JP4690648B2 - Injectable polysaccharide-cell composition - Google Patents

Description

(Background of the Invention)
The present invention relates generally to the field of using polysaccharide hydrogel-cell compositions in the field of medical medicine. More particularly, it relates to a method for correcting vesicoureteral reflux, incontinence, and other deficiencies.

(Skull facial contour malformation)
Correcting craniofacial contour malformations, whether traumatic, congenital, or orthopedic, currently requires invasive surgical methods. Furthermore, malformations that require augmentation often require the use of inanimate material prostheses, which have the problems of infection and protrusion. The least invasive method of adding additional autogenous cartilage or bone to the skull face skeleton minimizes surgical trauma and requires the use of inanimate prostheses Absent. If transplantation by injection can be performed and isolated cells can be implanted in large quantities, the skull-facial bone-cartilage skeleton can be augmented with autogenous tissue without extensive surgery.

  Unfortunately, attempts to inject discrete cells by subcutaneous injection or transplant the isolated tissue within a body region such as the peritoneum have not been successful. Cells are cleared relatively quickly, perhaps by phagocytosis and cell death.

  As described in US Pat. No. 5,041,138 to Vacanti et al., Cells can be implanted on a polymer matrix and can also be implanted to form a cartilage structure. However, this requires surgical implantation of the matrix and shaping of the matrix prior to implantation in order to form the desired anatomy.

(Bladder-ureter reflux phenomenon)
The vesicoureteral reflux phenomenon is a state in which the ureteric bud develops abnormally because the ureteral bud enters the bladder during embryonic development and growth. Because the ureteral pathway through the musculature of the bladder is shortened, ureteral resistance is reduced, resulting in urine flowing back from the bladder reservoir to the ureter and kidney. In this state, bacteria that may occasionally be present in the bladder due to retrograde urethral transport can reach the kidney and cause recurrent pyelonephritis. Furthermore, constant back pressure into the urinary kidneys and renal pyramids results in mechanical damage to the renal parenchyma. As examined by Atala and Casale, Infections in Urology, 39-43 (March / April 1990), urinary bladder urethral reflux phenomenon can cause renal parenchyma Loss and, in some cases, can cause renal failure. In 1960, 70% of patients with renal failure were described as having a major etiology of vesicourethral reflux. With the advent of new diagnoses and treatments, patients with vesicourethral reflux are now less than 1% of the renal failure patient population.

  In the past, vesicourethral reflux was usually diagnosed by urinary cystography after the child had repeatedly developed pyelonephritis. As reported by Atala and Casale, prenatal and postnatal sonography has become more frequently used, making hydronephrosis more detectable and, moreover, radiation Medical work-up and early detection are promoted. The vesicourethral reflux phenomenon is graded by severity. Grade 1 regurgitation is when urine is found to regurgitate only from the bladder to the ureter; in grade 2 regurgitation, the urine regurgitates until the ureter and renal cup dilation. Grades 4 and 5 reflux are heavier, indicating ureter twisting and further blunting and dilation of the renal cup, respectively.

  Treatment of vesicourethral reflux has been well established over the past decade. Initially, it was believed that all reflux patients needed surgery. Eventually, another treatment method was proposed that only medical treatment with antibiotics was sufficient. Currently, treatment of reflux is well established to depend on many factors, including the severity of reflux, the associated congenital anomalies, and the social situation of the child (parental compliance with medical treatment) Yes. Medical care is usually recommended for patients with grade 1 and 2 reflux. In this case, it is usually solved automatically as the bladder / ureter shape changes with growth. Grade 3 reflux is generally managed in medical care if it does not persist, or if it is in an antibiotic-suppressed state but no breakthrough infection occurs. Surgical treatment is usually required for grade 4 and grade 5 reflux.

  Medical care means that the patient is treated with daily inhibitory antibiotics. Such patients generally have urine culture with a catheter once every three months, ultrasound and serum analysis once every six months, urinary vesicourethrography with an annual fluoroscope or nucleus, and two years. A close follow-up consisting of a single DMSA kidney scan is required. Surgical treatment consists of an abdominal surgery incising the lower abdomen, entering the bladder and moving the ureter to form a new ureteral submucosal tunnel; thereby extending the ureteral muscle support, Their resistance increases. Such patients include general intratracheal anesthesia during 4-5 hours of surgery, epidural catheters for intraoperative and postoperative pain control, bladder catheters for urination, pericystic drainage, and 5 to 6 days of hospitalization is required. Antibiotic treatment and bladder antispasmodics are needed after surgery.

Open surgery for regurgitation provides superior results by skilled surgeons, which may be painful and fixed in lower abdominal incisions, bladder spasms, hematuria, and in certain children, postoperatively Causes well-known morbidity, including frequent urination. Matouschek, E .; : Die Behung des des veikorenalen Refluxes durch transuterial Einspritzung von polytetrafluoroelast to experience urine, as reported by urinary surgery, Urologic, 20: 263 (1981) Interest in endoscopic injection of Teflon ^™ (polytetrafluoroethylene) paste has spread. According to this method, a cystoscope is inserted into the urinary bladder, a needle is inserted through the cystoscope and placed under direct observation under a refluxing ureter in the submucosal space, and an open ureter Inject the paste until the mouth shape changes to a half-moon shaped slit. Teflon ^™ paste injected with an endoscope corrects reflux by acting as a bulk material that increases ureteral resistance. However, shortly after the introduction of this treatment, there was controversy over the use of Teflon ^™ paste. Malizia et al., “Migration and Granulomatosis after Periurethral Injection of Polymer (Polytetrafluoroethylene),” JAMA, 251: 3277 (1984), is an animal study that shows granuloma formation and brain, lung, Showed the movement of particles to the lymph nodes. The migration of polytetrafluoroethylene and the formation of granuloma is also described by Claes et al. Urol. 142: 821 (1989). The safety of Teflon ^™ ® for human use has been a problem, and thus the paste has been banned by the FDA.

  However, there are some distinct advantages to treating vesicoureteral reflux with an endoscope. Atala et al., “Endoscopic treatment of vesicoureteral reflux phenomenon with a spontaneously removable balloon system”, J. Am. Urol. 148: 724 (1992), this method is simple and can be completed within 15 minutes. It has a low morbidity and a success rate of over 85% and can be performed on an outpatient basis. The goal for those studying this is to find other implants that are safe for human use.

  Bovine skin collagen preparations have been used to treat reflux with an endoscope. However, Leonard et al., “Endoscopic injection of glutaraldehyde-crosslinked bovine skin collagen for correction of vesicoureteral reflux”, J. Am. Urol. 145: 115 (1991), only 58.5% of patients were cured at 1 year follow-up. Collagen implantation volume decreases with time, and as a result, reflux recurs at a high rate of over 90% within 3 years. Due to the high failure rate of this substance, there is a high risk that careless patients will suffer kidney damage after treatment.

  Buckley et al., “Endoscopic correction of vesicoureteral reflux phenomenon with injectable silicone microparticles”, J. Am. Urol. 149: 259A (1993), in one study in Europe, a paste consisting of silicone texture microparticles suspended in a hydrogel was applied to the subureter to correct reflux. And has an initial success rate of 91%. However, Henly et al., “Particulate silicones for use in periurethral injection: study of local tissue effects and exploration of migration”, J. Am. Urol. 147: 376A (1992), movement of particles to distant locations has been observed in animal models. About 30% of the silicone particles have a diameter of 100 μm or less. This suggests that 30% of the silicone particles have the potential to migrate to distant organs by the macrophage system. The manufacturer of this technology tried to get FDA approval but was unsuccessful and went bankrupt as a result.

  Laparoscopic correction of regurgitation is based on animal models (Atala et al., "Laposcopy correction of vesicoureteral reflux phenomenon", J. Urol., 150: 748 (1993)) and humans (Atala, "vesicoureteral reflux phenomenon. Laparoscopic treatment ", Dial.Ped.Urol., 14: 212 (1993)" and is technically feasible, but requires at least two surgeons skilled in laparoscopes Surgical time is longer than open surgery, and surgery is switched from extraperitoneal to intraperitoneal approach, and the longer operating time is more expensive due to the cost of disposable laparoscopic devices.

Despite more than a decade since the Teflon ( ^TM ) controversy, research in this area has made little progress. The ideal material for reflux endoscopy is that it is injectable, non-antigenic, non-migrating, volume-stable and safe for human use (Atala et al., 1992). .

(Urinary incontinence)
Urinary incontinence is the most common and least curable of all GU diseases. Urinary incontinence, or retention of urine and inability to excrete urine without knowledge, depends on the interaction of two sets of muscles. One is the detrusor, which is a longitudinal fiber complex that forms a coating of muscle outside the bladder. The detrusor is activated by parasympathetic nerves. The second muscle is the bladder sphincter, which is smooth / striated muscle. For micturition, it is necessary to contract the detrusor of the bladder at the same time that the sphincter is relaxed at will. As humans age, the ability to control the sphincter voluntarily is lost, as does overall muscle condition decline with age. This can also occur when an abrupt event, such as paraplegia, “cuts” the parasympathetic nervous system and causes loss of sphincter control. In another patient, urinary incontinence exhibits different levels of severity and is classified according to level.

  The most common incontinence is urge incontinence, especially in older people. This type of incontinence is a significantly shorter sign followed by rapid urination. This type of incontinence is caused by overactive detrusor muscles and is usually treated by “toilet training” or medication. On the other hand, reflex incontinence shows no signs and is usually the result of a parasympathetic disorder such as spinal cord injury.

Stress incontinence is most common in older women, but can be seen in women of any age. It is also commonly seen in pregnant women. This type of incontinence is more than half of the total number of incontinences. This is seen in men, but not so much. Stress incontinence is characterized by urine leaking under stress conditions such as sneezing, laughing or physically exercising. There are five recognized categories of severity of stress incontinence, referred to as types 0, 1, 2a, 2b, and 3. Type 3 is the most serious and requires a diagnosis of intrinsic sphincter dysfunction, i.e. ISD (Contemporary Urology, March 1993). Many commonly performed procedures include weight loss, exercise, medication, and, in extreme cases, surgical intervention. The two most common surgical procedures either lift the bladder neck to prevent leakage, or build an inner layer from a prosthetic material such as the patient's own body tissue or PTFE to place pressure on the urethra. Includes either giving. Another method is to use a prosthetic device such as an artificial bladder for an external device such as an intravaginal balloon or penile forceps. For the treatment of type 3 stress incontinence, Teflon ^™ or collagen paste is injected around the sphincter to “beef up” the area around the sphincter and improve muscle condition This is a recent trend. However, none of the above treatment methods are very effective over a period of one year or more.

  Overflow incontinence is caused by anatomical disturbances or inactive detrusor muscles in the bladder. It is characterized by an inflated bladder that leads to frequent urine leakage. This type of incontinence is treated in the short term by catheterization and in the long term by drug therapy. Enuresis or nocturnal enuresis is a problem in children and is controlled by pads with various alarm devices and sensors. Enuresis is not considered a serious problem unless it persists beyond the age of 4,5. The net result is truly functional incontinence that occurs in patients with chronic impairments in either motor function or mental function. Such patients are usually treated with a diaper, incontinence pad or continuous catheterization (BBI, 1985 Report 7062).

  Accordingly, it is an object of the present invention to provide cell injection methods and compositions for forming cellular tissues and cartilage structures.

  A further object of the present invention is to provide a cellular tissue comprising a non-cellular material, such that it is later degraded and removed to leave the same tissue or cartilage as the tissue or cartilage naturally produced histologically and chemically, and It is to provide a composition that forms a cartilage structure.

  Another object of the present invention is to provide methods and materials for treating vesicoureteral reflux, incontinence, and other disorders, which will naturally and permanently treat the disorders. is there.

  It is a further object of the present invention to provide methods and materials for treating vesicoureteral reflux, incontinence, and other disorders that are quick, convenient, safe, and relatively non-invasive. is there.

  1. In one aspect, the present invention relates to a method for transplanting a tissue into an animal, the step of mixing a biodegradable, biocompatible hydrogel solution and separated cells, and transplanting the mixture to an animal. A method comprising the steps of:

  2. In a preferred embodiment, the method of claim 1 is provided wherein the hydrogel solution is cured prior to implantation into an animal.

  3. In a preferred embodiment, the method of claim 1 is provided wherein the hydrogel is injected into the animal as a cell suspension and then it is cured.

  4). In a preferred embodiment, the hydrogel is a polysaccharide, a protein, a polyphosphazine, a polyethylene oxide-polypropylene glycol block copolymer, a poly (acrylic acid) (poly) (Methacrylic acid), copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate), and sulfonated polymers It is selected from the group consisting of, a method according to 1.

  5. In a preferred embodiment, the method of claim 4 is provided wherein the polymer solution is selected from the group consisting of fibrin, collagen, hyaluronic acid, and other naturally occurring polymers.

  6). In a preferred embodiment, the method of claim 4 is provided wherein the hydrogel is cured by exposure to a factor selected from the group consisting of ions, pH changes, and temperature changes.

  7). In a preferred embodiment, the hydrogel is copper, calcium, aluminum, magnesium, strontium, barium, tin, and bifunctional, tri- A cation selected from the group consisting of functional and tetrafunctional organic cations; and a group selected from the group consisting of low molecular weight dicarboxylic acids, sulfate ions, and carbonate ions Item 7. The method according to Item 6, wherein the method is cured by interacting with an ion selected from the group consisting of anions.

  8). In a preferred embodiment, the method according to Item 4 above, wherein the hydrogel is further stabilized by crosslinking with a polyion.

  9. In a preferred embodiment, the cells are selected from the group consisting of cartilage forming chondrocytes and other cells, bone forming osteoblasts and other cells, muscle cells, fibroblasts and organ cells. The method according to Item 1, is provided.

  10. In a preferred embodiment, the method of claim 1 is provided wherein the hydrogel is molded prior to implantation to form a particular shape.

  11. In a preferred embodiment, the method of claim 1 is provided wherein the hydrogel is molded to form a specific shape after mixing with the cells and implantation into the animal.

  12 In a preferred embodiment, the method according to Item 1, wherein the defect is vesicoureteral reflux.

  13. In a preferred embodiment, the method of paragraph 1 above, wherein the patient is suffering from incontinence.

  14 In a preferred embodiment, the method according to Item 1, wherein the defect is in the chest.

  15. In a preferred embodiment, the method according to Item 1, wherein the defect is in the upper gastrointestinal tract is provided.

  16. In one aspect, the present invention provides a composition for forming new tissue in an animal, comprising a hydrogel solution mixed with separated cells.

  17. In a preferred embodiment, the composition of claim 16 is provided wherein the hydrogel solution is cured prior to implantation into an animal.

  18. In a preferred embodiment, the composition of claim 16, wherein the hydrogel solution is injected into the animal as a cell suspension and then it is cured.

  19. In a preferred embodiment, the polymer is a polysaccharide, protein, polyphosphazine, polyethylene oxide-polypropylene glycol block copolymer, poly (acrylic acid), poly (methacrylic acid), a copolymer of acrylic acid and methacrylic acid, poly (vinyl acetate), Item 17. The composition according to Item 16, wherein the composition is selected from the group consisting of a sulfonated polymer.

  20. In a preferred embodiment, the suspension of item 19 is provided, wherein the polymer is selected from the group consisting of fibrin, collagen, hyaluronic acid, and other naturally occurring polymers.

  21. In a preferred embodiment, the composition of claim 18, wherein the hydrogel is combined with a factor selected from the group consisting of ions, pH changes and temperature changes in an amount effective to crosslink the polymer after injection. Offer things.

  22. In a preferred embodiment, the hydrogel is a cation selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin, and difunctional, trifunctional, and tetrafunctional organic cations; and low molecular weight Item 22. The composition according to Item 21, which is administered together with an ion selected from the group consisting of a dicarboxylic acid, a sulfate ion, and an anion selected from the group consisting of carbonate ions.

  23. In a preferred embodiment, the composition according to item 22 above, wherein the hydrogel is further stabilized by crosslinking with a polyion.

  24. In a preferred embodiment, the cells are selected from the group consisting of cartilage forming chondrocytes and other cells, bone forming osteoblasts and other cells, muscle cells, fibroblasts and organ cells. Item 16. A composition according to item 16, is provided.

(Summary of the present invention)
Slowly polymerized, biocompatible and biodegradable hydrogels are shown to be useful as a means to introduce large numbers of isolated cells into a patient to produce organ equivalents or tissue like cartilage. It was done. This gel promotes grafting and provides a three-dimensional template for the growth of new cells. The resulting tissue is compositionally and histologically the same as the naturally occurring tissue. In one embodiment, the cells are suspended in a hydrogel solution and injected directly into the patient site. Here, the hydrogel hardens to form a matrix, and the cells are dispersed inside the matrix. In a second embodiment, the cells are suspended in a hydrogel solution and poured or injected into a mold to have the desired anatomical shape and then hardened to form a matrix, the matrix comprising: Cells are dispersed inside and transplanted to the patient. Eventually, the hydrogel will break down, leaving only the resulting tissue.

  This method can be used in a variety of reconstruction techniques, including custom cell grafts to reconstruct three-dimensional tissue defects, and general tissue transplantation.

  Methods for the treatment of vesicoureteral reflux, incontinence and other disorders are described. Here, bladder muscle cells are mixed with a liquid polymeric substance to form a cell suspension that is injected into the impaired area in an amount effective to obtain a tissue that heals the disorder, for example, thereby Provides the control necessary for the passage of urine. As described in the Examples, human bladder muscle samples or chondrocytes are obtained and processed and the cells are mixed with alginate. It is designed to coagulate at a controlled rate when contacted with calcium salt, and then the cells are injected into the desired site where they proliferate and cure the disorder. The examples show effects in mice and pigs.

(Detailed description of the invention)
The technology of tissue engineering using biocompatible polymer scaffolds is a means of creating an alternative to the currently used prosthetic materials in craniomaxillofacial surgery, as well as Promising as a means of forming organ equivalents that replace diseased, defective, or damaged tissue. However, polymers used to make these scaffolds are difficult to mold, such as polyacetic acid, polyorthoesters, and polyanhydrides, And since it is hydrophobic, as a result, it is inferior to cell adhesion. Furthermore, all manipulations of these polymers must be performed prior to implantation of the polymeric material.

  Calcium alginate and certain other polymers can form ionic hydrogels that are malleable and can be used to encapsulate cells. In a preferred embodiment described herein, the hydrogel is produced by crosslinking an anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with a calcium cation. This intensity is increased by increasing either the calcium ion concentration or the alginate concentration. The alginate solution is mixed with the cells to be transplanted to form an alginate suspension. In one embodiment, the suspension is then injected directly into the patient prior to curing the suspension. This suspension then cures in a short time. In a second embodiment, the suspension is poured or poured into a mold where it hardens to form the desired anatomical form with the cells dispersed therein.

(Cell source)
The cells can be obtained directly from a donor, from a cell culture of cells from the donor, or from an established cell culture line. In a preferred embodiment, cells of the same type and preferably the same immunological profile are obtained from a patient, or a close relative, by biopsy, and then obtained under standard conditions (eg as in Example 1 below). Incubate until the culture is confluent and use as needed. When using cells that are prone to induce an immune response, such as human muscle cells from immunologically different individuals, the recipient may optionally need other immunosuppression, such as steroids and cyclosporine. The agent can be used deliberately to be immunosuppressed. However, in the most preferred embodiment, the cell is autologous.

  In a preferred embodiment, the cells are obtained directly from the donor, washed, and combined with the polymeric material and implanted directly. The cells are cultured using techniques known to those skilled in the art of tissue culture.

  Cells obtained by biopsy are collected and expanded while being passaged as needed to remove contaminating cells. The isolation of chondrocytes and muscle cells is shown in the examples.

  Cell attachment and viability can be assessed by scanning electron microscopy, histology, and quantitative assessment by radioisotope. The function of the transplanted cells can be determined using a combination of the above techniques and functional assays. For example, in the case of hepatocytes, in vivo liver function studies can be performed by placing a cannula into the recipient's normal bile duct. The bile can then be increased and collected. Bile pigments can be analyzed by high performance liquid chromatography, by examining underivatized tetrapyrrole, or by diazotized azodipyrroles ethyl anthranilate and P-glucuronidase. Can be analyzed by thin layer chromatography after conversion to azodipyrroles by reaction with or without treatment. Diconjugated and monoconjugated bilirubin can also be measured by thin layer chromatography after alkaline methanol degradation of conjugated bile pigments. In general, as the number of functioning transplanted hepatocytes increases, the level of bound bilirubin increases. Simple liver function tests can also be performed with blood samples, such as albumin production. Similar organ function studies can be performed using techniques known to those skilled in the art, as needed to measure the degree of cell function after transplantation. For example, pancreatic islet cells are introduced in a manner similar to that used specifically for hepatocyte transplantation to achieve glucose regulation by treating appropriate insulin secretion to treat diabetes. Can do. Other endocrine tissues can also be transplanted. Studies using labeled glucose, as well as studies using protein assays, can be performed to quantify the amount of cells on the polymer scaffold. These cell volume studies are then correlated with cell function studies to determine the appropriate cell volume. In the case of chondrocytes, function is defined as providing adequate structural support to the surrounding attached tissue.

  These techniques allow multiple cell types, including genetically modified cells, to be used for efficient migration and transplantation of multiple cells for the purpose of creating new tissues or tissue equivalents. Can be used to provide within a three-dimensional scaffold for promotion. It can also be used for immunoprotection of cell grafts by eliminating the host immune system while new tissues or tissue equivalents are growing.

  Examples of cells that can be transplanted as described herein include chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organs Cells are included. As used herein, “organ cells” include hepatocytes, islets of Langerhans cells, cells derived from the intestines, cells derived from the kidneys, and other cells that act primarily to synthesize and secrete or metabolize substances. .

(Addition of biologically active substances to the hydrogel)
To facilitate cell transplantation and grafting, the polymeric matrix can be combined with a humoral factor. For example, the polymeric matrix can be combined with pro-angiogenic factors, antibiotics, anti-inflammatory analgesics, growth factors, compounds that induce differentiation, and other factors known to those skilled in the art of cell culture.

  For example, the humoral factor can be mixed with the cell-alginate suspension in a sustained release form prior to forming the implant or transplant. Alternatively, the hydrogel can be modified and bound to humoral factors or signal recognition sequences before being combined with the isolated cell suspension.

(Polymer solution)
In the preferred embodiments described herein, malleable calcium alginate and certain other polymers that can form ionic hydrogels are used to encapsulate cells. This hydrogel is produced by cross-linking anionic salts of alginic acid, a carbohydrate polymer isolated from seaweed, with calcium cation, which has a strength of calcium ion concentration or alginate concentration. Increase by increasing either. An alginate solution is mixed with the cells to be transplanted to form an alginate suspension. The suspension is then injected directly into the patient before the suspension is cured. This suspension then hardens in a short time, depending on the presence of physiological concentrations of calcium in vivo.

Polymeric material mixed with cells for implantation into the body should form a hydrogel. A hydrogel is defined as a material that forms a three-dimensional open lattice structure that incorporates water molecules to form a gel when an organic polymer (natural or synthetic) is crosslinked by covalent, ionic, or hydrogen bonds. . Examples of materials that can be used to form hydrogels include polysaccharides such as alginate, polyphosphazine, and polyacrylate (which are ionically crosslinked), or Pluronics ^™ or Included are block copolymers such as Tetronics ^™ , polyethylene oxide-polypropylene glycol block copolymers, which crosslink with temperature or pH. Other materials include proteins such as fibrin, polyvinylpyrrolidone, hyaluronic acid, and polymers such as collagen.

  In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, charged side groups, or monovalent ionic salts thereof. have. Examples of polymers having acidic side groups capable of reacting with cations are poly (phosphazenes), poly (acrylic acids), poly (methacrylic acids) (poly). (Methacrylic acids)), copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate) (poly (vinyl acetate)), and sulfonated polymers such as sulfonated polystyrene. Copolymers with acidic side groups formed by reaction of acrylic acid or methacrylic acid with vinyl ether monomers or polymers may also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

  Examples of polymers having basic groups that can react with anions are poly (vinylamines), poly (vinylpyridine) (poly (vinyl pyridine)), poly (vinylimidazole) (poly (vinyl)). imidazole)), and some imino substituted polyphosphazenes. The ammonium or quaternary salts of these polymers can also be formed from backbone nitrogen or pendant imino groups. Examples of basic side groups are amino groups and imino groups.

  Alginate can be ionically crosslinked by divalent cations in water at room temperature to form a hydrogel matrix. Because of these mild conditions, alginate is most commonly used for encapsulation of hybridoma cells (eg, Lim US Pat. No. 4,452,883). In Lim's process, an aqueous solution containing the biological material to be encapsulated is suspended in a solution of a water-soluble polymer, this suspension forming droplets, the droplets being multivalent cations. The individual microcapsules are constituted by contact with each other, and then the surface of the microcapsules is cross-linked by the polyamino acid to form a semipermeable membrane around the encapsulated material.

  Polyphosphazenes are polymers that have a backbone composed of nitrogen and phosphorous that are alternately separated by single and double bonds. Each phosphorus atom is covalently bonded to two side chains (“R”). The repeating unit of polyphosphazene has the general structure (1):

ここで、ｎは整数である。

Here, n is an integer.

Polyphosphazenes suitable for cross-linking are mainly acidic and have side chains that can form salt bridges with divalent or divalent cations. Examples of preferred acidic side groups are carboxylic acid groups and sulfonic acid groups. Hydrolytically stable polyphosphazenes are formed from monomers having carboxylic acid side groups and crosslinked by divalent or trivalent cations such as Ca ²⁺ or Al ³⁺ . Polymers that degrade by hydrolysis can be synthesized by incorporating monomers with imidazole side groups, amino acid ester side groups, or glycerol side groups. For example, a polyanionic poly [bis (carboxylatophenoxy)] phosphazene (poly [bis (carboxylatophenoxy)] phosphozene) (PCPP) can be synthesized, which is a polyvalent cation dissolved in an aqueous medium at or below room temperature. To form a hydrogel matrix.

  Bioerodible polyphosphazenes contain at least two different types of side chains: acidic side groups that can form salt bridges with multivalent cations, and side groups that are hydrolyzed under in vivo conditions (eg, imidazole). Groups, amino acid esters, glycerol, and glucosyl). The term bioerodible or biodegradable is used herein to describe the desired application (usually in vivo therapy) once exposed to a physiological solution at a pH of 6-8 between about 25 ° C and 38 ° C. ) Means a polymer that can be dissolved or degraded within an acceptable period of time (less than about 5 years, and most preferably less than about 1 year). Corrosion of the polymer occurs due to side chain hydrolysis. Examples of side chains to be hydrolyzed are unsubstituted and substituted imidazoles and amino acid esters, where these groups are attached to the phosphorus atom through an amino bond (both R groups are in this manner). The polyphosphazene polymer attached at is known as polyaminophosphazene). For polyimidazolidophosphazenes, some of the “R” groups on the polyphosphazene backbone are imidazole rings, which are attached to the backbone phosphorus through the ring nitrogen atom. Other “R” groups are organic residues that do not participate in hydrolysis (eg, methyl phenoxy groups or other groups shown in the scientific paper of Allcock et al., Macromolecule 10: 824-830 (1977)). It can be.

  Methods for the synthesis and analysis of various types of polyphosphazenes include:

に記載され、これらの手法は、本明細書中に参考として特に援用される。

These techniques are specifically incorporated herein by reference.

  Other methods for synthesizing the above polymers are known to those skilled in the art. See, for example, Concise Encyclopedia of Polymer Science and Polymer Amines and Ammonium Salts, E.I. See Gothals (Pergamen Press, Elmsford, NY 1980). Many polymers are commercially available, such as poly (acrylic acid).

  A water-soluble polymer with charged side groups is a polyvalent ion that is oppositely charged with this polymer (multivalent cations if the polymer has acidic side groups and multivalent ions if the polymer has basic side groups). It is crosslinked by reaction with an aqueous solution containing an anion). Preferred cations for cross-linking polymers with acidic side groups to form hydrogels are copper, calcium, aluminum, magnesium, strontium, barium, And divalent or trivalent cations such as tin,

うな２、３または４官能性の有機カチオンもまた使用し得る。これらのカチオンの塩の水性溶液をポリマーに添加し、軟質で、高度に膨潤したヒドロゲルおよび膜を形成する。カチオンの濃度が高い程、または価数が高いほど、ポリマーの架橋度は高くなる。０．００５Ｍという低濃度からポリマーを架橋させることが示されている。高濃度は、塩の溶解度によって規定される。

Such 2, 3 or 4 functional organic cations may also be used. An aqueous solution of these cationic salts is added to the polymer to form a soft, highly swollen hydrogel and membrane. The higher the cation concentration or the higher the valence, the higher the degree of cross-linking of the polymer. It has been shown to crosslink polymers from concentrations as low as 0.005M. High concentration is defined by the solubility of the salt.

  Preferred anions for crosslinking the polymer to form hydrogels are divalent and low molecular weight dicarboxylic acids (eg, terephthalic acid, sulfate ion, and carbonate ion). A trivalent anion, as described for cations, an aqueous solution of these anions is added to the polymer to form a soft, highly swollen hydrogel and membrane.

  Various polycations can be used to make the polymer hydrogel complex and thereby stabilize the polymer hydrogel into a semi-permeable surface membrane. Examples of materials that can be used are polymers having basic reactive groups such as amine groups or imine groups, the preferred molecular weight being between 3,000 and 100,000, and polyethylenimine and Polymers such as polylysine. These are commercially available. One polycation is poly (L-lysine). Examples of synthetic polyamines are polyethyleneimine, poly (vinylamine) (poly (vinylamine)), and poly (allylamine). These are also natural polycations such as the polysaccharide, chitosan.

Polyanions that can be used to form a semipermeable membrane by reaction with basic surface groups on the polymer hydrogel include polymers and copolymers of acrylic acid, methacrylic acid, and other acrylic acid derivatives, such as sulfonated polystyrene. , Polymers having pendant SO ₃ H groups, and polystyrene having carboxylic acid groups.

(Cell suspension)
Preferably the polymer is added to an aqueous solution, preferably a 0.1 M potassium phosphate solution, at a physiological pH to a concentration that forms a hydrogel of the polymer, for example 0.5 to 2% by weight for alginate. During this time, it is preferably dissolved at a concentration of 1%. Isolated cells are suspended in the polymer solution at a concentration between 1 million and 50 million cells / ml, most preferably between 10 million and 20 million cells / ml.

(Transplantation method)
The techniques described herein can be used to introduce many different cell types and achieve different tissue structures. In a preferred embodiment, the cells are mixed with the hydrogel solution and injected directly into the site where it is desired to implant the cells prior to hydrogel hardening. However, the matrix can also be shaped to suit a particular application and implanted into one or more different areas of the body. This application is particularly appropriate when a specific structural design is desired or when promoting the growth and proliferation of cells in a location where the area into which the cells are to be transplanted does not have a specific structure or support. is there.

  The site or multiple sites where the cells are to be transplanted is determined based on the individual requirements, as well as the number of cells required. For cells with organ function, such as hepatocytes or islet cells, the mixture can be injected into the mesentery, subcutaneous tissue, peritoneum, intraperitoneal space, and intramuscular space. For cartilage formation, cells are injected into the site where cartilage formation is desired. In addition, a solution to be injected can be molded using an external mold. In addition, by controlling the rate of polymerization, implants that have been injected with cell-hydrogels to mold clay can be molded.

  Alternatively, the mixture can be poured into a mold, the hydrogel can harden, and the material can then be implanted.

  This suspension can be observed by syringe barrels and needles in specific areas where fillers are desired, i.e. secondary muscle atrophy areas due to congenital or acquired diseases or trauma, burns, etc. It can be injected directly into the soft tissue malformation. Infusion of this suspension into the upper torso of a patient with secondary muscle atrophy of neuropathy would be an example of this.

  This suspension is also injected as a filler for hard tissue defects such as bone or cartilage defects, congenital or acquired disease states, or secondary hard tissue defects such as trauma, burns, etc. obtain. An example of this would be the injection of this suspension into the peripheral region of the skull where bone deformities are secondary to trauma. Infusion in these examples can be made directly into the required area using a needle and syringe under local or general anesthesia.

  This suspension can also be injected percutaneously by direct pulpation, for example by placing a needle inside the vas deferens, and occluding the vas deferens with the injected filling, Thus, the patient can be made infertile. The suspension can also be injected with a catheter or needle using fluoroscopic or holographic computed tomography, magnetic resonance imaging or radiological guidance. This allows placement or injection of this material by vascular or percutaneous access to a particular organ or other tissue region of the body, wherever a filler is needed.

  In addition, this substance can be injected into any intraperitoneal or extraperitoneal or thoracic organ through a laparoscope, thoracoscope. For example, the suspension can be injected into the gastroesophageal transition for the treatment of gastroesophageal reflux. This is done by injecting this material into the esophageal part of the gastroesophageal region using a thoracoscope, or by injecting this material into the gastric part of the gastroesophageal region, or by a combined approach. .

  Cystoureteral reflux is one of the most common congenital defects that affects approximately 1% of the population in children. Although not all patients require surgical treatment, surgical treatment is still one of the most common treatments performed in children.

  More than 600 ureteral retransplants occur annually at Children's Hospital in Boston, Massachusetts. In other words, if the endoscopic treatment described herein is used in place of open surgery, this facility alone will give approximately 3600 inpatient hospital days per year. It will be saved.

  In addition to the use for endoscopic treatment of reflux, the injectable autologous muscle cell system is also used in other medical conditions such as urinary and rectal incontinence, dysphonia, plastic reconstruction. As well as the treatment of any condition where an injectable permanent biomaterial is required.

  As described herein, injectable biodegradable polymers as delivery vehicles for muscle cells or chondrocytes are useful for the treatment of reflux and incontinence. In a preferred embodiment, biopsy material is obtained from a vesicoureteral reflux patient under anesthesia, the isolated cells are mixed with alginate, and the cell-alginate solution is endoscopically as shown in FIG. 1a. Inject into the sub-uretal region to treat reflux. The time to clotting of the alginate-cell solution can be manipulated by changing the calcium concentration and the temperature at which the cells are added to the alginate. The use of autologous cells eliminates the immune response. The solidification of the alginate prevents its movement until it is broken down. The suspension can be injected with a cystoscopic needle that allows direct access to the area of interest using a cystoscope, eg, for the treatment of cystoureteral reflux or urinary incontinence. In addition to the use of cell-polymer suspensions for the treatment of reflux and incontinence, this suspension can also be applied to reconstructive surgery and any human body that requires a biocompatible permanent injectable material. It can be applied to the site as well. This suspension can be injected under an endoscope. For example, it can be injected with a laryngoscope for injection into the vocal cords for treatment of vocal disturbances, or with a hysteroscope for injection into the oviduct as a method of infertility to the patient, or the sphincter region around the rectum For the injection of this substance into the rectum, it is injected by a rectoscope, which increases sphincter resistance and allows the patient to control defecation.

  This technique can also be used for other purposes. For example, a custom-made cell transplant can be used to reconstruct a three-dimensional tissue defect. For example, human ear molds can be made and chondrocyte-hydrogel replicas can be formed and transplanted to restore lost ears. The cells can also be transplanted in the form of a three-dimensional structure that can be transported by injection.

  The invention will be further understood by reference to the following non-limiting examples.

Example 1: Preparation of calcium-alginate-chondrocyte mixture and injection into mice to form cartilage structures
A calcium alginate mixture was obtained by combining calcium sulfate (poorly soluble calcium salt) with 1% sodium alginate dissolved in 0.1 M potassium phosphate buffer (pH 7.4). This mixture was kept in a liquid state at 4 ° C. for 30 minutes to 45 minutes. Chondrocytes isolated from the calf forelimb articular surface are added to the mixture to produce a final cell density of 1 × 10 ⁷ / ml (representing a cell density of about 10% of human child articular cartilage). I let you.

  The calcium alginate-chondrocyte mixture was injected with a 100 μl aliquot of 22 gauge needle under the pannus scab tunnel on the back of nude mice.

  Nude mice were examined 24 hours after surgery and all injection sites were firm with no apparent diffusion of the mixture upon palpation. Samples were collected after 12 weeks of in vivo incubation. In the overall examination, the calcium alginate-chondrocyte specimen showed a pearly milky white color and was firm against palpation. The specimen weighed 0.11 ± 0.01 g (starting weight 0.10 g). From the specimen, the surrounding tissue was easily dissected and removed, and the inflammatory response was minimal. Histologically, the specimens were stained with hematoxylin and eosin, and a hiatus was shown in the base affinity basophil ground glass substrate.

  The control specimen of calcium alginate without chondrocytes had a dough firmness 12 weeks after injection and there was no histological evidence of chondrogenesis.

  This study shows that injectable calcium alginate matrix can provide a three-dimensional scaffold for successful implantation and chondrocyte implantation. Chondrocytes transplanted in this way form a volume of cartilage similar to the initially injected cartilage after 12 weeks of in vivo incubation.

(Example 2: Effect of cell density on cartilage formation)
Varying the number of chondrocytes isolated from the articular surface of the calf forelimb, mixed with 1.5% sodium alginate solution, 0.0, 0.5, 1.0, and 5.0 × 10 ⁶ A final cell density of chondrocytes / ml (approximately 0.0, 0.5, 1.0, and 5.0% cell density of human child articular cartilage) was generated. An aliquot of the chondrocyte-alginate solution was transferred to a 9 mm diameter round mold and polymerized at room temperature by diffusion of calcium chloride solution through the semipermeable membrane at the bottom of the mold. The gel formed a disk measuring 2 mm in height and 9 mm in diameter.

Disks with fixed cell density of 5 × 10 ⁶ cells / ml were also formed with varying sodium alginate concentration and molar concentration of calcium chloride solution.

  All discs were placed in the subcutaneous pocket on the back of nude mice. Samples were taken at 8 and 12 weeks and examined for global and histological evidence of cartilage formation.

Examination of specimens after 8 and 12 weeks showed that a minimum cell density of 5 × 10 ⁶ chondrocytes / ml is required for cartilage formation observed only 12 weeks after transplantation.

  In the overall examination, the specimen was disc-shaped and weighed 0.13 ± 0.01 g (starting weight 0.125 g). From the specimen, the surrounding tissue was easily dissected and removed, and the inflammatory response was minimal. Histologically, specimens were stained with hematoxylin and eosin and a hiatus was shown in the base affinity priming glass material.

  Cartilage formation is independent of the calcium chloride concentration used for gel polymerization. Cartilage was observed in specimens with alginate concentrations varied from 0.5% to 4.0%; however, at the lowest alginate concentration tested (0.5%), cartilage was only seen by microscopic examination. There wasn't.

Cartilage can grow into a predetermined disc shape in 12 weeks using calcium alginate as a support matrix in the subcutaneous pocket. Chondrogenesis is not inhibited by either polymerization at high calcium concentrations or the presence of high alginate concentrations, but requires a minimum cell density of 5 × 10 ⁶ cells / ml.

  The ability to create a calcium alginate-chondrocyte gel with a given shape indicates that this technique can be used for custom designs and grow cartilage scaffolds for skull face reconstruction. Such a scaffold has the potential to replace many of the currently used prosthetic devices.

Example 3: Preparation of implantable preformed cell-polymer mixture
A 250 μl aliquot of the isolated chondrocyte suspension was mixed with 750 μl of 2% (w / v) sodium alginate solution (0.1 M K ₂ HPO ₄ , 0.135 M NaCl, pH 7.4). A 125 μl aliquot was placed in a 9 mm diameter cell culture infusion with a semipermeable membrane with a pore size of 0.45 μm. The cell-alginate mixture was contacted with a 30 mM CaCl ₂ bath and polymerized at 37 ° C. for 90 minutes. After 90 minutes, the cell-alginate gel construct was removed from the mold and it had a diameter of 9 mm and a height of 2 mm. Insert a disk into the wells of 24-well tissue culture plates, and the presence of 5% CO _2, Hamm of F-12 culture medium (Gibco, Grand Island, N.Y. ) And 10% fetal calf serum (Gibco, Grand In 0.5 ml of a solution containing Island, NY), L-glutamine (292 μg / ml), penicillin (100 U / ml), streptomycin (100 μg / ml) and Incubated with ascorbic acid (5 μg / ml) for 48 hours at 37 ° C.

  Using this method, bovine chondrocyte-alginate discs were prepared and then implanted into the dorsal subcutaneous pocket of athymic mice using standard aseptic techniques. After 1, 2 and 3 months, athymic mice were sacrificed and the gel / chondrocyte construct was removed, weighed and placed in an appropriate fixative. Cell-polymer complexes were examined by histological analysis.

Chondrogenesis was observed histologically after 3 months of in vivo incubation at an initial chondrocyte density of 5 × 10 ⁶ cells / ml.

The above protocol was modified by using a predetermined CaCl ₂ concentration range and a sodium alginate concentration range. Chondrogenesis was observed using 15 mM, 20 mM, 30 mM, and 100 mM CaCl ₂ baths and 0.5%, 1.0%, 1.5%, 2.0%, and 4.0% sodium alginate solutions.

  By changing the mold from which the cell-alginate construct is made, the shape of the implant can be made to order. Furthermore, the mold need not be semi-permeable so that calcium ions can be mixed directly with the cell-alginate solution before entering the mold. An important feature is that the construct can be shaped into a predetermined shape prior to implantation.

Example 4: Preparation of injectable osteoblast hydrogel mixture
Using the methodology described above, bovine osteoblasts were substituted for chondrocytes and injected into animals using a hydrogel matrix.

  After 12 weeks of in vivo incubation, histologically showed the presence of early bone formation.

Example 5: Use of a hydrogel to form an immune defense matrix around the cells to be transplanted
By creating a cell-alginate construct as described above, cells encapsulated with a hydrogel matrix can be isolated stereologically from the host immune system, thus allogeneic cells. This allows the formation of new tissues or organs without immunosuppression.

  Bovine chondrocytes in alginate suspension were transplanted into normal immune competent mice. In vivo incubation histology after 6 weeks indicates the presence of cartilage formation. A gross examination of this specimen showed no cartilage characteristics. The literature states that cartilage is not formed in similar chondrocyte xenografts without alginate.

  The following examples show that human bladder muscle cell-alginate suspension is injectable, non-migrating and seems to retain its volume, and that autologous muscle cells are endoscopically vesicourethral reflux endoscopes. That it is useful in clinical treatment.

Example 6: Isolation and characterization of human bladder muscle cells grown in vitro
(Cell culture :)
From human tissue. Human bladder tissue specimens were obtained from surgical removal at Children's Hospital in Boston and processed within 1 hour. Samples varied in size in the range of 1 cm ² to 4 cm ² . Samples were transferred to Hank's balanced salt solution containing 10 mM HEPES and IOOKIU apoprotein.

  Smooth muscle cells. Muscle cells were obtained from small pieces of detrusor muscle chopped to a diameter of about 0.5 mm and used as explants in 100 mm tissue culture dishes containing 10 mL DMEM supplemented with 10% fetal calf serum. . Approximately 30 extracorporeal grafts of similar size were added to each dish. The medium was changed twice a week. Growth was routinely observed 72 hours after placing the explant in culture. When the culture was 80% confluent, the cells were trypsinized and passaged. A cell population highly enriched in elongated, striated cells was routinely obtained using this method. Cell lines were stained with a-actin antibody to confirm their muscle phenotype.

  result. A fusiform, striated cell population is obtained from a biopsy specimen of the bladder by dissecting its muscle layer and then explanting the muscle fragment in DMEM supplemented with 10% bovine serum. Obtained. The phenotype of this muscle was confirmed by immunostaining with a monoclonal antibody that specifically recognizes a-actin. These data show that a highly proliferated population of smooth muscle can be obtained from small biopsy specimens. The muscle cells can also be temporarily cultured in a serum-free defined medium so that the cells do not contain any impurities.

(Example 7: Injection of cell suspension into mice)
Using a 22 gauge needle, 20 nude mice were injected with 500 μl of muscle cells and alginate solution. Each mouse had two injection sites consisting of a control and 10 million human bladder cell solution per cc alginate (32 injection sites). Controls were injections of alginate alone or bladder muscle cells alone. Animals were sacrificed at 2, 4, 6, and 8 weeks after transplantation. A histological examination of the injection area showed evidence of muscle formation at the muscle / alginate injection site. Immunohistological analysis using an anti-desmin antibody showed that the cells maintained a program of muscle differentiation. Examination of the injection site over time showed that the alginate was gradually replaced by muscle. The size of the muscle-alginate complex appeared to be uniform and stable. In the two control groups (alginate alone and muscle cell alone), muscle cells were not evident. Histological analysis of distant organs showed no evidence of bladder muscle cell or alginate migration or granulomas formation.

(Example 8: Correction of vesicoureteral reflux in pigs using bladder muscle cells transplanted with alginate gel)
(Materials and methods)
(Animal model of vesicoureteral reflux)
Pigs were used in this study due to bladder and kidney similarities between pigs and humans. Hanford mini-pig was used because of its small size and simplicity. Bilateral vesicoureteral reflux was created in 4 minipigs using an open bladder technique. This technique is described by Vacanti et al., “Synthetic polymers inoculated with chondrocytes provide a new chondrogenic template,” Plastic and Recon. Surg. 88: 753 (1991), which consists of unwrapping the ureters inside all bladders.

  After 3 months of this procedure, the presence of bilateral reflux has been demonstrated by Vacanti et al., “Chondrocytes, by conventional bladder radiography with iodinated contrast agents and by ultrasonography with sonicated albumin. "Tissue-processed growth of new cartilage in molding of human ears using synthetic polymers inoculated with". Mat. Res. Soc. Proc. 252: 367 (1992). Excretion urography was performed to detect any evidence of failure.

(Collection of cells)
Bladder muscle sections were obtained from each animal. Muscle cells were harvested and plated separately in vitro. After growth, the cells were individually quantified and concentrated to 20 × 10 ⁶ cells per cc.

(Autologous bladder myocytes-calcium alginate suspension)
2% w / v sodium alginate (0.1 M K ₂ PO ₄ , 0.135 M NaCl, pH 7.4, Protan, Portsmouth, NH) was made and sterilized in ethylene oxide. A 1.5 ml aliquot of 20 × 10 ⁶ cells / ml bladder muscle cell suspension was added to the same volume of sodium alginate to a final alginate concentration of 1%. A suspension of bladder muscle cells-sodium alginate was kept at 32 ° C. Calcium sulfate (0.2 g / ml) was added to the bladder muscle cell-sodium alginate suspension immediately before injection. This mixture was vortexed to mix and kept in ice until injection. The gelling process begins with the addition of calcium sulfate and the suspension is in a liquid state for about 40 minutes.

(Experimental research)
Minipigs were anesthetized with 25 ml / kg ketamine, and 1 ml / kg acylpromazine by intramuscular injection. Another anesthesia was obtained by intramuscular administration of 25 mg / kg ketamine and 10 mg / kg xylazine. The animal was placed on its back. A 15.5 French cystoscope was inserted into the bladder and a 21 gauge needle was inserted into the subureteral region of the right reflux ureter. Approximately 2-3 ml of autologous bladder muscle cell-alginate suspension (40-60 × 10 ⁶ bladder muscle cells) was viewed through an endoscope while lifting the ureteral opening and injected through the needle. The left ureteral opening was left untreated and served as a control. A series of cystography, cystoscope, and excretory urography studies were performed at 8-week intervals until sacrifice. Minipigs were sacrificed at 8 weeks (1), 16 weeks (1) and 26 weeks (2) after treatment. The bladder injection site was excised and examined visually and under a microscope. Samples were stained with hematoxylin and eosin, and Alcian blue at pH 1.0 and 2.5. Histological analysis of the vesicoureter, regional lymph nodes, kidney, liver, and spleen was performed.

(result)
Four minipigs were subjected to reflux on both sides. All 4 animals were found to have bilateral reflux without evidence of failure after 3 months of treatment. Bladder muscle cells were derived from each minipig and grown in vitro. The animals were then subjected to reflux endoscopic repair only on the right side using an injectable autologous bladder muscle cell-alginate gel solution.

  Cystoscopy and radiography were performed at 2, 4, and 6 months after treatment. Cystoscopy showed a smooth bladder wall. Cystography showed no evidence of reflux on the treated side and evidence of persistent reflux in the control ureter that was not corrected in all animals. All animals received successful healing of reflux in the repaired ureter without showing evidence of hydronephrosis on excretory urography.

  A general examination of the bladder infusion site at the time of sacrifice showed the presence of bladder myocyte-alginate complex in the subureteral region. Microscopic analysis of the tissue around the injection site showed no inflammation. Tissue sections of the bladder, ureter, lymph node, kidney, liver, and spleen showed no evidence of alginate migration and granuloma formation.

(Summary of experimental data)
Autologous bladder muscle cells can be easily collected, expanded in vitro, and cystoscopically injected. The cells survive and form a non-immunogenic muscle nucleus. This system can correct backflow without showing any evidence of failure.

  The following examples show that the chondrocyte-polymer suspension is injectable, non-migrating and appears to retain its volume, and useful for endoscopic treatment of vesicoureteral reflux Indicates that As shown in Example 1, the alginate-bovine chondrocyte xenograft was found to contain viable chondrocytes for as long as 90 days after transplantation into athymic mice. The formed new cartilage retains the approximate shape and dimensions of the injected mold. Cell-polymer constructs are essential because when chondrocytes or alginate alone are injected, chondrogenesis does not occur.

(Example 9: Mouse transplantation material and method of chondrocytes in alginate gel)
Hyalin cartilage was obtained from the articular surface of the calf shoulder and chondrocytes were harvested. The chondrocyte suspension was concentrated to 20, 30, and 40 × 10 ⁶ cells / cc and mixed with dry alginate powder to form a gel. Twelve athymic mice were injected subcutaneously with the chondrocyte / alginate solution. Each mouse had 4 injection sites consisting of control 10, 15, and 20 × 10 ⁶ chondrocytes (48 injection sites). Mice were sacrificed at 2, 4, 6, and 12 weeks after injection.

  Histological examination of the injection sites showed evidence of cartilage formation in 34 of 36 experimental injection sites. A general examination of the injection site during the growth phase showed that the polymer gel was progressively replaced by cartilage. The final size of the cartilage formed was related to the concentration of the initial chondrocytes injected and appeared to be uniform and stable within each category. There was no evidence of cartilage formation in the 12 controls. Histological analysis of distant organs showed no evidence of cartilage or alginate gel migration or granuloma formation.

(Materials and methods)
Animals-Young adult athymic nu / nu mice were used as cell receptors. Animals were individually housed, accessible to food and water as desired, and maintained at 12 hour lighting and dark intervals. Anesthesia was performed with methoxyflurane by administration of an anesthesia inhaler.

  Polymer-dried alginate pressed powder (Dentsply International; Milford, DE) was used as the delivery vehicle. Alginate is a copolymer of gluconic acid and mannuronic acid, meaning a gel when mixed with calcium salts and water at a controlled rate. Calcium phosphate and calcium sulfate are contained in pure polymer powder and control gelation kinetics. The powder was sterilized in ethylene oxide and sealed in aluminum overnight until injection.

Cell harvesting-hyaline cartilage was obtained from the articular surface of the calf shoulder within 6 hours after sacrifice. The shoulder is washed in a 10% solution of providine-iodine and the chondrocytes are prepared according to the technique described in Klagsbrun, “Preparation of large-scale chondrocytes” Methods in Enzymology, 58: 560 (1979). And collected under sterile conditions. Isolated cells were quantified using a hemocytometer and the chondrocyte suspension was concentrated to 20, 30, and 40 × 10 ⁶ cells / cc.

Cell delivery-Chondrocyte suspension was mixed with dry alginate powder to form a gel. Using a 21 gauge needle, 12 nude mice were injected with 600 microliters of chondrocyte / alginate solution. Each mouse had 4 injection sites consisting of control 10, 15, and 20 × 10 ⁶ chondrocytes (48 injection sites). An injection of alginate gel alone served as a control in 6 mice. As another control, 6 mice were injected subcutaneously with 600 microliters of cell suspension containing 10, 15, and 20 × 10 ⁶ chondrocytes alone and no alginate in the same area.

  Graft recovery—Mice were sacrificed at 2, 4, 6, and 12 weeks after injection. The grafts were excised according to a tissue plane that easily separates the graft from the surrounding tissue, weighed, fixed with 10% neutral buffered formalin and embedded in paraffin. Tissue sections were also obtained from regional lymph nodes, kidney, bladder, ureter, lung, spleen, and liver. Tissue sections were stained with hematoxylin and eosin. Global and histological examinations were performed.

(result)
FIG. 1a is a schematic diagram of the general method used. Histological examination of the injection site showed evidence of cartilage formation in 34 of 36 chondrocyte / alginate grafts. The mild inflammatory response appeared to have resolved by 4 weeks. This consisted of an inflammatory response indicative of acute and chronic foreign body reactions. Fibroblast infiltration was seen up to 2 weeks after injection. A general examination of the injection site during the growth phase showed that the polymer gel was progressively replaced by cartilage. A general examination showed a rubbery to hard cartilage structure that appeared normal. The final size of the cartilage formed appeared to be related to the initial volume injected and the concentration of chondrocytes and appeared to be uniform within each category. The weight of the recovered cartilage structure appeared to be persistently stable. With six polymer gel control injections (without chondrocytes), there was no visible evidence of chondrogenesis. In the second control group (chondrocyte suspension alone), chondrogenesis was not evident anywhere. Histological analysis of the peri-injection site and distant organs showed no evidence of cartilage or alginate gel migration.

(Example 10: Correction of vesicoureteral reflux in pigs using chondrocytes transplanted with alginate gel)
(Materials and methods)
Animal model of vesicoureteral reflux. Pigs were used in this study due to the similarities between the bladder and kidneys of pigs and humans. A Hanford minipig was used due to its smaller size convenience. Bilateral vesicoureteral reflux was made in 4 minipigs using open bladder technique. The technique is described in Vacanti et al., “Synthetic polymers inoculated with chondrocytes provide a new template for chondrogenesis” Plastic and Recon. Surg. 88: 753 (1991) and consists of uncovering the entire intravesical ureter.

  Three months after treatment, the presence of bilateral reflux has been reported by Vacanti et al., “Processed tissue growth of new cartilage in human ear formation using synthetic polymers inoculated with chondrocytes” Mat. Res. Soc. Proc. 252: 367 (1992) as assessed by conventional bladder X-ray contrast using an iodine contrast agent and ultrasonography using sonicated albumin. An excretory urography was performed to detect any evidence of failure.

  Cell harvest. Hyaline cartilage was obtained from the ear surface from each minipig. Ears were washed with a 10% solution of Providin-Iodine and chondrocytes were analyzed by Atala et al., “Endoscopic treatment of vesicoureteral reflux using a self-removable balloon system” Urol. 148: 724 (1992).

The isolated cells were treated with 10% fetal bovine serum (Gibco), 5 microgram / ml ascorbic acid, 292 microgram / ml glutamine, 100 microgram / ml streptomycin, 40 nanogram / ml vitamin D3. And grown in vitro in a solution of Hamms F-12 medium (Gibco, Grand Island, NY) containing 100 units / ml penicillin. Cells were incubated at 37 ° C. in the presence of 5% CO ₂ . Five to eight weeks after the first harvest, chondrocytes were trypsinized and quantified using a hemocytometer. Each minipig-derived chondrocyte suspension was concentrated in minimal essential medium-199 (Gibco) to 40 × 10 ⁶ cells / ml.

Autologous chondrocyte calcium alginate suspension. 2 w / v% sodium alginate (0.1 M K ₂ PO ₄ , 0.135 M NaCl, pH 7.4, Protan, Portsmouth, NH) was made and sterilized in ethylene oxide. A 1.5 ml aliquot of 40 × 10 ⁶ cells / ml chondrocyte suspension was added to an equal volume of sodium alginate solution to a final alginate concentration of 1%. The chondrocyte-sodium alginate suspension was maintained at 32 ° C. Immediately before injection, calcium sulfate (0.2 g / ml) was added to the chondrocyte-sodium alginate suspension. The mixture was vortexed and stored in ice until injection. The gelling process begins with the addition of calcium sulfate and the suspension is in a liquid state for about 40 minutes.

Experimental study. Minipigs were anesthetized with an intramuscular injection of 25 ml / kg ketamine and 1 ml / kg acylpromazine. Another anesthesia was obtained with intramuscular administration of 25 ml / kg ketamine and 10 mg / kg xylazine. Place the animal on its back. Using a 15.5 French cystoscope introduced into the bladder, a 22 gauge needle was inserted into the subureteral region of the right reflux ureter. About 2-3 ml of autologous cartilage-alginate suspension (40-60 × 10 ⁶ chondrocytes) was injected through the needle while lifting the ureteral opening as viewed with an endoscope. The left ureteral opening was left untreated and served as a control. A series of cystography, cystoscopy, and excretory urography studies were performed at 8-week intervals until sacrifice. Minipigs were sacrificed at 8 (1), 16 (1), and 26 (2) weeks after treatment. The bladder injection site was excised and examined visually and microscopically. Specimens were stained with hematoxylin and eosin and stained with alcian blue at pH 1.0 and 2.5. Histological analysis of the bladder, ureter, local lymph node, kidney, liver and spleen was performed.

(result)
Four minipigs experienced bilateral reflux. All 4 animals were found to have bilateral reflux without any evidence of failure at 3 months after treatment. Chondrocytes were harvested from the left ear surface of each minipig and grown in vitro for 5-8 weeks to give a final concentration of 50-150 × 10 ⁶ viable cells per animal. The animals were then subjected to reflux repair using an endoscope with an autologous chondrocyte-alginate gel solution that was injectable only on the right side.

  Cystoscopy and radiography were performed at 2, 4, and 6 months after treatment. Cystoscopy showed a smooth bladder wall. Cystography in all animals showed no reflux on the treated side and evidence of persistent reflux in the uncorrected control ureter. All animals had successful healing of the repaired ureters with no evidence of hydronephrosis on excretory urography.

  A general examination of the bladder injection site at the time of sacrifice showed a rubbery to hard cartilage structure well defined in the subureteral region. Histological examination of these specimens using hematoxylin and eosin staining showed evidence of cartilage formation. The polymer gel was progressively replaced by cartilage over time. Aldehyde fucinalcian blue staining suggested the presence of chondroitin sulfate. Microscopic analysis of the surrounding tissue at the injection site showed no inflammation. Tissue sections from bladder, ureter, lymph node, kidney, liver and spleen showed no evidence of chondrocyte or alginate migration or granuloma formation.

(Summary of experimental data)
Chondrocytes can grow easily and can grow in culture. Novel chondrogenesis can be achieved in vitro and in vivo using chondrocytes cultured on synthetic biodegradable polymers. In these experiments, the cartilage matrix replaced alginate as a biodegradable polysaccharide polymer. Six minipigs experienced bilateral reflux. All 6 animals were found to have bilateral reflux without evidence of failure at 3 months after treatment. Chondrocytes were harvested from the surface of the left ear of each minipig and grown to a final concentration of 50-150 × 10 ⁶ viable cells per animal. The animals were then subjected to reflux repair using an endoscope with an autologous chondrocyte-alginate gel solution that was injectable only on the right side.

  Cystoscopy and radiography were performed at 2, 4, and 6 months after treatment. Cystoscopy showed a smooth bladder wall. Cystography in all animals showed no reflux on the treated side and evidence of persistent reflux in the uncorrected control ureter. All animals had successful healing with a repaired ureter without evidence of hydronephrosis on excretory urography. The harvested ears had evidence of cartilage regrowth within one month of chondrocyte repair.

  A general examination of the bladder injection site at the time of sacrifice showed a rubbery to hard cartilage structure well defined in the subureteral region. Histological examination of these specimens with hematoxylin and eosin showed evidence of normal cartilage formation. The polymer gel was progressively replaced by cartilage over time. Aldehyde fucin Alcian blue staining suggested the presence of chondroitin sulfate. Microscopic analysis of the surrounding tissue at the injection site showed no inflammation. Tissue sections from bladder, ureter, lymph node, kidney, lung, liver and spleen showed no evidence of chondrocyte or alginate migration or granuloma formation.

  Modifications and variations of the compositions and methods of the invention may be apparent to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to be within the scope of the following claims.

FIG. 1a shows that a polymer-chondrocyte suspension is injected into an area of the ureteral opening as a control of vesicoureteral reflux or in the area around the ureter to manage incontinence A scheme is shown; a photomicrograph shows an alginate polymer suspension, new cartilage formed following injection of the suspension, and a chondrocyte control injected in the absence of polymer. FIG. 1b shows that the polymer-chondrocyte suspension is injected into an area of the ureteral opening as a control of vesicoureteral reflux or in the area around the ureter to manage incontinence A scheme is shown; a photomicrograph shows an alginate polymer suspension, new cartilage formed following injection of the suspension, and a chondrocyte control injected in the absence of polymer. FIG. 1c shows that the polymer-chondrocyte suspension is injected into a region of the ureteral opening as a control of vesicoureteral reflux or in the region around the ureter to manage incontinence A scheme is shown; a photomicrograph shows an alginate polymer suspension, new cartilage formed following injection of the suspension, and a chondrocyte control injected in the absence of polymer. FIG. 1d shows that a polymer-chondrocyte suspension is injected into an area of the ureteral opening as a control of vesicoureteral reflux or in the area around the ureter to manage incontinence A scheme is shown; a photomicrograph shows an alginate polymer suspension, new cartilage formed following injection of the suspension, and a chondrocyte control injected in the absence of polymer.

Claims (19)

An injectable composition comprising an alginate solution and separated cells for forming new tissue in an animal, the composition being cross-linked with a three-dimensional open lattice. -Lattice) is injected into the animal under conditions where the three-dimensional open lattice captures water molecules to form a hydrogel with cells dispersed therein, where the cells are chondrocytes Selected from the group consisting of non-cartilage forming cells, osteoblasts and other bone forming cells, muscle cells, fibroblasts, and organ cells,
Here, cross-linking is initiated prior to injection into the animal,
A composition characterized in that the cells are suspended in the solution at a concentration between 1 million cells / ml and 50 million cells / ml. The composition of claim 1, wherein the alginate solution is crosslinked by exposure to ions. An ion selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin, and a cation selected from the group consisting of difunctional, trifunctional and tetrafunctional organic cations; The composition of claim 1, which is cross-linked by interaction. The composition of claim 1, wherein the hydrogel is further stabilized by crosslinking with polyions. The composition of claim 1, wherein the hydrogel is molded after injection into the animal to form a particular shape. A device for forming new tissue in an animal, comprising: a solution formed from alginate mixed with separated cells; and a condition under which the alginate solution is crosslinked to form a three-dimensional open lattice. In combination with a means for injecting the alginate-cell solution into the animal, wherein the three-dimensional open lattice captures water molecules to form a hydrogel with cells dispersed therein, wherein The cells are selected from the group consisting of cells that form cartilage other than chondrocytes, osteoblasts and other bone-forming cells, muscle cells, fibroblasts, and organ cells;
Here, cross-linking is initiated prior to injection into the animal,
The device, wherein the cells are suspended in the solution at a concentration between 1 million cells / ml and 50 million cells / ml. The device of claim 6, wherein the alginate solution is combined with an amount of ions effective to crosslink the alginate after implantation. The alginate is selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin, and a cation selected from the group consisting of difunctional, trifunctional, and tetrafunctional organic cations; The device of claim 7, which is combined. For the treatment of vesicoureteral reflux or loss prohibition, a composition according to any one of claims 1-5, wherein said cells of said composition, form cartilage other than chondrocytes A composition selected from the group consisting of cells, osteoblasts and other bone-forming cells , fibroblasts, and organ cells. 6. A composition according to any one of claims 1-5 for the treatment of gastroesophageal reflux, wherein the cells of the composition are muscle cells. 6. A composition according to any one of claims 1 to 5 for the treatment of hard tissue defects. 6. A composition according to any one of claims 1 to 5 for the treatment of soft tissue defects. The composition according to any one of claims 1 to 5, for inducing male infertility. For the treatment of vesicoureteral reflux or loss prohibition, a device according to any one of claims 6-8, wherein the cells the cells of the device, to form cartilage other than chondrocytes A device selected from the group consisting of osteoblasts and other bone-forming cells , fibroblasts, and organ cells. 9. A device according to any one of claims 6-8 for the treatment of gastroesophageal reflux, wherein the cells of the device are muscle cells. 9. A device according to any one of claims 6 to 8 for the treatment of hard tissue defects. 9. A device according to any one of claims 6 to 8 for the treatment of soft tissue defects. 9. A device according to any one of claims 6 to 8 for inducing male infertility. The device of claim 6 for the treatment of vesicoureteral reflux.

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