Patent 6,878,383

The invention provides a composition for promoting regeneration of tissue which has degenerated in a subject as a result of a disease or disorder and a method of using the composition is provided. The composition comprises a biodegradable acellular matrix, and passaged autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, integrated within the matrix. Also provided is an injectable composition comprising an acellular filler material (e.g., any type of collagen) and passaged autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, for correcting defects in skin, such as wrinkles or scars, and for augmenting tissue in the subject, particularly facial tissue.

Description of the Invention

FIELD OF THE INVENTION

The present invention concerns the regeneration of tissues in a subject that have degenerated as a result of a disease or disorder in the subject. More particularly, the present invention concerns novel compositions for use in surgical and nonsurgical techniques that promote regeneration of tissue whose mass has been diminished due to a disease or disorder in a subject, correct defects in the skin of subjects, or augment tissue in subjects. Also disclosed is the use of a novel composition in conjunction with a biodegradable acellular matrix for ameliorating defects in the tissues, and methods for using the novel composition.

BACKGROUND OF THE INVENTION

"Periodontal disease" is the term commonly used to describe inflammatory disease of the periodontium, i.e., the tissue surrounding and securing teeth to the jawbone. The condition is characterized by inflammatory and degenerative processes that develop at the gingival margin (gingivitis) and lead to a progressive breakdown and resorption of the periodontal ligament and bone (periodontitis), oftentimes resulting in severe diminution of the periodontium. Periodontal disease is the leading cause of tooth loss in adults after middle age. [Anderson's Pathology, p. 2000, John M. Kissane ed., 9th ed. (1992)].

Periodontal disease results from the accumulation of bacterial plaque in the gap between the gingiva and the tooth. While anaerobic bacteria are the primary etiologic agents, the destructive process is believed to be mediated in large part by immunologic reactions of the host. As the disease progresses, a periodontal pocket is established below the gingival margin, thus prolonging and promoting the inflammatory process. Successive inflammatory reactions result in the progressive erosion of the tooth-supporting tissues, i.e., the collagenous fibers making up the periodontal ligament and the bone pocket in which the tooth sits. [Reviewed in Anderson's Pathology, pp. 1999-2000, John M. Kissane ed., 9th ed. (1992); Shafer et al., A Textbook of Oral Pathology, 4th ed. (1983)].

Periodontal disease can be diagnosed by checking the gingiva for inflammation, probing the depths of periodontal pockets, checking clinical attachment level, and assessing bone loss by means of autoradiography. [Jeffcoat, M. K., et al., J. Am. Dent. Assoc., 128:713-724 (1997)].

A number of techniques, both surgical and nonsurgical, have been developed to treat periodontal disease. Particularly with respect to severe periodontitis, none of the currently available treatments are wholly satisfactory.

For relatively mild cases of periodontitis, practitioners have traditionally employed nonsurgical mechanical debridement (i.e. scaling and root planing) to remove the bacterial plaque whose accumulation perpetuates the disease, thereby reducing inflammation. Mechanical debridement can be accomplished using manual, sonic or ultrasonic instruments. Scaling and root planing have been shown to decrease gingival inflammation, decrease probe depth, and promote maintenance of clinical attachment level. However, without resorting to surgical procedures, access to root surfaces and bony defects is restricted, and only limited debridement is possible. [Jeffcoat, M. K., et al., J. Am. Dent. Assoc. 128:713-724 (1997)].

As a result, nonsurgical scaling and root planing is insufficient to treat more severe cases of periodontitis, and it is necessary to resort to more aggressive surgical techniques. Surgical techniques comprise reflecting the gingival tissues to provide access to root surfaces and bone defects, in order that mechanical debridement may be accomplished directly. Following debridement, the gingival tissue is sutured back in position. Currently available surgical approaches entail substantial patient discomfort and fail to consistently provide satisfactory outcome.

There are a number of non-surgical, non-mechanical approaches to treating periodontal disease, including supragingival and subgingival irrigation and the application of chemical and antimicrobial agents. Yet none of these approaches have achieved more than marginal success [Jeffcoat, M. K., et al., J. Am. Dent. Assoc., 128:713-724 (1997)]. In particular, there are a number of deleterious side effects associated with the use of antibiotics, along with risks such as drug sensitivity and the emergence of antibiotic-resistant pathogens.

Another approach to combating destruction of the periodontal tissue has focused on inhibiting the matrix metalloproteases responsible for this destruction. Tetracyclines in particular have shown promise as inhibitors of extracellular collagenases, but cause the same side effects associated with antibiotics in general. Modified forms of tetracycline have been developed which are non-antimicrobial and retain their ability to inhibit collagenases, but these chemically modified tetracyclines are not commercially available. [Ciancio, G. C. et al., J. Am. Dent. Assoc. 123:34-43 (1992)].

Because cell proliferation, cell migration and matrix synthesis are prerequisites for periodontal regeneration, some researchers have attempted to use tissue growth factors, for example insulin-like growth factor, platelet-derived growth factor, and transforming growth factor to promote periodontal regeneration.

In summary, none of the nonmechanical approaches to treating periodontitis have been able to offer more than modest, short term enhancement of traditional mechanical debridement. As noted in a 1997 review of techniques used in treating periodontal disease, "scaling and root planning accompanied by oral hygiene procedures remains the first mode of treatment for adult periodontitis." [Jeffcoat, M. K. et al., J. Am. Dent. Assoc., 128:713-724 (1997)].

A great deal of research has been directed to methods of regenerating periodontal tissue lost as a consequence of periodontal disease, but as yet no wholly satisfactory method is available. For the most part, efforts have focused on surgical approaches that fill the defects with a variety of materials (bone grafting) or use guided tissue regeneration.

Bone grafting techniques involve the use of natural bone or synthetic bone materials. Natural bone grafts are typically either autografts (grafts transferred from one position in the body of a patient to another position in the body of the same patient) or allografts (grafts transferred from one person to another). Clinicians using natural bone grafts have had limited success in inducing new bone growth. Problems associated with the use of autografts include the need for a second surgical site and, in some cases, fresh grafts may be associated with root resorption. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc. 128:713-724 (1997)].

Freeze-dried, demineralized bone has been used as an allograft and shown to promote bone formation. However, the predictability and the amount of bone fill achieved varies. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc., 128:713-724 (1997)]. Since allografts are transferred from one person to another, the potential exists that viruses or other pathogens might be transferred to the patient.

Synthetic bone materials which have been investigated include plaster, calcium carbonates, and ceramics such as hydroxyapatite. Clinical trials have demonstrated that the use of synthetic grafts has resulted in improvements in probing depth and attachment level. Histologic findings, however, indicate that, in general, synthetic grafts act primarily as space fillers, with little if any regeneration. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc., 128:713-724 (1997)].

Guided tissue regeneration is a surgical approach based on placing a membrane barrier under a soft tissue flap above the area of bone loss to enhance wound healing potential. [Ciancio, G. C. et al., J. Am. Dent. Assoc., 123:34-43 (1992]). Investigators have studied both resorbable and nonresorbable membranes. A significant disadvantage of using a nonresorbable membrane is the requirement of a second surgical procedure after approximately six weeks to remove the membrane. Furthermore, in about 40%-50% of the cases, such membranes become infected in the patient. While less evidence is available for resorbable membranes than for nonresorbable membranes, improvements in clinical attachment levels have been shown for both types of membranes compared with debridement alone. Most favorable results are reported for Class II furcations in the mandible and for intrabony defects. Less favorable results have been reported in maxillary molar and Class III (through and through) furcation defects. [Jeffcoat, M. K. et al., J. Am. Dent. Assoc., 128:731-724 (1997)].

In summary, none of the currently available treatments for periodontal disease is wholly satisfactory, particularly with regards to regenerating periodontal tissue lost as a result of periodontitis.

The oral mucosa is the tissue lining the oral cavity. There are a number of conditions that can result in defects in the oral mucosa, for example trauma, dermatoses, recurrent aphthous stomatitis, and infections. [Flint, S., The Practitioner 235:56-63 (1991)]. There is currently no wholly satisfactory means of correcting these defects.

Furthermore, there have been efforts to develop and use compositions and methods to correct defects in skin, such as scars and wrinkles, or to augment the tissue of a subject in order to improve the appearance of the skin, particularly facial skin. The principal method employed to correct such defects involves injecting a filler composition into the dermal layer of the skin proximate to the defect or desired tissue augmentation. Examples of non-biological filler compositions used in these roles include mineral oil, paraffin, silicone fluid, autologous fat, gelatin powder mixes, polymethylmethacrylate microspheres, cross-linked polydimethylsiloxane, ""TEFLON"" paste, reconstituted bovine collage, and autologous human collagen.

However, the use of these compositions comprises inherent limitations. For example, the use of mineral oil, paraffin and similar oils and waxes has resulted in complications such as local chronic edema, lymphadenopathy, scarring and ulcerations (Devore et al., Effectiveness of injectable filler materials for smoothing wrinkle lines and depressed scars. Medical Progress Through Technology 20:243-250 (1994 which is hereby incorporated by reference in its entirety).

The use of reconstituted bovine collagen to correct defects or augment tissue also possesses inherent limitations. For example, it has been reported that reconstituted bovine collage is only moderately effective, and is associated with infrequent, but controversial, adverse reactions. In addition, it is rapidly broken down and resorbed in vivo, providing only a temporary correction of a skin defect or augmentation. More importantly, reconstituted bovine collagen may elicit an immune response in the subject. Id.

As explained above, gelatin matrix implant such as that sold under the mark "FRIBEL", is a composite material of porcine gelatin powder and o-aminocaproic acid which are dispersed in 0.9% (by volume) sodium chloride solution and an aliquot of the recipient's plasma mixed in a 1:1 ratio, is also used to correct skin defects and augment tissue. However, this material also possess inherent limitations. Specifically gelatin matrix does not appear to have applications in the treatment of wrinkle lines. Moreover, since a large bore needle (27 gauge or greater) is used to inject the gelatin matrix into the subject's skin, treatment with gelatin results in greater discomfort and pain to the subject as opposed to the injection of other fillers. Id.

In addition, the use of autologous fat injections to correct a skin defect or augment tissue in a subject, while eliminating the potential of eliciting an immune response, also possesses disadvantages. More specifically, prior to its injection, fat must be processed by skilled clinicians in aseptic conditions to maintain sterility. In addition, the injections are not dermal but are subcutaneous or subdermal. Also, a very large bore needle (as large as 16 gauge) is needed to inject the fat into a subject, resulting in great pain, moderate bruising, and formation of visible puncture holes. Moreover, fat injections are subject to rapid resorption, and must be repeated in order to maintain skin augmentation or defect correction.

The use of autologous, injectable dermal collagen to correct defects or augment tissue has also met with limited success. For example, if large concentrations of collagen are injected, a 27 gauge needle or larger is used, resulting in the infliction of pain on the subject. Furthermore, serial injections are required in order to compensate for the gradual resorption of autologous collagen.

Hence, what is needed is an efficient non-surgical composition that promotes the regeneration of tissues of the gums or the palate and bone that have degenerated as a result of periodontal disease or trauma.

Moreover, what is needed is a composition and method for promoting regeneration of tissue that does not elicit an immune response in the subject at the site of desired tissue regeneration.

What is also needed is a composition that can be used in non-surgical methods to correct defects in skin, such as scars or wrinkles, and augment tissue in a subject, particularly facial tissue, which is not rapidly resorbed by the body so that additional injections are required.

U.S. Pat. No. 5,591,444, U.S. Pat. No. 5,660,850, and U.S. Pat. No. 5,665,372 are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides a composition for promoting regeneration of tissue in a subject that has degenerated as a result of a disease or disorder, and a method of using the composition that does not suffer from the shortcomings of other methods described above. The present invention is based on the inventors' discovery of the successful use of autologous fibroblasts, with and without various forms of matrix, filler or carrier material, to regenerate tissue in a subject, correct skin defects in the subject, or augment tissue in the subject. Fibroblasts are connective-tissue cells involved in tissue repair. When a tissue is injured, nearby fibroblasts migrate into the wound, proliferate, and produce large amounts of collagenous matrix, which helps to isolate and repair the damaged tissue. [Alberts et al., Molecular Biology of the Cell, p. 987, 2nd ed., (1992)].

Broadly, the present invention extends to a method for regenerating a subject's tissue (a) that has degenerated as a result of a disease or disorder or (b) that has a defect, comprising the steps of providing a pharmaceutical composition comprising autologous, passaged fibroblasts, identifying a site of tissue degeneration, and injecting an effective amount of the composition into tissue at the site of tissue degeneration so that the tissue is augmented and regeneration of tissue is promoted.

Injection of a pharmaceutical composition of the present invention can be into tissues of the subject comprising the periodontal pocket and/or the periodontal tissue adjacent to the area of degeneration or into tissue subadjacent to a defect in the oral mucosa, or into the tissue of the palate of a human subject, in order promote regeneration of tissue in the oral mucosa, the gingiva, or the palate. Typical defects in the oral mucosa or palate that can be corrected with this embodiment of the present invention include those caused by trauma, dermatoses, recurrent aphthous stomatitis, and infections, or a disease or disorder. Moreover, the present invention can be used to correct defects in the skin, such as scars, wrinkles, laugh lines, rhytids, stretch marks, depressed scars, cutaneous depressions of non-traumatic origin, acne scarring, or subcutaneous atrophy from acne, trauma, congenital malformation, or aging. Moreover, the invention can be used to treat defects such a hypoplasia of the lips, or labial folds. In addition, the composition can be used to repair a defect, disorder or disease of bone, e.g., bones such as, for example, facial bones including orbits, mandibles, maxillae, zygomatic bones, crania, and nasal bones. Bone diseases, disorders, or defects, include, for example, tooth extraction-related bone defects or those due to periodontal disease.

A disease or disorder which results in tissue degeneration in a subject that can be treated with the present invention includes, but is not limited to, defects of the oral mucosa, periodontal disease, trauma to the oral mucosa (e.g., extraction of a tooth), diabetes, cutaneous ulcers, or venous stasis. Moreover, periodontal disease can include periodontal degeneration, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa.

The present invention further extends to a method of forming a composition comprising autologous, passaged fibroblasts which are substantially free of immunogenic proteins, such as culture medium serum-derived proteins, and are histocompatible with a subject. This method comprises the steps of collecting a biopsy of dermis from a subject, isolating the autologous fibroblasts contained in the biopsy from extracellular matrix and other cells contained in the biopsy, culturing the autologous fibroblasts in a culture medium that permits expansion of the autologous fibroblasts, incubating the autologous fibroblasts in a protein free medium for at least about 2 hours between about 30^oC. and about 37.5^oC., and exposing the incubated autologous fibroblasts to a proteolytic enzyme so as to suspend the fibroblasts. An example of a culture medium that permits expansion of autologous fibroblasts comprises between 0.0% and about 20% serum, wherein the serum can be either human or non-human. Also, the biopsy from dermis can comprise tissue from the gums, palate or skin of the subject. Hence autologous fibroblasts from the gums, plate or skin have applications in the present invention.

In another embodiment, the passaged autologous fibroblasts can be added to a pharmaceutically acceptable carrier to form a pharmaceutical composition. In producing a pharmaceutical composition of the present invention, immunogenic proteins, e.g., culture medium serum-derived proteins, are removed from the autologous fibroblasts, thereby avoiding an immunological reaction in a subject when such cells are reintroduced to the subject proximate to the site of tissue degeneration or defect.

In addition, the present invention extends to a device for delivering a pharmaceutical composition of the present invention to a site proximate to the site of tissue degeneration or defect in a subject, wherein the device comprises a hypodermic syringe having a syringe chamber, a piston disposed therein, an orifice communicating with the chamber, a pharmaceutical composition comprising autologous passaged fibroblasts and a pharmaceutically acceptable carrier, such that the pharmaceutical composition is disposed in the chamber, and a hypodermic needle is fixed to the orifice.

Tissues which have suffered degeneration or have a defect that can be treated with a device of the present invention include the oral mucosa, the gingival mucosa, and the palatal mucosa. Moreover, diseases or disorders which can be treated with this device include periodontal degeneration, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa. Other defects that can be treated with this invention include those listed above.

The present invention further extends to a composition for repairing tissue that has degenerated in a subject as result of a disease, disorder or defect in the subject, wherein the composition comprises a biodegradable acellular matrix, and autologous passaged fibroblasts (derived, for example, from the gums, palate, or skin of the subject) and is substantially free of immunogenic proteins, e.g., culture medium xenogeneic (e.g., fetal bovine) serum-derived proteins, wherein the autologous fibroblasts are integrated into the biocompatible biodegradable acellular matrix. In an embodiment of the present invention, the biocompatible biodegradable acellular matrix comprises exogenous proteins, such as any type of collagen. In addition, the biodegradable acellular matrix can be comprised of any type of collagen and glycosaminoglycans (GAG) cross-linked with, for example, glutaraldehyde, or any type of collagen.

In yet another example, the biodegradable acellular matrix comprises one or more of gelatin, polyglycolic acid, cat gut, demineralized bone, or hydroxyapatite. Other appropriate matrices consist of bone from which substantially all (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or even 100% by weight) organic material has been removed (referred to herein as "anorganic bone"); such matrices can, optionally, include exogenous collagen in various amounts (e.g., about 1%, about 2%, about 5%, about 10%, or about 20% by dry weight).

Also, diseases, disorders or defects resulting in degeneration of tissue in a subject which can be treated with the present invention, comprise defects of the oral mucosa, trauma (e.g., extraction of a tooth) to the oral mucosa or oral bones such as the maxillary or mandibular bones, periodontal disease, diabetes, cutaneous ulcers, or venous stasis. In addition, examples of periodontal disease which result in tissue degeneration include, but are not limited to, periodontal degeneration, gingivitis, or non-healing wounds of the palatal mucosa or gingival mucosa, or bone degeneration. Other defects that can be treated with this invention include skin defects, such as scars, wrinkles, laugh lines, stretch marks, depressed scars, cutaneous depressions of non-traumatic origin, acne scarring, or subcutaneous atrophy from acne, trauma, congenital malformation, or aging. Moreover, the invention can be used to treat defects such a hypoplasia of the lips, labial folds, or bone defects, e.g., defects of bones such as, for example, facial bones including orbits, mandibles, maxillae, zygomatic bones, crania, and nasal bones.

Also encompassed by the invention is a method for making a composition for the repair of tissue that has degenerated in a subject as result of a disease, disorder, or defect in the subject. The method comprises: providing a suspension of autologous, passaged fibroblasts; providing a biodegradable acellular matrix; incubating the suspension of autologous passaged fibroblasts with the biodegradable acellular matrix such that the autologous passaged fibroblasts integrate within the biodegradable acellular matrix; and removing substantially all culture medium serum-derived proteins from said biodegradable acellular matrix and said integrated fibroblasts to form a composition for promoting the repair of tissue. Sufficient autologous, passaged fibroblasts integrate within the biodegradable acellular matrix to substantially fill the space on and within the biodegradable acellular matrix available for cells. As used herein, "substantially" fill with passaged autologous fibroblasts means to fill to a level sufficient to prevent an amount of cell proliferation that degrades a collagen matrix to a practically deleterious level, as a person skilled in the art can readily determine for a particular embodiment.

The biodegradable acellular matrix used in this method can contain exogenous protein such as, for example, any type of collagen, e.g., any type of collagen and glycosaminoglycans, cross-linked with, for example, glutaraldehyde. The biodegradable acellular matrix can contain one or more of the following substances: gelatin, polyglycolic acid, cat gut, demineralized bone, hydroxyapatite, gelatin, polyglycolic acid, cat gut, or anorganic bone with or without any of range of concentrations of exogenous collagen (see below).

The disease, disorder, or defect to be treated can be a defect of an oral mucosa, trauma to an oral mucosa, periodontal disease, diabetes, a cutaneous ulcer, venous stasis, a scar of skin, or a wrinkle of skin. Alternatively, the disease or disorder can be periodontal disease, and the periodontal disease can be periodontal degeneration, gingivitis, or a non-healing wound of a palatal mucosa or a gingival mucosa.

In this method the step of providing a suspension of autologous, passaged fibroblasts can involve: collecting a biopsy of dermis or palate of the subject; separating dermal autologous fibroblasts from the biopsy; culturing the dermal autologous fibroblasts in a culture medium containing (a) between 0.0% and about 20% human or non-human serum and (b) a reagent that prevents the growth of mycoplasma; and exposing the incubated dermal autologous fibroblasts to a proteolytic enzyme so as to suspend fibroblasts. The step of collecting a biopsy of dermis can involve collecting a biopsy from gums, palate or skin of the subject. The reagent can contain tylosin and, optionally, one or more of the following compounds: gentamicin, ciprofloxacine, alatrofloxacine, azithromycin, or tetracycline.

The present invention further extends to a method of using a composition for promoting regeneration of tissue, wherein the method comprises providing passaged autologous fibroblasts integrated into a biodegradable acellular matrix, identifying a site (i) of tissue degeneration due to a disease or disorder in the subject or (ii) a defect in the tissue of the subject, and placing the composition on the site so that the tissue is repaired. Autologous passaged fibroblasts used herein can comprise fibroblasts from the gums, palate or skin of the subject.

Diseases, disorders, or defects which can be treated with this method include, but are not limited to, defects of the oral mucosa, trauma to the oral mucosa (e.g., extraction of a tooth), periodontal disease, diabetes, cutaneous ulcers, or venous stasis. Examples of periodontal disease that can be treated with the present invention comprise periodontal degeneration, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa. Moreover, defects (e.g., skin defects such as scars or wrinkles) can be treated with the composition of the present invention. In a preferred embodiment, such defects are treated with a composition comprising fibroblasts from the palate. Any of the above listed diseases, disorders, or defects can also be treated by these methods.

Furthermore, biodegradable acellular matrices having applications in the present invention may comprise exogenous proteins. Examples of such matrices include matrices comprising any type of collagen, or any type of collagen and glycosaminoglycans (GAG) cross-linked with, for example, glutaraldehyde.

Other examples of biodegradable acellular matrices having applications in the present invention include one or more of gelatin, polyglycolic acid, cat gut, demineralized bone (e.g., demineralized human bone), or hydroxyapatite. Other appropriate matrices consist of bone from which substantially all (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or even 100% by weight) organic material has been removed (referred to herein as "anorganic bone"); such matrices can, optionally, include exogenous collagen, in various amounts (e.g., about 1%, about 2%, about 5%, about 10%, or about 20% by dry weight).

The present invention further extends to an injectable composition for correcting a defect in skin of a subject, or augmenting tissue of a subject, said injectable composition comprising passaged, autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, and a biodegradable, acellular injectable filler material. Passaged autologous fibroblasts having applications in an injectable composition of the present invention are from gums, palate or skin of the subject.

Furthermore, the present invention extends to an injectable composition as described above, wherein the biodegradable, acellular injectable filler material comprises endogenous proteins. In particular, the acellular injectable filler material of an injectable composition of the present invention comprises an injectable dispersion of autologous collagen fibers having a concentration in the composition of at least 24 mg/ml of composition.

In addition, the present invention extends to an injectable composition as described above, wherein the biodegradable acellular injectable filler material comprises exogenous proteins, such as any type of collagen. An example of an exogenous collagen having applications in an injectable composition of the present invention is reconstituted bovine collagen fibers cross-linked with glutaraldehyde.

Furthermore, the filler material can comprise any type of solubilized gelatin either alone, or in combination with other materials. In a particular example, the filler material comprises porcine gelatin powder and o-aminocaproic acid dispersed in sodium chloride solution and an aliquot of plasma from the subject to be injected with the composition. Preferably the ratio of sodium chloride to serum is 1:1 by volume. Other examples of materials having applications in the present invention as biodegradable, acellular injectable filler material include, but are not limited to polyglycolic acid or cat gut.

The present invention further extends to a method for correcting a defect in skin of a subject, or augmenting tissue of a subject, wherein the method comprises injecting an effective amount of an injectable composition comprising autologous passaged fibroblasts substantially free of immunogenic proteins (e.g., culture medium serum-derived proteins) and a biodegradable, acellular injectable filler material, into the skin of the subject at the site of the skin defect or desired tissue augmentation, so that regeneration of tissue at the site is promoted at the site.

Moreover, the present invention extends to a method for correcting a defect in skin of a subject, or augmenting tissue of a subject, the method comprising the steps of injecting autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, into the subject at a site of a skin defect or desired tissue augmentation, and subsequently injecting a biodegradable, acellular injectable filler material into the site. In a particular embodiment of this method of the present invention, the duration between injecting the autologous fibroblasts into the subject and injecting the biodegradable acellular injectable filler into the subject is about two weeks.

Autologous fibroblasts having applications in methods of the present invention for correcting a defect in skin of a subject, or augmenting tissue of a subject can be obtained from the gums, palate of skin of the subject.

The present invention further extends to a method for correcting a defect in skin of a subject, or augmenting tissue of the subject, as described above, wherein the biodegradable, acellular injectable filler material comprises endogenous proteins. For example, the biodegradable acellular injectable filler material can comprise an injectable dispersion of autologous collagen fibers, preferably at a concentration of at least 24 mg of autologous fibers per ml of composition.

Furthermore, the present invention extends to a method for correcting a defect in skin or other tissues of a subject (such as those described above), or augmenting tissue of the subject, also as described above, wherein the biodegradable, acellular filler material of the composition comprises exogenous proteins such as, for example, any type of collagen. An example of collagen having applications in a method of the present invention comprises reconstituted bovine collagen fibers cross-linked with, for example, glutaraldehyde.

Other examples of biodegradable, acellular injectable filler material for use in a method for correcting a defect in skin or a subject, or augmenting tissue of the subject include, but are not limited to solubilized gelatin, polyglycolic acid, or cat gut sutures. More specifically, an example of acellular injectable filler material having applications in the present invention comprises porcine gelatin powder and aminocaproic acid dispersed in sodium chloride solution, and an aliquot of plasma from the subject. Preferably, the ratio of sodium chloride solution to the aliquot of serum is 1:1 by volume. Furthermore, the sodium chloride solution comprises 0.9% sodium chloride by volume.

In addition, the present invention extends to a method for correcting a defect in skin of a subject, or augmenting tissue of a subject, as described above, wherein the ratio of autologous fibroblasts substantially free of immunogenic proteins (e.g., culture medium serum-derived proteins) biodegradable, acellular injectable filler material is approximately 1:1 by volume.

Accordingly, it is an object of the present invention to provide a composition for augmenting tissue, or promoting regeneration of tissue such as the oral mucosa, the gingival mucosa, or the palatal mucosa or skin, which has degenerated as a result of a disease or disorder. Examples of such disorders include periodontal disease, trauma, dermatoses, recurrent aphthous stomatitis, infections, scars, or wrinkles and the others listed above.

It is another object of the present invention to provide a composition for augmenting tissue, or promoting regeneration of tissue, wherein the composition is histocompatible with a subject, thereby avoiding elicitation of an immune response and inflammation in the tissues of the subject near the site of degeneration of tissue.

It is yet another object of the present invention to provide method of promoting tissue regeneration that does not require surgery.

It is yet still another object of the present invention to promote regeneration of tissue in a subject without the use of antibiotics in the subject, and hence prevent the emergence of antibiotic resistant pathogens and deleterious side effects associated with antibiotics in the subject.

It is another object to provide an injectable composition for correcting defects in skin, such as scars or wrinkles, or for augmenting tissue in a subject, particularly facial tissue (such as lips), which includes passaged autologous fibroblasts that can withstand resorption so that subsequent injections are not needed, and to prevent the elicitation of an immune response in the subject.

It is yet another object of the present invention to provide methods for correcting defects in skin, such as scars or wrinkles, or for augmenting tissue in a subject, that employs the injectable composition set forth above, that inflicts limited pain on the subject, and does not elicit an immune response in the subject, and is not rapidly resorbed.

Claim 1 of 16 Claims

What is claimed is:

1. A method of repairing bone tissue in a subject with a bone defect, wherein said method comprises:

a) providing a composition,

wherein the composition comprises (i) a biodegradable acellular matrix and (ii) autologous fibroblasts,

wherein the composition is substantially free of proteins derived from xenogeneic serum of xenogeneic serum-containing culture medium,

wherein said biodegradable acellular matrix, prior to combination with said autologous fibroblasts, comprises one or more substances selected from the group consisting of collagen, glycosaminoglycans, gelatin, polyglycolic acid, cat gut, demineralized bone, hydroxyapatite, and anorganic bone, and

wherein and said autologous fibroblasts are added to the matrix at high density and then cultured for up to 4 days, such that sufficient autologous fibroblasts integrate within said biodegradable acellular matrix to substantially fill the space on and within said biodegradable acellular matrix available for cells;

b) identifying a site of a bone defect in said subject; and

c) placing the composition on the site so that said bone defect is repaired.

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