WO1999043270A1 - Augmentation and repair of dermal, subcutaneous, and vocal cord tissue defects - Google Patents

Abstract

This application concerns a method for corrective surgery of defects being amenable to rectification by the augmentation of tissue subjacent to the defect. The method involves retrieving viable cells from a subject, a neonate or human fetus. The cells are then cultured in vitro and placed into a tissue of the subject, the tissue being located in a position subjacent to the defect to be rectified. Alternatively, the cells may be cultured in a collagen matrix or suspended in a collagen matrix prior to being placed in a position subjacent to the defect to be rectified. In a further embodiment, the cells are cultured and the in vitro produced extracellular matrix is collected and placed in a position subjacent to the defect to be rectified. A method for the correction of vocal cord defects is also disclosed.

Description

AUGMENTATION AND REPAIR OF DERMAL, SUBCUTANEOUS, AND VOCAL CORD TISSUE DEFECTS

FIELD OF INVENTION

The field of the present invention is the long-term augmentation and/or repair of defects in dermal, subcutaneous, or vocal cord tissue.

BACKGROUND OF THE INVENTION I. IN VITRO CELL CULTURE

The majority of in vitro vertebrate cell cultures are grown as monolayers on an artificial substrate which is continuously bathed in a nutrient medium. The nature of the substrate on which the monolayers may be grown may be either a solid (e.g., plastic) or a semi-solid (e.g., collagen or agar). Currently,disposable plastics have become a preferred substrate for cell culture.

While the growth of cells in two-dimensions is frequently used for the preparation and examination of cultured cells in vitro, it lacks the characteristics of intact, in vivo tissue which, for example, includes cell-cell and cell-matrix interactions. Therefore, in order to characterize these functional and morphological interactions, various investigators have examined the use of three-dimensional substrates in such forms as a collagen

gel (Yang et al, Cancer Res. 47:1027 (1981); Douglas et al, In Vitro 7(5:306 (1980); Yang et al ., Proc. Nafl Acad. Sci. 2088 (1980) ), cellulose sponge (Leighton et al, J. Nat'l Cancer Inst. 72:545 (1951 ) ), collagen-coated cellulose sponge (Leighton et al, Cancer Res. 25:286 (1968) ), and GELFOAM^® (Sorour et al, J. Neurosurq. 43:742 (1975) ). Typically, these aforementioned three-dimensional substrates are inoculated with the cells to be cultured, which subsequently penetrate the substrate and establish a "tissue-like" histology similar to that found in vivo. Several attempts to regenerate "tissue-like" histology from dispe rsed monolayers of cells utilizing three-dimensional substrates have been reported. For example, three-dimensional collagen substrates have been utilized to culture a variety of cells including breast epithelium (Yang, Cancer Res. 47:1021 (1981 ) ), vascular epithelium (Folkman et al, Nature 288 :551 (1980) ), and hepatocytes (Sirica et al, Cancer Res. 76:3259 (1980) ). However, long- term culture and proliferation of cells in such systems has not yet been achieved. Prior to the present invention, a three-dimensional substrate had not been utilized in the autologous in vi tro culture of cells or tissues derived from the dermis, fascia, or lamina propria.

II. AUGMENTATION AND/OR REPAIR OF DERMAL AND SUBCUTANEOUS TISSUES

In the practice of cosmetic and reconstructive plastic surgery, it is frequently necessary to employ the use of various injectable materials to augment and/or repair defects of the subcutaneous or dermal tissue, thus effecting an aesthetic result. Non-biological injectable materials (e g., paraffin) were first utilized to correct facial contour defects as early as the late nineteenth century. However, numerous complications and the generally unsatisfactory nature of long-term aesthetic results caused the procedure to be rapidly abandoned. More recently, the use of injectable silicone became prevalent in the 1960's for the correction of minor defects, although various inherent complications also limited the use of this substance. Complications associated with the utilization of injectable liquid silicone include local and systemic inflammatory reactions, formation of scar tissue around the silicone droplets, rampant and frequently distant, unpredictable migration throughout the body, and localized tissue breakdown. Due to these potential complications, silicone is not currently approved for general clinical use. Although the original proponents of silicone injection have continued experimental programs utilizing specially manufactured "Medical Grade" silicone (e.g., Dow Coming's MDX 4.401 1^®) with a limited number of subjects, it appears highly unlikely that its use will be generally adopted by the surgical community. See e.g., Spira and Rosen, Clin. Plastic Surgery 20: 181 ( 1993); Matton et al, Aesthetic Plastic Surgery 9:133 (1985).

It has also been suggested to compound extremely small paniculate species in a lubricious material and inject such micro-particulate media subcutaneously for both soft and hatd tissue augmentation and repair. However, success has been heretofore limited. For example, bioreactive materials such as hydroxyapatite or cordal granules (osteo conductive) have been utilized for the repair of hard tissue defects. Subsequent undesirable micro-particulate media migration and serious granulomatous reactions frequently occur with the injection of this material. These undesirable effects are well-documented with the use of such materials as polytetrafluoro- ethylene (TEFLON^®) spheres of small-diameter (~ 90% of particles having diameters of <30μm) in glycerin. See e.g., Malizia et al, JAMA 257:3277 ( 1984). Additionally, the use of very small-diameter particulate spheres (~l -20μm) or small elongated fibrils (~l-30μm in diameter) of various materials in a biocompatible fluid lubricant as injectable implant composition are disclosed in U.S. Patent No. 4,803,075. However, while these aforementioned materials create immediate augmentation and/or repair of defects, they also have a tendency to migrate and be reabsorbed from the original injection site.

The poor results initially obtained with the use of non-biological injectable materials prompted the use of various non-immunogenic, proteinaceous materials (e.g., bovine collagen and fibrin matrices). Prior to human injection, however, the carboxyl- and amino-terminal peptides of bovine collagen must first be enzymatically degraded, due to its highly immunogenic nature. Enzymatic degradation of bovine collagen yields a material, atelocollagen, which can be used in limited quantities in patients pre-screened to exclude those who are immunoreactive to this substance. The methodologies involved in the preparation and clinical utilization of atelocollagen are disclosed in U.S. Patent Nos. 3,949,073; 4,424,208; and 4,488,91 1. Atelocollagen has been marketed as ZYDERM^® brand atelocollagen solution in concentrations of 35 mg/ml and 65 mg/ml.

Although atelocollagen has been widely employed, the use of ZYDERM solution has been associated with the development of antibovine antibodies in approximately 90% of patients and with overt immunological complications in 1 -3% of patients. See DeLustro et al, Plastic and Reconstructive Surgery 79:581 (1987) .

Injectable atelocollagen solution also was shown to be absorbed from the injection site, without replacement by host material, within a period of weeks to months. Clinical protocols calling for repeated injections of atelocollagen are, in practice, primarily limited by the development of immunogenic reactions to the bovine collagen. In order to mitigate these limitations, bovine atelocollagen was further processed by cross-linking with 0.25% glutaraldehyde, followed by filtration and mechanical shearing through fine mesh. The methodologies involved in the preparation and clinical utilization of this material are disclosed in U.S. Patent Nos. 4,582,640 and 4,642,1 17. The modified atelocollagen was marketed as ZYPLAST^® brand cross-linked bovine atelocollagen. The propertied advantages of cross-linking were to provide increased resistance to host degradation, however this was offset by an increase in solution viscosity. In addition, cross-linking of the bovine atelocollagen was found to decrease the number of host cells which infiltrated the injected collagen site. The_ increased viscosity, and in particular irregular increased viscosity resulting in "lumpiness," not only rendered the material more difficult to utilize, but also made it unsuitable for use in certain circumstances. See e.g., U.S. Patent No. 5,366,498. In addition, several investigators have reported that there is no or marginally- increased resistance to host degradation of ZYPLAST cross-linked bovine atelocollagen in comparison to that of the non-cross-linked ZYDERM atelocollagen solution and that the overall longevity of the injected material is, at best, only 4-6 months. See e.g., Ozgentas et al, Ann. Plastic Surgery 33:171 (1994); and Matti and Nicolle, Aesthetic Plastic Surgery 14:227 (1990).

Moreover, bovine atelocollagen cross-linked with glutaraldehyde may retain this agent as a high molecular weight polymer which is continuously hydrolyzed, thus facilitating the release of monomeric glutaraldehyde. The monomeric form of glutaraldehyde is detectable in body tissues for up to 6 weeks after the initial injection of the cross-linked atelocollagen. The cytotoxic effect of glutaraldehyde on in vitro fibroblast cultures is indicative of this substance's not being an ideal cross-linking agent for a dermal equivalent which is eventually infiltrated host cells and in which the bovine atelocollagen matrix is rapidly degraded, thus resulting in the release of monomeric glutaraldehyde into the bodily tissues and fluids. Similarly,chondroitin-6-sulfate (GAG), which weakly binds to collagen at neutral pH, has also been utilized to chemically modify bovine protein for tissue graft implantation. See Hansborough and Boyce, JAMA 136:2125 ( 1989). However, like glutaraldehyde, GAG may be released into the tissue causing unforeseen long-term effects on human subjects. GAG has been reported to increase scar tissue formation in wounds, which is to be avoided in grafts. Additionally, a reduction of collagen blood clotting capacity may also be deleterious in the application in bleeding wounds, as fibrin clot contributes to an adhesion of the graft to the surrounding tissue.

The limitations which are imposed by the immunogenicity of both modified and non-modified bovine atelocollagen have resulted in the isolation of human collagen from placenta (see e.g., U.S. Patent No. 5,002,071 ); from surgical specimens (see e.g., U.S. Patent Nos. 4,969,912 and 5,332,802); and cadaver (see e.g., U.S. Patent No. 4,882,166). Moreover, processing of human-derived collagen by cross-linking and similar chemical modifications is also required, as human collagen is subject to analogous degradative processes as is bovine collagen. Human collagen for injection, derived from a sample of the patient's own tissue, is currently available and is marketed as AUTOLOGEN^®. It should be noted, however, that there is no quantitative evidence which demonstrates that human collagen injection results in lower levels of implant degradation than that which is found with bovine collagen preparations. Furthermore, the utilization of autologous collagen preparation and injection is limited to those individuals who have previously undergone surgery, due to the fact that the initial culture from which the collagen is produced is derived from the tissue removed during the surgical procedure. Therefore, it is evident that, although human collagen circumvents the potential for immunogenicity exhibited by bovine collagen, it fails to provide long-term therapeutic benefits and is limited to those patients who have undergone prior surgical procedures.

An additional injectable material currently in use as an alternative to atelocollagen augmentation of the subjacent dermis consists of a mixture of gelatin powder, e-aminocaproic acid, and the patient's plasma marketed as FIBREL^®. See Multicenter Clinical Trial, J. Am. Acad. Dermatoloqy 16: 1 155 (1987). The action of the FIBREL product appears to be dependent upon the initial induction of a sclerogenic inflammatory response to the augmentation of the soft tissue via the subcutaneous injection of the material. See e.g., Gold, J. Dermatologic Surg. Oncology, 20:586 (1994). Clinical utilization of the FIBREL product has been reported to often result in an overall lack of implant uniformity, (i.e., "lumpiness") and longevity, as well as complaints of patient discomfort associated with its injection. See e.g., Millikan et al., J. Dermatoloqic. Surg. Oncology, 17:223 (1991 ). Therefore, in conclusion, none of the currently utilized protein-based injectable materials appears to be totally satisfactory for the augmentation and/or repair of the subjacent dermis and soft tissue.

The various complications associated with the utilization of the aforementioned materials have prompted experimentation with the implantation (grafting) of viable, living tissue to facilitate augmentation and/or repair of the subjacent dermis and soft tissue. For example, surgical correction of various defects has been accomplished by initial removal and subsequent re-implantation of the excised adipose tissue either by injection (see e.g., Davies et al., Arch, of Otolaryngoloqy-Head and Neck Surgery 121 :95 ( 1995); McKinney & Pandya, Aesthetic Plastic Surgery 18:383 ( 1994); and Lewis, Aesthetic Plastic Surgery, 17: 109 (1993)) or by the larger scale surgical-implantation (see e.g., Ersck, Plastic & Reconstructive Surgery 87:219 ( 1991 ) ) . To perform both of the aforementioned techniques a volume of adipose tissue equal or greater than is required for the subsequent augmentation or repair procedure must be removed from the patient. Thus, for large scale repair procedures (e.g., breast reconstruction) the amount of adipose tissue which can be surgically-excised from the patient may be limiting. In addition, other frequently encountered difficulties with the aforementioned methodologies include non-uniformity of the injectate, unpredictable longevity of the aesthetic effects, and a 4-6 week period of post-injection inflammation and swelling. In contrast, in a preferred embodiment, the present invention utilizes the surgical engraftment of autologous adipocytes which have been cultured on a solid support typically derived from, but not limited to, collagen or isolated extracellular matrix. The culture may be established from a simple skin biopsy specimen and the amount of adipose tissue which can be subsequently cultured in vitro is not limited by the amount of adipose tissue initially excised from the patient.

Living skin equivalents have been examined as a methodology for the repair and/or replacement of human skin. Split thickness autographs' epidermal autographs (cultured autogenic keratinocytes), and epidermal allographs (cultured allogenic keratinocytes) have been used with a varying degree of success. However, unfortunately, these forms of treatment have all exhibited numerous disadvantages. For example, split thickness autographs generally show limited tissue expansion, require repeated surgical operations, and give rise to unfavorable aesthetic results. E pidermal autographs require long periods of time to be cultured, have a low success ('"take") rate of approximately 30-48%, frequently form spontaneous blisters, exhibit contraction to 60-70% of their original size, are vulnerable during the first 15 days of engraftment, and are of no use in situations where there is both epidermal and dermal tissue involvement. Similarly, epidermal allografts (cultured allogenic keratinocytes) exhibit many of the limitations which are inherent in the use of epidermal autographs. Additional methodologies have been examined which involve the utilization of irradiated cadaver dermis. However, this too has met with limited success due to, for example, graft rejection and unfavorable aesthetic results. Living skin equivalents comprising a dermal layer of rodent fibroblast cells cast in soluble collagen and an epidermal layer of cultured rodent keratinocytes have been successfully grafted as allografts onto Sprague Dawley rats by Bell et al., J. Investigative Dermatology 81 :2 (1983). Histological examination of the engrafted tissue revealed that the epidermal layer had fully differentiated to form desmonosomes, tonofilaments, keratohyalin, and a basement lamella. However, subsequent attempts to reproduce the 1 iving skin equivalent using human fibroblasts and keratinocytes has met with only limited success. In general, the keratinocytes failed to fully differentiate to form a basement lamella and the dermo-epidermal junction was a straight line.

The present invention includes the following methodologies for the repair and/or augmentation of various skin defects: (1) the injection of autologously cultured dermal or fascial fibroblasts into various layers of the skin or injection directly into a "pocket" created in the region to be repaired or augmented, or (2) the surgical engraftment of "strands" derived from autologous dermal and fascial fibroblasts which are cultured in such a manner as to form a three-dimensional "tissue-like" structure similar to that which is found in vivo.

Moreover, the present invention also differs on a two-dimensional level in that "true" autologous culture and preparation of the cells is performed by utilization of the patient's own cells and serum for in vi tro culture.

III. VOCAL CORD TISSUE AUGMENTATION AND/OR REPAIR Phonation is accomplished in humans by the passage of air past a pair of vocal cords located within the larynx. Striated muscle fibers within the larynx, comprising the constrictor muscles, function so as to vary the degree of tension in the vocal cords, thus regulating both their overall rigidity and proximity to one another to produce speech. However, when one (or both) of the vocal cords becomes totally or partially immobile, there is a diminution in the voice quality due to inability to regulate and maintain the requisite tension and proximity of the damaged cord in relation to that of the operable cord. Vocal cord paralysis may be caused by cancer, surgical or mechanical trauma, or similar afflictions which render the vocal cord incapable of being properly tensioned by the constrictor muscles.

One therapeutic approach which has been examined to allow phonation involves the implantation or injection of biocompatible materials. It has long been recognized that a paralyzed or damaged vocal cord may be repositioned or supported so as to remain in a fixed location relative to the operable cord such that the unilateral vibration of the operable cord produces an acceptable voice pattern. Hence, various surgical have been developed which involve the formation of the thyroid cartilage and subsequently providing a means for the support and/or repositioning of the paralyzed vocal cord.

For example, injection of TEFLON into the paralyzed vocal cord to increase its inherent "bulk" has been described. See e.g., von Leden et al., Phonosurgery 3:175 (1989). However, this procedure is now considered unacceptable due to the inability of the injected TEFLON to close large glottic gaps, as well as its tendency to induce inflammatory reactions resulting in the formation of fibrous infiltration into the injected cord. See e.g., Maves et al., Phonosurgery: Indications and Pitfalls 98:577 (1989). Moreover, removal of the injected TEFLON may be quite difficult should it subsequently be desired or become necessary.

Another methodology for supporting the paralyzed vocal cord which has been employed involves the utilization of a custom-fitted block of siliconized rubber (SILASTIC^®). In order to ensure the proper fit of the implant, the surgeon hand carves SILASTIC block during the procedure in order to maximize the ability of the patient to phonate. The patient is kept under local anesthesia so that he or she can produce sounds to test the positioning of the implant. Generally, the implanted blocks are formed into the shape of a wedge which is totally implanted within the thyroid cartilage or a flanged plug which can be moved back-and-forth within the opening in the thyroid cartilage to fine-tune the voice of the patient.

Although SILASTIC implants have proved to be superior over TEFLON injections, there are several areas of dissatisfaction with the procedure including difficulty in the carving and insertion of the block, the large amount of time required for the procedure, and a lack of an efficient methodology for locking the block in place within the thyroid cartilage. In addition, vocal-cord edema, due to the prolonged nature of the procedure and repeated voice testing during the operation, may also prove problematic in obtaining optimal voice quality.

Other methodologies which have been utilized in the treatment of vocal cord paralysis and damage include GELFOAM^® hydroxyapatite, and porous ceramic implants, as well as injections of silicone and collagen. See, e.g., Kaufman, Laryngoplastic Phonosurqery (1988). However, these materials have also proved to be less than ideal due to difficulties in the sizing and shaping of the solid implants as well as the potential for subsequent immunogenic reactions. Therefore, there still remains a need for the development of a methodology which allows the efficacious treatment of vocal cord paralysis and/or damage.

SUMMARY OF THE INVENTION

The present invention discloses a methodology for the longterm augmentation and/or repair of dermal, suboutaneous, or vocal cord tissue by the injection or direct surgical placement/implantation of: (1) autologous cultured fibroblasts derived from connective tissue, dermis, or fascia; (2) lamina propria tissue; (3) fibroblasts derived from the lamina propria or (4) adipocytes. The fibroblast cultures utilized for the augmentation and/or repair of skin defects are derived from either connective tissue, dermal, and/or fascial fibroblasts. Typical defects of the skin which can be corrected with the injection or direct surgical placement of autologous fibroblasts or adipocytes include rhytids, stretch marks, depressed scars, cutaneous depressions of traumatic or non-traumatic origin, hypoplasia of the lip, and/or scarring from acne vulgaris. Typical defects of the vocal cord which can be corrected by the injection or direct surgical placement of lamina propria or autologous cultured fibroblasts from lamina propria include scarred, paralyzed, surgically or traumatically injured, or congenitally underdeveloped vocal cord(s).

The use of autologous cultured fibroblasts derived from the dermis, fascia, connective tissue, or lamina propria mitigates the possibility of an immunogenic reaction due to a lack of tissue histocompatibility. This provides vastly superior post-surgical results. In a preferred embodiment of the present invention, fibroblasts of connective tissue, dermal, or fascial origin as well as adipocytes are derived from full biopsies of the skin. Similarly, lamina propria tissue or fibroblasts obtained from the lamina propria are obtained from vocal cord biopsies. It should be noted that the aforementioned from the individual who will subsequently undergo the surgical procedure, thus mitigating the potential for an immunogenic reaction. These tissues are then expanded in vitro utilizing standard tissue culture methodologies. Additionally, the present invention further provides a methodology of rendering the cultured cells substantially free of potentially immunogenic serum-derived proteins by late-stage passage of the cultured fibroblasts, lamina propria tissue, or adipocytes in serum-free medium or in the patient's own serum. In addition, immunogenic proteins may be markedly reduced or eliminated by repeated washing in phosphate-buffered saline (PBS) or similar physiologically-compatible buffers.

DESCRIPTION OF THE INVENTION I. HISTOLOGY OF THE SKIN The skin is composed of two distinct layers: the epidern a specialized epithelium derived from the ectoderm, and beneath this, the dermis, a vascular dense connective tissue, a derivative of mesoderm. These two layers are firmly adherent to one another and form a region which varies in overall thickness from approximately 0.5 to 4 mm in different areas of the body. Beneath the dermis is a layer of loose connective tissue which varies from areolar to adipose in character. This is the superficial fascia of gross anatomy, and is sometimes referred as the hypodermis, but is not considered to be part of the skin. The dermis is connected to the hypodermis by connective tissue fibers which pass from one layer to the other.

A. EPIDERMIS The epidermis, a stratified squamous epithelium, is composed of cells of two separate and distinct origins. The majority of the epithelium, of ectodermal origin, undergoes a process of keratinization resulting in the formation of the dead superficial layers of skin. The second component comprises the melanocytes which are involved in the synthesis of pigmentation via melanin. The latter cells do not undergo the process of keratinization. The superficial keratanized cells are continuously lost from the surface and must be replaced by cells that arise from the mitotic activity of cells of the basal layers of the epidermis. Cells which result from this proliferation are displaced to higher levels, and as they move upward they elaborate keratin, which eventually replaces the majority of the cytoplasm. As the process of keratinization continues the cell dies and is finally shed. Therefore, it should be appreciated that the structural organization of the epidermis into layers reflects various stages in the dynamic process of cellular proliferation and differentiation. B. DERMIS

It is frequently difficult to quantitatively differentiate the limits of the dermis as it merges into the underlying subcutaneous layer (hypodermis). The average thickness of the dermis varies from 0.5 to 3 mm and is further subdivided into two strata - the papillary layer superficially and the reticular layer beneath. The papillary layer is composed of thin collagenous, reticular, and elastic fibers arranged in an extensive network. Just beneath the epidermis, reticular fibers of the dermis form a close network into which the basal processes of the cells of the stratum germinativum are anchored. This region is referred to as the basal lamina.

The reticular layer is the main fibrous bed of the dermis. Generally, the papillary layer contains more cells and smaller and finer connective tissue fibers than the reticular layer. It consists of coarse, dense, and interlacing collagenous fibers, in which are intermingled a small number of reticular fibers and a large number of elastic fibers. The predominant arrangement of these fibers is parallel to the surface of the skin. The predominant cellular constituent of the dermis are fibroblasts and macrophages. In addition, adipose cells may be present either singly or, more frequently, in clusters. Owing to the direction of the fibers, lines of skin tension, Langer's lines, are formed. The overall direction of these lines is of surgical importance since incisions made parallel with the lines tend to gape less and heal with less scar tissue formation than incisions made at right-angles or obliquely across the lines. Pigmented, branched connective tissue cells, chromatophores, may also be present. These cells do not elaborate pigment but, instead, apparently obtain it from melanocytes.

Smooth muscle fibers may also be found in the dermis. These fibers are arranged in small bundles in connection with hair follicles (arrectores pilorum muscles) and are scattered throughout the dermis in considerable numbers in the skin of the nipple, penis, scrotum, and parts of the perineum. Contraction of the muscle fibers gives the skin of these regions a wrinkled appearance. In the face and neck, fibers of some skeletal muscles terminate in delicate elastic fiber networks of the dermis.

C. ADIPOSE TISSUE/ADIPOCYTES Fat cells, or adipocytes, are scattered in areolar connective tissue.

When adipocytes form large aggregates, and are the principle cell type, the tissue is designated adipose tissue. Adipocytes are fully differentiated cells and are thus incapable of undergoing mitotic division. New adipocytes therefore, which may develop at any time within the connective tissue, arise as a result of differentiation of more primitive cells. Although adipocytes, prior to the storage of lipid, resemble fibroblasts, it is likely that they arise directly from undifferentiated mesenchymal tissue.

Each adipocyte is surrounded by a web of fine reticular fibers; in the spaces between are found fibroblasts, lymphoid cells, eosinophils, and some mast cells. The closely spaced adipocytes form lobules, separated by fibrous septa. In addition, there is a rich network of capillaries in and between the lobules. The richness of the blood supply is indicative of the high rate of metabolic activity of adipose tissue.

It should be appreciated that adipose tissue is not static There is a dynamic balance between lipid deposit and withdrawal. The lipid contained within adipocytes may be derived from three sources. Adipocytes, under the influence of the hormone insulin, can synthesize fat from carbohydrate. They can also produce fat from various fatty acids which are derived from the initial breakdown of dietary fat. Fatty acids may also be synthesized from glucose in the liver and transported to adipocytes as serum lipoproteins. Fats derived from different sources also differ chemically. Dietary fats may be saturated or unsaturated, depending upon the individual diet. Fat which is synthesized from carbohydrate is generally saturated. Withdrawals of fat result from enzymatic hydrolysis of stored fat to release fatty acids into the blood stream. However, if there is a continuous supply of exogenous glucose, then fat hydrolysis is negligible. The normal homeostatic balance is affected by hormones, principally insulin, and by the autonomic nervous system, which is responsible for the mobilization of fat from adipose tissue.

Adipose tissue may develop almost anywhere areolar tissue is prevalent, but in humans the most common sites of adipose tissue _ accumulation are the subcutaneous tissues (where it is referred to as the panniculus adiposus), in the mesenteries and omenta, in the bone marrow, and surrounding the kidneys. In addition to its primary function of storage and metabolism of neutral fat, in the subautaneous tissue, adipose tissue also acts as a shock absorber and insulator to prevent excessive heat loss or gain through the skin.

II. HISTOLOGY OF THE LARYNX AND VOCAL CORDS

The larynx is that part of the respiratory system which connects the pharynx and trachea. In addition to its function as part of the respiratory system, it plays an important role in phonation (speech). The wall of the larynx is composed of a "skeleton" of hyaline and elastic cartilages, collagenous connective tissue, striated muscle, and mucous glands. The major cartilages of the larynx (the thyroid, cricoid, and arytenoids) are hyaline, whereas the smaller cartilages (the corniculates, cuneiforms, and the tips of the arytenoids) are elastic, as is the cartilage of the epiglottis. The aforementioned cartilages, together with the hyoid bone, are connected by three large, flat membranes: the thyrohyoid, the quadrates, and the cricovocal. These are composed of dense fibroconnective tissue in which many elastic fibers are present, particularly in the cricovocal membrane. The true and false vocal cords (vocal-and vestibular ligaments) are, respectively, the free upper boarders of the cricovocal (cricothyroid) and the free lower boarders of the quadrate (aryspiglottic) membranes. Extending laterally on each side between the true and false cords are the sinus and saccule of the larynx, a small slit-like diverticulum. Behind the cricoid and arytenoid cartilages, the posterior wall of the pharynx is formed by the striated muscle of the pharyngeal constrictor muscles.

The epithelium of the mucous membrane of the larynx varies with location. For example, over the vocal folds, the lamina propria of the stratified squamous epithelium is extremely dense and firmly bound to the underlying connective tissue of the vocal ligament. While there is no true submucosa in the larynx, the lamina propria of the mucous membrane is thick and contains large numbers of elastic fibers. III. METHODOLOGIES

A. IN VITRO CELL CULTURE OF FIBROBLASTS OR LAMINA

PROPRIA

While the present invention may be practiced by utilizing any type of non-differentiated mesenchymal cell found in the skin which can be expanded in in vi tro culture, fibroblasts derived from dermal, connective tissue, fascial, lamina proprial tissue adipocytes, and/or extracellular tissues (matrix) derived from the cells are utilized in a preferred embodiment due to their relative of isolation and in vi tro expansion in tissue culture. In general, tissue culture techniques which are suitable for the propagation of non-differentiated mesenchymal cells may be used to expand the aforementioned cells/tissue and practice present invention as further discussed below. See e.g., Culture of Animal Cells: A Manual of Basic Techniques, Freshney, R. I., ed., (Alan R. Liss & Co., New York 1987); Animal Cell Culture: A Practical Approach, Freshney, R.I. ed., (IRL Press, Oxford, England (1986), whose references are incorporated herein by reference. The utilization of autologous engraftment is a preferred therapeutic methodology due to the potential for graft rejection associated with the use of allograft-based engraftment. Autologous grafts (i.e., those derived directly from the patient ensure histocompatibility by initially obtaining a tissue sample via biopsy directly from the patient who will be undergoing the corrective surgical procedure and then subsequently culturing fibroblasts derived from the dermal, connective tissue, fascial, or lamina proprial regions contained therein.

While the following sections will primarily discuss the autologous culture of fibroblasts of connective tissue, dermal, or fascial origins, in vitro culture of lamina propria tissue may also be established utilizing analogous methodologies. An autologous fibroblast culture is preferably initiated by the following methodology. A full-thickness biopsy of the skin (-3x6 mm) is initially obtained through, for example, a punch biopsy procedure. The specimen is repeatedly washed with antibiotic and anti-fungal agents prior to culture. Through a process of sterile microscopic dissection, the keratinized tissue-containing epidermis and subcutaneous adipocyte-containing tissue is removed, thus ensuring that the resultant culture is substantially free of non-fibroblast cells (e.g., adipocytes and keratinocytes). The isolated adipocytes-containing tissue may then be utilized to establish adipocyte cultures. Alternately, whole tissue may be cultured and fibroblast-specific growth medium may be utilized to "select" for these cells.

Two methodologies are generally utilized for the autologous culture of fibroblasts in the practice of the present invention - mechanical and enzymatic. In the mechanical methodology, the fascia, dermis, or connective tissue is intially dissected out and finely divided with scalpal or scissors. The finely minced pieces of the tissue are initially placed in 1-2 ml of medium in either a 5 mm petri dish (Costar), a 24 multi-well culture plate (Corning), or other appropriate tissue culture vessel.

Incubation is preferably performed at 37 deg. C in a 5% C02 atmosphere and the cells are incubated until a confluent monolayer of fibroblasts has been obtained. This may require up to 3 weeks of incubation. Following the establishment of confluence, the monolayer is trypsinized to release the adherent fibroblasts from the walls of the culture vessel. The suspended cells are collected by centrifugation, washed in phosphate-buffered saline, and resuspended in culture medium and placed into larger culture vessels containing the appropriate complete growth medium.

In a preferred embodiment of the enzymatic culture methodology, pieces of the finely minced tissue are digested with a protease for varying periods of time. The enzymatic concentration and incubation time are variable depending upon t individual tissue source, and the initial isolation of the fibroblasts from the tissue as well as the degree of subsequent outgrowth of the cultured cells are highly dependent upon these two factors. Effective proteases include, but are not limited to, trypsin, chymotrypsin, papain, chymopapain, and similar proteolytic enzymes. Preferably, the tissue is incubated with 200-1000 U/ml of collagenase type II for a time period ranging from 30 minutes to 24 hours, as collagenase type II was found to be highly efficacious in providing a high yield of viable fibroblasts. Following enzymatic digestion, the cells are collected by centrifugation and resuspended into fresh medium in culture flasks.

Various media may be used for the initial establishment of an in vitro culture of human fibroblasts. Dulbecco's Modified Eagle Medium (DMEM, Gibco/BRL Laboratories) with concentrations of fetal bovine serum (FBS), cosmic calf serum (CCS) or the patient's own serum varying from 5-20% (v/v) ~ with higher concentrations resulting in faster culture growth ~ are readily utilized for fibroblast culture. It should be noted that substantial reductions in the concentration of serum (i.e., 0. 5% v/v) results in a loss of cell viability in culture. In addition, the complete culture medium typically contains Lglutamine, sodium bicarbonate, pyridoxine hydrochloride, lg/liter glucose, and gentamycin sulfate. The use of the patient's own serum mitigates the possibility of subsequent immunogenic reaction due to the presence of constituent antigenic proteins in the other serums.

Establishment of a fibroblast cell line from an initial human biopsy specimen generally requires 2 to 3. 5 weeks in total. Once the initial culture has reached confluence, the cells may be passaged into new culture flasks following trypsinization by standard methodologies known within the relevant field. Preferably, for expansion, cultures are "split" 1 :3 or 1 :4 into T-150 culture flasks (Corning) yielding ~5xl 0⁷ cells/culture vessel. The capacity of the T-150 culture flask is typically reached following 5- 8 days of culture at which time the cultured cells are found to be confluent or near confluent.

Cells are preferably removed for freezing and long-term storage during the early passage stages of culture, rather thane the later stages due to the fact that human fibroblasts are capable of undergoing a finite numbers of passages. Culture medium containing 70% DMEM growth medium, 10% (v/v) serum, and 20% (v/v) tissue culture grade dimethyleulfoxide (DM SO, Gibco/BRL) may be effectively utilized for freezing of fibroblast cultures. Frozen cells can subsequently be used to inoculate secondary cultures to obtain additional fibroblasts for use inthe original patient, thus doing away with the requirement to obtain a second biopsy specimen.

To minimize the possibility of subsequent immunogenic reactions in the engraftment patient, the removal of the various antigenic constituent proteins contained within the serum may be facilitated by collection of the fibroblasts by centrifugation, washing the cells repeatedly in phosphate-buffered saline (PBS) and then either re-suspending or culturing the washed fibroblas for a period of 2-24 hours in serum-free medium containing requisite growth factors which are well known in the field. Culture media include, but are not limited to, Fibroblast Basal Medium (FBM). Alternately, the fibroblasts may be cultured utilizing the patient's own serum in the appropriate growth medium. After the culture has reached a state of confluence or sub- confluence, the fibroblasts may either be processed for injection or further cultured to facilitate the formation of a three-dimensional "tissue" for subsequent surgical engraftment. Fibroblasts utilized for injection consist of cells suspended in a collagen gel matrix or extracellular matrix. The collagen gel matrix is preferably comprised of a mixture of 2 ml of a collagen solution containing 0.5 to 1.5 mg/ml collagen in 0. 05% acetic acid, 1 ml of DMEM medium, 270 μl of 7.5% sodium bicarbonate, 48 microliters of 100 micrograms/ml solution of gentamycin sulfate, and up to 5x10⁶ fibroblast cell/ml of collagen gel. Following the suspension of the fibroblasts in the collagen gel matrix, the suspension is allowed to solidify for approximately 15 minutes at room temperature or 37 deg C in a 5% C02 atmosphere. The collagen may be derived from human or bovine sources, or from the patient and may be enzymatically- or chemically-modified (e.g., atelocollagen). Three-dimensional "tissue" is formed by initially suspending the fibroblasts in the collagen gel matrix as described above. Preferably, in the culture of three-dimensional tissue, full-length collagen is utilized, rather than truncated or

24

modified collagen derivatives. The resulting suspension is then placed into a proprietary "transwell" culture system which is

typically comprised of culture well in which the lower growth

medium is separated from the upper region of the culture well by

a microporous membrane. The microporous membrane typically

possesses a pore size ranging from 0.4 to 8 μm in diameter and is

constructed from materials including, but not limited to, polyester, nylon, nitrocellulose, cellulose acetate, polyacrylamide, cross-linked dextrose, agarose, or other similar

materials. The culture well component of the transwell culture

system may be fabricated in any desired shape or size (e.g.,

square, round, ellipsoidal, etc.) to facilitate subsequent surgical tissue engraftment and typically holds a volume of culture medium ranging from 200 μl to 5 ml. In general, a

concentration ranging from 0.5 x 10⁶ to 10 x 10⁶ cells/ml, and

preferably 5 x 10⁶ cells/ml, are inoculated into the

collagen/fibroblast -containing suspension as described above.

Utilizing a preferred concentration of cells (i.e., 5 x 10⁶

cells/ml) , a total of approximately 4-5 weeks is required for the

formation of a three-dimensional tissue matrix. However, this

time may vary with increasing or decreasing concentrations of

inoculated cells. Accordingly, the higher the concentration of cells utilized the less time due to a higher overall rate of cell proliferation and replacement of the exogenous collagen with

endogenous collagen and other constituent materials which form

the extracellular matrix synthesized by the cultured fibroblasts.

Constituent materials which form the extracellular matrix

include, but are not limited to, collagen, elastin, (fibrinλ fibrinogen, proteases, fibronectin, laminin, fibrellins, and other similar proteins. It should be noted that the potential

for immunogenic reaction in the engrafted patient is markedly

reduced due to the fact that the exogenous collagen used in establishing the initial collagen/fibroblast -containing suspension is gradually replaced during subsequent cu t re by

endogenous collagen and extracellular matrix materials

synthesized by the fibroblasts.

B. IN VITRO CULTURE OF ADIPOCYTES

Adipocytes require a "feeder-layer" or other type of solid

support on which to grow. One potential solid support may be

provided by utilization of the previously discussed collagen gel

matrix. Alternately, the solid support may be provided by

cultured extracellular matrix. In general, the in vi tro culture

of adipocytes is performed by the mechanical or enzymatic 26

disaggregation of the adipocytes from adipose tissue derived from

a biopsy specimen. The adipocytes are "seeded" onto the surface

of the aforementioned solid support and allowed to grow until

near-confluence is reached. The adipocytes are removed by gentle

scraping of the solid surface. The isolated adipocytes are then

cultured in the same manner as utilized for fibroblasts as

previously discussed in Section III A.

C. ISOLATION OF THE EXTRACELLULAR MATRIX

The extracellular matrix (ECM) may be isolated in either a cellular or acellular form. Constituent materials which form the

ECM include, but are not limited to, collagen, elastin, fibrin,

fibrinogen, proteases, fibronectin, laminin, fibrellins, and

other similar proteins. ECM is typically isolated by the initial

culture of cells derived from skirfabr vocal cord biopsy specimens

as previously described. After the cultured cells have reached a

minimum of 25-50% sub-confluence, the ECM may be obtained by

mechanical, enzymatic, chemical, or denaturant treatment.

Mechanical collection is performed by scraping the ECM off of the

plastic culture vessel and re-suspending in phosphate -buffered

saline (PBS) . If desired, the constituent cells are lysed or

ruptured by incubation in hypotonic saline containing 5 mM EDTA. Preferably, however, scraping followed by PBS re-suspension is generally utilized. Enzymatic treatment involves brief incubation with a proteolytic enzyme such as trypsin. Additionally, the use of detergents such as sodium dodesyl sulfate (SDS) or treatment with denaturants such as urea or dithiotheritol (DTT) followed by dialysis against PBS, will also facilitate the release of the ECM from surrounding associated tissue .

The isolated ECM may then be utilized as a "filler" material in the various augmentation or repair procedures disclosed in the present application. In addition, the ECM may possess certain cell growth- or metabolism-promoting characteristics.

D. IN VITRO CULTURE OF FETAL OR JUVENILE CELLS OR TISSUES In another preferred embodiment, rather than utilizing the patient's own tissue, all of the aforementioned cells, cell

typically possesses differs in both quantity and characteristics

from that of the ECM derived from senescent or late-passage

cells. The cellular or acellular ECM derived from fetal or

juvenile sources may be used as a "filler" material in the various augmentation or repair procedures disclosed in the present application. In addition, the fetal or juvenile ECM nay

possess certain cell growth- or metabolism-promoting

characteristics.

E. INJECTION OF AUTOLOGOUS CULTURED ^"THERMAL/FASCIAL FIBROBLASTS

To augment or repair derma-l dale -ts, autologously cultured

fibroblasts are injected initially into the lower dermis, next in

the upper and middle dermis, and finally in the subcutaneous

regions of the skin as to form raised areas or "wheals." The

fibroblast suspension is injected via a syringe with a needle

ranging frog 30 to 18 gauge, with the gauge of the needle being

dependent upon such factors as the overall viscosity of the

fibroblast suspension and the type of anesthetic utilized.

Preferably, needles ranging from 22 to 18 gauge and 30 to 27

gauge are used with general and local anesthesia, respectively. To inject the fibroblast suspension into the lower dermis, the needle ^'is placed at approximately a 45° angle to the skin

with the bevel of the needle directed downward. To place the

fibroblast suspension into the middle dermis the needle is placed

at approximately a 20-30° angle. To place the suspension into

the upper dermis, the needle is placed almost horizontally (i.e.,

-10-15° angle) . Subcutaneous injection is accomplished by initial placement of the needle into the subcutaneous tissue and

injection of the fibroblast suspension during subsequent needle withdrawal. In addition, it should be noted that the needle is preferably inserted into the skin from various directions such

that the needle tract will be somewhat different with -each

subsequent injection. This technique facilitates a greater

amount of total skin area receiving the injected fibroblast

suspension.

Following the aforementioned injections, the skin should be

expanded and possess a relatively taut feel. Care should be

taken so as not to produce an overly hard feel to the injected

region. Preferably, depressions or rhytids appear elevated

following injection and should be "overcorrected" by a slight

degree of over-injection of the fibroblast suspension, as typically some degree of settling or shrinkage will occur post-operatively.

In some scenarios, the injections may pass into deeper

tissue layers. For example, in the case of lip augmentation or

repair, a preferred manner of injection is accomplished by

initially injecting the fibroblast suspension into the dermal and

subcutaneous layers as previously described, into the skin above the lips at the vermillion border. In addition, the vertical

philtrum may also be injected. The suspension is subsequently injected into the deeper tissues of the lip, including the

muscle, in the manner described for subcutaneous injection.

F. SURGICAL PLACEMENT OF AUTOLOGOUSLY CULTURED DERMAL/FASCIAL FIBROBLAST STRANDS

In a preferred methodology utilized to augment or repair the

skin and/or lips by the surgical placement of autologously

cultured dermal and/or fascial fibroblast strands, a needle (the

"passer needle") is selected which is larger in diameter and

greater in length than the area to be repaired or augmented .

The passer needle is then placed into the skin and threaded down

the length of the area. Guide sutures are placed at both ends

through the dermal or fascial fibroblast strand. One end of the guide suture is fixed to a Keith needle which is subsequently placed through the passer needle. The guide suture is brought

out through the skin on the side furthest (distal point) from the

initial entry point of the passer needle. The dermal or fascial

fibroblast graft is then pulled into the passer needle and its

position may be adjusted by pulling on the distal point guide suture or, alternately, the guide suture closest to the passer needle entry point. While the dermal or fascial strand is held

in place by the distal point suture, the passer needle is pulled

backward and removed, thus resulting in the final placement of

the^' graft following the final cutting of the remaining suture. Generally, the fascial or dermal graft is placed into the subcutaneous layer of the skin. However, in some situations, it

may be placed either more deeply or superficially.

If the area to be repaired or augmented is either smaller or

larger than would be practical to fill with the aforementioned

needle method, a subcutaneous "pocket" may be created with a

myringotomy knife, scissors, or other similar instrument. A

piece of dermis or fascia is then threaded into this area by use

of guide sutures and passer needle, as described above. 32

G. INJECTION OF CELLS OR OTHER SUBSTANCES INTO THE VOCAL CORDS OR LARYNX

Generally, it is not possible to inject cellular matter or

other substances directly into the vocal cord epithelium due to

its extreme thinness. Accordingly, injections are usually made

into the lamina propria layer or the muscle itself.

Generally, lamina propria tissue (finely minced if required

for injection), fibroblasts derived from lamina propria tissue,

or gelatinous substances are utilized for injection. The

preferable methodology consists of injection directly into the space containing the lamina propria, specifically into Reinke ' s

space. Injection is accomplished by use of laryngeal injection

needles of the smallest possible gauge which will accommodate the

injectate without the use of extraneous pressure during the

actual injection process. This is a subjective process as to the

overall "feel" and the use of too much pressure may irreparably

damage the injected cells. The material is injected via a

syringe with a needle ranging from 30 to 18 gauge, with the gauge

of the needle being dependent upon such factors as the overall

viscosity of the injectate and the type of anesthetic utilized.

Preferably, needles ranging from 22 to 18 gauge and 30 to 27

gauge are used with general and local anesthesia, respectively. O 99/43270

33

If required, several injections may be performed along the length of the vocal cord.

To medialize a vocal cord with autologously cultured fascial

or dermal fibroblasts, the materials are preferably injected

directly into the tissue lateral or at the lateral edge of the

vocal cord. The fibroblasts may be injected into scar, Reinke 's

space, or muscle, depending upon the specific vocal cord pathology. Preferably, it would be injected into the muscle Tne procedure may be performed under general, local,

topical, monitored, or with no anesthesia, depending upon patient

compliance and tolerance, the amount of injected material, and

εr.e type of injection performed.

If a greater degree of augmentation is required, a "pocket" may be created by needle dissection. Alternately, laryngeal

icrodisection, using knives and dissectors, may be performed

The desired material is then placed into the pocket with

laryngeal forceps, or directly injected, depending upon the size

of the pocket, the size of the graft material, the anesthesia,

and the open access. If the pocket is left open after the

procedure, it is preferably closed with sutures, adhesive, or a

laser, depending upon the size and availability of these

materials and the individual preferences of the surgeon. While embodiments and applications of the present invention have been described in some detail by way of illustration and

example for purposes of clarity and understanding, it would be

apparent to those individuals whom are skilled within the

relevant art that many additional modifications would be possible

without departing from the inventive concepts contained herein.

Claims

WHAT IS CLAIMED IS:

1. A method for corrective surgery in a human subject of a defect being amenable to rectification by. the augmentation of tissue subjacent to the defect comprising the steps of: a) retrieving a plurality of viable cells from the subject; b) culturing the viable cells in vi tro; and c) placing an effective volume of the in vitro cultured cells into a tissue of the subject , the tissue being located in a position subjacent to the defect to be rectified.

2. The method of claim 1 wherein the in vi tro cultured cells are placed in the tissue of the subject by a method selected from injection, engraftment, engraftment by threading and direct placement.

3. The method of claim 1 wherein the in vi tro cultured cells are fibroblasts.

4. The method of claim 3 wherein said fibroblasts are derived from a tissue selected from the group consisting of dermis, fascia, and connective tissue.

5. The method of claim 1 wherein the in vitro cultured cell are adipocytes.

6. The method of claim 1 wherein the cultured cells are suspended in a collagen or modified collagen solution prior to injection.

7. The method of claim 1 wherein the cells are cultured in a collagen or modified collagen matrix.

8. The method of claim 7 wherein the cells and the matrix are placed in the tissue by injection.

9. The method of claim 7 wherein the cells and the extracellular matrix are placed in the tissue by engraftment.

10. The method of claim 1 wherein serum from the subject or serum-free medium is used for in vitro culture of the cells.

1 1. The method of claim 1 wherein the in vitro cultured cells are washed in phosphate buffered saline before being placed into the tissue of the subject.

12. The method of claim 1 wherein the defect is selected from the group consisting of a rhytid, stretch mark, depressed scar, cutaneous depression, hypoplasia of the lip, and scarring from acne vulgaris.

13. The method of claim 12 wherein the defect is sselected from the group consisting of a stretch mark, wrinkle, depressed scar, cutaneous depression, hypoplasia of the lip, prominent nasolabial fold, prominent melolabial fold, and scarring from acne vulgaris.

14. The method of claim 1 wherein the in vi tro cultured cells are placed by injection into: a) the lower dermis; b) the middle dermis; c) the upper dermis; or d) the subcutaneous region of the skin; or. e) any combination of the foregoing within the tissue and/or surrounding the tissue.

15. An in vi tro produced extracellular matrix composition, which is either substantially pure or combined with cells embedded in the matrix and is obtained from the process comprising the steps of: a) culturing cells in vi tro in a culture vessel for a time sufficient for the cells to produce extrace-fular matrix; b) separating the extracellular matrix from the culture vessel and in addition, if the composition is substantially pure, separating the extracellular matrix produced by the cultured cells from such cells; and c) collecting the extracellular matrix.

16. A method for corrective surgery in a human subject of a defect being amenable to rectification by the augmentation of tissue subjacent to the defect comprising the steps of: a) retrieving a plurality of viable cells from the subject; b) culturing the cells in vitro in a culture vessel for a time sufficient for the cells to produce extracellular matrix; c) separating the extracellular matrix produced by the cells from the culture vessel; d) collecting the extracellular matrix; and e) placing the collected extracellular matrix into a tissue subjacent to the defect.

17. The method of claim 16 wherein the extracellular matrix is exposed to a hypotonic solution prior to being placed into the tissue subjacent to the defect.

18. A method for the long-term augmentation of subcutaneous or dermal tissue in a human subject which comprises:

(a) providing a suspension of autologous, passaged dermal fibroblasts, substantially free of immunogenic proteins;

(b) identifying a defect that is susceptible to amelioration by augmentation of the subadjacent subcutaneous or dermal tissue; and (c) injecting an effective volume of the suspension into the subadjacent tissue so that the tissue is augmented.

19 . The method of claim 18, wherein the defect is a rhytid, stretch mark, a depressed scar, a cutaneous depression of non-traumatic origin or an underdevelopment of the lip.

20. The method of claim 18, which further comprises the steps of:

(a) biopsying the dermis of the subject;

(b) passaging the dermal fibroblasts from the dermal biopsy in a culture medium comprising between 5% and 20% non-human serum, so as to provide dermal fibroblasts substantially free of adipocytes, keratinocytes and extracellular matrix;

(c) incubating the passaged dermal fibroblasts in a serum-free medium; and

(d)exposing the incubated fibroblasts to a proteolytic enzyme so as to suspend the fibroblasts.

21. A device for repairing a dermal defect in a subject comprising

(a) a hypodermic syringe having a syringe chamber, a piston disposed therein, and an orifice communicating with the chamber;

(b) a suspension comprising:

( 1 ) dermal fibroblasts derived from the subject, said fibroblasts being substantially free of cells other than fibroblasts and substantially free of proteins that are immunogenic in the subject, and (2) a pharmaceutically acceptable carrier solution, said suspension being disposed in the chamber; and

(c) a hypodermic needle affixed to the orifice.

22. A method of making a device of claim 21, comprising the steps of:

(a) biopsying the dermis of the subject;

(b) passaging the dermal fibroblasts from the dermal biopsy in a culture medium comprising between 5% and 20%) of non-human serum, serum from the subject or serum-free medium, so as to provide dermal fibroblasts substantially free of cells other than fibroblasts;

(c) incubating the passaged dermal fibroblasts in a serum-free medium; (d) exposing the incubated fibroblasts to a proteolytic enzyme so as to suspend the fibroblasts; and

(e) adding the suspended fibroblasts to an acceptable carrier solution so that a suspension is formed and placing said suspension into the syringe chamber.

23. The method of claim 1 wherein the defect is present in the vocal cord of the subject.

24. The method of claim 23 wherein the autologous in vitro cultured cells are placed in a site of the vocal cord selected from the group comprising a scar, Reinke' s space, a muscle of the vocal cord, and the lamina propria.

25. The method of claim 16 wherein the defect is present in the vocal cord of the subject.