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|Publication number
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|Application number
|Publication date
|Nov 2, 1999
|Filing date
|Aug 28, 1997
|Priority date
|Apr 30, 1993
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|Anthony Atala
|Original Assignee
|Children's Medical Center Corporation
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|Patent Citations (7), Non-Patent Citations (30), Referenced by (29), Classifications (37), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of U.S. Ser. No. 08/228,678, entitled "Injectable Bladder Muscle Cells-Polymer Suspension for Treatment of Vesicoureteral Reflux" filed Apr. 18, 1994 by Anthony Atala, now U.S. Pat. No. 5,667,778, which is a continuation-in-part of U.S. Ser. No. 08/056,140 entitled "Injectible Polysaccharide Cells Compositions filed Apr. 30, 1993 by Keith T. Paige, Linda G. Cima, Charles A. Vacanti and Anthony Atala now U.S. Pat. No. 5,709,854.
The present invention is generally in the area of medical treatments, and specifically relates to a method for correcting vesicoureteral reflux, incontinence and other defects.
Vesicoureteral Reflux.
Vesicoureteral reflux is a condition wherein there is an abnormal development of the ureteral bud as it enters the bladder during embryologic 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. In addition, the constant back pressure of the urine into the calyces and renal pyramids results in mechanical damage to the renal parenchyma. If untreated, urinary vesicoureteral reflux can cause loss of renal parenchyma, and in some instances, renal failure, as reviewed by Atala and Casale, Infections in Urology 39-43 me (March/April 1990). In 1960, 70% of the patients with renal failure were described as having vesicoureteral reflux as the primary etiology. With the advent of new diagnostic and treatment modalities, patients with vesicoureteral reflux now account for less than 1% of the renal failure population.
In the past, 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.
The treatment of vesicioureteral reflux has been well established over the last decade. Initially it was believed that all patients with reflux would require surgery. Another school of management soon proposed that only medical therapy with antibiotics was required. It is now well established that the treatment of reflux depends on many factors, including the severity of reflux, associated congenital abnormalities, and the social situation of the child (parental compliance with medical treatment). Medical treatment is usually recommended for patients with grade 1 and 2 reflux, which usually resolve on their own as the bladder/ureteral configuration changes with growth. Grade 3 reflux is generally managed with medical therapy unless it persists or breakthrough infections occur while on antibiotic suppression. Surgical treatment is usually required for grade 4 and 5 reflux.
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. These patients require a general endotracheal anesthetic for a four to five hour surgery, an epidural catheter for both intraoperative and postoperative pain control, a bladder catheter for drainage, a perivesical drain, and a five to six day hospital stay. Antibiotic therapy and bladder antispasmodics are required post-operatively.
Although open surgical procedures for the correction of reflux have excellent results in the hands of experienced surgeons, it is associated with a well recognized morbidity, including pain and immobilization of a lower abdominal incision, bladder spasms, hematuria, and post-operative voiding frequency in some children. In an effort to avoid open surgical intervention, widespread interest was initiated by Matouschek's clinical experience with the endoscopic injection of Teflon™ (polytetrafluoroethylene) paste subureterally in 1984, as reported in Matouschek, E.: Die Behandlung des vesikorenalen Refluxes durch transueterale Einspritzung von polytetrafluoroethylenepast. Urologe, 20:263 (1981). With this technique, 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 Teflon™ paste is injected until the gaping ureteric orifice configuration changes into a half-moon slit. The Teflon™ paste, injected endoscopically, corrects the reflux by acting as a Ebulking material which increases ureteral resistance. However, soon after the introduction of this treatment, a controversy regarding the use of Teflon™ paste ensued. Malizia et al. Migration and granulomatous reaction after periurethral injection of polymer (polytetrafluoroethylene)" JAMA, 251:3277 (1984), showed granuloma formation and particulate migration to the brain, lungs, and lymph nodes in animal studies. Polytetrafluoroethylene migration and granuloma formation have also been reported in humans by Claes et al., "Pulmonary migration following periurethral polyetrafluoroethylene injection for urinary incontinence" J. Urol., 142:821 (1989). The safety of Teflon™ for human use was questioned, and the paste was thereafter banned by the FDA.
However, there are definite advantages in treating vesicoureteral reflux endoscopically. The method is simple and can be completed in less than fifteen minutes, it has a success rate of greater than 85% with low morbidity and it can be performed in an outpatient basis, as reported by Atala et al, "Endoscopic treatment of vesicoureteral reflux with a self-detachable balloon system" J. Urol. 148:724 (1992). The goal of several investigators has been to find alternate implant materials which would be safe for human use.
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, "Endoscopic injection of glutaraldehyde cross-linked bovine dermal collagen for correction of vesicoureteral reflux" J. Urol. 145:115 (1991). The collagen implant volume decreases with time, which results in high percentage of recurrence of reflux, over 90% within 3 years. The high failure rate with this substance presents a high risk to the unaware patient of developing renal damage after treatment.
A paste consisting of textured microparticles of silicone, suspended in a hydrogel, has been injected subureterally to correct reflux with an initial success rate of 91% in one European study, as reported by Buckley at al., "Endoscopic correction of vesicoureteric reflux with injectable silicone microparticles" J. Urol. 149: 259A (1993). However, distant particle migration has been observed in animal models, as reported by Henly et al., "Particulate silicone for use in periurethral injections: a study of local tissue effects and a search for migration" J. Urol. 147:376A (1992). Approximately thirty percent of the silicone particles have a diameter which is less than 100 μm. This suggests that thirty percent of the silicone particles have a potential for distant organ migration through the macrophage system. The manufacturer of this technology tried unsuccessfully to obtain FDA approval, and subsequently filed for bankruptcy.
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. However, 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.
Despite the fact that over a decade has transpired since the Teflon™ controversy, little progress has been made in this area of research. The ideal substance for the endoscopic treatment of reflux should be injectable, non-antigenic, non-migratory, volume stable, and safe for human use (Atala et al, 1992).
Urinary Incontinence.
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.
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). There are many popular treatments including weight loss, exercise, medication and in more extreme cases, surgical intervention. 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. For treatment of type 3 stress incontinence, there has been a recent trend toward injection of Teflon™ 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).
It is therefore an object of the present invention to provide a method and material for treating vesicoureteral reflux, incontinence, and other defects which results in a natural and permanent cure to the defect.
It is a further object of the present invention to provide a method and material for treating vesicoureteral reflux, incontinence, and other defects which is quick, simple, safe, and relatively non-invasive.
A method of treatment of vesicoureteral reflux, incontinence and other defects is described wherein bladder muscle cells are mixed with a liquid polymeric material, such as alginate which can be solidified in vivo, to form a cell suspension, which is injected into the area of the defect, in an amount effective to yield a muscle area that corrects the defect, for example, which provides the required control over the passage of urine.
As described in the examples, human bladder muscle specimens are obtained and processed within one hour after surgical removal. In the preferred embodiment, muscle cells are mixed with a biodegradable liquid polymer such as alginate, a biodegradable copolymer of gluronic and mannuronic acid, which is designed to solidify at a controlled rate when contacted with calcium salts. The cells are then injected at the desired site where they proliferate and correct the defect. Examples demonstrate efficacy in mice.
FIG. 1 is a schematic of injection of a polymer-muscle cell suspension into a region for control of vesicoureteral reflux.
A biodegradable polymer, embedded with muscle cells, serves as a synthetic substrate for the injectable delivery and maintenance of muscle architecture in humans that satisfies all the requirements for an ideal injectable substance. A biopsy of the bladder can be easily and quickly performed cystoscopically followed by muscle cell processing and endoscopic injection of the autologous muscle cell/polymer suspension for the treatment reflux, incontinence and other defects.
Studies show that muscle cells can be easily harvested and combined with alginate in vitro, the suspension can be easily injected cystoscopically and the muscle tissue formed is able to correct vesicoureteral reflux without any evidence of obstruction. The ideal injectable substance for the endoscopic treatment of reflux should be a natural bulking agent which is non-antigenic, non-migratory, and volume stable. Autologous muscle cells seem to fulfill all of these requirements. Since the muscle cells are autologous, this method of treatment does not require FDA approval. The procedure can be performed under 15 minutes, with a short period of a mask anesthetic, in the outpatient unit, without any need for a hospital stay. Neither vesical nor perivesical drainage is required. Since the whole procedure is done endoscopically and the bladder is not entered surgically, there is no postoperative discomfort whatsoever. The patient can return to a normal level of activity almost immediately.
Source of Cells
In the preferred embodiment, 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 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 immunosuppressed 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 obtained by biopsy are harvested and cultured, passaging as necessary to remove contaminating non-bladder muscle cells.
Polymer Solutions
In the preferred embodiment described herein, 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.
The polymeric material which is mixed with cells for implantation into the body should form a hydrogel. 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. Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are crosslinked jonically, or block copolymers such as Pluronics™ or Tetronics™, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively. Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen.
In general, 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. Examples of 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. 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 with basic side groups that can be reacted with anions are poly(vinyl amines), poly(vinyl pyridine), poly(vinyl imidazole), and some imino substituted polyphosphazenes. 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 dross-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. Pat. No. 4,352,883 to Lim. In the Lim process, 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): ##STR1## where n is an integer.
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 Ca2+ or Al3+. Polymers can be synthesized that degrade by hydrolysis by incorporating monomers having imidazole, amino acid ester, or glycerol side groups. For example, a polyanionic poly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized, which is cross-linked with dissolved multivalent cations in aqueous media at room temperature or below to form hydrogel matrices.
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. The term bioerodible or biodegrable, as used herein, 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. Examples of 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). For 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).
Methods for synthesis and the analysis of various types of polyphosphazenes are described by Allcock, H. R.; et al., Inorg. Chem. 11, 2584 (1972); Allcock, et al., Macromolecules 16, 715 (1983); Allcock, et al., Macromolecules 19, 1508 (1986); Allcock, et al., Biomaterials, 19, 500 (1988); Allcock, et al., Macromolecules 21, 1980 (1988); Allcock, et al., Inorg. Chem. 21(2), 515-521 (1982); Allcock, et al., Macromolecules 22, 75 (1989); U.S. Pat. Nos. 4,440,921, 4,495,174 and 4,880,622 to Allcock, et al.; U.S. Pat. No. 4,946,938 to Magill, et al.; and Grolleman, et al., J. Controlled Release 3, 143 (1986), the teachings of which are specifically incorporated herein by reference.
Methods for the synthesis of the other polymers described above are known to those skilled in the art. See, for example Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such as poly(acrylic acid), are commercially available.
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., R3 N+ -\/\/\/-+ NR3 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. The higher the 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. Examples of 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. These are commercially available. One polycation is poly(L-lysine); examples of synthetic polyamines are: polyethyleneimine, poly(vinylamine), and poly(allyl amine). There are also natural polycations such as the polysaccharide, chitosan.
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 SO3 H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups.
Cell Suspensions
Preferably 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 bladder muscle 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.
Injection of Cells
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, Mass. 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.
In addition to its use for the endoscopic treatment of reflux, 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.
As described herein, an injectable biodegradable polymer as a delivery vehicle for muscle cells is useful in the treatment of reflux and incontinence. In the preferred embodiment, a bladder muscle biopsy is obtained under anesthesia from a patient with vesicoureteral reflux, the isolated muscle cells are mixed with alginate, and the muscle cell-alginate solution is injected endoscopically in the sub-ureteral region to correct reflux, as shown in FIG. 1. The time to solidification of the alginate-cell solution may be manipulated by varying the concentration of calcium as well as the temperature at which the muscle cells are added to the alginate. The use of autologous bladder muscle cells 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. In addition to the use of the muscle cell-polymer suspension for the treatment of reflux and 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.
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. 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. An example of this would be an injection into the area surrounding the skull where a bony deformity exists secondary to trauma. 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 would allow for placement or injection of this substance either by vascular access or percutaneous access to specific organs or other tissue regions in the body, wherever a bulking agent would be required.
Further, this substance could be injected through a laparoscopic or thoracoscope to any intraperitoneal or extraperitoneal or thoracic organ. For example, 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.
The present invention will be further understood by reference to the following non-limiting examples. The examples demonstrate that human bladder muscle cell-alginate suspensions are injectable, non-migratory, and appear to conserve their volume and that autologous bladder muscle cells are useful in the endoscopic treatment of vesicoureteral reflux.
Cell Culture:
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 cm2 to four cm2. 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. When cultures were 80% confluent, the cells were trypsinized and passaged. Cell populations highly enriched in elongated, striated cells were routinely obtained using this method. Cell strains were stained with a-actin antibody to verify their muscle phenotype.
Results. Populations of fusiform, striated cells could be obtained from the bladder biopsy specimens by dissection of the muscle layer followed by explant culture of the muscle fragments in DMEM supplemented with 10% calf-serum. Immunostaining with a monoclonal antibody which specifically recognized a-actin verified the muscle phenotype. These data indicate that highly proliferative populations of smooth muscle can be obtained from small biopsy specimens. The muscle cells can also be grown transiently in serum-free defined media which renders the cells free from any impurities.
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 (alginate alone or muscle cells alone) there were no muscle cells evident. Histologic analysis of distant organs showed no evidence of bladder muscle cells or alginate migration or granuloma formation.
Materials and Methods
Animal model of vesicoureteral reflux. 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. Surg. 88:753 (1991).
Three months after the procedure, the presence of bilateral reflux was assessed by conventional radiographic cystography using an iodinated contrast agent, and by sonography using sonicated albumin, as described by Vacanti, et al., "Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes" Mat. Res. Soc. Proc. 252:367 (1992). Excretory urography was performed to detect any evidence of obstruction.
Cell Harvest. A segment of bladder muscle was obtained from each animal. Muscle cells were harvested and plated separately in vitro. After expansion, the cells were individually quantitated and concentrated to 20�106 cells per cc.
Autologous bladder muscle cell-calcium alginate suspension. Two percent weight/volume sodium alginate (0.1 M K2 PO4, 0.135 M NaCl, pH 7.4, Protan, Portsmouth, N.H.) was made and sterilized in ethylene oxide. A 1.5 ml aliquot of 20�106 cells/ml bladder muscle cell suspension was added to an equal volume of sodium alginate solution for a final alginate concentration of 1%. The bladder muscle cell-sodium alginate suspension was kept at 32� C. Immediately prior to injection, calcium sulfate (0.2 g/ml) was added to the bladder muscle cell-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.
Experimental study. 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. Approximately 2 to 3 ml of the autologous bladder muscle cell-alginate suspension (40-60�106 bladder muscle cells) were injected through the needle, while lifting of the ureteral orifice was endoscopically visualized. 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.
Four mini-swine underwent bilateral creation of reflux.
All four were found to have bilateral reflux without evidence of obstruction at three months following the procedure. Bladder muscle cells were from each mini-swine and expanded in vitro. The animals then underwent endoscopic repair of reflux with the injectable autologous bladder muscle cell-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.
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.
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.
Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.
|Cited Patent
|Filing date
|Publication date
|May 24, 1984
|Jun 16, 1987
|Connaught Laboratories Limited
|Microencapsulation of living tissue and cells
|Apr 17, 1989
|Aug 20, 1991
|Massachusetts Institute Of Technology
|Neomorphogenesis of cartilage in vivo from cell culture
|Apr 29, 1988
|Oct 1, 1991
|Samuel Itay
|Compositions for repair of cartilage and bone
|Jan 2, 1992
|Mar 15, 1994
|Southwest Research Institute
|Composition and method of promoting hard tissue healing
|Apr 22, 1993
|Aug 9, 1994
|Robert A. Ersek
|Treatment of urological and gastric fluid reflux disorders by injection of mmicro particles
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|Chitosan matrices for encapsulated cells
|Jan 27, 1994
|Sep 29, 1994
|A composition and a method for tissue augmentation
|Atala et al. "Injectable Alginate Seeded With Human Bladder Muscle Cells As A Potential Treatment For Vesicoueteral Reflux", American Urological Association, Eighty-Ninth Annual Meeting, May 14-19, 1994 and Journal of Urology 151:362 A (May 1994).
|Atala et al. Injectable Alginate Seeded With Human Bladder Muscle Cells As A Potential Treatment For Vesicoueteral Reflux , American Urological Association, Eighty Ninth Annual Meeting, May 14 19, 1994 and Journal of Urology 151:362 A (May 1994).
|Atala, Anthony and Anthony J. Casale, "Management of Primary Vesicoureteral Reflux," Infections in Urology 39-43 (Mar./Apr. 1990).
|Atala, Anthony and Anthony J. Casale, Management of Primary Vesicoureteral Reflux, Infections in Urology 39 43 (Mar./Apr. 1990).
|Atala, Anthony, et al. "Endoscopic treatment of vesicoureteral reflux with a self-detachable balloon system" J. Urol. 148-724 (1992).
|Atala, Anthony, et al. Endoscopic treatment of vesicoureteral reflux with a self detachable balloon system J. Urol. 148 724 (1992).
|Atala, Anthony, et al., "Endoscopic Treatment of Reflux with Autologous Bladder Muscle Cells," American Academy of Pediatrics meeting held in Dallas, Texas on Oct. 23, 1994, Abstract.
|Atala, Anthony, et al., "Injectable Alginate Seeded with Chondrocytes as a Potential Treatment for Vesicoureteral Reflux," Annual Meeting of the Section on Urology, American Academy of Pediatrics, Oct. 10-15, 1992, subsequently published in the Journal of Urology, 150:745-7477 (Aug. 1993), Abstract.
|Atala, Anthony, et al., "Laparoscopic correction of vesicoureteral reflux" J. Urol. 150:748 (1993).
|Atala, Anthony, et al., Endoscopic Treatment of Reflux with Autologous Bladder Muscle Cells, American Academy of Pediatrics meeting held in Dallas, Texas on Oct. 23, 1994, Abstract.
|Atala, Anthony, et al., Injectable Alginate Seeded with Chondrocytes as a Potential Treatment for Vesicoureteral Reflux, Annual Meeting of the Section on Urology, American Academy of Pediatrics, Oct. 10 15, 1992, subsequently published in the Journal of Urology, 150:745 7477 (Aug. 1993), Abstract.
|Atala, Anthony, et al., Laparoscopic correction of vesicoureteral reflux J. Urol. 150:748 (1993).
|Buckley, J. F., et al., "Endoscopic correction of vesicoureteric reflux with injectable silicone microparticles" J. Urol. 149 Abstract 259A (1993).
|Buckley, J. F., et al., Endoscopic correction of vesicoureteric reflux with injectable silicone microparticles J. Urol. 149 Abstract 259A (1993).
|Claes, H. et al., "Pulmonary migration following periurethral polytrafluoroethylene injection for urinary incontinence" J. Urol., 142:821 (1989).
|Claes, H. et al., Pulmonary migration following periurethral polytrafluoroethylene injection for urinary incontinence J. Urol., 142:821 (1989).
|Contemporary Urology (Mar. 1993).
|Henly, David R., et al., "Particulate silicone for use in periurethral injections: a study of local tissue effects and a search for migration" J. Urol. 147 Abstract 376A (1992).
|Henly, David R., et al., Particulate silicone for use in periurethral injections: a study of local tissue effects and a search for migration J. Urol. 147 Abstract 376A (1992).
|Jones, Exp. Neurol. 66(3):602 610 (1979).
|Jones, Exp. Neurol. 66(3):602-610 (1979).
|Leonard, Michael P. et al. "Endoscopic injection of glutaraldehyde cross-linked bovine dermal collagen for correction of Vesicoureteral reflux" J. Urol. 145:115 (1991).
|Leonard, Michael P. et al. Endoscopic injection of glutaraldehyde cross linked bovine dermal collagen for correction of Vesicoureteral reflux J. Urol. 145:115 (1991).
|Malizia, Anthony A., et al. "Migration and granulomatous reaction after periurethral injection of polymer (polytetrafluoroethylene)" JAMA , 251:3277 (1984).
|Malizia, Anthony A., et al. Migration and granulomatous reaction after periurethral injection of polymer (polytetrafluoroethylene) JAMA , 251:3277 (1984).
|Matouschek, E.: Die Behandlung des vesikorenalen Refluxes durch transueterale Einspritzung von polytetrafluoroethylenepast, Urologe, 20:263 (1981).
|Page, Keith T., et al., "De Novo Cartilage Generation Utilizing Calcium Alginate-Chondrocyte Constructs," 1993 Plastic Surgery Research Council meeting held in Houston, Texas between Apr. 28, 1993 and May 1, 1993, Abstract.
|Page, Keith T., et al., De Novo Cartilage Generation Utilizing Calcium Alginate Chondrocyte Constructs, 1993 Plastic Surgery Research Council meeting held in Houston, Texas between Apr. 28, 1993 and May 1, 1993, Abstract.
|Yarom et al., Virchows Arch B Cell Pathol. Incl. Mol. Pathol. 41(1 2):171 180 (1982).
|Yarom et al., Virchows Arch B Cell Pathol. Incl. Mol. Pathol. 41(1-2):171-180 (1982).
|Citing Patent
|Filing date
|Publication date
|Aug 13, 2001
|Mar 9, 2004
|Promethean Surgical Devices Llc
|Situ bulking device
|Apr 9, 2002
|May 23, 2006
|Promethean Surgical Devices Llc
|Treatment for Gastroesophageal disease
|Mar 22, 2005
|Dec 18, 2007
|Promethean Surgical Devices
|Treatment for gastroesophageal disease
|Aug 9, 2001
|Oct 27, 2009
|Temple University - Of The Commonwealth System Of Higher Education
|Obesity controlling method
|Apr 29, 2003
|Jun 15, 2010
|Temple University Of The Commonwealth System Of Higher Education
|Obesity controlling method
|Jan 29, 2009
|Sep 20, 2011
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Feb 13, 2009
|Dec 20, 2011
|The Cleveland Clinic Foundation
|Molecular enhancement of extracellular matrix and methods of use
|Feb 27, 2009
|Mar 20, 2012
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Jan 29, 2009
|Mar 20, 2012
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Sep 15, 2008
|Jun 26, 2012
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Apr 2, 2013
|The Cleveland Clinic Foundation
|Methods to treat urinary incontinence
|Nov 5, 2010
|Aug 5, 2014
|Spotlight Technology Partners Llc
|Fragmented hydrogels
|May 16, 2006
|Apr 7, 2015
|Tigenix, S.A.U.
|Use of adipose tissue-derived stromal stem cells in treating fistula
|Jul 23, 2014
|Mar 22, 2016
|Spotlight Technology Partners Llc
|Hydrogel compositions
|Aug 15, 2012
|Aug 16, 2016
|Wake Forest University Health Sciences
|Muscle tissue regeneration using muscle fiber fragments
|Apr 19, 2002
|Aug 5, 2004
|Rainer Marksteiner
|Production and use of a suspension composition comprising an ultrasound contrast medium
|Mar 22, 2005
|Nov 10, 2005
|Milbocker Michael T
|Treatment for gastroesophageal disease
|Jun 24, 2005
|Mar 2, 2006
|Universidad Autonoma De Madrid Ciudad Universitaria de Cantoblanco
|Use of adipose tissue-derived stromal stem cells in treating fistula
|Mar 29, 2004
|Nov 23, 2006
|Regentec Ltd.
|Porous matrix
|Jun 5, 2008
|Dec 18, 2008
|Anna Gosiewska
|Tissue Fragment Compositions for the Treatment of Incontinence
|Sep 15, 2008
|Feb 12, 2009
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Jan 29, 2009
|Jun 4, 2009
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Jan 29, 2009
|Jun 4, 2009
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Feb 13, 2009
|Aug 13, 2009
|The Cleveland Clinic Foundation
|Molecular enhancement of extracellular matrix and methods of use
|Feb 27, 2009
|Oct 8, 2009
|The Cleveland Clinic Foundation
|Hydroxyphenyl cross-linked macromolecular network and applications thereof
|Nov 5, 2009
|The Cleveland Clinic Foundation
|Compositions and methods to treat urinary incontinence
|Dec 28, 2009
|Nov 25, 2010
|Lifecore Biomedical, Llc
|Stress urinary incontinence treatment
|Aug 15, 2012
|Jun 25, 2014
|Wake Forest University Health Sciences
|Muscle tissue regeneration using muscle fiber fragments
|Jun 5, 2008
|Dec 24, 2008
|Ethicon, Inc.
|Tissue fragment compositions for the treatment of incontinence
|U.S. Classification
|424/93.7, 424/423
|International Classification
|Cooperative Classification
|European Classification
|Mar 15, 1999
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Free format text: RELEASE OF SECURITY INTEREST;ASSIGNORS:ESSEX WOODLANDS HEALTH VENTURES FUND, L.P.;H&Q HEALTHCARE INVESTORS;PIEDMONT VENTURE PARTNERS, LIMITED PARTNERSHIP;AND OTHERS;REEL/FRAME:010539/0060
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text:
Jim Giacomazzi, who guided West Texas A&M to the 1991 NCAA Division II national volleyball title, has been named head volleyball coach at Wayland Baptist.
Giacomazzi, who resides in Canyon, will be introduced at 1:30 p.m. today at the Hutcherson Center in Plainview.
Also, Brian Whitlock has been promoted from assistant track coach to head track coach. He is also the cross country coach.
- Globe-News Staff
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text:
Now Playing
Teaching 2.0: Is Tech In The Classroom Worth The Cost?
The growth of tech and Internet use in schools has brought one unexpected benefit: Kids can get lectures from high-level scholars via Skype, and interact with them over the Internet. This opens up a larger question, though, of how to measure if the new technologies are improving learning.


text:
Anton van der Gaag wrote:
> What am I doing wrong?
You're trying to delete something that can't be deleted!
> And how can I revoke a key that I do not use anymore?
What have you done so far and what software are you using?
Do you think people have crystal balls here?
But generally just import you revocation certificate for the key in question
and then upload the key back to the PGP network.
Jabber: email@example.com
PGP ID: 0x304563A8


text:
Development: How to avoid the TextCha on the Wiki / live.gnome.org
To avoid needing to fill in that TextCha, make sure your wiki name is listed on the following page: https://live.gnome.org/TrustedEditorGroup Anyone on the page can add new people, so just ask someone on there to add you. I added a bunch of names to the initial page (from RecentChanges). In case you're are not listed the old rules still apply: When editing the wiki please do the following: Fill in the word 'gnome' at the top of the page! This is called a TextCha and I added it to avoid all the spammers. Unfortunately the wiki doesn't authenticate any email addresses, so this crappy solution is the best I could figure out. If this stops the scripts, I'll work on relaxing the TextCha (allowing people to add themselves to a page to avoid the TextCha).
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text: Can not complete authoring process due to some error in Win DVD creator 2 I get this message- "Can not complete authoring process due to some error" In Win DVD Creator 2 version. Now is this problem a hardware problem? Software Problem ? DVD-R media problem ? I am using Maxell DVD + r disk . HP DVD Burner 400C combo cd+rw dvd+rw burner on a Compaq Presario Xp Computer. Compaq Presario SR 1030NX What could be the problem? Thanks, Bun Mui


text:
This comparative literature panel focuses on intersections of mirror(ing), gender AND race, in comparative approaches to contemporary literature in languages other than English. Comparative approaches of contemporary literature on race AND gender in languages other than English are preferred (life writing, novel, testimony, poetry and hybrid texts welcome).
Discussions revolving around psychoanalysis and the mirror; coping with trauma and witnessing; the role of the mirror in fashioning racialized or undoing stigmatized identities; mirrors in life writing;(m)othering and mirroring;GLBTA identities and the mirror;reflections and postcolonial or decolonial literary criticism and feminisms;reflections on non-Western practices of healing and therapy, etc, are all welcome.
Submit a 300-word abstract, with title and five keywords, and mini-bio (100 words) to Sarah Soanirina Ohmer, firstname.lastname@example.com.
Please include with your abstract:
Name and Affiliation
Email address
Postal address
Telephone number
A/V requirements (if any; $10 handling fee with registration)
44th Annual Convention, Northeast Modern Language Association (NeMLA)
March 21-24, 2013
Boston, Massachusetts
Host Institution: Tufts University
The Convention will be located centrally near Boston Commons and the Theatre District at the Hyatt Regency.
Interested participants may submit abstracts to more than one NeMLA session; however,panelists can only present one paper (panel or seminar). Convention participants may present a paper at a panel and also present at a creative session or participate in a roundtable.


text:
Uzza means "strength" and is a fallen angel.
Check Out Our Top 10 Demon Movies You Must See!
Demon: Uzza
Back to Demon Names starting with "U"
Demons Total: 372
Hell Horror takes no responsibility for any views expressed about these spirits/gods and casts no judgement about any one's specific religion/beliefs. This page is meant strictly as reference material.


text:
After guiding the Central High School girls varsity basketball program for just one season, Mike Horton has decided to step down.
Horton led Central to an 11-9 record and a berth in the District 6-AA playoffs last February. His resignation, he said, had nothing to do with personnel issues.
He said he enjoyed his time coaching the players and his relationships with the players' parents. He also said he got good support and cooperation from his employer, Smith Transport of Roaring Spring, where he works in the safety department.
"The players were great, the parents and my employer were very supportive,'' Horton, 54, said. "There isn't one specific reason (for the resignation). There are a lot of small reasons - none that have to do with the players or their parents.
"I've spoken to the players, and I'll leave what I said to them between myself and the players,'' Horton added. "I've also had a few health concerns in the last couple weeks that put my decision to resign over the edge.''
Horton is happy with what was accomplished during his coaching tenure in the Central program, which included two years as the junior high school girls head basketball coach and one year as an assistant varsity coach before he took over as the varsity coach last winter.
"I think we left the program in better shape than when I began four years ago,'' said Horton, a 1979 graduate of Central.
Central's policy when there is a resignation at the top coaching position is to open all the coaching staff positions from the varsity down through the junior high school levels.
Horton hopes that his varsity assistant coach last year, Rob Mock, gets considered for the head coaching position.
"I hope the school board and administrators give Rob Mock serious consideration,'' Horton said. "I worked with him both at the junior high and high school levels, and he was very valuable to me and will continue the positive approach that we both believe in.''
Central athletic director Chuck Gojmerac lauded Horton for his contributions to the girls basketball program.
"He did a fine job, and he was really dedicated to, and had a true commitment to, the sport of basketball,'' Gojmerac said of Horton. "We'll probably start advertising the position later this week, and we'll keep the application process going for about a two-week period. We would like to have a new head coach in place by the September (school) board meeting.''
Horton will continue to root for the Lady Dragons no matter who takes over the head coaching role.
"I am very thankful for the time that I shared with them, and I will be their biggest fan,'' he said.


text:
February 18, 2013, 10:08 PM
Local pawn shop has an Iver Johnson single action 44-40 for sale. has some bluing wear but the action is tight. They are asking $350 but might go less... Does anyone have one of these and if so what can you tell me about them? Good guns? junk?


text:
Separate names with a comma.
Discussion in 'Rants & Raves' started by CHEZ, Mar 18, 2012.
has somebody decided to do the spring post cleaning?
twice they have been cleared today
If you jump from device to device the forum gets confused and wipes out to days posts.
Lol @ Chez
Scott's right
no scott! been at vhome on laptop all day! helping a lad out! never turned it off or vlogged out! v dodgey


text:
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text:
There are no other The Ascott Limited properties in this city
Because life is about living
Europa-Allee 23
60327 Frankfurt am Main, Germany
A stay in Apart'hotel Citadines City Centre Frankfurt is a wonderful opportunity to immerse yourself in the heart of the major crossroads that is Frankfurt am Main. Nicknamed Mainhattan, the city proudly displays its skyscrapers, which are dominated by the Commerzbank Tower, symbol of its economic prestige. This is a city that thinks big and climbs high, which is also the best way to visit it!
Don't miss: Ascend the Main Tower, which reaches a height of around 200m. Set on the 56th floor, its terrace offers a breath-taking panoramic view of the city and surrounding areas.
Just west of the City Centre, the Apart´Hotel is situated near the Messe Frankfurt, the Concert Hall "Festhalle" and the Congress Center Kap Europa. This is the ideal place to go whether you're in the city on business or on a leisure break. Just opposite is the Skyline Plaza shopping centre with its 170 shops, restaurants, a relaxing roof garden and the Meridian Spa. Also close by is Frankfurt Central Station, connecting Frankfurt with major German towns and cities and European capitals.
Our reception is open 24 hours a day. The serviced apartments hotel has a car park (charges apply), a meeting room, gym and breakfast room, as well as a business corner.
Frankfurt has a modern and original cultural identity. Visit the amazing Palmengarten botanical gardens and the Tropicarium. And don't miss the town hall, known as the"Römer", located on the Römerberg square, home to many fairs and the Christmas market.
Take a walk along the "Museum embankment" and discover the masterpieces of the Städel Museum, one of Germany's largest and most famous art museums.
If you like to cook, don´t miss the Kleinmarkthalle: this food market is bustling and full of stalls selling quality food from all over. A must: taste Mrs Schreiber´s Fleischwurst!
At the end of the day, head to the top floor of the Zeilgalerie to admire the view over the town.
Whether you're on a trip for business or pleasure, the Apart´Hotel offers accommodation with all mod cons to meet your every need, from the classic rooms to studios, 2 or 3-room apartments, all modern and practical in design. All types include a fully-equipped kitchenette with the exception of the classic room, air conditioning, a TV with cable channels and free high-speed internet.


text:
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Sudoku Epic
Sudoku Epic Full Description
Sudoku puzzle game and solver for Windows. Hint features include scan and markup
Full Description
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29, October 2013
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Product Info
Sudoku Epic is a Windows version of the famous game, sudoku. You can generate puzzles in sizes from 4x4 to 16x16 and difficulties from very easy to very hard. The Assistant can help you with scanning, markup and revealing certain numbers, allowing you to focus on the logic. You can also enter puzzles from newspapers and solve them, or see the solution. Additionally you can store puzzles and continue solving them at a later time.
What's new in current version
Some improvements and bug fixes.
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if you have questions or issues regarding this product.
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Sudoku Dream
Sudoku puzzle game and solver for Windows. Hint features include scan, markup and reveal cell. You can also enter puzzles from newspapers and solve them, or see the solution. Sizes from 4x4 to 16x16 and difficulties from very easy to very hard.
Kakuro Dream
Kakuro puzzle game and solver for windows. Hint features include scan, markup and reveal cell. You can also enter puzzles from newspapers and solve them, or see the solution. Sizes from 6x6 to 14x14 and difficulties from easy to hard.
Kakuro Epic
Kakuro puzzle game and solver for windows. Hint features include scan, markup and reveal cell. You can also enter puzzles from newspapers and solve them, or see the solution. Sizes from 6x6 to 14x14 and difficulties from easy to hard.
SUDOKUjm will let you generate, edit, create unlimited sudoku puzzles. It will also help you solve those or any other puzzles with user-friendly and intuitive steps.
A humanistic Sudoku solver that explains the logic required to solve the puzzle. Will easily create and quickly solve Sudoku puzzles. It can act as a solving assistant by keeping the pencil marks accurate, but leaving the logic and fun to you.
Sudoku Studio
Sudoku Studio will let you enter your own Sudoku puzzle, or it can generate a new one for you at various levels of difficulty. Then race against time or your friends to solve the puzzle and get a high score. Sudoku Studio can solve any Sudoku puzzle.
Sudoku Puzzles Ace
Sudoku Puzzles Ace is a new program based on Sudoku - a Japanese numbers game initially introduced in 1986. The program is able to generate millions of different random Sudoku puzzles and has "Hint" and "Solve" features to ease the solution process.
How quickly can you decode the popular number puzzle?
3D SuDoku Pro
This version has OpenGL compatible 3D graphics and sounds which make the gameplay more pleasant than ever. The game has such features as hints, mistake spotting and it automatically saves the game for you whenever you exit and loads it when you start
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ProEar Website
Welcome to www.MiraCellProEar.com! We are currently making updates and expect to have even greater functionality soon.
Our current Tradeshow schedule is available under the Event Calendar link.
To find a Hearing Professional who carries ProEar, check the store locator, under the 'Buy ProEar' tab on the top menu. If your business listing is incorrect or missing, just give us a call and we'll correct it.
Watch for additional improvements over the coming weeks.
What is ProEar?
ProEar is a carefully selected blend of plant extracts that provides fast relief for irritated, itching ears while providing the nutrients your ear needs to restore and maintain healthy tissue. It contains vitamins (A, E, D3) and other nutrients that nourish the ear. It quickly absorbs into the ear tissue, so you never have any greasy, oily residue to clean off. Everything in it is a food grade ingredient because you're 'feeding' your skin with good ingredients.
New Name for MiraCell
Introducing ProEarTM! You knew it as MiraCell, but it's now relabeled at ProEarTM to avoid confusion with other company products. MiraCell, Inc has expanded it's product line with a full set of skin care products. We are pleased to announce that we now have Hand & Food Support Cream, Serious Skin Support Cream, Scar & Stretch Mark Support Cream, Lip Calm (in Natural, Pina Colada, Mint and Berry) and Lip Support (Rich in L-Lysine).
Don't worry, even though 'MiraCell' is now renamed as ProEarTM, it's still the same great product, with the best set of ingredients we could find to put in a bottle.


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Date of last delinquency
The date when your credit was last contractually delinquent for one or more months
The date of last delinquency shows up on your credit report. Even if you make a partial payment of a debt, the creditor can still report you to a credit bureau as delinquent.
See: Delinquency
Money owed to an individual or a business
See: Acceptable debt
A legal document that transfers ownership (title) of a property
See: Grant deed, Quitclaim deed
Deed of trust
A document that gives the deed of the property to the lender until the buyer has paid the loan in full
In many states, a deed of trust is used in place of a mortgage. Essentially a deed is like a mortgage except that a deed of trust involves a third party called a trustee, usually a title insurance company. A deed of trust transfers the title (ownership) of the property to the trustee while you still retain the right to live in and use the property. The lender or trustee holds the original deed of trust until you repay the loan. Unlike a mortgage, a deed of trust also gives the lender the right to foreclose on your property without taking you to court first.
Compare: Mortgage
When the borrower fails to uphold his part of the agreement
Default happens when a home owner fails to comply with the terms of the note and mortgage such as failing to make payments on the mortgage or keep the property insured.
See: Foreclosure
Deficiency judgement
The amount for which a borrower is personally responsible for if a foreclosure sale does not cover the total amount owed on the mortgage
If you somehow default on a mortgage, the lender may foreclose on your property, but if the amount the house sells for does not cover the balance on the loan (including any fees) the lender may also be able to sue you. Only some states give the lender the right to a deficiency judgment.
See: Judicial foreclosure, Trustee's sale
Delayed adjustable rate mortgage
An ARM that has a longer initial period than usual
Delayed adjustable rate mortgages (Delayed ARMs) have a fixed initial interest rate after which adjustments are made, usually annually, for the life of the loan. A delayed ARM gives you fixed monthly payments for a longer period of time than traditional ARMs. Often, people choose this type of loan if they plan to sell the property before the ARM starts to fluctuate. Delayed ARMs are also called intermediate ARMs.
See: Adjustable rate mortgage
Being behind on paying back a creditor
If you've missed one or more payments on any debt, your account is considered delinquent.
See: Date of last delinquency
A sum of money that a buyer gives to the seller when making an offer on a home
To a seller, a deposit shows that the buyer is serious about buying the property. A deposit is not required by law. It is smart to put down as little as possible because it is possible the deposit may be lost if the deal falls through due to fault on the part of the buyer. If the deal works out, your deposit is then applied to your total closing costs to purchase the home. The deposit is also called earnest money.
Deposit receipt
A document that can perform as both the receipt for a deposit and the purchase agreement
Some states use a deposit receipt to outline a buyer's offer on a home, including the description of the property and how it will be financed along with how the deposit money is handled in the event the deal breaks down. If the seller accepts the offer and signs the document, the deposit receipt becomes the legal purchase agreement for the deal. The deposit receipt is also called a sales contract.
See: Deposit
Discount point
An exchange, where by, more is paid in closing costs to receive a lower interest rate on the loan
The basic idea of discount points is to pay up-front in order to save over the life of the loan. One discount point equals one percent of the loan amount. So, if you pay 2 discount points on a $200,000 loan you would pay $4,000 up-front at closing. Each discount point you pay will typically lower your loan's rate by .25%, but the amount can vary based on term, loan amount, fixed or ARM etc. Discount points may be a good idea if you plan to hold onto your home for a long period of time. This allows you to offset the costs of paying for the points. Some sellers will pay some of the discount points as a way to make their homes more attractive to buyers.
Discount points lower your interest rate. The longer that you hold a mortgage, the longer you will enjoy the savings of the lower interest rate.
See: Point, Origination fee, Buydown
Disposable income
Your net income after taxes and regular monthly bills is the income you are able to spend
To calculate your disposable income, start with your salary and subtract taxes and any regular monthly bills you pay.
Down payment
The portion of the house purchase price that the buyer agrees to pay up front with personal funds
A required 20% down payment is standard among lenders. There are financing options to assist you in paying the down payment. Three common choices are: (1) mortgage insurance, where you pay a fixed monthly premium in return for a lower down payment; (2) government-insured loans that let you put down less, but limit how much you can borrow; and (3) piggyback loans, which require a 10% down payment. You get 2 loans, one for 80% of the purchase price and the other, usually at a higher rate, for 10% of the purchase price.
See: Mortgage Insurance (MI)
Due-on-sale clause
Part of a loan agreement that gives a lender the right to demand repayment of a mortgage when the property is sold
Lenders include a due-on-sale clause in a mortgage to prevent buyers from taking over a seller's existing mortgage. A due-on-sale clause is a type of acceleration clause.
See: Acceleration clause, Assumable mortgage


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Sunovion specializes in improving lives of patientsSunovion science revolves around our patients. Rethinking the way medicines are engineered. With a specialized focus on two treatment areas.
Central Nervous System (CNS) disorders can affect people at almost any stage of life. This is why there's a real and urgent need for more treatment options. See what we're working on.
More and more Americans are living with respiratory diseases—among them, asthma, allergies and chronic obstructive pulmonary disease (COPD). Find out what we're doing to help.


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Elsie's Pave #Heart Key #Necklace
Elsie's Pave Heart Key Necklace – Final Sale
Designer inspired Elsie's pave heart key necklace is our romantic key pendant yet. The fine sterling silver chain holds a large key which has pave set CZs covering the entire front surface.
The top of the key features a flower/clover shape made from three heart outlines clustered together. Pave set CZs also accent the pendant's bale for even more sparkle.
Save an extra 25% off final sale items simply by using the code – AFFFEBCLR25 – at checkout. Offer ends February 28, 2013.


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Aid Through Trade was founded in 1993 by former Peace Corps volunteer and teacher, Damian Jones. While in the Peace Corps, Damian became inspired by the artisans of the Kathmandu Valley and throughout the country. He also had always been intrigued by the use of glass beads in the Nepalese culture.
Upon his return to the United States, Damian began to conceive the idea of working with artisans in a fair, dignified manner as a way to improve their lives. In 1993 and 1994 he took three trips back to Nepal to actualize his vision of "fair trade" as a path to sustainable economic and personal development for artisans.
In 1993, he also began the U.S. marketing and in 1994 Aid Through Trade is proud to have become a founding member of the Fair Trade Federation.
In Nepal, Damian visited many artisans and artisan groups in various parts of the country. He observed that women's lives changed dramatically when they had an income.
Shortly after starting Aid Through Trade, it became clear that the "sustainable" part of the equation would only come through design (continual design and re-design). Aid Through Trade shifted its emphasis to becoming a fashion design company with ethics!
Aid through Trade is recognized in the industry for its signature glass beaded Roll-On™ bracelet and incomparable color combinations. The artisans delight in coming to the exclusive studio and office in Nepal where they enjoy prestige, sisterhood, dignity, fair remuneration, a retirement account and other benefits.


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The first month of 2013 is officially over. Since I'm trying to keep better track of what I play and buy, I thought it'd be good to summarize the month. So here we go:
Games Beaten
-Kirby Mass Attack (DS)
I started Mass Attack last summer and then got distracted by other things. There are more medals hidden in the levels that I could look for but I'm done with it for now. Good game.
-Radiant Historia (DS)
I can't say enough good things about this RPG. The time travel plot is engaging and mostly free of JRPG clichés, the battle system is fun and strategic, and the soundtrack by Yoko Shimomura is phenomenal. If you like the 16-bit RPGs of yore or just a darn good game, make sure to check this one out.
-Shinobi (3DS)
I had a lot of fun with this one. I've been slowly putting a review together, so I won't say too much now. Hopefully I'll have it done to post soon.
-Double Dragon Neon (PSN)
Played through this with my wife. It wasn't her favorite kind of game but she stuck with me and turned out to be pretty good at it, keeping up with me fairly easily. We beat the Normal difficulty and while I'd love to try the next one (you can keep your character stats as you go), I'm not gonna ask my wife to play it again right now. It's a fun game, though, and doesn't take itself seriously at, a definite plus is this case. It has a lot of fun 'cause the '80s were rad!
Games Bought
This is the part I'm trying to cut down on. Hopefully there will be fewer games to list here in the coming months or my goal is sunk. Some of these are because I'm a PlayStation Plus member—namely Darksiders and Mega Man 9 and 10 and I got The Unfinished Swan and NiGHTS for 75% off—and most of the others came from a splurge at the start of the month. I'm not planning on buying anything else till BioShock Infinite comes out. We'll see in a month's time if I made it.
-Baldur's Gate: Dark Alliance (PS2)
-Puzzle Quest: Galactrix (DS)
-Robocalypse (DS)
-Spectrobes (DS)
-Assassin's Creed (360)
-Dynasty Warriors 7 (360)
-Halo: Anniversary (360)
-Darksiders (PS3)
-Mega Man 9 (PSN)
-Mega Man 10 (PSN)
-NiGHTS into Dreams (PSN)
-The Unfinished Swan (PSN)
-Mario Kart 7 (3DS)
-Guild Wars 2 (Mac)
Now Playing
-Resident Evil 5 (PS3)
Playing this online with my brother. We just found Jill so I think we must be nearly done.
-Guild Wars 2 (Mac)
I'm playing this with my wife. I doubt we'll ever be done with it but I'm really enjoying it, which is surprising because I'm not a big fan of MMOs.
-Mario Kart 7 (3DS)
This came preloaded on my awesome 3DS XL. I've played a good and will keep going for quite a while, I'm sure. It's the perfect game to have available on the system all the time.
-Kid Icarus: Uprising (3DS)
Picked this back up this month after a long hiatus. Again, I think I'm close to the end (just destroyed the Reset Bomb depot, for those of you who've played it) so I hope to finish it soon. I like this game a lot but man, the ground combat controls are wonky.
-Jeanne D'Arc (PSP)
I started this tactical RPG just this week. I like it so far—fantastical history setting (the Hundred Years War), good writing, nice graphics, no perma death—but I'm not very far in yet. My save file tells me I'm in Chapter 2, story mission #8. I don't know how many chapters or missions there are but I've heard it's a pretty short game, as far as these sorts usually are anyway, and that suits me just fine.