time to cause free silver ions from said substrate to alter a moφhology of at least a portion of said cells to produce a population of changed cells.

2. The method as recited in claim 1, wherein said material contains silver in a mechamcally stable form, silver ions being releasable from said material when said matenal is wetted, further comprising the step of wetting said material with an aqueous liquid so that at least some of said silver ions migrate into said culture medium.

3. The method as recited in claim 1, wherein said material further comprises a nonconducting, nonallergenic, non-adherent, mechamcally stable fabnc.

4. The method as recited in claim 1 , wherein said material further comprises silver nylon fabnc.

5. The method as recited in claim 1, wherein at least a portion of said silver is crystalline in form.

6. The method as recited in claim 1 , wherein said cells are adult mammalian cells.

7. The method as recited in claim 2, further comprising the initial steps of: introducing an initial quantity of said mammalian cells into a vessel containing an initial culture medium; growing said initial quantity of cells to produce a cell population having a selected population density ; and transferπng at least a portion of said cell population to said culture medium.

8. The method as recited in claim 1 , wherein said contacting step is earned out for a sufficient period to time to cause dedifferentiation of at least a portion of said mammalian cells.

9. The method as recited in claim 1, wherein said contacting step is earned out for a sufficient period of time to cause at least a portion of said mammalian cells to dedifferentiate to form stem cells

10. The method as recited in claim 1, further compπsing the next step of contacting said changed cells with cells of a selected type, thereby causing at least some of said changed cells to redifferentiate to form additional cells of said selected type.

11. The method as recited in claim 1 , wherein said culture medium contains collagen, and wherein at least a portion of said free silver ions bind to said collagen to form a silver- collagen complex, said complex inducing expansion of said population of changed cells.

12. A cell population, said cell population produced by a process comprising the steps of: introducing a quantity of mammalian cells into a culture medium; and contacting at least a portion of said mammalian cells with a silver-containing matenal for a sufficient period of time to cause free silver ions from said substrate to change a moφhology of at least some of said mammalian cells to produce said cell population.

13. The method as recited in claim 12, wherein said silver ions are releasable from said material when said material is wetted, further comprising the step of wetting said matenal with an aqueous liquid so that at least some silver ions migrate into said culture medium.

14. The cell population as recited in claim 12, wherein said silver-containing matenil contains crystalline silver.

15. The cell population as recite din claim 12, wherein said contacting step is earned out for a sufficient period of time to cause dedifferentiation of at least a portion of said mammalian cells to form said cell population.

16. The cell population as recited in claim 12, wherein said silver-containing substrate is silver nylon fabnc.

17. The cell population as recited in claim 12, wherein said culture medium contains collagen, and wherein at least a portion of said free silver ions bind to said collagen to form a silver-collagen complex, said complex inducing expansion of said cell population.

18. The cell population as recited in claim 15, wherein said process further comprises the next step of contacting at least a portion of said cell population with cells ol a selected type, thereby causing at least some of said cell population to redifferentiate to form additional cells of said selected type.

19. A device for inducing moφhological changes in mammalian cells, said device compπsing: a substrate; and a quantity of crystalline silver attached to said substrate, at least a portion of said silver releasing ions when wetted with an aqueous liquid.

20. The device as recited in claim 19, wherein said substrate includes nylon fabnc.