EP0708662A1 - Injizierbare polysaccarid-zell-zusammensetzungen - Google Patents

Injizierbare polysaccarid-zell-zusammensetzungen

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Publication number
EP0708662A1
EP0708662A1 EP94919101A EP94919101A EP0708662A1 EP 0708662 A1 EP0708662 A1 EP 0708662A1 EP 94919101 A EP94919101 A EP 94919101A EP 94919101 A EP94919101 A EP 94919101A EP 0708662 A1 EP0708662 A1 EP 0708662A1
Authority
EP
European Patent Office
Prior art keywords
cells
hydrogel
group
alginate
reflux
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP94919101A
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English (en)
French (fr)
Inventor
Linda Griffith-Cima
Anthony Atala
Charles A. Vacanti
Keith T. Paige
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boston Childrens Hospital
Massachusetts Institute of Technology
Original Assignee
Boston Childrens Hospital
Massachusetts Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/056,140 external-priority patent/US5709854A/en
Application filed by Boston Childrens Hospital, Massachusetts Institute of Technology filed Critical Boston Childrens Hospital
Priority to EP08075891A priority Critical patent/EP2025353A2/de
Publication of EP0708662A1 publication Critical patent/EP0708662A1/de
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials

Definitions

  • the present invention is generally in the area of using polysaccharide hydrogel-cell compositions in the area of medical treatments, and specifically relates to a method for correcting vesicoureteral reflux, incontinence and other defects.
  • Craniofacial contour deformities Craniofacial contour deformities, whether traumatic, congenital, or aesthetic, currently require invasive surgical techniques for correction. Furthermore, deformities requiring augmentation often necessitate the use of alloplastic prostheses which suffer from problems of infection and extrusion.
  • a minimally invasive method of delivering additional autogenous cartilage or bone to the craniofacial skeleton would minimize surgical trauma and eliminate the need for alloplastic prostheses. If one could transplant via injection and cause to engraft large numbers of isolated cells, one could augment the craniofacial osteo-cartilaginous skeleton with autogenous tissue, but without extensive surgery.
  • Cells can be implanted onto a polymeric matrix and implanted to form a cartilaginous structure, as described in U.S. Patent No.
  • Vesicoureteral reflux is a condition wherein there is an abnormal development of the ureteral bud as it enters the bladder during e bryologic development. The shortened course of the ureter through the bladder musculature decreases the ureteral resistance and allows for urine to reflux from the bladder reservoir back up into the ureter and into the kidney. With this condition, bacteria which may occasionally be present in the bladder through retrograde urethral transport, can reach the kidneys and cause recurrent pyelonephritis.
  • Vesicoureteral reflux was usually diagnosed with a voiding cystogram after the child presented with repeated episodes of pyelonephritis. With the increased use of prenatal and postnatal sonography, hydronephrosis is more detectable, prompting further radiologic workup and earlier detection, as reported by Atala and Casale. Vesicoureteral reflux is graded depending on the severity. Grade 1 reflux signifies that urine is seen refluxing from the bladder up to the ureter only; in grade 2 reflux, urine refluxes into the ureter and calyceal dilatation. Grade 4 and 5 reflux are more severe, showing ureteral tortuosity and further calyceal blunting and dilatation, respectively.
  • Medical treatment implies that the patient is treated with daily suppressive antibiotics. A close follow-up is required in these patients, generally consisting of a catheterized urine culture every three months, an ultrasound exam and serum analysis every six months, a fluoroscopic or nuclear voiding cystourethrogram every year, and a DMSA renal scan every two years.
  • Surgical treatment consists of an open surgery wherein a low abdominal incision is made, the bladder is entered, the ureters are mobilized and new ureteral submucosal tunnels are created; thereby extending the muscular backing of the ureter which increases their resistance.
  • a cystoscope is inserted into the bladders, a needle is inserted through the cystoscope and placed under direct vision underneath the refluxing ureter in the submucosal space, and TeflonTM paste is injected until the gaping ureteric orifice configuration changes into a half-moon slit.
  • TeflonTM paste injected endoscopically, corrects the reflux by acting as a bulking material which increases ureteral resistance.
  • a controversy regarding the use of TeflonTM paste ensued. Malizia et al.
  • Bovine dermal collagen preparations have been used to treat reflux endoscopically. However, only 58.5% of the patients were cured at one year follow-up, as described by Leonard et al,
  • Laparoscopic correction of reflux has been attempted in both an animal model (Atala et al, "Laparoscopic correction of vesicoureteral reflux” J. Urol. 150:748 (1993)) and humans (Atala, "Laparoscopic treatment of vesicoureteral reflux” Dial. Ped. Urol. 14:212 (1993)) and is technically feasible.
  • at least two surgeons with laparoscopic expertise are needed, the length of the procedure is much longer than with open surgery, the surgery is converted from an extraperitoneal to an intraperitoneal approach, and the cost is higher due to both increased operative time and the expense of the disposable laparoscopic equipment.
  • Urinary Incontinence is the most common and the most intractable of all GU maladies. Urinary incontinence, or the inability to retain urine and not void urine involuntarily, is dependent on the interaction of two sets of muscles. One is the detrusor muscle, a complex of longitudinal fibers forming the external muscular coating of the bladder. The detrusor is activated by parasympathetic nerves. The second muscle is the smooth/striated muscle of the bladder sphincter. The act of voiding requires the sphincter muscle be voluntarily relaxed at the same time that the detrusor muscle of the bladder contracts. As a person ages, his ability to voluntarily control the sphincter muscle is lost in the same way that general muscle tone deteriorates with age. This can also occur when a radical event such as paraplegia "disconnects" the parasympathetic nervous system causing a loss of sphincter control. In different patients, urinary incontinence exhibits different levels of severity and is classified accordingly.
  • incontinence The most common incontinence, particular in the elderly, is urge incontinence. This type of incontinence is characterized by an extremely brief warning following by immediate urination. This type of incontinence is caused by a hyperactive detrusor and is usually treated with "toilet training" or medication. Reflex incontinence, on the other hand, exhibits no warning and is usually the result of an impairment of the parasympathetic nerve system such as a spinal cord injury.
  • Stress incontinence is most common in elderly women but can be found in women of any age. It is also commonly seen in pregnant women. This type of incontinence accounts for over half of the total number of cases. It is also found in men but at a lower incidence. Stress incontinence is characterized by urine leaking under conditions of stress such as sneezing, laughing or physical effort. There are five recognized categories of severity of stress incontinence, designated as types as 0, 1, 2a, 2b, and 3. Type 3 is the most severe and requires a diagnosis of intrinsic
  • Sphincter Deficiency or ISD Contemporary Urology, March 1993.
  • ISD Contemporary Urology, March 1993.
  • the two most common surgical procedures involve either elevating the bladder neck to counteract leakage or constructing a lining from the patient's own body tissue or a prosthetic material such as PTFE to put pressure on the urethra.
  • Another option is to use prosthetic devices such as artificial sphincters to external devices such as intravaginal balloons or penile clamps.
  • TeflonTM or collagen paste around the sphincter muscle in order to "beef up" the area and improve muscle tone. None of the above methods of treatment, however, are very effective for periods in excess of a year.
  • Overflow incontinence is caused by anatomical obstructions in the bladder or underactive detrustors. It is characterized by a distended bladder which leads to frequent urine leakage. This type of incontinence is treated acutely by catheterization and long-term by drug therapy. Enuresis or bed-wetting is a problem in pediatrics and is controlled by various alarming devices and pads with sensors. Enuresis is not considered a serious problem unless it lasts beyond the age of four or five. Finally, there is true functional incontinence which occurs in patients with chronic impairment either of mobility or mental function. Such patients are usually treated by the use of diapers, incontinence pads or continuous catheterization (BBI, 1985 Report 7062) . Accordingly, it is an object of the present invention to provide a method and compositions for injection of cells to form cellular tissues and cartilaginous structures.
  • cells are suspended in a hydrogel solution which is poured or injected into a mold having a desired anatomical shape, then hardened to form a matrix having cells dispersed therein which can be implanted into a patient. Ultimately, the hydrogel degrades, leaving only the resulting tissue.
  • This method can be used for a variety of reconstructive procedures, including custom molding of cell implants to reconstruct three dimensional tissue defects, as well as implantation of tissues generally.
  • a method of treatment of vesicoureteral reflux, incontinence and other defects is described wherein bladder muscle cells are mixed with a liquid polymeric material, to form a cell suspension, which is injected into the area of the defect, in an amount effective to yield a tissue that corrects the defect, for example, which provides the required control over the passage of urine.
  • human bladder muscle specimens or chondrocytes are obtained and processed, the cells are mixed with alginate, which is designed to solidify at a controlled rate when contacted with calcium salts, and the cells are then injected at the desired site where they proliferate and correct the defect. Examples demonstrate efficacy in mice and pigs. Brief Description of the Drawings
  • Figures la-Id are a schematic of injection of a polymer-chondrocyte suspension into a region of the ureteral orifice for control of vesicoureteral reflux or into the peri-urethral region to manage incontinence; with photomicrographs showing the alginate polymer suspension, new cartilage formed following injection of the suspension, and a cartilage cell control injected in the absence of polymer.
  • Calcium alginate and certain other polymers can form ionic hydrogels which are malleable and can be used to encapsulate cells.
  • the hydrogel is produced by cross-linking the anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with calcium cations, whose strength increases with either increasing concentrations of calcium ions or alginate.
  • the alginate solution is mixed with the cells to be implanted to form an
  • BECJREDSHEET(RULE91) iSA/EP alginate suspension is injected directly into a patient prior to hardening of the suspension. The suspension then hardens over a short period of time. In a second embodiment, the suspension is injected or poured into a mold, where it hardens to form a desired anatomical shape having cells dispersed therein.
  • Source of Cells can be obtained directed from a donor, from cell culture of cells from a donor, or from established cell culture lines.
  • cells of the same species and preferably immunological profile are obtained by biopsy, either from the patient or a close relative, which are then grown to confluence in culture using standard conditions, for example, as described below in Example 1 and used as needed. If cells that are likely to elicit an immune reaction are used, such as human muscle cells from immunologically distinct individual, then the recipient can be i munosuppressed as needed, for example, using a schedule of steroids and other immunosuppressant drugs such as cyclosporine. However, in the most preferred embodiment, the cells are autologous.
  • cells are obtained directly from a donor, washed and implanted directly in combination with the polymeric material.
  • the cells are cultured using techniques known to those skilled in the art of tissue culture.
  • Cells obtained by biopsy are harvested and cultured, passaging as necessary to remove contaminating cells. Isolation of chondrocytes and muscle cells are demonstrated in the examples. Cell attachment and viability can be assessed using scanning electron microscopy, histology, and quantitative assessment with radioisotopes.
  • the function of the implanted 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 common bile duct. Bile can then be collected in increments.
  • Bile pigments can be analyzed by high pressure liquid chromatography looking for underivatized tetrapyrroles or by thin layer chromatography after being converted to azodipyrroles by reaction with diazotized azodipyrroles ethylanthranilate either with or without treatment with P-glucuronidase.
  • Diconjugated and monoconjugated bilirubin can also be determined by thin layer chromatography after alkalinemethanolysis of conjugated bile pigments. In general, as the number of functioning transplanted hepatocytes increases, the levels of conjugated bilirubin will increase. Simple liver function tests can also be done on blood samples, such as albumin production.
  • Analogous organ function studies can be conducted using techniques known to those skilled in the art, as required to determine the extent of cell function after implantation.
  • islet cells of the pancreas may be delivered in a similar fashion to that specifically used to implant hepatocytes, to achieve glucose regulation by appropriate secretion of insulin to cure diabetes.
  • Other endocrine tissues can also be implanted.
  • Studies using labelled glucose as well as studies using protein assays can be performed to quantitate cell mass on the polymer scaffolds. These studies of cell mass can then be correlated with cell functional studies to determine what the appropriate cell mass is.
  • function is defined as providing appropriate structural support for the surrounding attached tissues.
  • This technique can be used to provide multiple cell types, including genetically altered cells, within a three-dimensional scaffolding for the efficient transfer of large number of cells and the promotion of transplant engraftment for the purpose of creating a new tissue or tissue equivalent. It can also be used for immunoprotection of cell transplants while a new tissue or tissue equivalent is growing by excluding the host immune system.
  • Examples of cells which can be implanted as described herein include chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organ cells.
  • organ cells includes hepatocytes, islet cells, cells of intestinal origin, cells derived from the kidney, and other cells acting primarily to synthesize and secret, or to metabolize materials.
  • the polymeric matrix can be combined with humoral factors to promote cell transplantation and engraftment.
  • the polymeric matrix can be combined with angiogenic factors, antibiotics, antiinflammatories, growth factors, compounds which induce differentiation, and other factors which are known to those skilled in the art of cell culture.
  • humoral factors could be mixed in a slow-release form with the cell-alginate suspension prior to formation of implant or transplantation.
  • the hydrogel could be modified to bind humoral factors or signal recognition sequences prior to combination with isolated cell suspension.
  • calcium alginate and certain other polymers that can form ionic hydrogels which are malleable are used to encapsulate cells.
  • the hydrogel is produced by cross-linking the anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with calcium cations, whose strength increases with either increasing concentrations of calcium ions or alginate.
  • the alginate solution is mixed with the cells to be implanted to form an alginate suspension. Then the suspension is injected directly into a patient prior to hardening of the suspension. The suspension then hardens over a short period of time due to the presence in vivo of physiological concentrations of calcium ions.
  • a hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open- lattice structure which entraps water molecules to form a gel.
  • materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block copolymers such as PluronicsTM or TetronicsTM, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively.
  • polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen.
  • these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
  • aqueous solutions such as water, buffered salt solutions, or aqueous alcohol solutions
  • polymers with acidic side groups that can be reacted with cations are poly(phosphazenes) , poly(acrylic acids) , poly(methacrylic acids) , copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate) , and sulfonated polymers, such as sulfonated polystyrene.
  • Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.
  • acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.
  • the ammonium or quaternary salt of the polymers can also be formed from the backbone nitrogens or pendant imino groups.
  • Examples of basic side groups are amino and imino groups.
  • Alginate can be ionically cross-linked with divalent cations, in water, at room temperature, to form a hydrogel matrix. Due to these mild conditions, alginate has been the most commonly used polymer for hybridoma cell encapsulation, as described, for example, in U.S. Patent No. 4,352,883 to Lim.
  • an aqueous solution containing the biological materials to be encapsulated is suspended in a solution of a water soluble polymer, the suspension is formed into droplets which are configured into discrete microcapsules by contact with multivalent cations, then the surface of the microcapsules is crosslinked with polyamino acids to form a semipermeable membrane around the encapsulated materials.
  • Polyphosphazenes are polymers with backbones consisting of nitrogen and phosphorous separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two side chains ("R") .
  • the repeat unit in polyphosphazenes has the general structure (1) :
  • the polyphosphazenes suitable for cross- linking have a majority of side chain groups which are acidic and capable of forming salt bridges with di- or trivalent cations.
  • Examples of preferred acidic side groups are carboxylic acid groups and sulfonic acid groups.
  • Hydrolytically stable polyphosphazenes are formed of monomers having carboxylic acid side groups that are crosslinked by divalent or trivalent cations such as Ca 2+ or Al 3+ . Polymers can be synthesized that degrade by hydrolysis by incorporating monomers having imidazole, amino acid ester, or glycerol side groups.
  • PCPP polyanionic poly[bis(carboxylatophenoxy) ]phosphazene
  • Bioerodible polyphosphazines have at least two differing types of side chains, acidic side groups capable of forming salt bridges with multivalent cations, and side groups that hydrolyze under in vivo conditions, e.g., imidazole groups, amino acid esters, glycerol and glucosyl.
  • bioerodible or biodegradable means a polymer that dissolves or degrades within a period that is acceptable in the desired application (usually in vivo therapy) , less than about five years and most preferably less than about one year, once exposed to a physiological solution of pH 6-8 having a temperature of between about 25°C and 38°C. Hydrolysis of the side chain results in erosion of the polymer.
  • hydrolyzing side chains are unsubstituted and substituted imidizoles and amino acid esters in which the group is bonded to the phosphorous atom through an amino linkage (polyphosphazene polymers in which both R groups are attached in this manner are known as polyaminophosphazenes) .
  • polyphosphazene polymers in which both R groups are attached in this manner are known as polyaminophosphazenes
  • polyimidazolephosphazenes some of the "R" groups on the polyphosphazene backbone are imidazole rings, attached to phosphorous in the backbone through a ring nitrogen atom.
  • Other "R” groups can be organic residues that do not participate in hydrolysis, such as methyl phenoxy groups or other groups shown in the scientific paper of Allcock, et al., Macromolecule 10:824-830 (1977).
  • the water soluble polymer with charged side groups is crosslinked by reacting the polymer with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups.
  • the preferred cations for cross-linking of the polymers with acidic side groups to form a hydrogel are divalent and trivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, although di-, tri- or tetra-functional organic cations such as alkylammonium salts, e.g., R 3 N + ⁇ / ⁇ / ⁇ /- + NR 3 can also be used.
  • Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels and membranes.
  • concentration of cation or the higher the valence, the greater the degree of cross-linking of the polymer. Concentrations from as low as 0.005 M have been demonstrated to cross-link the polymer. Higher concentrations are limited by the solubility of the salt.
  • the preferred anions for cross-linking of the polymers to form a hydrogel are divalent and trivalent anions such as low molecular weight dicarboxylic acids, for example, terepthalic acid, sulfate ions and carbonate ions.
  • Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels and membranes, as described with respect to cations.
  • a variety of polycations can be used to complex and thereby stabilize the polymer hydrogel into a semi-permeable surface membrane.
  • materials that can be used include polymers having basic reactive groups such as amine or imine groups, having a preferred molecular weight between 3,000 and 100,000, such as polyethylenimine and polylysine.
  • poly(L-lysine) examples of synthetic polyamines are: polyethyleneimine, poly(vinylamine) , and poly(allyl amine) .
  • polysaccharide examples of synthetic polyamines
  • chitosan examples of synthetic polyamines
  • Polyanions that can be used to form a semi- permeable membrane by reaction with basic surface groups on the polymer hydrogel include polymers and copolymers of acrylic acid, methacrylic acid, and other derivatives of acrylic acid, polymers with pendant S0 3 H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups.
  • Cell Suspensions include polymers and copolymers of acrylic acid, methacrylic acid, and other derivatives of acrylic acid, polymers with pendant S0 3 H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups.
  • the polymer is dissolved in an aqueous solution, preferably a 0.1 M potassium phosphate solution, at physiological pH, to a concentration forming a polymeric hydrogel, for example, for alginate, of between 0.5 to 2% by weight, preferably 1%, alginate.
  • the isolated cells are suspended in the polymer solution to a concentration of between 1 and 50 million cells/ml, most preferably between 10 and 20 million cells/ml.
  • the techniques described herein can be used for delivery of many different cell types to achieve different tissue structures.
  • the cells are mixed with the hydrogel solution and injected directly into a site where it is desired to implant the cells, prior to hardening of the hydrogel.
  • the matrix may also be molded and implanted in one or more different areas of the body to suit a particular application. This application is particularly relevant where a specific structural design is desired or where the area into which the cells are to be implanted lacks specific structure or support to facilitate growth and proliferation of the cells.
  • the site, or sites, where cells are to be implanted is determined based on individual need, as is the requisite number of cells.
  • the mixture can be injected into the mesentery, subcutaneous tissue, retroperitoneum, properitoneal space, and intramuscular space.
  • the cells are injected into the site where cartilage formation is desired.
  • the mixture can be injected into a mold, the hydrogel allowed to harden, then the material implanted.
  • the suspension can be injected via a syringe and needle directly into a specific area wherever a bulking agent is desired, i.e., a soft tissue deformity such as that seen with areas of muscle atrophy due to congenital or acquired diseases or secondary to trauma, burns, and the like.
  • a bulking agent i.e., a soft tissue deformity such as that seen with areas of muscle atrophy due to congenital or acquired diseases or secondary to trauma, burns, and the like.
  • An example of this would be the injection of the suspension in the upper torso of a patient with muscular atrophy secondary to nerve damage.
  • the suspension can also be injected as a bulking agent for hard tissue defects, such as bone or cartilage defects, either congenital or acquired disease states, or secondary to trauma, burns, or the like.
  • the injunction in these instances can be made directly into the needed area with the use of a needle and syringe under local or general anesthesia.
  • the suspension could also be injected percutaneously by direct palpation, such as by placing a needle inside the vas deferens and occluding the same with the injected bulking substance, thus rendering the patient infertile.
  • the suspension could also be injected through a catheter or needle with fluoroscopic, sonographic, computed tomography, magnetic resonance imaging or other type of radiologic guidance.
  • this substance could be injected through a laparoscopic or thoracoscope to any intraperitoneal or extraperitoneal or thoracic organ.
  • the suspension could be injected in the region of the gastro-esophageal junction for the correcting of gastroesophageal reflux. This could be performed either with a thoracoscope injecting the substance in the esophageal portion of the gastroesophageal region, or via a laparoscope by injecting the substance in the gastric portion of the gastroesophageal region, or by a combined approach.
  • Vesicoureteral reflux is one of the most common congenital defects in children, affecting approximately 1% of the population. Although all patients do not require surgical treatment, it is still one of the most common procedure performed in children. Over 600 ureteral reimplants are performed yearly at Children's Hospital in Boston, Massachusetts. This translates to an approximately saving of 3600 inpatient hospital days per year at this institution alone, if the endoscopic treatment described herein is used instead of open surgery.
  • the system of injectable autologous muscle cell may also be applicable for the treatment of other medical conditions, such as urinary and rectal incontinence, dysphonia, plastic reconstruction, and wherever an injectable permanent biocompatible material is needed.
  • an injectable biodegradable polymer as a delivery vehicle for muscle cells or chondrocytes is useful in the treatment of reflux and incontinence.
  • a biopsy is obtained under anesthesia from a patient with vesicoureteral reflux, the isolated cells are mixed with alginate, and the cell-alginate solution is injected endoscopically in the sub-ureteral region to correct reflux, as shown in Figure la.
  • the time to solidification of the alginate-cell solution may be manipulated by varying the concentration of calcium
  • RECTIFIED SHEET (RULE 91) ISA. ⁇ P as well as the temperature at which the cells are added to the alginate.
  • the use of autologous cells precludes an immunologic reaction. Solidification of the alginate impedes its migration until after it is degraded.
  • the suspension can be injected through a cystoscopic needle, having direct visual access with a cystoscope to the area of interest, such as for the treatment of vesico-ureteral reflux or urinary incontinence.
  • the suspension can also be applied to reconstructive surgery, as well as its application anywhere in the human body where a biocompatible permanent injectable material is necessary.
  • the suspension can be injected endoscopically, for example through a laryngoscope for injection into the vocal chords for the treatment of dysphonia, or through a hysteroscope for injection into the fallopian tubes as a method of rendering the patient infertile, or through a proctoscope, for injection of the substance in the perirectal sphincter area, thereby increasing the resistance in the sphincter area and rendering the patient continent of stool.
  • This technology can be used for other purposes.
  • custom-molded cell implants can be used to reconstruct three dimensional tissue defects, e.g., molds of human ears could be created and a chondrocyte-hydrogel replica could be fashioned and implanted to reconstruct a missing ear.
  • Cells can also be transplanted in the form of a thee-dimensional structure which could be delivered via injection.
  • Example l Preparation of a Calciu -Alginate- chondrocyte mixture and injection into mice to form cartilaginous structures.
  • a calcium alginate mixture was obtained by combining calcium sulfate, a poorly soluble calcium salt, with a 1% sodium alginate dissolved in a 0.1 M potassium phosphate buffer solution (pH 7.4). The mixture remained in a liquid state at 4°C for 30-45 min. Chondrocytes isolated from the articular surface of calf forelimbs were added to the mixture to generate a final cellular density of 1 x 10 7 /ml (representing approximately 10% of the cellular density of human juvenile articular cartilage) . The calcium alginate-chondrocyte mixture was injected through a 22 gauge needle in 100 ⁇ l aliquots under the pannus cuniculus on the dorsum of nude mice.
  • the nude mice were examined 24 hours post- operatively, and all injection sites were firm to palpation without apparent diffusion of the mixture. Specimens were harvested after 12 weeks of in vivo incubation. On gross examination, the calcium alginate-chondrocyte specimens exhibited a pearly opalescence and were firm to palpation. The specimens weighed 0.11 ⁇ 0.01 gms (initial weight 0.10 gms). The specimens were easily dissected free of surrounding tissue and exhibited minimal inflammatory reaction. Histologically, the specimens were stained with hematoxylin and eosin and demonstrated lacunae within a basophilic ground glass substance.
  • Control specimens of calcium alginate without chondrocytes had a doughy consistency 12 weeks after injection and had no histologic evidence of cartilage formation.
  • Chondrocytes transplanted in this manner form a volume of cartilage after 12 weeks of in vivo incubation similar to that initially injected.
  • Example 2 Effect of cell density on cartilage formation.
  • Varying numbers of chondrocytes isolated from the articular surface of calf forelimbs were mixed with a 1.5% sodium alginate solution to generate final cell densities of 0.0, 0.5, 1.0, and 5.0 x 10 6 chondrocytes/ml (approximately 0.0, 0.5, 1.0, and 5.0% of the cellular density of human juvenile articular cartilage) .
  • An aliquot of the chondrocyte-alginate solution was transferred to a circular mold 9 mm in diameter and allowed to polymerize at room temperature by the diffusion of a calcium chloride solution through a semi- permeable membrane at the base of the mold.
  • the gels formed discs measuring 2 mm in height and 9 mm in diameter.
  • Discs of a fixed cellular density of 5 x 10 6 cells/ml were also formed in which the concentration of the sodium alginate and the molarity of the calcium chloride solutions were varied.
  • Cartilage can be grown in a subcutaneous pocket to a pre-determined disc shape using calcium alginate gel as a support matrix in 12 weeks. Cartilage formation is not inhibited by either polymerization with high calcium concentrations or the presence of high alginate concentrations but does require a minimum cellular density of 5 x 10° cells/ml.
  • the ability to create a calcium alginate- chondrocyte gel in a given shape demonstrates that it is possible to use this technique to custom design and grow cartilaginous scaffolds for craniofacial reconstruction. Such scaffolds have the potential to replace many of the prosthetic devices currently in use.
  • Example 3 Preparation of ImpIan able Premolded Cell-polymer mixtures.
  • the cell-alginate gel constructs were removed from the mold and had a diameter of 9 mm and a height of 2 mm.
  • the discs were placed into the wells of 24-well tissue culture plates and incubated at 37°C in the presence of 5% C0 2 with 0.5 ml of a solution containing Hamm's F-12 culture media (Gibco, Grand Island, N.Y.) and 10% fetal calf serum (Gibco, Grand Island, N.Y.) with L-glutamine (292 ⁇ g/ml), penicillin (100 U/ml) , streptomycin (100 ⁇ g/ml) and ascorbic acid (5 ⁇ g/ml) for 48 hrs.
  • bovine chondrocyte- alginate discs were prepared, then implanted in dorsal subcutaneous pockets in athymic mice using standard sterile technique. After one, two, and three months, athymic mice were sacrificed, and the gel/chondrocyte constructs removed, weighed and placed in appropriate fixative. The cell-polymer complexes were studied by histochemical analysis.
  • Cartilage formation was observed histologically after three months of in vivo incubation at an initial chondrocyte density of 5 x 10 6 cell/ml.
  • the above protocol was modified by using a range of CaCl 2 concentration and a range of sodium alginate concentrations. Cartilage formation was observed using 15, 20, 30, and 100 mM CaCl 2 baths and 0.5, 1.0, 1.5, 2.0, and 4.0% sodium alginate solutions.
  • the shape of the implant can be customized. Additionally, the mold need not be semipermeable as calcium ion can be directly mixed with the cell-alginate solution prior to being placed within a mold. The key feature is that the construct can be fashioned into a given shape prior to implantation.
  • Example 4 Preparation of injectable osteoblasts- hydrogel mixtures.
  • bovine osteoblasts have been substituted for chondrocytes and injected into animals using a hydrogel matrix.
  • Example 5 Use of the hydrogel matrix to form an immunoprotective matrix around the implanted cells.
  • hydrogel matrix By fashioning a cell-alginate construct as described above, one can use the hydrogel matrix to sterically isolate the encapsulated cells from the host immune system, and thereby allow allogenic cell transplants to form new tissues or organs without immunosuppression.
  • Bovine chondrocytes in an alginate suspension were transplanted into normal immune- competent mice. Histology after six weeks of in vivo incubation shows the presence of cartilage formation. Gross examination of the specimens does not demonstrate features of cartilage. Literature states that similar chondrocyte xenografts without alginate do not form cartilage.
  • Human Tissue Origin Human bladder tissue specimens were obtained and processed within one hour after surgical removal at Children's Hospital, Boston. The specimens varied in size, ranging from one cm 2 to four cm 2 . The specimens were transported to Hanks balanced salt solution containing 10 mM HEPES and 100 KIU apoprotein. Smooth Muscle Cells. Muscle cells were obtained by mincing small detrusor muscle fragments to approximately 0.5 mm diameter and using these as explants in 100 mm tissue culture dishes containing 10 mL of DMEM supplemented with 10% fetal calf serum. Approximately 30 explants of similar size were added to each dish. Medium was changed twice a week. Outgrowth was routinely observed at 72 hr after explants were placed in culture.
  • Example 7 Injection of cell suspensions into mice. Using a 22 gauge needle, 20 nude mice were injected with a 500 microliter solution of muscle cells and alginate. Each mouse had two injection sites consisting of either control, or a solution of ten million human bladder cells per cc of alginate (32 injection sites) . Injections of alginate alone or bladder muscle cells alone served as controls. Animals were sacrificed at two, four, six and eight weeks after implantation. Histologic examination of the injection areas demonstrated evidence of muscle formation in the muscle/alginate injection sites. Immunohistochemical analysis using an anti-desmin antibody indicated that the cells maintained a program of muscular differentiation. Examination of the injection sites with increasing periods of time, showed that the alginate was progressively replaced by muscle. The size of the muscle-alginate complex appeared to be uniform and stable. In both control groups
  • Example 8 Correction of vesicoureteral reflux in pigs using bladder muscle cells implanted in an alginate gel.
  • the gelling process was initiated with the addition of calcium sulfate, which allowed the suspension to remain in a liquid state for approximately 40 minutes.
  • Mini-pigs were anesthetized with intramuscular injections of 25 ml/kg ketamine and 1 ml/kg acylpromazine. Additional anesthesia was obtained with an intramuscular administration of 25 mg/kg ketamine and 10 mg/kg of xylazine. Animals were placed in a supine position. With a 15.5 French cystoscope introduced into the bladder, a 21 gauge needle was inserted in the subureteral region of the right refluxing ureter.
  • Cystoscopic and radiographic examinations were performed at two, four, and six months after treatment. Cystoscopic examinations showed a smooth bladder wall. Cystograms showed no evidence of reflux on the treated side and persistent reflux in the uncorrected control ureter in all animals. All animals had a successful cure of reflux in the repaired ureter without evidence of hydronephrosis on excretory urography. At the time of sacrifice, gross examination of the bladder injection site showed the presence of bladder muscle cell-alginate composites in the subureteral region. Microscopic analyses of the tissues surrounding the injection site showed no inflammation. Tissue sections from the bladder, ureters, lymph nodes, kidneys, liver and spleen showed no evidence of alginate migration or granuloma formation. Summary of Experimental Data
  • Autologous bladder muscle cells can be readily harvested, expanded in vitro , and injected cystoscopically. The cells survive and form a muscle nidus which is non-antigenic. This system is able to correct reflux without any evidence of obstruction.
  • chondrocyte-polymer suspensions are injectable, non-migratory, and appear to conserve their volume, and are useful in the endoscopic treatment of vesicoureteral reflux.
  • alginate-bovine chondrocyte cell allografts were found to contain viable cartilage cells after implantation times for as long as 90 days in athymic mice. The new cartilage formed retains the approximate configuration and dimensions of the injected template.
  • the cell-polymer construct is essential in that injection of free chondrocytes or alginate alone does not result in cartilage formation.
  • Example 9 Implantation of chondrocytes in alginate gel into mice.
  • Hyaline cartilage was obtained from the articular surfaces of calf shoulders and chondrocytes were harvested. Chondrocyte suspensions were concentrated to 20, 30, and 40 X 10 6 cells per cc and mixed with dry alginate powder to form a gel. Twelve athymic mice were injected subcutaneously with a chondrocyte/alginate solution. Each mouse had four injection sites consisting of control, 10, 15, and 20 X 10 6 chondrocyte cells (48 injection sites) . Mice were sacrificed at 2, 4, 6, and 12 weeks after injection.
  • mice Females - Young adult athymic nu/nu mice were used as cell recipients. The animals were housed individually, allowed access to food and water as desired, and maintained on 12 hours of light and dark intervals. Anesthesia was performed with methoxyflurane by cone administration.
  • Polymers - Dry alginate impression powder (Dentsply International; Milford, DE) was used as the delivery vehicle.
  • Alginate a copolymer of gluronic and mannuronic acid, is designed to gel at a controlled rate when mixed with calcium salts and water.
  • Calcium phosphates and sulfates are included in the pure polymer powder to control the gelation kinetics.
  • the powder was sterilized in ethylene oxide and sealed in aluminum foil until injection.
  • Cell Harvest - Hyaline cartilage was obtained from the articular surfaces of calf shoulders within six hours of sacrifice.
  • chondrocytes were harvested under sterile conditions using a technique described by Klagsbrun, "Large scale preparation of chondrocytes" Methods in Enzymolocry, 58:560 (1979) .
  • the isolated cells were quantitated using a hemocytometer, and the chondrocyte suspension was concentrated to 20, 30, and 40 X 10 6 cells per cc.
  • chondrocyte cell suspensions were mixed with dry alginate powder to form a gel.
  • 12 nude mice were injected with a 600 microliter chondrocyte/alginate solution.
  • Each mouse had four injection sites consisting of control, 10, 15, and 20 X 10 6 chondrocytes (48 injection sites).
  • Injection of alginate gel alone served as control in six mice.
  • As another control six mice were injected subcutaneously in the same region with 600 microliters cell suspensions containing 10, 15, and 20 X 10 6 chondrocytes alone, without alginate.
  • Implant Recovery - Mice were sacrificed at 2, 4, 6, and 12 weeks after injection. The implants were excised following a tissue plane that easily separated the implant from the surrounding tissue, weighted, fixed in 10 percent neutral buffered formalin, and imbedded in Paraffin. Tissue sections were also obtained from the regional lymph nodes, kidneys, bladder, ureters, lungs, spleen, and liver. Tissue sections were stained with hematoxylin and eosin. Gross and histologic examination were performed. Results
  • Figure la is a schematic of the general method which was used. Histologic examination of
  • ISA/EP injection sites demonstrated evidence of cartilage formation in 34 of the 36 chondrocyte/alginate implants.
  • a mild inflammatory response appeared to be resolving by four weeks. This consisted of an inflammatory response that exhibited an acute phase and a chronic foreign body reaction. Fibroblast infiltration were seen up to two weeks after injection. Examination of the injection sites with increasing periods of time, showed that the polymer gels were progressively replaced by cartilage.
  • Example 10 Correction of vesicoureteral reflux in pigs using chondrocytes implanted in an alginate gel.
  • the pig was used for this study because of the similarities between porcine and human bladders and kidneys.
  • the Hanford mini-pig was used for the convenience of its smaller size.
  • Bilateral vesicoureteral reflux was created in four mini- swine using the open bladder technique, which consists of unroofing the entire intravesical ureter, as described by Vacanti, et al., "Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation" Plastic and Recon. Surq. 88:753 (1991).
  • Hyaline cartilage was obtained from the auricular surfaces of each mini- swine. The ears were washed with providine-iodine 10% solution and chondrocytes were harvested under sterile conditions using the technique, Atala, et al., "Endoscopic treatment of vesicoureteral reflux with a self-detachable balloon system" J. Urol. 148:724 (1992).
  • the isolated cells were expanded in vitro in a solution of Ham s F-12 media (Gibco, Grand Island, NY) with 10% fetal calf serum (Gibco) , 5 micrograms/ml ascorbic acid, 292 micrograms/ml glutamine, 100 micrograms/ml streptomycin, 40 nanograms/ml vitamin D3 and 100 units/ml penicillin.
  • the cells were incubated at 37°C in the presence of 5% C0 2 .
  • the chondrocytes were trypsinized and quantitated using a hemocytometer.
  • the chondrocyte suspension from each mini-swine was concentrated to 40 X 10 6 cells/ml in minimal essential media - 199 (Gibco) .
  • Autologous chondrocyte-calcium alginate suspension Two percent weight/volume sodium alginate (0.1 M K 2 P0 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 x 10 6 cells/ml chondrocyte suspension was added to an equal volume of sodium alginate solution for a final alginate concentration of 1%.
  • the chondrocyte-sodium alginate suspension was kept at 32°C.
  • 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 was initiated with the addition of calcium sulfate, which allowed the suspension to remain in a liquid state for approximately 40 minutes.
  • mice were anesthetized with intramuscular injections of 25 ml/kg ketamine and 1 ml/kg acylpromazine. Additional anesthesia was obtained with an intramuscular administration of 25 mg/kg ketamine and 10 mg/kg of xylazine. Animals were placed in a supine position. With a 15.5 French cystoscope introduced into the bladder, a 22 gauge needle was inserted in the subureteral region of the right refluxing ureter. Approximately 2-3 ml of the autologous cartilage-alginate suspension (40-60 x 10 6 chondrocytes) were injected through the needle, while lifting of the ureteral orifice was endoscopically visualized.
  • autologous cartilage-alginate suspension 40-60 x 10 6 chondrocytes
  • the left ureteral orifice remained untreated and served as a control.
  • Serial cystograms, cystoscopy, and excretory urographic studies were performed at eight week intervals until sacrifice.
  • the mini-pigs were sacrificed at eight (1), 16 (1), and 26 (2) weeks after treatment.
  • the bladder injection sites were resected and examined macroscopically and microscopically. Specimens were stained with hematoxylin and eosin, and alcian blue at a pH of 1.0 and 2.5. Histological analyses of the bladder, ureters, regional lymph nodes, kidneys, liver, and spleen were performed.
  • Chondrocytes can be readily grown and expanded in culture. Neocartilage formation can be achieved in vitro and in vivo using chondrocyte cultured on synthetic biodegradable polymers. In these experiments, the cartilage matrix replaced the alginate as the polysaccharide polymer underwent biodegradation. Six mini-swine underwent bilateral creation of reflux. All six were found to have bilateral reflux without evidence of obstruction at three months following the procedure. Chondrocyte were harvested from the left auricular surface of each mini-swine and expanded to a final concentration of 50-150 X 10 6 viable cells per animal. The animals then underwent endoscopic repair of reflux with the injectable autologous chondrocyte-alginate gel solution on the right side only.
  • Cystoscopic and radiographic examinations were performed at two, four, and six months after treatment. Cystoscopic examinations showed a smooth bladder wall. Cystograms showed no evidence of reflux on the treated side and persistent reflux in the uncorrected control ureter in all animals. All animals had a successful cure of reflux in the repaired ureter without evidence of hydronephrosis on excretory urography. The harvested ears had evidence of cartilage regrowth within one month of chondrocyte retrieval.

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US20100196433A1 (en) * 2005-06-02 2010-08-05 Williams Stuart K Prevascularized devices and related methods
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