CA1308033C - Injectable soft tissue augmentation materials from the placenta and their method of manufacture - Google Patents
Injectable soft tissue augmentation materials from the placenta and their method of manufactureInfo
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- CA1308033C CA1308033C CA000562773A CA562773A CA1308033C CA 1308033 C CA1308033 C CA 1308033C CA 000562773 A CA000562773 A CA 000562773A CA 562773 A CA562773 A CA 562773A CA 1308033 C CA1308033 C CA 1308033C
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- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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- 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
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- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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Abstract
Injectable Soft Tissue Augmentation Materials from the Placenta and Their Method of Manufacture Abstract of the Disclosure Disclosed is a soft tissue augmentation material from human placenta homogenized to pass through a surgical needle, preferably a 30 gauge surgical needle, and cross-linking collagen molecules of the material by gamma irradiation. Also disclosed are methods of making the injectable soft tissue augmentation material and using it in human beings.
Description
13~1~Q33 Injectable Soft Tissue Auqmentation Materials from the Placenta and Their Method of Manufacture Fiel_ of the Invention The present invention is in the field of injectable material for soft tissue augmentation.
Backqround of the Invention The idea of using an injectable material for soft tissue augmentation developed soon after the invention of the hypodermic needle. Various products have been injected into the human body for correction of soft tissue defects including paraffin, petrolatum, vegetable oils, lanolin, bees wax, silicone, and more recently, collagen.
Paraffin was first used by Gersury in 1~99l who injected it into the scrotum of a young man to replace resected testicles. He later used paraffin to correct facial contour defects. During the period of 1900-1914, the use of paraffin injections became popularized.
Heidingsfeld in 19062 described the paraffinoma.
Injected paraffin forms many widely diffused droplets in the tissues. Phagocytosis by macrophages and giant cells occurs, followed by hyalin necrosis of fibrovascular septa, proliferation of fibroblasts, and development of scar tissue containing oil globules and cysts lined by foreign body giant cells.
Clinically, edema and scar formation occurred, sometimes followed by ulceration. The use of injectable paraffin was discontinued in the United States around World War I, but continued to be used in the Far East until the 1950's.
Silicones were the next material to be injected into humans on a large scale. Conway and Goulian reported on the use of silicone injections into the breast and face in 19633. Some people were using the "Sakurai" formula, a ~ 31~8~'33 silicone fluid, adulterated with an additive for better fixation4. Dow Corning developed a more purified "medical grade 360 li~uid silicone" and, although this product was not originally intended to be injected, it was soon used for human injections around the world.
The first reports of complica~ions of injected silicone including foreign body granulomas occurred in 19645. In 1965, Ben-Hur and Neuman6 described the siliconoma which was a tumor-like formation developing after the injection of silicone 360 in rats.
In 1965, the U.S. Food and Drug Administration (F.D.A.) determined that the clinical use of injectable silicone was a "drug use" and authorized seven investigators to employ silicone fluid as a soft tissue substitute. After lS reviewing their clinical experiences with injectable silicone - Dow Corning - MDX 4.4011, they concluded that silicone MDX 4.4011 can provoke an inflammatory reaction resulting in redness and ecchymosis which can be controlled with antibiotics and corticosteroids. Partial resorption and migration can occur but can be avoided by repeated injections of small amounts7r3~9~lo~ 2~l3 Alth complications have been reported with MDX 4.4011, many complications have been described after injection of liquid silicone with an unknown grade of purityl4. Although a few individuals are still injecting silicone with good results and few complications, the unforgiving nature of the material if used incorrectly will probably prevent its ~idespread use.
Bovine collagen has recently gained widespread use as an injectable material for soft tissue augmentation.
Collagen is the principal extracellular structural protein of the animal body. At least seven types of mammalian collagen have been described. Their common characteristic is a three-stranded helix, consisting of 3 polypeptide chains, *Trade Mark i~
~3~33 called alpha-chains. All chains have the same configuration, but differ in the composition and sequence of their aminoacids. This leads to different types of alpha-chains, but they all have glycine in every third position of the aminoacid sequence. This allows the helical conformation to occur.
Type I collagen is composed of ~ alphal-chains and one alpha2-chains and is the principal extracellular material of skin, tendon, and bone. When clinicians speak of "collagen,"
they are usually referring to Type I. Type II collagen is found in cartilage and the viotreous humor. Type III collagen is present in rapidly growing tissue, particularly juvenile and healing skin. Collagen Types IV and V are found in epithelial basement membranel4.
The major molecular species beside collagen that are found in the extracellular matrix include the noncollagenous structural glycoproteins, elastin, and the proteoglycans. The structural glycoproteins consist of fibronectin and laminin.
Fibronectin is found in both the plasma and tissue forms and is capable of interacting with other components of the extracellular matrix. Recently, Wartiovaara proposed that another fu~ction of fibronectin is to opsonize collagen or fibrin and, by this mechanism, to regulate the cellular diges~ion of these substratesl5. Laminin is found in all bas~ment membranes. Proteoglycans are characterized by a protein core linked to glycoaminoglycan side chainsl5~l6~l7~l8 When using collagen as a biomaterial, it is important to use it in its purest and crystalline form to eliminate the noncollagenous proteins that are far more potent antigens.
Once the inflammatory cycle is stimulated, the resorption of collagen occurs by the infiltrating inflammatory cells, principally macrophages and, to a lesser extent, granulocytes.
These cells contain collagenase which acts to digest ~3L?~
collagenl9. Houch and Chang demonstrated that skin collagen was chemotactic itself and became even more active by digestion with tissue collagenase into smaller peptide fragments20.
Chemotropism is the attraction of living protoplasm to chemical stimuli whereby the cells are attracted (positive hemotaxis) or repelled (negative chemotaxis) by acids, alkalis or other bodies exhibiting chemlcal properties. Postlethwaite et al21 showed that various types of collagens, their alpha-chains, as well as small peptides formed by collagenase digestion were all chemotactic to dermal fibroblasts. They concluded that the chemotactic migration of fibroblasts into the site of tissue injury or theoretically injected collagen can be regulated by the solubilized collagen or its degradation products. Thus, a collagen implant would not remain dormant in the tissue but a complex series of events may occur. First, the collagen implant could be invaded by inflammatory and fibroblasts and, while being continuously resorbed, it could promote an inflammatory reaction by chemotactic properties of its degradatio~ products. Thus, the area of collagen metabolism is not only important for collagen and other soft tissue injectable materials, but also to both normal and abnormal wound healing (i.e. hypertrophic scarring and keloids)19~20~21 The injectable collagen that recently gained widespread use was given marketing clearance as a device (not a drug) by the Food and Drug Administration in 1981. This material sold under the name Zyderm~ Collagen Implant is a purified bovine collage. About 95% consists of Type I collagen and the remaining 5%, Type III collagen. The collagen 3Q undergoes proteolytic hydrolysis of its telopeptide portion to decrease its antigenici~y. The material is suspended in physiologic saline buffer, and 0.3% lidocaine is added and the - .~3~8~33 material is packaged in syringes ready for injection through small gauge needles.
The most immediate concern to most plastic surgeons is the fate of bovine collagen after injection. Zyderm I~ with 3S mg/ml of collagen is rapidly degraded by tissue collagenases and resorbed within months. Zyderm II~ with 65 mg/ml of collagen and, thus, almost twice the concentration of collagen, is longer lasting but follows ~he same fate as Zyderm I~.
Zyplast~ was most recently introduced containing 35 mg~ml of collagen cross-linked with glutaraldehyde. Zyplast~ also is ultimately degraded over time. Kligman22 recently compared the biological fat of Zyderm~ collagen and Zyplast~
collagen when implanted into the back of human volunteers.
They reported that, while Zyderm~ collagen was apparently resorbed by host tissue within months of implantation, Zyplast~ was more persistent. Fibroblasts infiltrated the Zyplast~ collagen and deposited host collagen. Burke et al~3 reported that Zyderm~ collagen stimulates a response which results in implant degradation and replacement by newly generated host collagen.
One additional area of confusion about Zyderm I~
collagen, Zyderm II~ collagen and Zyplast~ collagen is the percentage of collagen in each. Zyderm I~ collagen and Zyplast~ collagen have 35 mg/ml, (3.5% collagen) while Zyderm II~ has 65 mg/ml (6.5% collagen).
A small percentage of patients receiving either Zyderm I~ collagen, Zyderm II~ collagen or Zyplast~ collagen, hereafter referred to as bovine collagen implants (BCI), have developed adverse reactions of an immune nature. The safe use of these implants is based on the reported low immunogenicity of the bovine collagen. It is, however, contraindicated in patients with a history of autoimmune disease. Skin tests are required before receiving the BCI. Only patients with negative ~Q~3~33 skin tests7after 4 weeks should have the treatment injections.
The Collagen Corporation indicates that approximately 3% of the patients have a positive skin test reaction characterized by edema, induration, erythema, pruritus or tenderness at the injection site, Adverse generalized treatment reactions have been ~uoted from less than 1% to greater than 5~. They are characterized by urticaria, myalgias, arthralgias and one anaphylactoid reaction23'24. A dramatic increase in the incidence of anti-BCI antibodies in the circulation of patients with adverse BCI-treatment reactions has been noted compared with serum samples from untreated individuals or treated patients suffering adverse reactions. BCI is a weak antigen but still is a foreign protein. It has been treated in such a way to cleave the telopeptides to make it less immunogenic but the helical portion of the molecule retains its antigenic loci. The dominant structures recognized by the cell-mediated immunological mechanism appear to reside within the triple helical body of the collagen molecules. There has been a major concern about repeated exposure of patients to these antigens and their long-term effects24'25'26.
In summary, due to the shortcomings of the BCI, including the lack of persistence, need for repeated injections and serious concern over adverse reactions, newer injectable materials for soft tissue augmentation are needed. The present invention is directed to injectable materials for soft tissue augmentation and their methods of manufacture which overcome shortcomings of BCI and other injectable materials of the prior art.
Description of Prior Art In addition to the prior art discussed above, U.S~
Patent No. 4,361,552 discloses a method of treating a wound or burn with an amnion dressing from any suitable animal species (human, cattle, pigs, etc.) fixed or stabilized by toxic 81~33 chemical agents, such as glutaraldehyde solutions or other fixing or tannirlg solutions, so that the proteins thereof are cross-linked, A nu~ber of U.S. patents are cited and listed in this patent.
U.K. Patent Application No. 8133375, published 22 ~une, 1983 discloses a substantially amnion-free preparation for use in the treatment of wounds derived from a culture medium in which amnion has been cultured, the amnion preferably being human amnion.
SummarY of the Invention The present invention is directed to an injectable soft tissue material in which the problems associa~ed with xenogeneic bovine sources have been eliminated, the problem o~
lack of persistence and need for repeated injection has been lS addressed to make it less susceptible to degradation, and for which the starting material is available in unlimited quantities and at low cost.
The injectable soft tissue augmentation material is from human placenta and consists of insoluble amnion, soluble amnion, soluble chorion and combinations thereof, homogenized to pass throug~ a surgical needle, Preferably, the homogenized material should pass through at least a 25 gauge and more preferably a 30 gauge surgical needle to avoid discomfort, and, if desired, an analgesic can be added. In one aspect of the invention, the material is irradiated at a minimum of 0.20 M rads for sterilization and cross~linking with a preferred range of from about 0.25 M rads to about 2.0 M rads. The presently preferred injectable soft tissue augmentation material is irradiated soluble amnion.
The method of the invention of making such an injectable soft tissue material comprises homogenizing a material from human placenta consisting of insoluble amnion, soluble amnion, soluble chorion and combinations thereof sufficiently so that it will pass through a surgical needle, : ~ -7-13~8~33 and preferably at least a 25 gauge and most preferably a 30 gauge surgical needle, sterilLzing the collagen molecules, preferably by gamma irradiation which also cross-links them. A minimum of .20 M
- rads is necessary to sterilize these materials, and the presently preferred range is from about 0.25 M rads to 2.0 M rads. If desired, an analgesic can be added to the homogenized injectable soft tissue material.
The method of the invention of correcting soft tissue contour defects is by injecting the injectable soft tissue material of the invention in a patient. Advantageously, a number of injections of small amounts of the sof~ tissue material are possible since even small amounts persist. This provides a greater host-implant interface and allows faster assimilation into the tissue since the center of the implant is not far from host tissue influences.
Accordingly, the present invention provides an injectable sterilized soft tissue material in which adverse allergic reactions are eliminated, which has long-term persistence and little inflammation, which can be injected through a preferably 30 gauge needle, and in one aspect of the invention gamma irradiation lS used to both sterilize the material and increase the amount of cross-linking of the collagen without using toxic cross-linking agents.
The present invention further provides an injectable sterilized soft tissue material from human placenta consisting of insoluble amnion, soluble amnion, soluble chorion, and combinations thereof, homogenized sufficiently to pass through a surgical needle, as for example a 30 gauge needle, and in one aspect of the invention is purified and cross-linked by gamma irradiatiGn without the use of toxic chemicals.
The present invention still further provides in a preferred embodiment the making of an injectible soft tissue material in which adverse allergic reactions are eliminated, in which gamma ~` 8-13~8~33 irradiation is used to both sterilize the material and increase the amount of cross-linking, and in which by varying the amount of cross-linking, the amount of tissue persistence can be regulated.
The present invention in a preferred embodiment provides a method of making a soft injectable augmentation material having the above-mentioned advantages by homogenizing insoluble amnion, soluble amnion, soluble chorion, or combinations thereof from a human placenta so that the material passes through a surgical needle, preferably a 30 gauge surgical needle, and cross-linking collagen molecules of the material by gamma irradiation of at least 0.20 M rads and, preferably, in the range of from 0.25 M rads to 2.0 M rads.
In another aspect the present invention provides a method : 15 of recontouring soft tissue by injecting into humans material from a human placenta comprising insoluble amnion, soluble amnion, soluble chorion, or combinations thereof, sterilized and homogenized to the e~tent that it can be injected by a surgical needle, and preferably a 30 gauge surglcal needle and which material is sterilized preferably by having its collagen molecules cross-linked by gamma irradiation.
The invention also provides in another aspect a method of recontouring soft tissue by injection into humans such injectable material in small amounts to provide good host implant interface and allow faster assimilation into the tissue and yet have good persistence.
Other and further features and advantages of the invention appear throughout the specification and claims.
Pr*sently Preerred Embodimènts o~ the Invention ; 3~ Human fetal membranes were selected as the starting material for the injectable soft tissue augmentation material into human beings for recontouring soft tissue since they ,~
130~'33 eliminate the allergic problems seen with xenogeneic sources, i.e. bovine, human placentas are available in relatively unlimited quantities and are a rich source of collagen, placentas are available at a low cost, and amnion has had a long-history of medical uses beginning almost 90 years ago for a wide variety of medical and surgical applications.
The fetal membranes are complex biochemical structures that can be developed into one or several different soft tissue injectable materials. These have physical and biological characteristics that can be modified to fulfill various clinical needs.
The preferred human fetal membranes are insoluble amnion, soluble amnion, soluble chorion or combinations of them. They are homogenized to pass through a surgical needle.
While injecting the soft tissue augmentation materials through a 25 gauge surgical needle is satisfactory for clinical purposes, to avoid discomfort, preferably the material is homogenized so that it passes can be injected through a 30 gauge surgical needle.
In one aspect o the invention, the injectable material is cross-linked b~ gamma irradiation without the use of chemical cross-linking agents, such as glutaraldehyde, which are toxic. The gamma irradiation should be a minimum o~ 20 M
rads to sterilize the material since all bacteria, fungi and viruses, including AIDS, are destroyed at 0.20 M rads.
Preferably, the material is irradiated from 0.25 to 2.0 ~ rads to sterilize and cross link the collagen molecules.
I~ desired, an analgesic, such as lidocaine can be added to the injectable material.
In making the soft injectable material, fresh placentae were collec~ed and the amnion is manually separated from tne chorion, such as by finger separation. Both the amnion and the chorion are then cleaned of any remaining blood 131~8~33 clots or debris. For short-term storage, the amnion and the chorion are placed in an antibiotic solution, for example, linomycin 3 gms/10 ml, amphotericin B 50 mg/10 ml, neomycin sulfate 0.5 gm/10 ml, polymyxin B sulfate 500,000 units/10 ml in 1 liter of normal saline until processed.
Collagen was extracted using limited proteolytic digestion with pepsin. In brief, tissue was homogenized in 0.5 M acetic acid, the pH was adjusted to 2.5 with HCl and the preparation was digested twice with pepsin (10 mg pepsin/gm wet weight tissue) overnight. A combination method of selecti~e precipitation from neutral salt solvent and acid solvents was used to purify the collagen. Purified collagen was reconstituted by dialysis against low ionic strength sodium phosphate buffer (pH 7.2) at 15-17C. Lidocaine was added to a final concentration of 0.3~. All procedures were carried out at 4-8C although other suitable ~emperatures can be used.
Insoluble Amnion Processinq A presently preferred method of processing the amnion comprises decanting the antibiotic from the amnion, adding 5 ml of cold distilled water to each amnion, and homogenizing the amnion approximately 15 minutes in polytron. The homogenized amnion is then centrifuged at 8,000 x 5 for 15 minutes at 4C, the supernatant is discarded, and the precipitant is washed to remove the lipids, such as 5 times with acetone. The precipitant is then weighed, and pepsin (Sigma, 1:10,000, from porcine stomach mucosa) 3.0 molar acetic acid per amnion was added, 15 ml or more if extra large amnions, and the precipitant was homogenized for approximately 5 minutes in polytron.
The mixture was allowed to stand for 18 hours at 4C, centrifuged at lOO,ooo x g for 1 hour at 4C, the supernatant was discarded, the precipitant weighed and then the pepsin and 81~33 homogenization steps were repeated and the supernatant discarded.
Soluble Amnion Processinq A presently pre~erred way of processing soluble amnions comprises rinsing the antibiotics from the amnions with deionized water, adding 5ml of cold distilled water to each amnion, homogenizing for approximately 15 minutes in polytron and centrifuging at 8,000 x g for 15 minutes at 4C. The supernatant was discarded and lipids were removed fro~ the precipitate by washing with acetone three times and precipitate was weighed.
Pepsin (Sigma, 1:10,000, from porcine stomach mucosa~
was added to the precipitate (1:100 w/w) and 100 ml of 0.5 molar acetic acid per amnion was added, more if the amnions are extra large, and then homogenized for approximately 10 minutes in polytron. The pepsin was allowed to extract collagen from the precipitate for 18 hours at 4C and then centrifuged at 100,000 x g for 1 hour at 4C retaining both the precipitate and the supernatant. The supernatant is again weighed, and the steps of pepsin and acetic acid addition, homogenization, pepsin extraction o~ collagen and centrifuging are then repeated.
The supernatants from the first and second extractions are combined and 10-molar NaOH is added dropwise to adjust the pH to from 7.0 to 7.2. The mixture is permitted to stand for 2 hours at 4C, centrifuged at 100,000 x g for 45 minutes at 4C
and the precipitate is discarded. NaCL to 3.0-molar is added to the supernatant and permitted to stand for 2 hours at 4C, centrifuged at 100,Q00 x g for 45 minutes at 4C and the precipitate is weighed and lidocaine to 0.3% is added.
~3~ 33 Soluble Amnion Processinq with Further Purification A presently preferred method of soluble amnion processing and further purification comprises rinsing the antibiotic from the amnion with deionized water, the amnions are cut to approximately 2 cm x 2 cm and washed briefly with acetone, soaked in 0.5 M acetic acid (pH adjusted to 2.5 with HCl), homogenized with polytron for about 15 minutes, pepsin is added (1:100 pepsin/set tissue) (l mg pepsin/l ml solution) and stirred at 4C overnight, centrifuged as indicated above, the supernatant being retained. Pepsin was again added as indicated previously and stirred at 4C overnight, centrifuged and the supernatant from both centrifuging steps were combined and NaCL was added to 2 M and permitted to stand overnight at 4C and again centrifuged, the supernatant discarded and the precipitate retained.
The precipitate was purified by dissolving it in 0.5 M
acetic acid, centrifuging, precipitate discarded, NaCL to 2 M
was added to the supernatant, and it was permitted to stand overnight at 4C, again centrifuged with the supernatant discarded. The resulting precipitate was dissolved in 0.5 M
acetic acid, again centrifuged, and the precipitate discarded.
The supernatant was dialysed against 0.02 M Na2HPO4 thoroughly for 48 hours with frequent dialysis fluid exchanges, centrifuged, the supernatant discarded, the precipitate weighed and solid lidocaine HCl was added to 0.30~ with mechanical agitation.
Chorion Processinq In a presently preferred method of processing soluble chorion the antibiotics were rinsed from the chorion with deionized water, the chorion was cut to approximately 2 cm x 2 cm units and washed briefly with acetone and then soa~ed into 0,5 M acetic acid that had been adjusted to pH 2.5 with HCl.
1.3~8~33 The tissue was then homogenized with polytron to fine particles for about 15 minutes, pepsin added and centrifuged as indicated above with the supernatant being retained. The pepsin and centrifuge steps were then repeated, the supernatant of each of these steps were combined and NaCL to 2 M was added and permitted to stand overnight at 4C and then centrifuged again with the supernatant discarded.
For purification, the precipitate was dissolved into 0.5 M acetic acid, centrifuged and the precipitate discarded.
NaCL to 2 M was added to the supernatant and permitted to stand overnight at 4C, then again centrifuged and the supernatant discarded. The precipitate was dissolved into 0.5 M acetic acid, centrifuged, dialysed against 0.02 M Na2HP04 thoroughly for 48 hours with frequent dialysis fluid exchanges, again centrifuged, the supernatant discarded and the precipitate weighed. Solid lidocaine HCl was added to 0.30% to the precipitate with mechanical agitation.
Cross-linkinq and Sterilizinq 15cc of each of the foregoing resulting precipitates was placed in 20 cc serum bottles with crimp closures and placed in CE137 radioactive source for varying lengths of time in order for them to receive 0.25 M rads, 0.5 M rads, 1.0 M rads, and 2.0 M rads which served the dual purpose of sterilizing the material and cross-linking the collagen.
ExamPle 1 Several groups of collagen extracts were reconstituted in phosphate buffered saline and placed in glass tubes and irradiated in 1779 curies Cesium gamma rays source at a dose of 1000 rads~minute. The dosage was 0.25 M rads. Irradiation was carried out at room temperature.
13~033 Table 1 The groups examined were as follows:
Grou~
1. Soluble amnion with 0.25 M rads.
Backqround of the Invention The idea of using an injectable material for soft tissue augmentation developed soon after the invention of the hypodermic needle. Various products have been injected into the human body for correction of soft tissue defects including paraffin, petrolatum, vegetable oils, lanolin, bees wax, silicone, and more recently, collagen.
Paraffin was first used by Gersury in 1~99l who injected it into the scrotum of a young man to replace resected testicles. He later used paraffin to correct facial contour defects. During the period of 1900-1914, the use of paraffin injections became popularized.
Heidingsfeld in 19062 described the paraffinoma.
Injected paraffin forms many widely diffused droplets in the tissues. Phagocytosis by macrophages and giant cells occurs, followed by hyalin necrosis of fibrovascular septa, proliferation of fibroblasts, and development of scar tissue containing oil globules and cysts lined by foreign body giant cells.
Clinically, edema and scar formation occurred, sometimes followed by ulceration. The use of injectable paraffin was discontinued in the United States around World War I, but continued to be used in the Far East until the 1950's.
Silicones were the next material to be injected into humans on a large scale. Conway and Goulian reported on the use of silicone injections into the breast and face in 19633. Some people were using the "Sakurai" formula, a ~ 31~8~'33 silicone fluid, adulterated with an additive for better fixation4. Dow Corning developed a more purified "medical grade 360 li~uid silicone" and, although this product was not originally intended to be injected, it was soon used for human injections around the world.
The first reports of complica~ions of injected silicone including foreign body granulomas occurred in 19645. In 1965, Ben-Hur and Neuman6 described the siliconoma which was a tumor-like formation developing after the injection of silicone 360 in rats.
In 1965, the U.S. Food and Drug Administration (F.D.A.) determined that the clinical use of injectable silicone was a "drug use" and authorized seven investigators to employ silicone fluid as a soft tissue substitute. After lS reviewing their clinical experiences with injectable silicone - Dow Corning - MDX 4.4011, they concluded that silicone MDX 4.4011 can provoke an inflammatory reaction resulting in redness and ecchymosis which can be controlled with antibiotics and corticosteroids. Partial resorption and migration can occur but can be avoided by repeated injections of small amounts7r3~9~lo~ 2~l3 Alth complications have been reported with MDX 4.4011, many complications have been described after injection of liquid silicone with an unknown grade of purityl4. Although a few individuals are still injecting silicone with good results and few complications, the unforgiving nature of the material if used incorrectly will probably prevent its ~idespread use.
Bovine collagen has recently gained widespread use as an injectable material for soft tissue augmentation.
Collagen is the principal extracellular structural protein of the animal body. At least seven types of mammalian collagen have been described. Their common characteristic is a three-stranded helix, consisting of 3 polypeptide chains, *Trade Mark i~
~3~33 called alpha-chains. All chains have the same configuration, but differ in the composition and sequence of their aminoacids. This leads to different types of alpha-chains, but they all have glycine in every third position of the aminoacid sequence. This allows the helical conformation to occur.
Type I collagen is composed of ~ alphal-chains and one alpha2-chains and is the principal extracellular material of skin, tendon, and bone. When clinicians speak of "collagen,"
they are usually referring to Type I. Type II collagen is found in cartilage and the viotreous humor. Type III collagen is present in rapidly growing tissue, particularly juvenile and healing skin. Collagen Types IV and V are found in epithelial basement membranel4.
The major molecular species beside collagen that are found in the extracellular matrix include the noncollagenous structural glycoproteins, elastin, and the proteoglycans. The structural glycoproteins consist of fibronectin and laminin.
Fibronectin is found in both the plasma and tissue forms and is capable of interacting with other components of the extracellular matrix. Recently, Wartiovaara proposed that another fu~ction of fibronectin is to opsonize collagen or fibrin and, by this mechanism, to regulate the cellular diges~ion of these substratesl5. Laminin is found in all bas~ment membranes. Proteoglycans are characterized by a protein core linked to glycoaminoglycan side chainsl5~l6~l7~l8 When using collagen as a biomaterial, it is important to use it in its purest and crystalline form to eliminate the noncollagenous proteins that are far more potent antigens.
Once the inflammatory cycle is stimulated, the resorption of collagen occurs by the infiltrating inflammatory cells, principally macrophages and, to a lesser extent, granulocytes.
These cells contain collagenase which acts to digest ~3L?~
collagenl9. Houch and Chang demonstrated that skin collagen was chemotactic itself and became even more active by digestion with tissue collagenase into smaller peptide fragments20.
Chemotropism is the attraction of living protoplasm to chemical stimuli whereby the cells are attracted (positive hemotaxis) or repelled (negative chemotaxis) by acids, alkalis or other bodies exhibiting chemlcal properties. Postlethwaite et al21 showed that various types of collagens, their alpha-chains, as well as small peptides formed by collagenase digestion were all chemotactic to dermal fibroblasts. They concluded that the chemotactic migration of fibroblasts into the site of tissue injury or theoretically injected collagen can be regulated by the solubilized collagen or its degradation products. Thus, a collagen implant would not remain dormant in the tissue but a complex series of events may occur. First, the collagen implant could be invaded by inflammatory and fibroblasts and, while being continuously resorbed, it could promote an inflammatory reaction by chemotactic properties of its degradatio~ products. Thus, the area of collagen metabolism is not only important for collagen and other soft tissue injectable materials, but also to both normal and abnormal wound healing (i.e. hypertrophic scarring and keloids)19~20~21 The injectable collagen that recently gained widespread use was given marketing clearance as a device (not a drug) by the Food and Drug Administration in 1981. This material sold under the name Zyderm~ Collagen Implant is a purified bovine collage. About 95% consists of Type I collagen and the remaining 5%, Type III collagen. The collagen 3Q undergoes proteolytic hydrolysis of its telopeptide portion to decrease its antigenici~y. The material is suspended in physiologic saline buffer, and 0.3% lidocaine is added and the - .~3~8~33 material is packaged in syringes ready for injection through small gauge needles.
The most immediate concern to most plastic surgeons is the fate of bovine collagen after injection. Zyderm I~ with 3S mg/ml of collagen is rapidly degraded by tissue collagenases and resorbed within months. Zyderm II~ with 65 mg/ml of collagen and, thus, almost twice the concentration of collagen, is longer lasting but follows ~he same fate as Zyderm I~.
Zyplast~ was most recently introduced containing 35 mg~ml of collagen cross-linked with glutaraldehyde. Zyplast~ also is ultimately degraded over time. Kligman22 recently compared the biological fat of Zyderm~ collagen and Zyplast~
collagen when implanted into the back of human volunteers.
They reported that, while Zyderm~ collagen was apparently resorbed by host tissue within months of implantation, Zyplast~ was more persistent. Fibroblasts infiltrated the Zyplast~ collagen and deposited host collagen. Burke et al~3 reported that Zyderm~ collagen stimulates a response which results in implant degradation and replacement by newly generated host collagen.
One additional area of confusion about Zyderm I~
collagen, Zyderm II~ collagen and Zyplast~ collagen is the percentage of collagen in each. Zyderm I~ collagen and Zyplast~ collagen have 35 mg/ml, (3.5% collagen) while Zyderm II~ has 65 mg/ml (6.5% collagen).
A small percentage of patients receiving either Zyderm I~ collagen, Zyderm II~ collagen or Zyplast~ collagen, hereafter referred to as bovine collagen implants (BCI), have developed adverse reactions of an immune nature. The safe use of these implants is based on the reported low immunogenicity of the bovine collagen. It is, however, contraindicated in patients with a history of autoimmune disease. Skin tests are required before receiving the BCI. Only patients with negative ~Q~3~33 skin tests7after 4 weeks should have the treatment injections.
The Collagen Corporation indicates that approximately 3% of the patients have a positive skin test reaction characterized by edema, induration, erythema, pruritus or tenderness at the injection site, Adverse generalized treatment reactions have been ~uoted from less than 1% to greater than 5~. They are characterized by urticaria, myalgias, arthralgias and one anaphylactoid reaction23'24. A dramatic increase in the incidence of anti-BCI antibodies in the circulation of patients with adverse BCI-treatment reactions has been noted compared with serum samples from untreated individuals or treated patients suffering adverse reactions. BCI is a weak antigen but still is a foreign protein. It has been treated in such a way to cleave the telopeptides to make it less immunogenic but the helical portion of the molecule retains its antigenic loci. The dominant structures recognized by the cell-mediated immunological mechanism appear to reside within the triple helical body of the collagen molecules. There has been a major concern about repeated exposure of patients to these antigens and their long-term effects24'25'26.
In summary, due to the shortcomings of the BCI, including the lack of persistence, need for repeated injections and serious concern over adverse reactions, newer injectable materials for soft tissue augmentation are needed. The present invention is directed to injectable materials for soft tissue augmentation and their methods of manufacture which overcome shortcomings of BCI and other injectable materials of the prior art.
Description of Prior Art In addition to the prior art discussed above, U.S~
Patent No. 4,361,552 discloses a method of treating a wound or burn with an amnion dressing from any suitable animal species (human, cattle, pigs, etc.) fixed or stabilized by toxic 81~33 chemical agents, such as glutaraldehyde solutions or other fixing or tannirlg solutions, so that the proteins thereof are cross-linked, A nu~ber of U.S. patents are cited and listed in this patent.
U.K. Patent Application No. 8133375, published 22 ~une, 1983 discloses a substantially amnion-free preparation for use in the treatment of wounds derived from a culture medium in which amnion has been cultured, the amnion preferably being human amnion.
SummarY of the Invention The present invention is directed to an injectable soft tissue material in which the problems associa~ed with xenogeneic bovine sources have been eliminated, the problem o~
lack of persistence and need for repeated injection has been lS addressed to make it less susceptible to degradation, and for which the starting material is available in unlimited quantities and at low cost.
The injectable soft tissue augmentation material is from human placenta and consists of insoluble amnion, soluble amnion, soluble chorion and combinations thereof, homogenized to pass throug~ a surgical needle, Preferably, the homogenized material should pass through at least a 25 gauge and more preferably a 30 gauge surgical needle to avoid discomfort, and, if desired, an analgesic can be added. In one aspect of the invention, the material is irradiated at a minimum of 0.20 M rads for sterilization and cross~linking with a preferred range of from about 0.25 M rads to about 2.0 M rads. The presently preferred injectable soft tissue augmentation material is irradiated soluble amnion.
The method of the invention of making such an injectable soft tissue material comprises homogenizing a material from human placenta consisting of insoluble amnion, soluble amnion, soluble chorion and combinations thereof sufficiently so that it will pass through a surgical needle, : ~ -7-13~8~33 and preferably at least a 25 gauge and most preferably a 30 gauge surgical needle, sterilLzing the collagen molecules, preferably by gamma irradiation which also cross-links them. A minimum of .20 M
- rads is necessary to sterilize these materials, and the presently preferred range is from about 0.25 M rads to 2.0 M rads. If desired, an analgesic can be added to the homogenized injectable soft tissue material.
The method of the invention of correcting soft tissue contour defects is by injecting the injectable soft tissue material of the invention in a patient. Advantageously, a number of injections of small amounts of the sof~ tissue material are possible since even small amounts persist. This provides a greater host-implant interface and allows faster assimilation into the tissue since the center of the implant is not far from host tissue influences.
Accordingly, the present invention provides an injectable sterilized soft tissue material in which adverse allergic reactions are eliminated, which has long-term persistence and little inflammation, which can be injected through a preferably 30 gauge needle, and in one aspect of the invention gamma irradiation lS used to both sterilize the material and increase the amount of cross-linking of the collagen without using toxic cross-linking agents.
The present invention further provides an injectable sterilized soft tissue material from human placenta consisting of insoluble amnion, soluble amnion, soluble chorion, and combinations thereof, homogenized sufficiently to pass through a surgical needle, as for example a 30 gauge needle, and in one aspect of the invention is purified and cross-linked by gamma irradiatiGn without the use of toxic chemicals.
The present invention still further provides in a preferred embodiment the making of an injectible soft tissue material in which adverse allergic reactions are eliminated, in which gamma ~` 8-13~8~33 irradiation is used to both sterilize the material and increase the amount of cross-linking, and in which by varying the amount of cross-linking, the amount of tissue persistence can be regulated.
The present invention in a preferred embodiment provides a method of making a soft injectable augmentation material having the above-mentioned advantages by homogenizing insoluble amnion, soluble amnion, soluble chorion, or combinations thereof from a human placenta so that the material passes through a surgical needle, preferably a 30 gauge surgical needle, and cross-linking collagen molecules of the material by gamma irradiation of at least 0.20 M rads and, preferably, in the range of from 0.25 M rads to 2.0 M rads.
In another aspect the present invention provides a method : 15 of recontouring soft tissue by injecting into humans material from a human placenta comprising insoluble amnion, soluble amnion, soluble chorion, or combinations thereof, sterilized and homogenized to the e~tent that it can be injected by a surgical needle, and preferably a 30 gauge surglcal needle and which material is sterilized preferably by having its collagen molecules cross-linked by gamma irradiation.
The invention also provides in another aspect a method of recontouring soft tissue by injection into humans such injectable material in small amounts to provide good host implant interface and allow faster assimilation into the tissue and yet have good persistence.
Other and further features and advantages of the invention appear throughout the specification and claims.
Pr*sently Preerred Embodimènts o~ the Invention ; 3~ Human fetal membranes were selected as the starting material for the injectable soft tissue augmentation material into human beings for recontouring soft tissue since they ,~
130~'33 eliminate the allergic problems seen with xenogeneic sources, i.e. bovine, human placentas are available in relatively unlimited quantities and are a rich source of collagen, placentas are available at a low cost, and amnion has had a long-history of medical uses beginning almost 90 years ago for a wide variety of medical and surgical applications.
The fetal membranes are complex biochemical structures that can be developed into one or several different soft tissue injectable materials. These have physical and biological characteristics that can be modified to fulfill various clinical needs.
The preferred human fetal membranes are insoluble amnion, soluble amnion, soluble chorion or combinations of them. They are homogenized to pass through a surgical needle.
While injecting the soft tissue augmentation materials through a 25 gauge surgical needle is satisfactory for clinical purposes, to avoid discomfort, preferably the material is homogenized so that it passes can be injected through a 30 gauge surgical needle.
In one aspect o the invention, the injectable material is cross-linked b~ gamma irradiation without the use of chemical cross-linking agents, such as glutaraldehyde, which are toxic. The gamma irradiation should be a minimum o~ 20 M
rads to sterilize the material since all bacteria, fungi and viruses, including AIDS, are destroyed at 0.20 M rads.
Preferably, the material is irradiated from 0.25 to 2.0 ~ rads to sterilize and cross link the collagen molecules.
I~ desired, an analgesic, such as lidocaine can be added to the injectable material.
In making the soft injectable material, fresh placentae were collec~ed and the amnion is manually separated from tne chorion, such as by finger separation. Both the amnion and the chorion are then cleaned of any remaining blood 131~8~33 clots or debris. For short-term storage, the amnion and the chorion are placed in an antibiotic solution, for example, linomycin 3 gms/10 ml, amphotericin B 50 mg/10 ml, neomycin sulfate 0.5 gm/10 ml, polymyxin B sulfate 500,000 units/10 ml in 1 liter of normal saline until processed.
Collagen was extracted using limited proteolytic digestion with pepsin. In brief, tissue was homogenized in 0.5 M acetic acid, the pH was adjusted to 2.5 with HCl and the preparation was digested twice with pepsin (10 mg pepsin/gm wet weight tissue) overnight. A combination method of selecti~e precipitation from neutral salt solvent and acid solvents was used to purify the collagen. Purified collagen was reconstituted by dialysis against low ionic strength sodium phosphate buffer (pH 7.2) at 15-17C. Lidocaine was added to a final concentration of 0.3~. All procedures were carried out at 4-8C although other suitable ~emperatures can be used.
Insoluble Amnion Processinq A presently preferred method of processing the amnion comprises decanting the antibiotic from the amnion, adding 5 ml of cold distilled water to each amnion, and homogenizing the amnion approximately 15 minutes in polytron. The homogenized amnion is then centrifuged at 8,000 x 5 for 15 minutes at 4C, the supernatant is discarded, and the precipitant is washed to remove the lipids, such as 5 times with acetone. The precipitant is then weighed, and pepsin (Sigma, 1:10,000, from porcine stomach mucosa) 3.0 molar acetic acid per amnion was added, 15 ml or more if extra large amnions, and the precipitant was homogenized for approximately 5 minutes in polytron.
The mixture was allowed to stand for 18 hours at 4C, centrifuged at lOO,ooo x g for 1 hour at 4C, the supernatant was discarded, the precipitant weighed and then the pepsin and 81~33 homogenization steps were repeated and the supernatant discarded.
Soluble Amnion Processinq A presently pre~erred way of processing soluble amnions comprises rinsing the antibiotics from the amnions with deionized water, adding 5ml of cold distilled water to each amnion, homogenizing for approximately 15 minutes in polytron and centrifuging at 8,000 x g for 15 minutes at 4C. The supernatant was discarded and lipids were removed fro~ the precipitate by washing with acetone three times and precipitate was weighed.
Pepsin (Sigma, 1:10,000, from porcine stomach mucosa~
was added to the precipitate (1:100 w/w) and 100 ml of 0.5 molar acetic acid per amnion was added, more if the amnions are extra large, and then homogenized for approximately 10 minutes in polytron. The pepsin was allowed to extract collagen from the precipitate for 18 hours at 4C and then centrifuged at 100,000 x g for 1 hour at 4C retaining both the precipitate and the supernatant. The supernatant is again weighed, and the steps of pepsin and acetic acid addition, homogenization, pepsin extraction o~ collagen and centrifuging are then repeated.
The supernatants from the first and second extractions are combined and 10-molar NaOH is added dropwise to adjust the pH to from 7.0 to 7.2. The mixture is permitted to stand for 2 hours at 4C, centrifuged at 100,000 x g for 45 minutes at 4C
and the precipitate is discarded. NaCL to 3.0-molar is added to the supernatant and permitted to stand for 2 hours at 4C, centrifuged at 100,Q00 x g for 45 minutes at 4C and the precipitate is weighed and lidocaine to 0.3% is added.
~3~ 33 Soluble Amnion Processinq with Further Purification A presently preferred method of soluble amnion processing and further purification comprises rinsing the antibiotic from the amnion with deionized water, the amnions are cut to approximately 2 cm x 2 cm and washed briefly with acetone, soaked in 0.5 M acetic acid (pH adjusted to 2.5 with HCl), homogenized with polytron for about 15 minutes, pepsin is added (1:100 pepsin/set tissue) (l mg pepsin/l ml solution) and stirred at 4C overnight, centrifuged as indicated above, the supernatant being retained. Pepsin was again added as indicated previously and stirred at 4C overnight, centrifuged and the supernatant from both centrifuging steps were combined and NaCL was added to 2 M and permitted to stand overnight at 4C and again centrifuged, the supernatant discarded and the precipitate retained.
The precipitate was purified by dissolving it in 0.5 M
acetic acid, centrifuging, precipitate discarded, NaCL to 2 M
was added to the supernatant, and it was permitted to stand overnight at 4C, again centrifuged with the supernatant discarded. The resulting precipitate was dissolved in 0.5 M
acetic acid, again centrifuged, and the precipitate discarded.
The supernatant was dialysed against 0.02 M Na2HPO4 thoroughly for 48 hours with frequent dialysis fluid exchanges, centrifuged, the supernatant discarded, the precipitate weighed and solid lidocaine HCl was added to 0.30~ with mechanical agitation.
Chorion Processinq In a presently preferred method of processing soluble chorion the antibiotics were rinsed from the chorion with deionized water, the chorion was cut to approximately 2 cm x 2 cm units and washed briefly with acetone and then soa~ed into 0,5 M acetic acid that had been adjusted to pH 2.5 with HCl.
1.3~8~33 The tissue was then homogenized with polytron to fine particles for about 15 minutes, pepsin added and centrifuged as indicated above with the supernatant being retained. The pepsin and centrifuge steps were then repeated, the supernatant of each of these steps were combined and NaCL to 2 M was added and permitted to stand overnight at 4C and then centrifuged again with the supernatant discarded.
For purification, the precipitate was dissolved into 0.5 M acetic acid, centrifuged and the precipitate discarded.
NaCL to 2 M was added to the supernatant and permitted to stand overnight at 4C, then again centrifuged and the supernatant discarded. The precipitate was dissolved into 0.5 M acetic acid, centrifuged, dialysed against 0.02 M Na2HP04 thoroughly for 48 hours with frequent dialysis fluid exchanges, again centrifuged, the supernatant discarded and the precipitate weighed. Solid lidocaine HCl was added to 0.30% to the precipitate with mechanical agitation.
Cross-linkinq and Sterilizinq 15cc of each of the foregoing resulting precipitates was placed in 20 cc serum bottles with crimp closures and placed in CE137 radioactive source for varying lengths of time in order for them to receive 0.25 M rads, 0.5 M rads, 1.0 M rads, and 2.0 M rads which served the dual purpose of sterilizing the material and cross-linking the collagen.
ExamPle 1 Several groups of collagen extracts were reconstituted in phosphate buffered saline and placed in glass tubes and irradiated in 1779 curies Cesium gamma rays source at a dose of 1000 rads~minute. The dosage was 0.25 M rads. Irradiation was carried out at room temperature.
13~033 Table 1 The groups examined were as follows:
Grou~
1. Soluble amnion with 0.25 M rads.
2~ Soluble amnion without radiation.
3. Insoluble amnion with 0.25 M rads.
4. Insoluble amnion without radiation.
5. Insoluble amnion + soluble amnion (1:1) with 0.25 M rads.
6. Insoluble amnion + soluble amnion (1:1) without radiation.
7. Soluble chorion with 0.25 M rads.
8. Soluble chorion without radiation.
9. Soluble chorion + soluble amnion (1:1) with 0.25 M rads.
10. Soluble chorion + soluble amnion (1:1) without radiation Samples from each group were sent for aerobic, anerobic, and fungal cultures. All groups showed no growth both before as well as after irradiation.
120 young Sprague-Dawley rats weighing between 200-300 grams were used for the animal injections. Twelve groups of animals were studied including groups injected with Zyderm II~ and Zyplast~. There were 10 rats in each group. 0.2 ml of injectable material was injected percutaneously into thelr backs below the panniculus carnosus in three separate sites. Samples were harvested from 5 rats in each qroup at one month and six months.
Speci~ens were~fixed in 10% buffered formalin and later embedded in paraffin. Serial sections were made and stained with hematoxylin-eosin (H&E). Results were compared using an independent staff pathologist.
Th~e histological evaluation of implants revealed similarities of all the experimental groups with the exception of the insoluble amnion which will be subsequently discussed.
Specifically, no inflammatory reaction was evident and no encapsulation occurred. Fibrocytic ingrowth is present at 30 days and is more advanced centripetally at 180 days.
Neovascularization was present in a variable extent at 180 1.3~8~33 days. The degree of both fibrocytic ingrowth and neovascularization was greater than that found with Zyderm II~ and Zyplast~ at 180 days.
The insoluble amnion group also demonstrated no inflammation or encapsulation but marked neovascularization and fibrocytic ingrowth. An additional observation was the presence of a few adipocytes at 3~ days. At 180 days the adipocytes that first appeared on the periphery of the implant had become dispersed throughout the implant. This phenomena has been observed by others27'~8.
A quantitative analysis (measuring exact dimensions of persisting implant) was very difficult to perform. Therefore, a preliminary qualitative study of persistence was performed.
Simply, the number of implants visibly present in the rats surviving at 6 months was counted. Table 2 demonstrates that the soluble amnion group showed very good persistence at 6 months. The nonirradiated soluble amnion had all implants present at 6 months and the irradiated soluble amnion had 11 out of 15. The insoluble group also showed good persistence in both the irradiated and nonirradiated groups. Some of the groups having dombinations of materials especially soluble chorion had less persistence after irradiation.
131~33 Table 2 PERSISTENCE OF SOFT TISSUE AUGMENTATION
ANIMAL EXPERIMENT GROUP 1-10, ZYDER~ and ZYPLAST~
Concentration Injections Present of CollagenNumber of Rats After 6 Months/
GrouP (mq/ml) Survival/Injected Total Number Iniected l* Sol. Am 51 5/5 . 11/15 2 Sol. Am 37.2 5/5 15/15 3* Ins. Am 23.5 5/5 9/15 4 Ins. Am 22 .2 5/5 13/15 5* Ins. Am & 40.4 4/5 3/12 Sol. Am 6 Ins. Am & 40.5 5/5 15/15 7* Sol. Ch. 40.3 5/5 2/15 8 Sol. Ch. 55.8 2/5 6/6 9* Sol. Am & 52.8 3/5 0/9 Sol. Ch.
Sol. Am & 58.6 5/5 15/15 Sol. Ch.
120 young Sprague-Dawley rats weighing between 200-300 grams were used for the animal injections. Twelve groups of animals were studied including groups injected with Zyderm II~ and Zyplast~. There were 10 rats in each group. 0.2 ml of injectable material was injected percutaneously into thelr backs below the panniculus carnosus in three separate sites. Samples were harvested from 5 rats in each qroup at one month and six months.
Speci~ens were~fixed in 10% buffered formalin and later embedded in paraffin. Serial sections were made and stained with hematoxylin-eosin (H&E). Results were compared using an independent staff pathologist.
Th~e histological evaluation of implants revealed similarities of all the experimental groups with the exception of the insoluble amnion which will be subsequently discussed.
Specifically, no inflammatory reaction was evident and no encapsulation occurred. Fibrocytic ingrowth is present at 30 days and is more advanced centripetally at 180 days.
Neovascularization was present in a variable extent at 180 1.3~8~33 days. The degree of both fibrocytic ingrowth and neovascularization was greater than that found with Zyderm II~ and Zyplast~ at 180 days.
The insoluble amnion group also demonstrated no inflammation or encapsulation but marked neovascularization and fibrocytic ingrowth. An additional observation was the presence of a few adipocytes at 3~ days. At 180 days the adipocytes that first appeared on the periphery of the implant had become dispersed throughout the implant. This phenomena has been observed by others27'~8.
A quantitative analysis (measuring exact dimensions of persisting implant) was very difficult to perform. Therefore, a preliminary qualitative study of persistence was performed.
Simply, the number of implants visibly present in the rats surviving at 6 months was counted. Table 2 demonstrates that the soluble amnion group showed very good persistence at 6 months. The nonirradiated soluble amnion had all implants present at 6 months and the irradiated soluble amnion had 11 out of 15. The insoluble group also showed good persistence in both the irradiated and nonirradiated groups. Some of the groups having dombinations of materials especially soluble chorion had less persistence after irradiation.
131~33 Table 2 PERSISTENCE OF SOFT TISSUE AUGMENTATION
ANIMAL EXPERIMENT GROUP 1-10, ZYDER~ and ZYPLAST~
Concentration Injections Present of CollagenNumber of Rats After 6 Months/
GrouP (mq/ml) Survival/Injected Total Number Iniected l* Sol. Am 51 5/5 . 11/15 2 Sol. Am 37.2 5/5 15/15 3* Ins. Am 23.5 5/5 9/15 4 Ins. Am 22 .2 5/5 13/15 5* Ins. Am & 40.4 4/5 3/12 Sol. Am 6 Ins. Am & 40.5 5/5 15/15 7* Sol. Ch. 40.3 5/5 2/15 8 Sol. Ch. 55.8 2/5 6/6 9* Sol. Am & 52.8 3/5 0/9 Sol. Ch.
Sol. Am & 58.6 5/5 15/15 Sol. Ch.
11 Zyderm II~ 65 2/5 6/6 12 Zyplast~ 35 4/5 12/12 Note: *Sample was irradiated with 0. 25 M rads gamma irradiation.
Numerous histological studies at intervals from one to six months have shown fibrocytic ingrowth as well as neovascularization with no inflammation. The observation of adipocyte incorporation into the insoluble amnion implants is very interesting but we are unable to explain why it occurs.
Stromal infiltration of fat has been previously observed by others27'2~ and is a strange phenomenom of an obscure nature. Accumulation of increased amounts of fat occasionally occurs within the interstitial tissue of certain tissues and organs. Robbins and Angel29 offer an explanation that presumably the multipotential fibroblasts in the interstitium become filled with lipid and in effect are converted to "obese"
fat cells. Stromal infiltration by fat tends to be associated with obesity or with atrophy of parenchymal cells, but the ~3~ ?33 correlations are imperfect. The heart and pancreas are most often involved.
The fact that there were only a few adipocytes at 30 days and an increased number at 180 days would seem to indicate a phenomenom occurred similar to that described above. The stimulation of new adipocyte deposition offers an interesting and new possible avenue to soft tissue augmentation.
Biochemical analysis has demonstrated the purity of the injectable human amnion and chorion collagen. The PAGE
electrophoresis and amino acid assay have shown HAC to have a typical collagen electrophoresis pattern and an amino acid composition of a mixture of Type I and III collagen.
The bacterial collagenase digestion results demonstrated that no noncollagen protein existed in injectable human amnion and chorion collagen. By immunoblotting examination, no residual fibronectin and laminin was detected in the collagen product of the present invention. We believe that the high purity of HAC contributed to the low immunological response in the rat model.
The proportion of type III collagen to type I collage is much larger in injectable human amnion collagen (43:57) than in Zyderm~ and Zyplast~ (5:95). Type III collagen has inter-chain disulfate bonds, whereas type I collagen does not, so after treatment by pepsin extraction, type I collagen exists in the form as a mixture of a, B and Y chains while type III collagen exists mainly in the Y form. It is postulated that the greater the cross-linking, ~he longer the persistence of type III collagen in amnion collagen would appear as a factor of importance in considering the most efficacious collagen for soft tissue augmentation.
Cell biology studies demonstrated that gamma irradiated human amnion collagen had no cytotoxic effect on cell growth and on cell behavior. Cells growing on irradiated ~3~8~33 human amnion collagen substrate have a normal morphological appearance and active pseudopodia. In cell number and [3H]
incorporated experiments, no significant differences were found among the cells growing on irradiated human amnion collagen and Vitrogen~, Zyderm~ and nonirradiated human amnion collagen.
The biochemical characterization of the various injectable human amnion collagens for purity assessment, and ratio of collagen types, included polyacrylamide gel electrophoresis (PAGE), densitometry, collagenase digestion, I amino acid analysis (AAA), immunoblotting, electron-microscopy (EM), collagenase sensitivity assay, and hydroxyproline assay.
No detailed description is given or deemed necessary of these various procedures since they are all conventional.
Good results are obtainable with gamma irradiation at a minimum of .20 M rads and in the range of .25 M rads and 2.0 M rads. Any combination of the amnions and chorion can be employed with good results.
The foregoing animal study is an excellent model for injection of the injectable material into human beings.
The present invention, therefore, is well suited and adapted to attain the objects and ends and has the features and advantages mentioned as well as others inherent therein.
While presently pre~erred examples have been given for the purpose of disclosure, changes can be made therein which are within the spirit of the invention as defined by the scope o~ the appended claims.
:~3/C~8(;~33 References 1. Gersuny, O: Ueber sine subcutane prothese. Z. Heilkunde.
9 : 1 , 1 9 0 0 .
2. Heidingsfeld, ML: Histopathology of paraffin prosthesis.
J Cutan Dis 24:513, 1958.
3. Conway, H; Goulian, D: Experience with an injectable silastic RrU as a subcutaneous prosthetic material. Plast Reconstr Surg 32:294, 1963.
4. Rees, TD; Platt, J; Ballantyne, DL: An investigation of cutaneous response to dimethylpolysiloxanes (silicone liquid) in animals and humans. A preliminary report.
Plast Reconstr Surg 35:131, 1965.
5. Sternberg, TH; Ashley, FL; Wines, LH; Lehman, R:
Gewebereaktionen auf injizierte Flussige Siliciumverbindungen. Hautarzt 15:281, 1964.
6. Ben-Hur, N; Neuman, Z: Siliconoma, another cutaneous response to the dimethylpolysiloxane. Plast Reconstr Surg 36:629, 1965.
7. Ashley, FL, Braley, S, McNail, EG: The current status of silicone injection therapy. Surg Clin North Am 51:501, 1971.
8. Ashley, FL, Thompson, DP, Henderson, T: Augmentation of surface contour by subcutaneous injections of silicone fluid. Plast Reconstr Surg 51:8, 1973.
9. Blocksma, R: Experience with dimethylpolysiolxane fluid in soft tissue augmentation. Plast Reconstr Surg 48:564, 1971.
10. Milojevie, B: Complications after silicone injection therapy in aesthetic plastic surgery. Aesth Plast Surg 6:203,1982.
11. Rees, TD, Ashley, FL: Treatment of facial atrophy with liquid silicone. Am J Surg 111:531, 1966.
12. Rees, TD, Coburn, FJ: Silicone treatment of partial lipodystrophy. JAMA 230:868, 1974.
Numerous histological studies at intervals from one to six months have shown fibrocytic ingrowth as well as neovascularization with no inflammation. The observation of adipocyte incorporation into the insoluble amnion implants is very interesting but we are unable to explain why it occurs.
Stromal infiltration of fat has been previously observed by others27'2~ and is a strange phenomenom of an obscure nature. Accumulation of increased amounts of fat occasionally occurs within the interstitial tissue of certain tissues and organs. Robbins and Angel29 offer an explanation that presumably the multipotential fibroblasts in the interstitium become filled with lipid and in effect are converted to "obese"
fat cells. Stromal infiltration by fat tends to be associated with obesity or with atrophy of parenchymal cells, but the ~3~ ?33 correlations are imperfect. The heart and pancreas are most often involved.
The fact that there were only a few adipocytes at 30 days and an increased number at 180 days would seem to indicate a phenomenom occurred similar to that described above. The stimulation of new adipocyte deposition offers an interesting and new possible avenue to soft tissue augmentation.
Biochemical analysis has demonstrated the purity of the injectable human amnion and chorion collagen. The PAGE
electrophoresis and amino acid assay have shown HAC to have a typical collagen electrophoresis pattern and an amino acid composition of a mixture of Type I and III collagen.
The bacterial collagenase digestion results demonstrated that no noncollagen protein existed in injectable human amnion and chorion collagen. By immunoblotting examination, no residual fibronectin and laminin was detected in the collagen product of the present invention. We believe that the high purity of HAC contributed to the low immunological response in the rat model.
The proportion of type III collagen to type I collage is much larger in injectable human amnion collagen (43:57) than in Zyderm~ and Zyplast~ (5:95). Type III collagen has inter-chain disulfate bonds, whereas type I collagen does not, so after treatment by pepsin extraction, type I collagen exists in the form as a mixture of a, B and Y chains while type III collagen exists mainly in the Y form. It is postulated that the greater the cross-linking, ~he longer the persistence of type III collagen in amnion collagen would appear as a factor of importance in considering the most efficacious collagen for soft tissue augmentation.
Cell biology studies demonstrated that gamma irradiated human amnion collagen had no cytotoxic effect on cell growth and on cell behavior. Cells growing on irradiated ~3~8~33 human amnion collagen substrate have a normal morphological appearance and active pseudopodia. In cell number and [3H]
incorporated experiments, no significant differences were found among the cells growing on irradiated human amnion collagen and Vitrogen~, Zyderm~ and nonirradiated human amnion collagen.
The biochemical characterization of the various injectable human amnion collagens for purity assessment, and ratio of collagen types, included polyacrylamide gel electrophoresis (PAGE), densitometry, collagenase digestion, I amino acid analysis (AAA), immunoblotting, electron-microscopy (EM), collagenase sensitivity assay, and hydroxyproline assay.
No detailed description is given or deemed necessary of these various procedures since they are all conventional.
Good results are obtainable with gamma irradiation at a minimum of .20 M rads and in the range of .25 M rads and 2.0 M rads. Any combination of the amnions and chorion can be employed with good results.
The foregoing animal study is an excellent model for injection of the injectable material into human beings.
The present invention, therefore, is well suited and adapted to attain the objects and ends and has the features and advantages mentioned as well as others inherent therein.
While presently pre~erred examples have been given for the purpose of disclosure, changes can be made therein which are within the spirit of the invention as defined by the scope o~ the appended claims.
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Claims (21)
1. An injectable soft tissue augmentation material comprising a sterilized mixture of type I and type III collagen extracted by proteolytic digestion from the group consisting of insoluble amnion, soluble amnion, soluble chorion from human placenta and combinations thereof, homogenized to pass through a surgical needle.
2. The injectable soft tissue augmentation material of Claim 1 where the collagen is homogenized to pass through a 30 gauge surgical needle.
3. The injectable soft tissue augmentation material of Claim 1 where the selected collagen is from soluble amnion.
4. The injectable soft tissue augmentation material of Claim 1 where the collagen is cross-linked by gamma irradiation.
5. The injectable soft tissue augmentation material of Claim 4 wherein the gamma irradiation is a minimum of 0.20 M rads.
6. The injectable soft tissue augmentation material of Claim 4 wherein the gamma irradiation is in the range of from 0.25 M rads to 2.0 M rads.
7. The injectable soft tissue augmentation material of Claim 1 which includes an analgesic.
8. A method of making an injectable soft tissue material comprising sterilizing a mixture of type I and type III collagen extracted by pepsin digestion selected from the group consisting of insoluble amnion, soluble amnion, soluble chorion from human placenta and combinations thereof, and homogenizing the material sufficiently to pass through at least a 25 gauge surgical needle.
9. The method of making the injectable soft tissue material of Claim 8 comprising cross-linking molecules of the collagen by gamma irradiation.
10. The method of Claim 9 wherein the gamma irradiation is a minimum of 0.20 M rads.
11. The method of Claim 9 wherein the gamma irradiation is in the range of .25 M rads to 2.0 M rads.
12. The method of making the injectable soft tissue material of Claim 8 including adding an analgesic to the homogenized collagen.
13. The use of the collagen of Claim 1 for augmenting soft tissue in a human being.
14. The use of the collagen of Claim 2 for augmenting soft tissue in a human being.
15. The use of the collagen of Claim 3 for augmenting soft tissue in a human being.
16. The use of the collagen of Claim 4 for augmenting soft tissue in a human being.
17. The use of the collagen of Claim 5 for augmenting soft tissue in a human being.
18. The use of the collagen of Claim 6 for augmenting soft tissue in a human being.
19. The use of the collagen of Claim 7 for augmenting soft tissue in a human being.
20. The injectable soft tissue augmentation material of Claim 1 wherein the proteolytic digestion is with pepsin.
21. The use of the collagen of claim 20 for augmenting soft tissue in a human being.
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GB8708009 | 1987-04-03 | ||
GB878708009A GB8708009D0 (en) | 1987-04-03 | 1987-04-03 | Injectable soft tissue augmentation materials |
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CA1308033C true CA1308033C (en) | 1992-09-29 |
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CA000562773A Expired - Lifetime CA1308033C (en) | 1987-04-03 | 1988-03-29 | Injectable soft tissue augmentation materials from the placenta and their method of manufacture |
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EP (1) | EP0285370B2 (en) |
JP (1) | JP2643976B2 (en) |
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AT (1) | ATE105481T1 (en) |
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CA (1) | CA1308033C (en) |
DE (1) | DE3889489T3 (en) |
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1988
- 1988-03-29 AT AT8888302782T patent/ATE105481T1/en not_active IP Right Cessation
- 1988-03-29 IE IE94688A patent/IE64226B1/en not_active IP Right Cessation
- 1988-03-29 NO NO881406A patent/NO176551C/en not_active IP Right Cessation
- 1988-03-29 ES ES88302782T patent/ES2052709T5/en not_active Expired - Lifetime
- 1988-03-29 EP EP88302782A patent/EP0285370B2/en not_active Expired - Lifetime
- 1988-03-29 DE DE3889489T patent/DE3889489T3/en not_active Expired - Lifetime
- 1988-03-29 CA CA000562773A patent/CA1308033C/en not_active Expired - Lifetime
- 1988-03-30 DK DK198801789A patent/DK173020B1/en not_active IP Right Cessation
- 1988-03-30 NZ NZ224085A patent/NZ224085A/en unknown
- 1988-03-30 IL IL85923A patent/IL85923A/en not_active IP Right Cessation
- 1988-03-31 AU AU14085/88A patent/AU609108B2/en not_active Expired
- 1988-03-31 FI FI881518A patent/FI92906C/en not_active IP Right Cessation
- 1988-03-31 ZA ZA882306A patent/ZA882306B/en unknown
- 1988-03-31 PT PT87150A patent/PT87150B/en not_active IP Right Cessation
- 1988-04-02 JP JP63082864A patent/JP2643976B2/en not_active Expired - Lifetime
- 1988-04-02 KR KR1019880003722A patent/KR960016207B1/en not_active IP Right Cessation
-
1989
- 1989-09-27 US US07/413,885 patent/US5002071A/en not_active Expired - Lifetime
-
1998
- 1998-06-23 HK HK98106036A patent/HK1006677A1/en not_active IP Right Cessation
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